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

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

    SciTech Connect

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

    1987-10-06

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

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

    PubMed Central

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

    2013-01-01

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

  3. Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l-isoleucine production.

    PubMed

    Ma, Wenjian; Wang, Jianli; Li, Ye; Hu, Xiaoqing; Shi, Feng; Wang, Xiaoyuan

    2016-11-01

    Three genes, gnd, pgl, and fbp, relevant to the pentose phosphate pathway (PPP) were overexpressed in Corynebacterium glutamicum IWJ001, leading to increase of l-isoleucine production. The transcriptional levels of gnd, pgl, and fbp significantly increased in IWJ001/pDXW-8-gnd-fbp-pgl. Compared with the control strain IWJ001/pDXW-8, intracellular NADPH/NADP(+) ratios in IWJ001/pDXW-8-gnd and IWJ001/pDXW-8-gnd-fbp cells grown for 36 H increased threefold and fourfold, respectively, indicating that overexpression of gnd and fbp redirected the carbon flux to PPP. Intracellular NADPH/NADP(+) ratio in IWJ001/pDXW-8-gnd-fbp-pgl grown for 36 H was similar to IWJ001/pDXW-8, suggesting that the NADPH produced by PPP could be quickly consumed for l-isoleucine production. 10.9 and 28.96 g/L of l-isoleucine was produced in IWJ001/pDXW-8-gnd-fbp-pgl in shake flask cultivation and fed-batch fermentation, respectively. In addition, IWJ001/pDXW-8-gnd-fbp-pgl grew fast, its dry cell weight reached 49 g/L after 48 H, whereas the start strain IWJ001/pDXW-8 reached only 40 g/L. After 96 H fermentation, l-isoleucine yield on glucose in IWJ001/pDXW-8-gnd-fbp-pgl reached 0.138 g/g. The results demonstrate that carbon flux redirection to PPP is an efficient approach to enhance l-isoleucine production in C. glutamicum.

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2015-03-01

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

  6. Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

    PubMed Central

    Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki

    2012-01-01

    Production of l-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the l-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall l-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of l-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for l-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD+ ratio significantly decreased, and glucose consumption and l-valine production drastically improved. Moreover, l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for l-valine production under oxygen deprivation conditions. The l-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM l-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and the l-valine productivity reached 1,940 mM after 48 h. PMID:22138982

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

    PubMed

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

    2015-11-01

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

  8. Exogenous l-Valine Promotes Phagocytosis to Kill Multidrug-Resistant Bacterial Pathogens

    PubMed Central

    Chen, Xin-hai; Liu, Shi-rao; Peng, Bo; Li, Dan; Cheng, Zhi-xue; Zhu, Jia-xin; Zhang, Song; Peng, Yu-ming; Li, Hui; Zhang, Tian-tuo; Peng, Xuan-xian

    2017-01-01

    The emergence of multidrug-resistant bacteria presents a severe threat to public health and causes extensive losses in livestock husbandry and aquaculture. Effective strategies to control such infections are in high demand. Enhancing host immunity is an ideal strategy with fewer side effects than antibiotics. To explore metabolite candidates, we applied a metabolomics approach to investigate the metabolic profiles of mice after Klebsiella pneumoniae infection. Compared with the mice that died from K. pneumoniae infection, mice that survived the infection displayed elevated levels of l-valine. Our analysis showed that l-valine increased macrophage phagocytosis, thereby reducing the load of pathogens; this effect was not only limited to K. pneumoniae but also included Escherichia coli clinical isolates in infected tissues. Two mechanisms are involved in this process: l-valine activating the PI3K/Akt1 pathway and promoting NO production through the inhibition of arginase activity. The NO precursor l-arginine is necessary for l-valine-stimulated macrophage phagocytosis. The valine-arginine combination therapy effectively killed K. pneumoniae and exerted similar effects in other Gram-negative (E. coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. Our study extends the role of metabolism in innate immunity and develops the possibility of employing the metabolic modulator-mediated innate immunity as a therapy for bacterial infections. PMID:28321214

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

    PubMed

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

    2015-08-01

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

  10. l-Leucine acts as a potential agent in reducing body temperature at hatching and affords thermotolerance in broiler chicks.

    PubMed

    Han, Guofeng; Yang, Hui; Bahry, Mohammad A; Tran, Phuong V; Do, Phong H; Ikeda, Hiromi; Furuse, Mitsuhiro; Chowdhury, Vishwajit S

    2017-02-01

    Thermal manipulation (TM) of incubation temperature causes metabolic alterations and contributes to improving thermotolerance in chicks post hatching. However, there has been no report on amino acid metabolism during TM and the part it plays in thermotolerance. In this study, we therefore first analyzed free amino acid concentrations in the embryonic brain and liver during TM (38.6°C, 6h/d during embryonic day (ED) 10 to ED 18). It was found that leucine (Leu), phenylalanine and lysine were significantly decreased in the embryonic brain and liver. We then chose l-Leu and other branched-chain amino acids (l-isoleucine (L-Ile) and l-valine (l-Val)) for in ovo injection on ED 7 to reveal their roles in thermoregulation, growth, food intake and thermotolerance in chicks. It was found that in ovo injection of l-Leu, but not of l-Ileu or l-Val, caused a significant decline in body temperature at hatching and increased food intake and body weight gain in broiler chicks. Interestingly, in ovo injection of l-Leu resulted in the acquisition of thermotolerance under high ambient temperature (35±1°C for 180min) in comparison with the control thermoneutral temperature (28±1°C for 180min). These results indicate that the free amino acid concentrations during embryogenesis were altered by TM. l-Leu administration in eggs caused a reduction in body temperature at hatching, and afforded thermotolerance in heat-exposed young chicks, further suggesting that l-Leu may be one of the key metabolic factors involved in controlling body temperature in embryos, as well as in producing thermotolerance after hatching.

  11. L-isoleucine-supplemented oral rehydration solution in the treatment of acute diarrhoea in children: a randomized controlled trial.

    PubMed

    Alam, N H; Raqib, R; Ashraf, H; Qadri, F; Ahmed, S; Zasloff, M; Agerberth, B; Salam, M A; Gyr, N; Meier, R

    2011-06-01

    Antimicrobial peptides represent an important component of the innate immune defenses of living organisms, including humans. They are broad-spectrum surface-acting agents secreted by the epithelial cells of the body in response to infection. Recently, L-isoleucine and its analogues have been found to induce antimicrobial peptides. The objectives of the study were to examine if addition of L-isoleucine to oral rehydration salts (ORS) solution would reduce stool output and/or duration of acute diarrhoea in children and induce antimicrobial peptides in intestine. This double-blind randomized controlled trial was conducted at the Dhaka Hospital of ICDDR,B. Fifty male children, aged 6-36 months, with acute diarrhoea and some dehydration, attending the hospital, were included in the study. Twenty-five children received L-isoleucine (2 g/L)-added ORS (study), and 25 received ORS without L-isoleucine (control). Stool weight, ORS intake, and duration of diarrhoea were the primary outcomes. There was a trend in reduction in mean +/- standard deviation (SD) daily stool output (g) of children in the L-isoleucine group from day 2 but it was significant on day 3 (388 +/- 261 vs. 653 +/- 446; the difference between mean [95% confidence interval (CI) (-)265 (-509, -20); p = 0.035]. Although the cumulative stool output from day 1 to day 3 reduced by 26% in the isoleucine group, it was not significant. Also, there was a trend in reduction in the mean +/- SD intake of ORS solution (mL) in the L-isoleucine group but it was significant only on day 1 (410 +/- 169 vs. 564 +/- 301), the difference between mean (95% CI) (-)154 (-288, -18); p = 0.04. The duration (hours) of diarrhoea was similar in both the groups. A gradual increase in stool concentrations of beta-defensin 2 and 3 was noted but they were not significantly different between the groups. L-isoleucine-supplemented ORS might be beneficial in reducing stool output and ORS intake in children with acute watery diarrhoea. A further

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

    PubMed

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

    2016-02-20

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

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

    PubMed

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

    2012-09-13

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

  14. Attenuation of paraquat-induced motor behavior and neurochemical disturbances by L-valine in vivo.

    PubMed

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

    2004-05-02

    Alterations of motor behavioral patterns and monoamine contents in the discrete rat brain areas after acute paraquat exposure (3, 5, 10, 20 mg/kg, s.c.) have been studied. The results showed that paraquat at the doses of 5, 10, and 20 mg/kg significantly reduced locomotive, stereotypic, and rotational behaviors. Significant decreases of norepinephrine (NE) contents in cortex and hypothalamus, as well as striatal contents of dopamine (DA) and its acidic metabolites, were detected. In addition, L-valine (200 mg/kg, i.p.) significantly attenuated paraquat-induced toxicity at moderate dose (5 mg/kg) but not at high dose (20 mg/kg). The results provide evidence that paraquat can enter the brain as illustrated by the alterations in the motor behavioral pattern and neurochemical contents. Furthermore, the attenuation effect of L-valine against systemic administration of paraquat-induced motor behaviors was detected, with a slightly protective effect on paraquat-induced neurochemical alterations.

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

    PubMed

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

    2016-10-01

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

  16. 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.

  17. Enthalpy characteristics of the dissolution of L-valine in water/formamide mixtures at 298.15 K

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

    The thermochemical dissolution of L-valine in solvent mixtures H2O + (formamide, N-methylformamide, and N, N-dimethylformamide) is studied at an organic component concentration of x 2 = 0-0.35 molar fractions and a temperature of 298.15 K. The experimental data are used to calculate standard enthalpies of dissolution, the transferring of L-valine from water to a mixed solvent, and the enthalpy coefficients of pairwise interactions ( h xy ) with organic solvent molecules. The correlation between the enthalpy characteristics of the dissolution of L-valine with the composition of aqueous organic mixtures and the nature of the organic solvent (its physicochemical properties) is determined. A comparative analysis of the values of h xy of a number of aliphatic L-amino acids in similar solvent mixtures with the hydrophobicity parameters of their side chains is performed.

  18. 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.

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

    PubMed

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

    2016-11-01

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

  20. Metabolic mechanism for L-leucine-induced metabolome to eliminate Streptococcus iniae.

    PubMed

    Du, Chao-Chao; Yang, Manjun; Li, Min-Yi; Yang, Jun; Peng, Bo; Li, Hui; Peng, Xuan-Xian

    2017-03-07

    Crucial metabolites that modulate hosts' metabolome to eliminate bacterial pathogens have been documented, but the metabolic mechanisms are largely unknown. The present study explores the metabolic mechanism for L-leucine-induced metabolome to eliminate Streptococcus iniae in tilapia. GC-MS based metabolomics was used to investigate tilapia liver metabolic profile in the presence of exogenous L-leucine. Thirty-seven metabolites of differential abundance were determined, and eleven metabolic pathways were enriched. Pattern recognition analysis identified serine and proline as crucial metabolites, which are the two metabolites identified in survived tilapias during S. iniae infection, suggesting the two metabolites play crucial roles in L-leucine-induced elimination of the pathogen by the host. Exogenous L-serine reduces mortality of tilapias infected by S. iniae, providing a robust proof for supporting the conclusion. Furthermore, exogenous serine elevates expression of genes Il-1β and Il-8 in tilapia spleen, but not TNFα, CXCR4 and Mx, suggesting the metabolite promotes a phagocytosis role of macrophages, which is consistent with the finding that L-leucine promotes macrophages to kill both Gram-positive and negative bacterial pathogens. Therefore, the ability of phagocytosis enhanced by exogenous L-leucine is partly attributed to elevation of serine. These results demonstrate a metabolic mechanism by which exogenous L-leucine modulates tilapias' metabolome to enhance innate immunity and eliminate pathogens.

  1. Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum.

    PubMed

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

    2011-09-01

    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 (13)C 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.

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    1996-03-01

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

  6. l-Isoleucine in a Choline Chloride/Ethylene Glycol Deep Eutectic Solvent: A Reusable Reaction Kit for the Asymmetric Cross-Aldol Carboligation.

    PubMed

    Fanjul-Mosteirín, Noé; Concellón, Carmen; Del Amo, Vicente

    2016-09-02

    l-Isoleucine is able to catalyze the cross-aldol reaction between cyclohexanone and aromatic aldehydes in a deep eutectic solvent consisting in choline chloride and ethylene glycol, rendering products with high diatereo- and enantioselectivity. This protocol is straightforward and green: the organocatalyst and the reaction medium can be recycled up to five times, allowing the preparation of different substrates with a single load of solvent and catalyst.

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

    PubMed

    Matheis, Katrin; Granvogl, Michael; Schieberle, Peter

    2016-01-27

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

  8. 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.

  9. L-leucine supplementation worsens the adiposity of already obese rats by promoting a hypothalamic pattern of gene expression that favors fat accumulation.

    PubMed

    Zampieri, Thais T; Torres-Leal, Francisco L; Campaña, Amanda B; Lima, Fabio B; Donato, Jose

    2014-04-02

    Several studies showed that l-leucine supplementation reduces adiposity when provided before the onset of obesity. We studied rats that were exposed to a high-fat diet (HFD) for 10 weeks before they started to receive l-leucine supplementation. Fat mass was increased in l-leucine-supplemented rats consuming the HFD. Accordingly, l-leucine produced a hypothalamic pattern of gene expression that favors fat accumulation. In conclusion, l-leucine supplementation worsened the adiposity of rats previously exposed to HFD possibly by central mechanisms.

  10. X-Ray and 35Cl NQR Studies on the Trichloroacetyl Group in Covalent and Ionic Compounds of L-Valine and DL-Valine

    NASA Astrophysics Data System (ADS)

    Ofial, A. R.; Dou, S.-q.; Krishnan, V. G.; Paulus, H.; Fuess, H.; Weiss, Al.

    1997-03-01

    The crystal structures and 35Cl NQR of trichloroacetyl-DL-valine (1), trichloroacetyl-L-valine (2), as well as the salts of trichloroacetic acid with DL-valine (3) and L-valine (4) have been investigated. Crystal data are for (1): Monoclinic, C2/c, a= 15.835(4) Å, b= 10.481 (3) Å, c = 14.046(4) Å, β = 103.28(1), Z = 8; (2): Orthorhombic, P212121, a = 12.117(3) Å, 6=10.896(3) Å, c= 8.718(2) Å, Z = 4; (3): Triclinic, P1¯, a= 17.269(3) Å, 6 = 8.504(3) Å, c = 10.427(4) A, a = 105.38(2), β = 96.98(2), γ = 96.24(2), Z = 2; (4): Monoclinic, P21, a = 10.378(4) Å, b = 20.349(8) Å, c= 11.890(5) Å, β = 95.28(2), Z = 8. The onset of rotational motion within the trichloroacetyl groups bleaches out 35Cl NQR lines between 115 K and 185 K for (1)-(4). While TCA-L-valine (1), TCA-DL-valine (2), and TCA(-) · DL-valine(+) (3) do not show any phase transition in the temperature range 77 K to 295 K, TCA(-) · L-valine(+) (4) shows more than one phase transition above 77 K before the three NQR signals bleach out at 164 K.

  11. Growth, spectral, linear and nonlinear optical characteristics of an efficient semiorganic acentric crystal: L-valinium L-valine chloride

    NASA Astrophysics Data System (ADS)

    Nageshwari, M.; Jayaprakash, P.; Kumari, C. Rathika Thaya; Vinitha, G.; Caroline, M. Lydia

    2017-04-01

    An efficient nonlinear optical semiorganic material L-valinium L-valine chloride (LVVCl) was synthesized and grown-up by means of slow evaporation process. Single crystal XRD evince that LVVCl corresponds to monoclinic system having acentric space group P21. The diverse functional groups existing in LVVCl were discovered with FTIR spectral investigation. The UV-Visible and photoluminescence spectrum discloses the optical and electronic properties respectively for the grown crystal. Several optical properties specifically extinction coefficient, reflectance, linear refractive index, electrical and optical conductivity were also determined. The SEM analysis was also carried out and it portrayed the surface morphology of LVVCl. The calculated value of laser damage threshold was 2.59 GW/cm2. The mechanical and dielectric property of LVVCl was investigated employing microhardness and dielectric studies. The second and third order nonlinear optical characteristics of LVVCl was characterized utilizing Kurtz Perry and Z scan technique respectively clearly suggest its suitability in the domain of optics and photonics.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  13. Structure of free radicals in irradiated acetyl-L-leucine single crystals at 77 K

    SciTech Connect

    Almanov, G.A.; Bogdanchikov, G.A.; Usov, O.M.

    1988-09-01

    By using the EPR method, two types of radicals are observed, which are formed in acetyl-L-leucine single crystals irradiated at 77K. These are alkyl type radicals (CH/sub 3/)/sub 2/CCH/sub 2/CH(NHCOCH/sub 3/)COOH and peptide group radicals. When the crystals are defrozen to room temperatures, the radicals of the second type disappear without formation of paramagnetic particles. Two possible structures of the peptide group radicals were studied by the INDO method. On defreezing to room temperature, the alkyl group radical is retained, while the peptide radical disappears without formation of paramagnetic particles. For the protonated form of the anion-radical, a better agreement is observed between the theoretically calculated and the experimentally obtained HFI constants. The quantum chemical analysis of the possible structures of the peptide group radicals indicates that the formation of the protonated form of the anion-radical is energetically favorable.

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-11-13

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

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

    PubMed

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

    2013-02-28

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

  20. The role of an L-leucine residue on the conformations of glycyl-L-leucine oligomers and its N- or C-terminal dependence: infrared absorption and Raman scattering studies.

    PubMed

    Okabayashi, Hiro-Fumi; Kanbe, Hide-Hiro; O'Connor, Charmian J

    2016-01-01

    The conformations of glycyl-L-leucine oligomers (GnL, residue number n = 3, 4, and 5) in the solid state were found to be similar to that of a polyglycine II (PGII). However, for L-leucyl-glycine oligomers (LGn; n = 3, 4, 5) in the solid state, LG3 and LG4 have already been confirmed to take a reverse-turn structure (LG3-type reverse-turn) while LG5 adopts a PGII-type helix. The present results provide evidence that the conformations of L-leucine-containing glycine oligomers depend strongly upon whether the L-leucine residue is placed in the N- or C-terminal position. For the aqueous G3L and G4L samples, we assumed that reverse-turn structures similar to the type II β-turn, rather than the LG3-type reverse-turn, are stabilized in concentrated solution, probably as the result of aggregation. Models to explain the mechanism of these phenomena are presented.

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

    PubMed

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

    2015-03-15

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

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

    PubMed Central

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

    2015-01-01

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

  3. 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

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

    PubMed

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

    2013-01-01

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

  5. Spectroscopic, thermal and structural studies of new L-leucine and D-leucine complexes with chloranilic acid

    NASA Astrophysics Data System (ADS)

    Pawlukojć, A.; Hetmańczyk, J.; Nowicka-Scheibe, J.; Maurin, Jan K.; Schilf, W.; Rozwadowski, Z.

    2017-04-01

    New molecular complexes of L-leucine and D-leucine with chloranilic acid have been synthesized. Crystal structures of these crystals have been solved; they crystallize in non-centrosymmetric monoclinic P21 space group. In the crystal, leucine molecules exist in protonated form (C6NO2H14+) and chloranilic acid molecules in deprotonated form (C6HCl2O4-). Electronic (UV-Vis) and vibrational absorption (VA) spectra for both materials were collected. Circular dichroism methods such as electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) were used to determine the absolute configuration of new complexes. In both methods a characteristic (-,+) Cotton patterns are observed. In ECD spectra absorption bands are observed at 212 nm for acetonitrile solution and at 202 nm for aqueous solution. VCD spectra (in DMSO-d6 solution) show (-,+) Cotton pattern with strong peaks at 1323 cm-1 (CH rocking mode). Differential scanning calorimetry (DSC) and thermogravimetric (TG) investigations were performed to explore thermal properties of new materials. In DSC curve the decomposition and combustion processes are observed in 220-227 °C. The decomposition process was described by use of TG method and quadruple mass spectrometer (QMS). NMR spectra of pure L-leucine and chloranilic acid as well as L-leucine - chloranilic acid complex in solutions (D2O and DMSO) and in solid state confirm the geometry of molecules in complex both in solution and in solid state.

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

    Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM K55.c, was coordinated by the Bureau International des Poids et Mesures (BIPM) in 2012. Twenty National Measurement Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of valine present as the main component in the comparison sample for CCQM-K55.c. The comparison samples were prepared from analytical grade L-valine purchased from a commercial supplier and used as provided without further treatment or purification. Valine was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of organic compounds of low structural complexity [molecular weight range 100-300] and high polarity (pKOW > -2). The KCRV for the valine content of the material was 992.0 mg/g with a combined standard uncertainty of 0.3 mg/g. The key comparison reference value (KCRV) was assigned by combination of KCRVs assigned from participant results for each orthogonal impurity class. The relative expanded uncertainties reported by laboratories having results consistent with the KCRV ranged from 1 mg/g to 6 mg/g when using mass balance based approaches alone, 2 mg/g to 7 mg/g using quantitative 1H NMR (qNMR) based approaches and from 1 mg/g to 2.5 mg/g when a result obtained by a mass balance method was combined with a separate qNMR result. The material provided several analytical challenges. In addition to the need to identify and quantify various related amino acid impurities including leucine, isoleucine, alanine and α-amino butyrate, care was required to select appropriate conditions for performing Karl Fischer titration assay for water content to avoid bias due to in situ formation of water by self-condensation under the assay conditions. It also proved to be a challenging compound for purity assignment by qNMR techniques

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

    PubMed

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

    2011-10-01

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

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

    PubMed

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

    2014-11-01

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

  10. Deuterium isotope effect on 13C chemical shifts of tetrabutylammonium salts of Schiff bases amino acids.

    PubMed

    Rozwadowski, Z

    2006-09-01

    Deuterium isotope effects on 13C chemical shift of tetrabutylammonium salts of Schiff bases, derivatives of amino acids (glycine, L-alanine, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-methionine) and various ortho-hydroxyaldehydes in CDCl3 have been measured. The results have shown that the tetrabutylammonium salts of the Schiff bases amino acids, being derivatives of 2-hydroxynaphthaldehyde and 3,5-dibromosalicylaldehyde, exist in the NH-form, while in the derivatives of salicylaldehyde and 5-bromosalicylaldehyde a proton transfer takes place. The interactions between COO- and NH groups stabilize the proton-transferred form through a bifurcated intramolecular hydrogen bond.

  11. Acetohydroxyacid synthases: evolution, structure, and function.

    PubMed

    Liu, Yadi; Li, Yanyan; Wang, Xiaoyuan

    2016-10-01

    Acetohydroxyacid synthase, a thiamine diphosphate-dependent enzyme, can condense either two pyruvate molecules to form acetolactate for synthesizing L-valine and L-leucine or pyruvate with 2-ketobutyrate to form acetohydroxybutyrate for synthesizing L-isoleucine. Because the key reaction catalyzed by acetohydroxyacid synthase in the biosynthetic pathways of branched-chain amino acids exists in plants, fungi, archaea, and bacteria, but not in animals, acetohydroxyacid synthase becomes a potential target for developing novel herbicides and antimicrobial compounds. In this article, the evolution, structure, and catalytic mechanism of acetohydroxyacid synthase are summarized.

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

    PubMed Central

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

    2014-01-01

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

  13. Prolonged oral treatment with an essential amino acid L-leucine does not affect female reproductive function and embryo-fetal development in rats.

    PubMed

    Mawatari, Kazunori; Katsumata, Toyohisa; Uematsu, Masanobu; Katsumata, Tomoyoshi; Yoshida, Junichi; Smriga, Miro; Kimura, Takeshi

    2004-09-01

    L-leucine, an essential amino acid, is one of the most popular ingredients in dietary supplements. To investigate a possibility of its embryo-fetal toxicity in rats, 11- to 12-week old dams were orally administered an aqueous solution of L-leucine at doses of 300 or 1000 mg/kg body weight on gestational days 7-17. Body weight and feed intake was evaluated throughout the whole course of pregnancy (days 0-20). L-Leucine did not influence body weight, but at a dose of 1000 mg/kg, slightly enhanced feed intake on days 14 and 18 of pregnancy. Caesarean section (day 20) revealed no influences on the litter size and weight of live-born fetuses, the number of corpora lutea, implantation index or the quality of placenta, and the minor increase in feed intake was considered irrelevant to the pregnancy outcomes. Fetuses were evaluated in a battery of external, visceral and skeletal examinations. No effects of L-leucine on gender ratio and external abnormalities, and no significant treatment-related variations in visceral and skeletal pathologies were observed. These results suggested that L-leucine, administered orally during organogenesis at doses up to 1000 mg/kg body weight, did not affect the outcome of pregnancy and did not cause fetotoxicity in rats.

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

    PubMed Central

    2015-01-01

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

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

    PubMed

    Mojibola, Adeolu; Dongmo-Momo, Gilles; Mohammed, Muzaffer; Aslan, Kadir

    2014-05-07

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

  16. Fluorescence and 19F NMR evidence that phenylalanine, 3-L-fluorophenylalanine and 4-L-fluorophenylalanine bind to the L-leucine specific receptor of Escherichia coli.

    PubMed Central

    Luck, L. A.; Johnson, C.

    2000-01-01

    The binding capacity of the L-leucine receptor from Escherichia coli was measured with L-phenylalanine and 4-fluoro-L-phenylalanine as substrates by fluorescence. The apparent dissociation constants (KD) for L-leucine, L-phenylalanine, and 4-fluoro-L-phenylalanine are 0.40, 0.18, and 0.26 respectively. 19F NMR data show protein-induced shifts for the 4-fluoro-L-phenylalanine peak and 3-fluoro-L-phenylalanine when receptor is present. Evidence points to the binding of only the L-isomers of these fluorine analogs. PMID:11206079

  17. Fermentative production of branched chain amino acids: a focus on metabolic engineering.

    PubMed

    Park, Jin Hwan; Lee, Sang Yup

    2010-01-01

    The branched chain amino acids (BCAAs), L-valine, L-leucine, and L-isoleucine, have recently been attracting much attention as their potential to be applied in various fields, including animal feed additive, cosmetics, and pharmaceuticals, increased. Strategies for developing microbial strains efficiently producing BCAAs are now in transition toward systems metabolic engineering from random mutagenesis. The metabolism and regulatory circuits of BCAA biosynthesis need to be thoroughly understood for designing system-wide metabolic engineering strategies. Here we review the current knowledge on BCAAs including their biosynthetic pathways, regulations, and export and transport systems. Recent advances in the development of BCAA production strains are also reviewed with a particular focus on L-valine production strain. At the end, the general strategies for developing BCAA overproducers by systems metabolic engineering are suggested.

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

    PubMed

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

    2012-09-13

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

  19. Differential effects of L-tryptophan and L-leucine administration on brain resting state functional networks and plasma hormone levels

    PubMed Central

    Zanchi, Davide; Meyer-Gerspach, Anne Christin; Suenderhauf, Claudia; Janach, Katharina; le Roux, Carel W.; Haller, Sven; Drewe, Jürgen; Beglinger, Christoph; Wölnerhanssen, Bettina K.; Borgwardt, Stefan

    2016-01-01

    Depending on their protein content, single meals can rapidly influence the uptake of amino acids into the brain and thereby modify brain functions. The current study investigates the effects of two different amino acids on the human gut-brain system, using a multimodal approach, integrating physiological and neuroimaging data. In a randomized, placebo-controlled trial, L-tryptophan, L-leucine, glucose and water were administered directly into the gut of 20 healthy subjects. Functional MRI (fMRI) in a resting state paradigm (RS), combined with the assessment of insulin and glucose blood concentration, was performed before and after treatment. Independent component analysis with dual regression technique was applied to RS-fMRI data. Results were corrected for multiple comparisons. In comparison to glucose and water, L-tryptophan consistently modifies the connectivity of the cingulate cortex in the default mode network, of the insula in the saliency network and of the sensory cortex in the somatosensory network. L-leucine has lesser effects on these functional networks. L-tryptophan and L-leucine also modified plasma insulin concentration. Finally, significant correlations were found between brain modifications after L-tryptophan administration and insulin plasma levels. This study shows that acute L-tryptophan and L-leucine intake directly influence the brain networks underpinning the food-reward system and appetite regulation. PMID:27760995

  20. 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.

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

    PubMed

    Li, Zhiyuan; Zhang, Liu; Li, Quanshun

    2015-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  3. Structure-property relations in crystalline L-leucine obtained from calorimetry, X-rays, neutron and Raman scattering.

    PubMed

    Façanha Filho, Pedro F; Jiao, Xueshe; Freire, Paulo T C; Lima, José A; dos Santos, Adenilson O; Henry, Paul F; Yokaichiya, Fabiano; Kremner, Ewout; Bordallo, Heloisa N

    2011-04-14

    We have studied the amino acid L-leucine (LEU) using inelastic neutron scattering, X-rays and neutron diffraction, calorimetry and Raman scattering as a function of temperature, focusing on the relationship between the local dynamics of the NH(3), CH(3), CH(2) and CO(2) moieties and the molecular structure of LEU. Calorimetric and diffraction data evidenced two novel phase transitions at about 150 K (T(1)) and 275 K (T(2)). The dynamical susceptibility function, obtained from the inelastic neutron scattering results, shows a re-distribution of the intensity of the vibrational bands that can be directly correlated with the phase transitions observed at T(1) and T(2), as well as with the already reported phase transition at T(3) = 353 K. Through the analysis of the Raman modes, the new structural arrangement observed below T(1) was related to conformational modifications of the CH and CH(3) groups, while the behavior of the N-H stretching vibration, ν(NH(3)), gave insight into the intermolecular N-H…O interactions. The observation of changes in the translational symmetry in the crystalline lattice, as well as anharmonic dynamics, allows for localized motions in LEU.

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

    PubMed Central

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

    2015-01-01

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

  5. Inhibition of Prostate Cancer Bone Metastasis by Synthetic TF Antigen Mimic/Galectin-3 Inhibitor Lactulose-l-Leucine1

    PubMed Central

    Glinskii, Olga V; Sud, Sudha; Mossine, Valeri V; Mawhinney, Thomas P; Anthony, Douglas C; Glinsky, Gennadi V; Pienta, Kenneth J; Glinsky, Vladislav V

    2012-01-01

    Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag) and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-l-leucine (Lac-l-Leu) to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-l-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-l-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-l-Leu resulted in a three-fold (P < .05) decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-l-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting β-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton. PMID:22355275

  6. 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.

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

    PubMed

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

    2016-05-01

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

  8. A novel l-leucine modified Sol-Gel-Carbon electrode for simultaneous electrochemical detection of homovanillic acid, dopamine and uric acid in neuroblastoma diagnosis.

    PubMed

    Khamlichi, Redouan El; Bouchta, Dounia; Anouar, El Hassane; Atia, Mounia Ben; Attar, Aisha; Choukairi, Mohamed; Tazi, Saloua; Ihssane, Raissouni; Faiza, Chaoukat; Khalid, Draoui; Khalid, Riffi Temsamani

    2017-02-01

    Neuroblastoma is a pediatric neuroblastic tumor arising in the sympathetic nervous crest cells. A high grade of Neuroblastoma is characterized by a high urinary excretion of homovanillic acid and dopamine. In this work l-leucine modified Sol-Gel-Carbon electrode was used for a sensitive voltammetric determination of homovanillic acid and dopamine in urine. The electrochemical response characteristics were investigated by cyclic and differential pulse voltammetry; the modified electrode has shown an increase in the effective area of up to 40%, a well-separated oxidation peaks and an excellent electrocatalytic activity. High sensitivity and selectivity in the linear range of 0,4-100μML(-1) of homovanillic acid and 10-120μML(-1) of dopamine were also obtained. Moreover, a sub-micromolar limit of detection of 0.1μM for homovanillic acid and 1.0μM for the dopamine was achieved. Indeed, high reproducibility with simple preparation and regeneration of the electrode surface made this electrode very suitable for the determination of homovanillic acid and dopamine in pharmaceutical and clinical preparations. The mechanism of homovanillic acid and the electrochemical oxidation at l-leucine modified Sol-Gel-Carbon electrode is described out the B3P86/6-31+G(d,p) level of theory as implemented in Gaussian software.

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

    PubMed

    Shlee, D

    1977-01-01

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

  10. Global Metabolic Profiling of Plasma Shows that Three-Year Mild-Caloric Restriction Lessens an Age-Related Increase in Sphingomyelin and Reduces L-leucine and L-phenylalanine in Overweight and Obese Subjects.

    PubMed

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

    2016-12-01

    The effect of weight loss from long-term, mild-calorie diets (MCD) on plasma metabolites is unknown. This study was to examine whether MCD-induced weight reduction caused changes in the extended plasma metabolites. Overweight and obese subjects aged 40-59 years consumed a MCD (approximately 100 kcal/day deficit, n=47) or a weight-maintenance diet (control, n=47) in a randomized, controlled design with a three-year clinical intervention period and plasma samples were analyzed by using UPLC-LTQ-Orbitrap mass spectrometry. The three-year MCD intervention resulted in weight loss (-8.87%) and significant decreases in HOMA-IR and TG. The three-year follow-up of the MCD group showed reductions in the following 13 metabolites: L-leucine; L-phenylalanine; 9 lysoPCs; PC (18:0/20:4); and SM (d18:0/16:1). The three-year MCD group follow-up identified increases in palmitic amide, oleamide, and PC (18:2/18:2). Considering the age-related alterations in the identified metabolites, the MCD group showed a greater decrease in L-leucine, L-phenylalanine, and SM (d18:0/16:1) compared with those of the control group. Overall, the change (Δ) in BMI positively correlated with the ΔTG, ΔHOMA-IR, ΔL-leucine, and ΔSM (d18:0/16:1). The ΔHOMA-IR positively correlated with ΔTG, ΔL-leucine, ΔL-phenylalanine, and ΔSM (d18:0/16:1). The weight loss resulting from three-year mild-caloric restriction lessens the age-related increase in SM and reduces L-leucine and L-phenylalanine in overweight and obese subjects. These changes were coupled with improved insulin resistance (ClinicalTrials.gov: NCT02081898).

  11. Global Metabolic Profiling of Plasma Shows that Three-Year Mild-Caloric Restriction Lessens an Age-Related Increase in Sphingomyelin and Reduces L-leucine and L-phenylalanine in Overweight and Obese Subjects

    PubMed Central

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

    2016-01-01

    The effect of weight loss from long-term, mild-calorie diets (MCD) on plasma metabolites is unknown. This study was to examine whether MCD-induced weight reduction caused changes in the extended plasma metabolites. Overweight and obese subjects aged 40-59 years consumed a MCD (approximately 100 kcal/day deficit, n=47) or a weight-maintenance diet (control, n=47) in a randomized, controlled design with a three-year clinical intervention period and plasma samples were analyzed by using UPLC-LTQ-Orbitrap mass spectrometry. The three-year MCD intervention resulted in weight loss (-8.87%) and significant decreases in HOMA-IR and TG. The three-year follow-up of the MCD group showed reductions in the following 13 metabolites: L-leucine; L-phenylalanine; 9 lysoPCs; PC (18:0/20:4); and SM (d18:0/16:1). The three-year MCD group follow-up identified increases in palmitic amide, oleamide, and PC (18:2/18:2). Considering the age-related alterations in the identified metabolites, the MCD group showed a greater decrease in L-leucine, L-phenylalanine, and SM (d18:0/16:1) compared with those of the control group. Overall, the change (Δ) in BMI positively correlated with the ΔTG, ΔHOMA-IR, ΔL-leucine, and ΔSM (d18:0/16:1). The ΔHOMA-IR positively correlated with ΔTG, ΔL-leucine, ΔL-phenylalanine, and ΔSM (d18:0/16:1). The weight loss resulting from three-year mild-caloric restriction lessens the age-related increase in SM and reduces L-leucine and L-phenylalanine in overweight and obese subjects. These changes were coupled with improved insulin resistance (ClinicalTrials.gov: NCT02081898). PMID:28053823

  12. Prevention of lethal murine graft versus host disease by treatment of donor cells with L-leucyl-L-leucine methyl ester

    SciTech Connect

    Charley, M.; Thiele, D.L.; Bennett, M.; Lipsky, P.E.

    1986-11-01

    Graft vs. host disease (GVHD) remains one of the main problems associated with bone marrow transplantation. The current studies were undertaken to determine whether treatment of the donor inoculum with the anticytotoxic cell compound L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) would alter the development of GVHD in a murine model. Irradiated recipient mice transplanted with a mixture of control bone marrow and spleen cells from naive semiallogeneic donors died rapidly from GVHD, whereas the recipients of cells incubated with 250 microM Leu-Leu-OMe all survived. In addition, Leu-Leu-OMe treatment of cells obtained from donors immunized against host alloantigens resulted in significantly prolonged survival. Phenotypic characterization of spleen cells from the various groups of mice that had received Leu-Leu-OMe-treated cells and survived consistently revealed the donor phenotype. Treatment of marrow cells with 250 microM Leu-Leu-OMe appeared to have no adverse effects on stem cell function. Erythropoiesis was undiminished, as assayed by splenic 5-iodo-2'-deoxyuridine-/sup 125/I uptake. Moreover, granulocytic and megakaryocytic regeneration were histologically equivalent in the spleens of recipients of control or Leu-Leu-OMe-treated cells. Treatment of the donor inoculum with Leu-Leu-OMe thus prevents GVHD in this murine strain combination with no apparent stem cell toxicity.

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2015-07-28

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

  17. New organic reference materials for hydrogen, carbon, and nitrogen stable isotope-ratio measurements: caffeines, n-alkanes, fatty acid methyl esters, glycines, L-valines, polyethylenes, and oils

    USGS Publications Warehouse

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

    2016-01-01

    An international project developed, quality-tested, and determined isotope−δ values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope−δ scales. The RMs span a range of δ2HVSMOW-SLAP values from −210.8 to +397.0 mUr or ‰, for δ13CVPDB-LSVEC from −40.81 to +0.49 mUr and for δ15NAir from −5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C16 n-alkanes, n-C20-fatty acid methyl esters (FAMEs), glycines, and l-valines, together with polyethylene powder and string, one n-C17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a 2H-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of δ2H values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain 13C and carbon-bound organic 2H-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies.

  18. The development and application of a single-cell biosensor for the detection of l-methionine and branched-chain amino acids.

    PubMed

    Mustafi, Nurije; Grünberger, Alexander; Kohlheyer, Dietrich; Bott, Michael; Frunzke, Julia

    2012-07-01

    The detection and quantification of specific metabolites in single bacterial cells is a major goal for industrial biotechnology. We have developed a biosensor based on the transcriptional regulator Lrp that detects intracellular l-methionine and branched-chain amino acids in Corynebacterium glutamicum. In assays, fluorescence output showed a linear relationship with cytoplasmic concentrations of the effector amino acids. In increasing order, the affinity of Lrp for the amino acids is l-valine, l-isoleucine, l-leucine and l-methionine. The sensor was applied for online monitoring and analysis of cell-to-cell variability of l-valine production by the pyruvate dehydrogenase-deficient C. glutamicum strain ΔaceE. Finally, the sensor system was successfully used in a high-throughput (HT) FACS screen for the isolation of amino acid-producing mutants after random mutagenesis of a non-producing wild type strain. These applications illustrate how one of nature's sensor devices - transcriptional regulators - can be used for the analysis, directed evolution and HT screening for microbial strain development.

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

    PubMed

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

    2015-12-15

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

  20. L-leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-regulation of system A amino acid transport.

    PubMed Central

    Peyrollier, K; Hajduch, E; Blair, A S; Hyde, R; Hundal, H S

    2000-01-01

    Amino acid availability is known to regulate diverse cell processes including the activation of p70 S6 kinase, initiation factors involved in mRNA translation, gene expression and cellular amino acid uptake. Essential amino acids, in particular the branched-chain amino acids (e.g. leucine), have been shown to be the dominant players in mediating these effects, although the precise nature by which they regulate these processes remain poorly understood. In this study we have investigated the mechanisms involved in the leucine-induced modulation of p70 S6 kinase and addressed whether this kinase participates in the up-regulation of the System A amino acid transporter in L6 muscle cells. Incubation of muscle cells that had been amino acid-deprived for 1 h with L-leucine (2 mM) led to a rapid (>2-fold) activation of p70 S6 kinase, which was suppressed by both wortmannin and rapamycin. Consistent with this finding, addition of leucine caused a rapid ( approximately 5-fold) but transient stimulation of phosphatidylinositol 3-kinase (PI3K). PI3K activation was inhibited by wortmannin and was not dependent upon insulin receptor substrate-1 activation. Unlike stimulation by insulin, activation of neither protein kinase B nor p42/p44 mitogen-activated protein kinase accompanied the leucine-induced stimulation of PI3K. However, the leucine-induced activation of PI3K and p70 S6 kinase did result in the concomitant inactivation of glycogen synthase kinase-3 (GSK-3). Leucine enhanced System A transport by approximately 50%. We have shown previously that this stimulation is protein-synthesis-dependent and in the current study we show that it was blocked by both wortmannin and rapamycin. Our findings indicate that PI3K and the mammalian target of rapamycin are components of a nutrient signalling pathway that regulates the activation of p70 S6 kinase and induction of System A in L6 cells. The activation of this pathway by leucine is also responsible for the inactivation of GSK-3

  1. 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 γ-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 α-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.

  2. Effect of L-Tryptophan and L-Leucine on Gut Hormone Secretion, Appetite Feelings and Gastric Emptying Rates in Lean and Non-Diabetic Obese Participants: A Randomized, Double-Blind, Parallel-Group Trial

    PubMed Central

    Meyer-Gerspach, Anne Christin; Häfliger, Simon; Meili, Julian; Doody, Alison; Rehfeld, Jens F; Drewe, Jürgen; Beglinger, Christoph; Wölnerhanssen, Bettina

    2016-01-01

    Background/Objectives Gut hormones such as cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) play a role as satiation factors. Strategies to enhance satiation peptide secretion could provide a therapeutic approach for obesity. Carbohydrates and lipids have been extensively investigated in relation to peptide release. In contrast, the role of proteins or amino acids is less clear. Our aim was to compare the effects of the amino acids L-tryptophan (L-trp) and L-leucine (L-leu) separately on gastric emptying and gut peptide secretion. Participants/Methods The study was conducted as a randomized (balanced), double-blind, parallel-group trial. A total of 10 lean and 10 non-diabetic obese participants were included. Participants received intragastric loads of L-trp (0.52 g and 1.56 g) and L-leu (1.56 g), dissolved in 300 mL tap water; 75 g glucose and 300 mL tap water served as control treatments. Results Results of the study are: i) L-trp at the higher dose stimulates CCK release (p = 0.0018), and induces a significant retardation in gastric emptying (p = 0.0033); ii) L-trp at the higher dose induced a small increase in GLP-1 secretion (p = 0.0257); iii) neither of the amino acids modulated subjective appetite feelings; and iv) the two amino acids did not alter insulin or glucose concentrations. Conclusions L-trp is a luminal regulator of CCK release with effects on gastric emptying, an effect that could be mediated by CCK. L-trp’s effect on GLP-1 secretion is only minor. At the doses given, the two amino acids did not affect subjective appetite feelings. Trial Registration ClinicalTrials.gov NCT02563847 PMID:27875537

  3. Permanganate oxidation of α-amino acids: kinetic correlations for the nonautocatalytic and autocatalytic reaction pathways.

    PubMed

    Perez-Benito, Joaquin F

    2011-09-08

    The reactions of permanganate ion with seven α-amino acids in aqueous KH(2)PO(4)/K(2)HPO(4) buffers have been followed spectrophotometrically at two different wavelengths: 526 nm (decay of MnO(4)(-)) and 418 nm (formation of colloidal MnO(2)). All of the reactions studied were autocatalyzed by colloidal MnO(2), with the contribution of the autocatalytic reaction pathway decreasing in the order glycine > l-threonine > l-alanine > l-glutamic acid > l-leucine > l-isoleucine > l-valine. The rate constants corresponding to the nonautocatalytic and autocatalytic pathways were obtained by means of either a differential rate law or an integrated one, the latter requiring the use of an iterative method for its implementation. The activation parameters for the two pathways were determined and analyzed to obtain statistically significant correlations for the series of reactions studied. The activation enthalpy of the nonautocatalytic pathway showed a strong, positive dependence on the standard Gibbs energy for the dissociation of the protonated amino group of the α-amino acid. Linear enthalpy-entropy correlations were found for both pathways, leading to isokinetic temperatures of 370 ± 21 K (nonautocatalytic) and 364 ± 28 K (autocatalytic). Mechanisms in agreement with the experimental data are proposed for the two reaction pathways.

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

    PubMed

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

    2010-03-21

    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.

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

    PubMed

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

    2013-03-01

    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.

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

    2014-01-01

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

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

    PubMed

    Robbins, Paul S; Alm, Steven R; Armstrong, Charlesd; 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.

  9. 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

  10. 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

  11. Gut microbiota of mice putatively modifies amino acid metabolism in the host brain.

    PubMed

    Kawase, Takahiro; Nagasawa, Mao; Ikeda, Hiromi; Yasuo, Shinobu; Koga, Yasuhiro; Furuse, Mitsuhiro

    2017-04-10

    Recently, it has been found that the gut microbiota influences functions of the host brain by affecting monoamine metabolism. The present study focused on the relationship between the gut microbiota and the brain amino acids. Specific pathogen-free (SPF) and germ-free (GF) mice were used as experimental models. Plasma and brain regions were sampled from mice at 7 and 16 weeks of age, and analysed for free d- and l-amino acids, which are believed to affect many physiological functions. At 7 weeks of age, plasma concentrations of d-aspartic acid (d-Asp), l-alanine (l-Ala), l-glutamine (l-Gln) and taurine were higher in SPF mice than in GF mice, but no differences were found at 16 weeks of age. Similar patterns were observed for the concentrations of l-Asp in striatum, cerebral cortex and hippocampus, and l-arginine (l-Arg), l-Ala and l-valine (l-Val) in striatum. In addition, the concentrations of l-Asp, d-Ala, l-histidine, l-isoleucine (l-Ile), l-leucine (l-Leu), l-phenylalanine and l-Val were significantly higher in plasma of SPF mice when compared with those of GF mice. The concentrations of l-Arg, l-Gln, l-Ile and l-Leu were significantly higher in SPF than in GF mice, but those of d-Asp, d-serine and l-serine were higher in some brain regions of GF mice than in those of SPF mice. In conclusion, the concentration of amino acids in the host brain seems to be dependent on presence of the gut microbiota. Amino acid metabolism in the host brain may be modified by manipulating microbiota communities.

  12. Bioactivity of food peptides: biological response of rats to bovine milk whey peptides following acute exercise

    PubMed Central

    Moura, Carolina Soares; Lollo, Pablo Christiano Barboza; Morato, Priscila Neder; Risso, Eder Muller; Amaya-Farfan, Jaime

    2017-01-01

    ABSTRACT Background: Several physiologically beneficial effects of consuming a whey protein hydrolysate (WPH) have been attributed to the greater availability of bioactive peptides. Aims: The aim was to investigate the effect of four branched-chain amino acid- (BCAA-)containing dipeptides, present in WPH, on immune modulation, stimulation of HSP expression, muscle protein synthesis, glycogen content, satiety signals and the impact of these peptides on the plasma free amino acid profiles. Methods: The animals were divided in groups: control (rest, without gavage), vehicle (water), L-isoleucyl-L-leucine (lle-Leu), L-leucyl-L-isoleucine (Leu-lle), L-valyl-Lleucine (Val-Leu), L-leucyl-L-valine (Leu-Val) and WPH. All animals were submitted to acute exercise, except for control. Results: lle-Leu stimulated immune response, hepatic and muscle glycogen and HSP60 expression, whereas Leu-Val enhanced HSP90 expression. All dipeptides reduced glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide, no changes were observed on leptin. All peptides inhibited NF-kB expression. The plasma amino acid time-course showed peptide-specific and isomer-specific metabolic features, including increases of the BCAAs. Conclusion: The data indicate that lle-Leu was effective to attenuate immune-suppression exercise-induced, promoted glycogen content and stimulated anti-stress effect (HSP). Furthermore, Leu-Val increased HSP90, p-4EBP1, p-mTOR and p-AMPK expression. The data suggest the involvement of these peptides in various beneficial functions of WPH consumption. PMID:28326005

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

    PubMed

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

    2010-02-01

    The unique aroma of melons (Cucumis melo L., Cucurbitaceae) is composed of many volatile compounds biosynthetically derived from fatty acids, carotenoids, amino acids, and terpenes. Although amino acids are known precursors of aroma compounds in the plant kingdom, the initial steps in the catabolism of amino acids into aroma volatiles have received little attention. Incubation of melon fruit cubes with amino acids and alpha-keto acids led to the enhanced formation of aroma compounds bearing the side chain of the exogenous amino or keto acid supplied. Moreover, L-[(13)C(6)]phenylalanine was also incorporated into aromatic volatile compounds. Amino acid transaminase activities extracted from the flesh of mature melon fruits converted L-isoleucine, L-leucine, L-valine, L-methionine, or L-phenylalanine into their respective alpha-keto acids, utilizing alpha-ketoglutarate as the amine acceptor. Two novel genes were isolated and characterized (CmArAT1 and CmBCAT1) encoding 45.6 kDa and 42.7 kDa proteins, respectively, that displayed aromatic and branched-chain amino acid transaminase activities, respectively, when expressed in Escherichia coli. The expression of CmBCAT1 and CmArAT1 was low in vegetative tissues, but increased in flesh and rind tissues during fruit ripening. In addition, ripe fruits of climacteric aromatic cultivars generally showed high expression of CmBCAT1 and CmArAT1 in contrast to non-climacteric non-aromatic fruits. The results presented here indicate that in melon fruit tissues, the catabolism of amino acids into aroma volatiles can initiate through a transamination mechanism, rather than decarboxylation or direct aldehyde synthesis, as has been demonstrated in other plants.

  14. Selective enhancement and suppression of frog gustatory responses to amino acids

    PubMed Central

    1981-01-01

    Properties of the receptor sites for L-amino acids in taste cells of the bullfrog (Rana catesbeiana) were examined by measuring the neural activities of the glossopharyngeal nerve under various conditions. (a) The frogs responded to 12 amino acids, but the responses to the amino acids varied with individual frogs under natural conditions. The frog tongues, however, exhibited similar responses after an alkaline treatment that removes Ca2+ from the tissue. The variation in the responses under natural conditions was apparently due to the variation in the amount of Ca2+ bound to the receptor membrane. (b) The responses to hydrophilic L-amino acids (glycine, L-alanine, L-serine, L- threonine, L-cysteine, and L-proline) were of a tonic type, but those to hydrophobic L-amino acids (L-valine, L-leucine, L-isoleucine, L- methionine, L-phenylalanine, and L-tyrptophan) were usually composed of both phasic and tonic components. (c) The properties of the tonic component were quite different from those of the phasic component: the tonic component was largely enhanced by the alkaline treatment and suppressed by the acidic treatment that increases binding of Ca2+ to the tissue. Also, the tonic component was suppressed by the presence of low concentrations of salts, or the action of pronase E, whereas the phasic component was unchanged under these conditions. These properties of the phasic component were quite similar to those of the response to hydrophobic substances such as quinine. These results suggest that the hydrophilic L-amino acids stimulate receptor protein(s) and that the hydrophobic L-amino acids stimulate both the receptor protein and a receptor site similar to that for quinine. (d) On the basis of the suppression of the responses to amino acids by salts, the mechanism of generation of the receptor potential is discussed. PMID:6972437

  15. Synthesis of Fluorescent Gelators and Direct Observation of Gelation with a Fluorescence Microscope.

    PubMed

    Hanabusa, Kenji; Ueda, Takuya; Takata, Shingo; Suzuki, Masahiro

    2016-11-14

    Fluorescein-, benzothiazole-, quinoline-, stilbene-, and carbazole-containing fluorescent gelators have been synthesized by connecting gelation-driving segments, including l-isoleucine, l-valine, l-phenylalanine, l-leucine residue, cyclo(l-asparaginyl-l-phenylalanyl), and trans-(1R,2R)-diaminocyclohexane. The emission behaviors of the gelators were investigated, and their gelation abilities studied against 15 solvents. The minimum gel concentration, variable-temperature spectroscopy, transmission electron microscopy, scanning electron microscopy, fluorescence microscopy (FM), and confocal laser scanning microscopy (CLSM) were used to characterize gelation. The intermolecular hydrogen bonding between the N-H and C=O of amide, van der Waals interactions and π-π stacking play important roles in gelation. The colors of emission are related to the fluorescence structures of gelators. Fibrous aggregates characterized by the color of their emission were observed by FM. 3D images are produced by the superposition of images captured by CLSM every 0.1 μm to a settled depth. The 3D images show that the large micrometer-sized aggregates spread out three dimensionally. FM observations of mixed gelators are studied. In the case of gelation, two structurally related gelators with the same gelation-driving segment lead to the gelators build up of the same aggregates through similar hydrogen-bonding patterns. When two gelators with structurally different gelation-driving segments induce gelation, the gelators build up each aggregate through individual hydrogen-bonding patterns. A fluorescent reagent that was incorporated into the aggregates of gels through van der Waals interactions was developed. The addition of this fluorescent reagent enables the successful observation of nonfluorescent gelators' aggregates by FM.

  16. Third system for neutral amino acid transport in a marine pseudomonad.

    PubMed Central

    Pearce, S M; Hildebrandt, V A; Lee, T

    1977-01-01

    Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system. PMID:856786

  17. Amino Acids Regulate mTORC1 by an Obligate Two-step Mechanism.

    PubMed

    Dyachok, Julia; Earnest, Svetlana; Iturraran, Erica N; Cobb, Melanie H; Ross, Elliott M

    2016-10-21

    The mechanistic target of rapamycin complex 1 (mTORC1) coordinates cell growth with its nutritional, hormonal, energy, and stress status. Amino acids are critical regulators of mTORC1 that permit other inputs to mTORC1 activity. However, the roles of individual amino acids and their interactions in mTORC1 activation are not well understood. Here we demonstrate that activation of mTORC1 by amino acids includes two discrete and separable steps: priming and activation. Sensitizing mTORC1 activation by priming amino acids is a prerequisite for subsequent stimulation of mTORC1 by activating amino acids. Priming is achieved by a group of amino acids that includes l-asparagine, l-glutamine, l-threonine, l-arginine, l-glycine, l-proline, l-serine, l-alanine, and l-glutamic acid. The group of activating amino acids is dominated by l-leucine but also includes l-methionine, l-isoleucine, and l-valine. l-Cysteine predominantly inhibits priming but not the activating step. Priming and activating steps differ in their requirements for amino acid concentration and duration of treatment. Priming and activating amino acids use mechanisms that are distinct both from each other and from growth factor signaling. Neither step requires intact tuberous sclerosis complex of proteins to activate mTORC1. Concerted action of priming and activating amino acids is required to localize mTORC1 to lysosomes and achieve its activation.

  18. Influence of amino acids on gastric adaptive relaxation (accommodation) in rats as evaluated with a barostat

    PubMed Central

    Uchida, Masayuki; Iwamoto, Chizuru

    2016-01-01

    Aim: The present study aimed to evaluate the effects of selected straight alkyl chain, hydroxylated chain and branched chain amino acids on gastric adaptive relaxation, as these have previously been shown to have differing effects on gastric emptying. Materials and Methods: Gastric adaptive relaxation was evaluated using a barostat in rats under urethane anesthesia. The pressure within the balloon, introduced from the mouth to the stomach, was changed stepwise from 1 to 8 mmHg. The increased volume just after the increase of balloon pressure was defined as distension-induced gastric adaptive relaxation (accommodation). Amino acids were administered orally or intravenously. Results: As compared with control rats administered with distilled water, those rats that were orally administered amino acids having straight alkyl chain and extra hydroxylated alkyl chain, such as glycine and l-serine, had significantly enhanced gastric adaptive relaxation, but administration of l-alanine and l-threonine did not. Branched chain amino acids, such as l-isoleucine, l-leucine and l-valine, also did not significantly influence gastric adaptive relaxation. Glycine and l-serine showed the same efficacy when administered intravenously. Conclusion: Among the amino acids evaluated in the present study, glycine and l-serine significantly enhanced gastric adaptive relaxation, suggesting that short alkyl chain amino acids may enhance gastric adaptive relaxation as compared with the other amino acids. These findings may suggest that glycine and l-serine would be useful in the therapy of functional dyspepsia, especially for early satiety, because the dysfunction of adaptive relaxation is one of the causes of early satiety. PMID:27558952

  19. Supplementation of branched-chain amino acids maintains the serum albumin level in the course of hepatocellular carcinoma recurrence.

    PubMed

    Kakazu, Eiji; Kondo, Yasuteru; Kogure, Takayuki; Ninomiya, Masashi; Kimura, Osamu; Iwata, Tomoaki; Morosawa, Tatsuki; Iwasaki, Takao; Shimosegawa, Tooru

    2013-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. Serum albumin (Alb) is an important prognostic factor for patients with HCC. Moreover, plasma levels of branched-chain amino acids (BCAA), L-valine, L-leucine, and L-isoleucine, are commonly decreased in patients with cirrhosis. Accordingly, formulations of BCAA has been used to maintain the Alb level and prevent ascites in patients with cirrhosis. The aim of this study is to investigate differences in the changes in Alb between a group that received a BCAA formulation (n = 29) and a group given a standard diet (n = 60) in the course of HCC recurrences. All patients experienced more than one hospitalization (mean: 2.6; range: 2-10) owing to recurrence. The plasma BCAA concentration and BCAA-to-tyrosine ratio (BTR), which is a good indicator of the severity of hepatic parenchymal injury in patients with cirrhosis, were significantly correlated with Alb. We defined the changes in BCAA and Alb between recurrences as ΔBCAA and ΔAlb, respectively, and stratified the patients in both groups based on number of recurrences (3 < early, 3-5 middle, or 5 > later). There was also a positive correlation between ΔBCAA and ΔAlb. Interestingly, in the group with BCAA, ΔAlb and ΔBCAA were significantly smaller, especially in the middle period (3-5 recurrences), than in the group without BCAA. These results indicate that the BCAA supplementation could maintain the BCAA and Alb levels in the middle period (3-5 recurrences). BCAA formulation is useful for hypoalbuminemia in the course of HCC recurrence.

  20. Effects of straight alkyl chain, extra hydroxylated alkyl chain and branched chain amino acids on gastric emptying evaluated using a non-invasive breath test in conscious rats

    PubMed Central

    Uchida, Masayuki; Kobayashi, Orie; Iwasawa, Kaori; Shimizu, Kimiko

    2016-01-01

    Aim: Some amino acids been known to influence gastric emptying. Thus we have evaluated the effects of straight alkyl chain, extra hydroxylated alkyl chain and branched chain amino acids on gastric emptying. Materials and Methods: Gastric emptying was evaluated in rats after feeding with Racol (nutrient formulae) containing [1-13C] acetic acid. Using a breath test, the content of 13CO2 in their expired air was measured by infrared analyzers. Rats were orally administered with test amino acids, while control rats were administered orally with distilled water. Results: The expired 13CO2 content in the expired air increased with time, peaked after about 30 min and decreased thereafter. Among the amino acids having an alkyl chain, l-serine, l-alanine and l-glycine, significantly decreased the 13CO2 content and Cmax, and delayed Tmax, suggesting inhibition and delay of gastric emptying. AUC120 min values of l-alanine and l-glycine also decreased significantly. l-Threonine significantly decreased 13CO2 content and delayed Tmax, but had no influence on Cmax and AUC120 min values, suggesting a delay of gastric emptying. l-Isoleucine and l-leucine and l-valine significantly decreased 13CO2 content, suggesting inhibition of the gastric emptying, but Cmax, Tmax and AUC120 min values were not significantly affected. Conclusion: The results show that the amino acids used in the present study had different effects on gastric emptying. Moreover, it was found that inhibition and delay of gastric emptying were clearly classifiable by analyzing the change in 13CO2 content of the expired air and the Cmax, Tmax and AUC120 min values. PMID:27169776

  1. Experimental evidence for three pheromone races of the scarab beetle Phyllophaga anxia (LeConte).

    PubMed

    Robbins, Paul S; Cash, Daniel B; Linn, Charles E; Roelofs, Wendell L

    2008-02-01

    This study offers experimental evidence for the existence of three pheromone races of the northern genitalic form of Phyllophaga anxia: one race in which females produce and males respond mainly to L-valine methyl ester, a second producing and responding to L-isoleucine methyl ester, and a third producing and responding to an intermediate range of blends of the two compounds. At Franklinville, NY, pheromone gland contents of females were analyzed using coupled gas chromatography-electroantennogram detection. Two types of females were found, one that produced greater than 99% L-valine methyl ester and another that produced greater than 99% L-isoleucine methyl ester. Capture-mark-release-recapture field tests with males at Franklinville established that most males were recaptured in traps baited with the same blends with which they were originally captured. The populations characterized at Franklinville, NY, have also been found at numerous locations from eastern Canada and the northeast and north central USA, sometimes in allopatry and sometimes in sympatry. At a site in Carver, MA, P. anxia males responded to blends of the methyl esters of L-valine and L-isoleucine, and Carver females produced blends similar to those to which the males responded. Populations responding to blends have been identified only from southeastern Massachusetts and Rhode Island. At a field site near Waterloo, NY, the addition of small proportions of L-isoleucine methyl ester to lures containing L-valine methyl ester did not affect trap captures, but higher proportions of L-isoleucine methyl ester were inhibitory, decreasing trap captures.

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

    SciTech Connect

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

    2014-08-01

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

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

    PubMed Central

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

    2008-01-01

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

  4. Falcatimonas natans gen. nov., sp. nov., a strictly anaerobic, amino-acid-decomposing bacterium isolated from a methanogenic reactor of cattle waste.

    PubMed

    Watanabe, Misa; Kaku, Nobuo; Ueki, Katsuji; Ueki, Atsuko

    2016-11-01

    A strictly anaerobic bacterial strain (WN011T) was isolated from a methanogenic reactor treating waste from cattle farms. Cells of the strain were Gram-stain-negative curved rods with a polar flagellum. Spores were not produced. The optimum temperature for growth was 35-37 °C and the optimum pH was 6.7. The strain did not utilize carbohydrates as growth substrates. The strain grew in PY medium and produced acetate, butyrate, isovalerate and H2 as well as propionate and isobutyrate as minor products. Amino acids (l-isoleucine, l-leucine, l-lysine, l-serine, l-threonine and l-valine) added to PY medium enhanced growth of the strain and increased the amounts of fermentation products. Oxidase, catalase and nitrate-reducing activities were negative. Hydrogen sulfide was produced. The genomic DNA G+C content was 38.8 mol%. Compounds related to iso-C15 : 0 (fatty acid, dimethylacetal and aldehyde) were detected as predominant components by the cellular fatty acids analysis. The diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. On the basis of 16S rRNA gene sequences, three clones from wastewater were very closely related to strain WN011T (up to 99.9 % sequence similarity). The most closely related described species were those in cluster XIVa of the class Clostridia such as Ruminococcus gauvreauii (93.8 % 16S rRNA gene sequence similarity), Clostridium fimetarium (93.5 %) and Clostridium bolteae(93.5 %). Based on the distinct differences in phylogenetic and phenotypic characteristics of strain WN011T from those of related species, it is concluded that strain WN011T represents a novel species of a new genus in the family Lachnospiraceae, for which the name Falcatimonas natans gen. nov., sp. nov. is proposed. The type strain of the type species is WN011T (=JCM 16476T=DSM 22923T).

  5. Heating improves poor compliance with branched chain amino acid-rich supplementation in patients with liver cirrhosis: A before-after pilot study.

    PubMed

    Itou, Minoru; Kawaguchi, Takumi; Taniguchi, Eitaro; Shiraishi, Satomi; Ibi, Ryoko; Mutou, Michiko; Okada, Teruyo; Uchida, Yuki; Otsuka, Momoka; Oriishi, Tetsuharu; Tanaka, Suiko; Takakura, Machiko; Mitsuyama, Keiichi; Tsuruta, Osamu; Sata, Michio

    2009-01-01

    Although branched chain amino acid (BCAA) supplementation improves malnutrition in cirrhotic patients, patient compliance with the administration of BCAA-rich supplements is poor due to their bitter taste. Since temperature is an important factor affecting taste, we examined the effect of heating on the stability of BCAAs and on the compliance of patients with liver cirrhosis with BCAA-rich supplement administration. A thermal denaturation test was first conducted, in which the BCAA-rich supplement Aminoleban® EN was heated to 37, 60, or 80°C for 30 or 60 min. The concentration of three amino acids, L-valine, L-leucine and L-isoleucine, was subsequently measured. The nutritional status of the cirrhotic patients was also evaluted. Patients presenting liver failure with a Child-Pugh class of A (n=2), B (n=2) or C (n=2) were hospitalized at Kurume University Hospital. Six patients with liver cirrhosis (HCV, n=3; HBV, n=1; alcohol, n=2) were enrolled. Venous blood samples were drawn in the morning after a 12-h overnight fast. The BCAA-rich supplement was administered to patients at room temperature (25°C) or heat loaded at 60°C for 10 min, with the temperature maintained above 45°C. Each patient was interviewed by a nationally registered dietitian regarding food consumption and intake of the BCAA-rich supplement immediately after each meal. Nutritional status was evaluated according to serum albumin levels, blood hemoglobin, prothrombin time and total lymphocyte count. No significant decrease was noted in valine, leucine or isoleucine levels following the heating of the BCAAs to 80°C. The caloric intake of the BCAA-rich supplement was significantly higher with administration after heating to 60°C, compared to caloric intake with administration at 25°C. In addition, heating of the BCAA-rich supplement significantly increased blood lymphocyte counts. In conclusion, heating did not affect the stability of the BCAAs, and may improve compliance with BCAA

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

    PubMed Central

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

    2014-01-01

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

  7. Conformation of dehydropentapeptides containing four achiral amino acid residues – controlling the role of L-valine

    PubMed Central

    Krzciuk-Gula, Joanna; Makowski, Maciej; Latajka, Rafał; Kafarski, Paweł

    2014-01-01

    Summary Structural studies of pentapeptides containing an achiral block, built from two dehydroamino acid residues (ΔZPhe and ΔAla) and two glycines, as well as one chiral L-Val residue were performed using NMR spectroscopy. The key role of the L-Val residue in the generation of the secondary structure of peptides is discussed. The obtained results suggest that the strongest influence on the conformation of peptides arises from a valine residue inserted at the C-terminal position. The most ordered conformation was found for peptide Boc-Gly-ΔAla-Gly-ΔZPhe-Val-OMe (3), which adopts a right-handed helical conformation. PMID:24778717

  8. L-Leucyl-L-Leucine Methyl Ester Treatment of Canine Marrow and Peripheral Blood Cells

    DTIC Science & Technology

    1988-11-01

    comple- OMe or MeOll treament of 7-day B-MIX generated CTL (day 7). merit, and incubated agan for 60 run. After washing twice in medium, CThe "mt mixed...Gir A-vermea-bost disease in dogs and man: C 5 10 15 20 25 30 3 the Seattlesexprience. lmmusiRevlSSSSft21&. Day post Trensplant 2. Stosh I. Critical...with Lsu-Leu-OMe-treate mtologous marrow. 3. Koragol R. Sprant J. LAWla graft-vermin-boat disease after bone The ordiinate indicte the absolute

  9. L-Leucine for gold nanoparticles synthesis and their cytotoxic effects evaluation.

    PubMed

    Berghian-Grosan, Camelia; Olenic, Liliana; Katona, Gabriel; Perde-Schrepler, Maria; Vulcu, Adriana

    2014-11-01

    This work reports the preparation of water-soluble leucine capped gold nanoparticles by two single-step synthesis methods. The first procedure involves a citrate reduction approach where the citrate is used as reducing agent and leucine as capping/stabilizing agent. Different sizes of gold nanoparticles, citrate reduced and stabilized by leucine, Leu-AuNPs-C, with the mean diameters in the range of 21-56 nm, were obtained by varying the macroscopic parameters such as: concentration of the gold precursor solution, Au (III):citrate molar ratio and leucine pH. In the second procedure, leucine acts both as reducing and stabilizing agent, allowing us to obtain spherical gold nanoparticles, Leu-AuNPs, with a majority of 80 % (with the mean diameter of 63 nm). This proves that leucine is an appropriate reductant for the formation of water-soluble and stable gold nanoparticles colloids. The characterization of the leucine coated gold nanoparticles was carried out by TEM, UV-Vis and FT-IR analysis. The cytotoxic effect of Leu-AuNPs-C and Leu-AuNPs was also evaluated.

  10. Hippuryl-L-histidyl-L-leucine, a substrate for angiotensin converting enzyme.

    PubMed

    Vrielink, A; Obel-Jorgensen, A; Codding, P W

    1996-05-15

    The tripeptide crystallizes as a zwitterion with a protonated histidyl ring and the C-terminus ionized and with five water molecules of hydration (C21H27N5O5(.5)H2O). The tripeptide adopts an all trans extended conformation with the histidine and phenyl rings parallel to one another. The C-terminus coils into a helical conformation. An intramolecular hydrogen bond between the C-terminus and the N delta atom of the histidine ring stabilizes the helical conformation. The principal torsion angles are phi 1 = -67.7 (8), psi 1 = 140.8 (5), omega 1 = 171.0 (6), phi 2 = -156.5 (5), psi 2 = 162.7 (5), omega 2 = 175.0 (5), phi 3 = -96.4 (6), psi T1 = 14.5 (8) and psi T2 = -164.6 (6) degrees [IUPAC-IUB Commission on Biochemical Nomenclature (1970). J. Mol. Biol. 52, 1-17]. The tripeptides are linked in infinite chains through a short intermolecular hydrogen bond between the C-terminal carboxylate group and the protonated histidy1 N epsilone atom.

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

    PubMed

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

    2011-09-01

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

  12. Role of Lipotropes in Mammary Carcinogenesis.

    DTIC Science & Technology

    1997-09-01

    6 1. Experimental M ethods ......................................... 6 2. R esults...Pyridoxine HC1 0.022 Calcium phosphate, 4.5 Riboflavin 0.022 dibasic L -Alanine 3.5 Thiamine HC1 0.022 L -Arginine HC1 12.1 Glycine 23.3 L -Asparagine 6.0 L ...histidine HC1.H 20 4.5 L -Aspartic acid 3.5 L -isoleucine 8.2 L -Cystine 3.5 L -leucine 11.1 L -Glutamic acid 40.0 L -lysine HC1 18.0 Mineral mix, 35.0 L

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

    PubMed

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

    2011-06-17

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

  14. 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.

  15. Vibrational analysis of amino acids and short peptides in hydrated media. I. L-glycine and L-leucine.

    PubMed

    Derbel, Najoua; Hernández, Belén; Pflüger, Fernando; Liquier, Jean; Geinguenaud, Frédéric; Jaïdane, Nejmeddine; Lakhdar, Zohra Ben; Ghomi, Mahmoud

    2007-02-15

    Raman scattering and Fourier-transform infrared (FT-IR) attenuated transmission reflectance (ATR) spectra of two alpha-amino acids (alpha-AAs), i.e., glycine and leucine, were measured in H2O and D2O (at neutral pH and pD). This series of observed vibrational data gave us the opportunity to analyze vibrational features of both AAs in hydrated media by density functional theory (DFT) calculations at the B3LYP/6-31++G* level. Harmonic vibrational modes calculated after geometry optimization on the clusters containing each AA and 12 surrounding water molecules, which represent primary models for hydration scheme of amino acids, allowed us to assign the main observed peaks.

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

    PubMed

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

    2015-05-22

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

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

    PubMed Central

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

    2015-01-01

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

  18. 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.

  19. Gly429 is the major determinant of uncompetitive inhibition of human germ cell alkaline phosphatase by L-leucine.

    PubMed Central

    Hummer, C; Millán, J L

    1991-01-01

    The catalytic activity of human placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (GCAP) can be inhibited, through an uncompetitive mechanism, by L-Phe. GCAP is also selectively inhibited by L-Leu. Site-directed mutagenesis of five of the 12 residues which are different in PLAP and GCAP revealed that Gly429 is the primary determinant of GCAP inhibition by L-Leu, and Ser84 and Leu297 play a modulatory role in the inhibition. PMID:2001256

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

    SciTech Connect

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

    1988-01-01

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

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

    SciTech Connect

    Farmanfarmaian, A. )

    1988-09-01

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

  2. 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.

  3. 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

  4. Controlled synthesis of amino acid-based pH-responsive chiral polymers and self-assembly of their block copolymers.

    PubMed

    Bauri, Kamal; Roy, Saswati Ghosh; Pant, Shashank; De, Priyadarsi

    2013-02-26

    Leucine/isoleucine side chain polymers are of interest due to their hydrophobicity and reported role in the formation of α-helical structures. The synthesis and reversible addition-fragmentation chain transfer (RAFT) polymerization of amino acid-based chiral monomers, namely Boc-L-leucine methacryloyloxyethyl ester (Boc-L-Leu-HEMA, 1a), Boc-L-leucine acryloyloxyethyl ester (Boc-L-Leu-HEA, 1b), Boc-L-isoleucine methacryloyloxyethyl ester (Boc-L-Ile-HEMA, 1c), and Boc-L-isoleucine acryloyloxyethyl ester (Boc-L-Ile-HEA, 1d), are reported. The controlled nature of the polymerization of the said chiral monomers in N, N-dimethylformamide (DMF) at 70 °C is evident from the formation of narrow polydisperse polymers, the molecular weight controlled by the monomer/chain transfer agent (CTA) molar ratio and the linear relationship between molecular weight and monomer conversion. The resulting well-defined polymers were used as macro-CTAs to prepare corresponding diblock copolymers by RAFT polymerization of methyl (meth)acrylate monomers. Deprotection of Boc groups in the homopolymers and block copolymers under acidic conditions produced cationic, pH-responsive polymers with primary amine moieties at the side chains. The optical activity of the homopolymers and block copolymers were studied using circular dichroism (CD) spectroscopy and specific rotation measurements. The self-assembling nature of the block copolymers to produce highly ordered structures was illustrated through dynamic light scattering (DLS) and atomic force microscopy (AFM) studies. The side chain amine functionality instills pH-responsive behavior, which makes these cationic polymers attractive candidates for drug delivery applications, as well as for conjugation of biomolecules.

  5. 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.

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

    NASA Technical Reports Server (NTRS)

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

    1988-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2006-06-01

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

  9. Supramolecular gelators based on benzenetricarboxamides for ionic liquids.

    PubMed

    Ishioka, Yumi; Minakuchi, Nami; Mizuhata, Minoru; Maruyama, Tatsuo

    2014-02-21

    Supramolecular gelators comprising 1,3,5-benzenetricarboxylic acids and amino acid methyl esters (glycine, L-alanine, L-valine, L-leucine, L-methionine, and L-phenylalanine) for ionic liquids were developed. Ten types of ionic liquids were gelated using the above-mentioned gelators at relatively low concentrations. Field emission-scanning electron microscopy and confocal laser scanning microscopy analyses revealed that these gelators self-assembled into an entangled fibrous structure in ionic liquids, leading to the gelation of the ionic liquids. Comparison studies, involving compounds analogous to the gelators, and Fourier transform infrared spectroscopy measurements suggested that hydrogen bonding played a key role in the self-assembly of the gelator molecules. The ionogels displayed reversible thermal transition characteristics and viscoelastic properties typical of a gel. The gelation of the ionic liquids studied under a wide range of gelator concentrations did not affect the intrinsic conductivity of the ionic liquids.

  10. Requirements for induction of the biodegradative threonine dehydratase in Escherichia coli.

    PubMed Central

    Egan, R M; Phillips, A T

    1977-01-01

    Synthesis of the biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, was prevented by dissolved oxygen concentrations of 6 micrometer or greater. This effect was shown to be exerted solely on synthesis, rather than being the result of enzyme inactivation in vivo. In addition to an anaerobic environment, maximum enzyme synthesis was dependent upon the presence of a complete complement of amino acids, with omission of L-threonine, L-valine, or L-leucine producing the largest decreases in enzyme formation. L-Threonine, the most essential of the amino acid requirements, could be partially replaced by DL-allothreonine or alpha-ketobutyrate. Half-maximal stimulation of enzyme synthesis occurred with 0.4 mM threonine in the medium. The roles of anaerobiosis and amino acids are interpreted as being in accord with the concept that threonine dehydratase functions in anaerobic energy production under conditions of amino acid sufficiency. PMID:334737

  11. Synthesis, structure, and spectroscopic properties of chiral oxorhenium(V) complexes incorporating polydentate ligands derived from L-amino acids: a density functional theory/time-dependent density functional theory investigation.

    PubMed

    Basak, Sucharita; Rajak, Kajal Krishna

    2008-10-06

    The oxorhenium(V) complexes [Re (V)O(L A)Cl 2] bearing the (N-2-pyridylmethyl) of l-valine (HL A (1)), l-leucine (HL A (2)), and l-phenylalanine (HL A (3)) and [Re (V)O(L B)Cl] containing the {(N-2pyridylmethyl)-(N-(5-nitro-2-hydroxybenzyl)} of l-valine (H 2L B (1)), l-leucine (H 2L B (2)), and l-phenylalanine (H 2L B (3)) are presented in this article. The complexes are isolated in enantiomeric pure form examined from X-ray structure determination. The complexes are characterized by spectroscopic and electrochemical methods. The molecular structures observed in the solid state are grossly preserved in solution ( (1)H, (13)C, and circular dichroism spectra). Gas-phase geometry optimization and the electronic structures of [Re (V)O(L A (1))Cl 2], [Re (V)O(L A (2))Cl 2], and [Re (V)O(L B (2))Cl] have been investigated with the framework of density functional theory. The absorption and circular dichroism spectra of the complexes were also calculated applying time-dependent density functional theory (TDDFT) using the conductor-like polarizable continuum solvent model to understand the origin of the electronic excitations. The chemical shift ( (1)H and (13)C) as well as (1)H- (1)H spin-spin coupling constant were also computed by the gauge-independent atomic orbital method, and the computed values are consistent with the experimental data.

  12. Dietary l-leucine supplementation of lactating rats results in a tendency to increase lean/fat ratio associated to lower orexigenic neuropeptide expression in hypothalamus.

    PubMed

    López, N; Sánchez, J; Picó, C; Palou, A; Serra, F

    2010-07-01

    The aim of this study was to assess the effects of dietary leucine supplementation in lactating dams, particularly on energy homeostasis through signaling mechanisms in the central nervous system. Dams were fed ad libitum with standard diet during pregnancy (control dams) or supplemented with 2% leucine (leucine-supplemented dams) from delivery onwards. Food intake, body weight and composition were periodically recorded. Hypothalamus was collected at the end of lactation, and the expression of neuropeptide Y (NPY), agouti-related protein (AgRP) pro-opiomelanocortin (POMC), cocaine and amphetamine regulated transcript (CART), insulin receptor (InsR), ghrelin receptor (GSHR), melanocortin receptor (MCR4), leptin receptor (Ob-Rb) and suppressor of cytokine signaling 3 (SOCS3) were analyzed. Dietary leucine supplementation to lactating rats increased plasma leucine by 56%, modulated body composition and contributed to a tendency of higher ratio of lean/fat mass content of dams during lactation, without affecting food intake, thermogenesis capacity or body or tissue/organs weights. No differences in body weight of offspring from control and leucine-supplemented dams were found. The expression of orexigenic peptides (NPY and AgRP) decreased in leucine-dams, whereas the expression of anorexigenic peptides (POMC and CART), the hypothalamic receptors of insulin, ghrelin, melanocortin and leptin and SOCS3 did not change by leucine supplementation. In conclusion, increased leucine intake during lactation may contribute to a healthier profile of body composition in dams, without compromising the growth and development of the progeny by a mechanism associated with lower expression of orexigenic neuropeptides in hypothalamus.

  13. 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

    DTIC Science & Technology

    1988-11-01

    Cnf’-GM srowt#. Lou-Leu-OMe sOW coated TLC plates (250 i&M, 10X20-cm, HPTLC Kieselgel 60 (Merck). MeOH treated and untreated marrow cells were tested ... ninhydrin in ethanol - lHs waen cocultured with citherU10’ utologous PBMC, Mo-enriched and then placing the plates in a 60*C oven for 30 min 11, values...and for the three control compounds tested . l M o Within the Leti-Leui-Ollie, there was a secondary spot that PWM 25 migrated with a Rf value equal to

  14. Regulation of Leucine Catabolism in Pseudomonas putida

    PubMed Central

    Massey, Linda K.; Conrad, Robert S.; Sokatch, John R.

    1974-01-01

    The generation time of Pseudomonas putida with l-leucine was 20 h in synthetic media but only 3 h with d-leucine. Slow growth in the presence of l-leucine was partially overcome by addition of 0.1 mM amounts of either d-valine, l-valine, or 2-ketoisovalerate. The activities of five enzymes which take part in the oxidation of leucine by P. putida were measured under various conditions of growth. Four enzymes were induced by growth with dl-leucine as sole source of carbon: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-methylcrotonyl-coenzyme A carboxylase, and 3-hydroxy-3-methylglutaryl-coenzyme A lyase. The segment of the pathway required for oxidation of 3-methylcrotonate was induced by growth on isovalerate or 3-methylcrotonate without formation of the preceding enzymes. The synthesis of carboxylase and lyase appeared to have been repressed by the addition of l-glutamate or glucose to cells growing on dl-leucine as the sole carbon source. Mutants unable to grow at the expense of isovalerate had reduced levels of carboxylase and lyase, whereas the levels of three enzymes common to the catabolism of all three branched-chain amino acids and those of two isoleucine catabolic enzymes were normal. PMID:4150714

  15. N- Trichloro- and dichloroacetyl amino acids and compounds of amino acids with halogeno acetic acids: 35Cl nuclear quadrupole resonance spectroscopy; crystal structure of N- trichloroacetyl- glycine, - DL-alanine, and - L-alanine

    NASA Astrophysics Data System (ADS)

    Dou, Shi-qi; Kehrer, Armin; Ofial, Armin R.; Weiss, Alarich

    1995-02-01

    The crystal structures of N- trichloroacetyl- glycine ( N- TCA- G), N-trichloroacetyl-dl-alanine ( N-TCA- dl-A ), and N-trichloroacetyl- l-alanine ( N-TCA- l-A ) were determined. In addition, the 35Cl NQR spectra of these N-trichloroacetyl amino acids, of N-trichloroacetyl- l-valine ( N-TCA- l-V ), and of N- dichloroacetyl- glycine and - L-alanine were measured, mostly as a function of temperature. Compounds of glycine and L-alanine with chlorodifluoroacetic acid, of glycine and L-leucine with monochloroacetic acid, of glycine and L-leucine with dichloroacetic acid, and of glycine and L-leucine with trichloroacetic acid were also studied using 35Cl NQR. The structures (in picometres and degrees) were found to be as follows. N- TCA- G: Pna2 1, Z = 8, a = 1641, b = 1002, c = 1018. N-TCA- dl-A : {C2}/{c}, Z = 8, a = 3280, b = 556, c = 1031, β = 96.68. N-TCA- l-A: P1 , Z = 2, a = 967, b = 949, c = 619, α = 74.97, β = 74.20, γ = 61.20. The 35Cl NQR frequencies (ν) were observed in the range 35-41 MHz, and decrease with increasing temperature. Some of the resonances bleach out at a temperature ( Tb) far below the melting temperature; this provides information about the crystal structures at 77 K. No phase transitions were observed by differential thermal analysis between 77 and 295 K. The crystal structures are discussed in connection with the NQR results, and conclusions are drawn about the structures of the compounds for which only 35Cl NQR data are available.

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

    PubMed

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

    2014-03-29

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

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  18. Optimization of a valine:isoleucine methyl ester pheromone blend and comparison of Robbins and Trécé traps for capture of Phyllophaga anxia (Coleoptera: Scarabaeidae) in Rhode Island.

    PubMed

    Alm, Steven R; Dawson, Charles G; Robbins, Paul

    2004-12-01

    Eight ratios of L-valine:L-isoleucine methyl esters were tested in Robbins traps for capture of Phyllophaga anxia (LeConte) adult males. The 90:10, 80:20, and 60:40 ratios of valine:isoleucine were the most effective blends for capture of beetles in Rhode Island. Females were captured in small numbers in some traps but not consistently to any particular blend. Other male Phyllophaga species captured included Phyllophaga fusca (Frölich), Phyllophaga forsteri (Burmeister), P. hirsuta (Knoch), and P. marginalis (LeConte). The number of these species collected was low, and it was not possible to determine whether they were attracted to any particular pheromone blend. Peak captures of P. anxia males occurred 31 May in 1999 and 2002 in Kingston, RI. The standard Japanese beetle trap manufactured by Trécé (Adair, OK) captured significantly more beetles than the Robbins trap. Because the Trécé trap is already marketed for Japanese beetles, a lure and trapping system can be adopted for P. anxia.

  19. Industrial production of amino acids by coryneform bacteria.

    PubMed

    Hermann, Thomas

    2003-09-04

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

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

    PubMed Central

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

    2011-01-01

    A prodrug strategy was applied to guanidino-containing analogs to increase oral absorption via hPEPT1 and hVACVase. L-Valine, L-isoleucine and L-phenylalanine esters of [3-(hydroxymethyl)phenyl]guanidine (3-HPG) were synthesized and evaluated for transport and activation. In HeLa/hPEPT1 cells, Val-3-HPG and Ile-3-HPG exhibited high affinity to hPEPT1 (IC50: 0.65 and 0.63 mM, respectively), and all three L-amino acid esters showed higher uptake (2.6- to 9-fold) than the parent compound 3-HPG. Val-3-HPG and Ile-3-HPG demonstrated remarkable Caco-2 permeability enhancement, and Val-3-HPG exhibited comparable permeability to valacyclovir. In rat perfusion studies, Val-3-HPG and Ile-3-HPG permeabilities were significantly higher than 3-HPG, and exceeded/matched the high-permeability standard metoprolol, respectively. All the L-amino acid 3-HPG esters were effectively activated in HeLa and Caco-2 cell homogenates, and were found to be good substrates of hVACVase (kcat/Km in mM−1·s−1: Val-3-HPG, 3370; Ile-3-HPG, 1580; Phe-3-HPG, 1660). In conclusion, a prodrug strategy is effective at increasing the intestinal permeability of polar guanidino analogs via targeting hPEPT1 for transport and hVACVase for activation. PMID:19957998

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

    PubMed

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

    2015-07-01

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

  2. Glutamate formation via the leucine-to-glutamate pathway of rat pancreas.

    PubMed

    Schachter, David; Buteau, Jean

    2014-06-01

    The leucine-to-glutamate (Leu→Glu) pathway, which metabolizes the carbon atoms of l-leucine to form l-glutamate, was studied by incubation of rat tissue segments with l-[U-(14)C]leucine and estimation of the [(14)C]glutamate formed. Metabolism of the leucine carbon chain occurs in most rat tissues, but maximal activity of the Leu→Glu pathway for glutamate formation is limited to the thoracic aorta and pancreas. In rat aorta, the Leu→Glu pathway functions to relax the underlying smooth muscle; its functions in the pancreas are unknown. This report characterizes the Leu→Glu pathway of rat pancreas and develops methods to examine its functions. Pancreatic segments effect net formation of glutamate on incubation with l-leucine, l-glutamine, or a mix of 18 other plasma amino acids at their concentrations in normal rat plasma. Glutamate formed from leucine remains mainly in the tissue, whereas that from glutamine enters the medium. The pancreatic Leu→Glu pathway uses the leucine carbons for net glutamate formation; the α-amino group is not used; the stoichiometry is as follows: 1 mol of leucine yields 2 mol of glutamate (2 leucine carbons per glutamate) plus 2 mol of CO2. Comparison of the Leu→Glu pathway in preparations of whole pancreatic segments, isolated acini, and islets of Langerhans localizes it in the acini; relatively high activity is found in cultures of the AR42J cell line and very little in the INS-1 832/13 cell line. Pancreatic tissue glutamate concentration is homeostatically regulated in the range of ∼1-3 μmol/g wet wt. l-Valine and leucine ethyl, benzyl, and tert-butyl esters inhibit the Leu→Glu pathway without decreasing tissue total glutamate.

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

    PubMed

    Zhang, Shengnan; Hinck, Andrew P; Fitzpatrick, Paul F

    2015-08-25

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

  4. 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.

  5. 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

  6. 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

    2010-12-08

    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.

  7. Investigation of Chiral Molecular Micelles by NMR Spectroscopy and Molecular Dynamics Simulation

    PubMed Central

    Morris, Kevin F.; Billiot, Eugene J.; Billiot, Fereshteh H.; Lipkowitz, Kenny B.; Southerland, William M.; Fang, Yayin

    2013-01-01

    NMR spectroscopy and molecular dynamics (MD) simulation analyses of the chiral molecular micelles poly-(sodium undecyl-(L,L)-leucine-valine) (poly-SULV) and poly-(sodium undecyl-(L,L)- valine-leucine) (poly-(SUVL)) are reported. Both molecular micelles are used as chiral selectors in electrokinetic chromatography and each consists of covalently linked surfactant chains with chiral dipeptide headgroups. To provide experimental support for the structures from MD simulations, NOESY spectra were used to identify protons in close spatial proximity. Results from the NOESY analyses were then compared to radial distribution functions from MD simulations. In addition, the hydrodynamic radii of both molecular micelles were calculated from NMR-derived diffusion coefficients. Corresponding radii from the MD simulations were found to be in agreement with these experimental results. NMR diffusion experiments were also used to measure association constants for polar and non-polar binaphthyl analytes binding to both molecular micelles. Poly(SUVL) was found to bind the non-polar analyte enantiomers more strongly, while the more polar analyte enantiomers interacted more strongly with poly(SULV). MD simulations in tum showed that poly(SUL V) had a more open structure that gave greater access for water molecules to the dipeptide headgroup region. PMID:23991355

  8. Leucine/isoleucine/valine-binding protein contracts upon binding of ligand.

    PubMed

    Olah, G A; Trakhanov, S; Trewhella, J; Quiocho, F A

    1993-08-05

    Small-angle x-ray scattering and computer modeling have been used to study the effects of ligand binding to the leucine/isoleucine/valine-binding protein, an initial component of the high-affinity active transport system for branched-chain aliphatic amino acids in Escherichia coli. Measurements were made with no ligand present and with either L-leucine or L-valine present. Upon binding of either leucine or valine, there is a decrease in the radius of gyration, from 23.2 +/- 0.2 to 22.2 +/- 0.2 A, and in the maximum particle dimension, from 82 +/- 3 to 73 +/- 3 A. The x-ray structure of the unbound form has been determined and gives a radius of gyration and a maximum dimension consistent with the values found for the solution structure in this study (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). The reduction in the radius of gyration and maximum dimension upon ligand binding can be accounted for by a substrate-induced cleft closure in a combined "hinge-twist" motion. Modeling of the substrate-bound state was done by comparison of this protein with another periplasmic binding protein (L-arabinose-binding protein), which possesses a similar two-lobe structure and for which the x-ray structure is known in its ligand-bound form.

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

    NASA Astrophysics Data System (ADS)

    Boghaei, Davar M.; Gharagozlou, Mehrnaz

    2007-07-01

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

  10. Transport of extraterrestrial biomolecules to the Earth: problem of thermal stability.

    PubMed

    Basiuk, V A; Douda, J; Navarro-Gonzalez, R

    1999-01-01

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

  11. Interaction of bovine serum albumin with N-acyl amino acid based anionic surfactants: Effect of head-group hydrophobicity.

    PubMed

    Ghosh, Subhajit; Dey, Joykrishna

    2015-11-15

    The function of a protein depends upon its structure and surfactant molecules are known to alter protein structure. For this reason protein-surfactant interaction is important in biological, pharmaceutical, and cosmetic industries. In the present work, interactions of a series of anionic surfactants having the same hydrocarbon chain length, but different amino acid head group, such as l-alanine, l-valine, l-leucine, and l-phenylalanine with the transport protein, bovine serum albumin (BSA), were studied at low surfactant concentrations using fluorescence and circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The results of fluorescence measurements suggest that the surfactant molecules bind simultaneously to the drug binding site I and II of the protein subdomain IIA and IIIA, respectively. The fluorescence as well as CD spectra suggest that the conformation of BSA goes to a more structured state upon surfactant binding at low concentrations. The binding constants of the surfactants were determined by the use of fluorescence as well as ITC measurements and were compared with that of the corresponding glycine-derived surfactant. The binding constant values clearly indicate a significant head-group effect on the BSA-surfactant interaction and the interaction is mainly hydrophobic in nature.

  12. 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.

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

    PubMed

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

    2008-10-01

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

  14. Self-assembly thermodynamics of pH-responsive amino-acid-based polymers with a nonionic surfactant.

    PubMed

    Bogomolova, Anna; Keller, Sandro; Klingler, Johannes; Sedlak, Marian; Rak, Dmytro; Sturcova, Adriana; Hruby, Martin; Stepanek, Petr; Filippov, Sergey K

    2014-09-30

    The behavior of pH-responsive polymers poly(N-methacryloyl-l-valine) (P1), poly(N-methacryloyl-l-phenylalanine) (P2), and poly(N-methacryloylglycyne-l-leucine) (P3) has been studied in the presence of the nonionic surfactant Brij98. The pure polymers phase-separate in an acidic medium with critical pHtr values of 3.7, 5.5, and 3.4, respectively. The addition of the surfactant prevents phase separation and promotes reorganization of polymer molecules. The nature of the interaction between polymer and surfactant depends on the amino acid structure in the side chain of the polymer. This effect was investigated by dynamic light scattering, isothermal titration calorimetry, electrophoretic measurements, small-angle neutron scattering, and infrared spectroscopy. Thermodynamic analysis revealed an endothermic association reaction in P1/Brij98 mixture, whereas a strong exothermic effect was observed for P2/Brij98 and P3/Brij98. Application of regular solution theory for the analysis of experimental enthalpograms indicated dominant hydrophobic interactions between P1 and Brij98 and specific interactions for the P2/Brij98 system. Electrophoretic and dynamic light scattering measurements support the applicability of the theory to these cases. The specific interactions can be ascribed to hydrogen bonds formed between the carboxylic groups of the polymer and the oligo(ethylene oxide) head groups of the surfactant. Thus, differences in polymer-surfactant interactions between P1 and P2 polymers result in different structures of polymer-surfactant complexes. Specifically, small-angle neutron scattering revealed pearl-necklace complexes and "core-shell" structures for P1/Brij98 and P2/Brij98 systems, respectively. These results may help in the design of new pH-responsive site-specific micellar drug delivery systems or pH-responsive membrane-disrupting agents.

  15. Proteiniclasticum ruminis gen. nov., sp. nov., a strictly anaerobic proteolytic bacterium isolated from yak rumen.

    PubMed

    Zhang, Kegui; Song, Lei; Dong, Xiuzhu

    2010-09-01

    Two strictly anaerobic, proteolytic bacterial strains, designated strain D3RC-2(T) and D3RC-3r, were isolated from a cellulose-degrading mixed culture enriched from yak rumen content. The strains were Gram-stain negative and non-spore-forming with cell sizes of 0.5-0.8 x 0.6-2.0 mum. The temperature range for growth was 24-46 degrees C (optimum 38-39 degrees C) and the pH range was between 5.6 and 8.7 (optimum 7.0-7.3). Both strains used soya peptone, tryptone, l-phenylalanine, l-leucine, l-methionine, l-serine, l-valine, l-threonine and l-histidine as carbon and nitrogen sources, but did not use any of the saccharides tested. The major fermentation products from PY medium were acetate, propionate and iso-butyrate. The DNA G+C contents of strains D3RC-2(T) and D3RC-3r were 41.0+/-0.1 mol% and 41.3+/-0.1 mol% (HPLC), respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains represented a new phyletic sublineage within the family Clostridiaceae, with <93.8 % 16S rRNA gene sequence similarity to recognized species. On the basis of the phenotypic, genotypic and physiological evidence, strains D3RC-2(T) and D3RC-3r are proposed as representing a novel species of a new genus, for which the name Proteiniclasticum ruminis gen. nov., sp. nov. is proposed. The type strain of the type species is D3RC-2(T) (=AS 1.5057(T)=JCM 14817(T)).

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

    PubMed

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

    2013-01-01

    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.

  17. Electrocatalytic oxidation and highly selective voltammetric determination of L-cysteine at the surface of a 1-[4-(ferrocenyl ethynyl)phenyl]-1-ethanone modified carbon paste electrode.

    PubMed

    Raoof, Jahan-Bakhsh; Ojani, Reza; Beitollahi, Hadi; Hosseinzadeh, Rahman

    2006-09-01

    A carbon paste electrode (CPE) chemically modified with 1-[4-(ferrocenyl ethynyl)phenyl]-1-ethanone (4-FEPEMCPE) was employed to study the electrocatalytic oxidation of L-cysteine using cyclic voltammetry, differential pulse voltammetry and double potential step chronoamperometry as diagnostic techniques. The diffusion coefficient (D = 7.863 x 10(-6) cm2 s(-1)) of L-cysteine was also estimated using chronoamperometry. The electron-transfer coefficient, alpha (= 0.40), for L-cysteine at the surface of 4-FEPEMCPE was determined using cyclic voltammetry technique. It was found that under an optimum pH (= 7.00), the oxidation of L-cysteine at the surface of such an electrode occurred at a potential of about 350 mV less positive than that of an unmodified CPE. The catalytic oxidation peak currents represented a linear dependence on the L-cysteine concentration. Linear analytical curves were obtained in the ranges of 9.0 x 10(-5) - 4.9 x 10(-3) M and 2.0 x 10(-5) - 2.8 x 10(-3) M of L-cysteine with correlation coefficients of 0.9981 and 0.9982 in cyclic voltammetry and differential pulse voltammetry, respectively. The detection limits (2 sigma) were determined to be 9.9 x 10(-6) M and 5 x 10(-6) M with cyclic voltammetry and differential pulse voltammetry, respectively. The influences of twenty other amino acids, such as glutamine, L-glutamic acid, L-glysine, L-histidine, L-isoleucine, L-leucine, L-arginine hydrochloride, L-aspargine, L-aspartic acid, S-carboxy methyl-L-cysteine, L-methionine, L-phenyl alanine, L-proline, L-serine, L-threonine, L-cystine, cysteamine and gluthathione, on the current response of the sensor were examined. The obtained results did not show any influence on the analytical signal of L-cysteine by these amino acids (except for cysteamine). The method was also used for the selective determination of L-cysteine in patient-blood plasma and some pharmaceutical preparations by using standard addition method.

  18. Optimum conditions for prebiotic evolution in extraterrestrial environments

    NASA Astrophysics Data System (ADS)

    Abbas, Ousama H.

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

  19. 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

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

    2012-10-01

    A series of studies were carried out to investigate the supplemental effects of dietary garlic extracts (GE) on whole body amino acids, whole body and muscle free amino acids, fatty acid composition and blood plasma changes in 6 month old juvenile sterlet sturgeon (Acipenser ruthenus). In the first experiment, fish with an average body weight of 59.6 g were randomly allotted to each of 10 tanks (two groups of five replicates, 20 fish/tank) and fed diets with (0.5%) or without (control) GE respectively, at the level of 2% of fish body weight per day for 5 wks. Whole body amino acid composition between the GE and control groups were not different (p>0.05). Among free amino acids in muscle, L-glutamic acid, L-alanine, L-valine, L-leucine and L-phenylalanine were significantly (p<0.05) higher in GE than in control. However, total whole body free amino acids were significantly lower in GE than in control (p<0.05). GE group showed higher EPA (C22:6n3) and DHA (C22:5n3) in their whole body than the other group (p<0.05). In the second experiment, the effects of dietary garlic extracts on blood plasma changes were investigated using 6 month old juvenile sterlet sturgeon averaging 56.5 g. Fish were randomly allotted to each of 2 tanks (300 fish/tank) and fed diets with (0.5%) or without (control) GE respectively, at the rate of 2% of body weight per day for 23 d. At the end of the feeding trial, blood was taken from the tail vein (n = 5, per group) at 1, 12, and 24 h after feeding, respectively. Blood plasma glucose, insulin and the other serological characteristics were also measured to assess postprandial status of the fish. Plasma glucose concentrations (mg/dl) between two groups (GE vs control) were significantly (p< 0.05) different at 1 (50.8 vs 62.4) and 24 h (57.6 vs 73.6) after feeding, respectively, while no significant difference (p>0.05) were noticed at 12 h (74.6 vs 73.0). Plasma insulin concentrations (μIU/ml) between the two groups were significantly (p<0

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    EPA Science Inventory

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

  2. Amino acid and peptide absorption after proximal small intestinal resection in the rat.

    PubMed Central

    Garrido, A B; Freeman, H J; Chung, Y C; Kim, Y S

    1979-01-01

    In experimental animals with massive proximal intestinal resection, in vivo ileal absorption of an amino acid mixture containing L-leucine and glycine as well as two different dipeptides, L-leucyl-glycine and glycyl-L-leucine, were compared. Both amino acid and dipeptide absorption were markedly enhanced in the ileal segments. However, the absorption rates from the two perfused dipeptides differed. L-leucine absorption from L-leucyl-glycine was much greater than from glycyl-L-leucine. Brush border amino-peptidase activities after resection were also increased but dissociation between absorption and hydrolytic activity occurred. This study indicates that certain dipeptides are handled differently by adapting ileal segments. Furthermore, the changes observed probably reflect mucosal cellular hyperplasia occurring in association with intestinal adaptation. PMID:428822

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

    NASA Technical Reports Server (NTRS)

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

    2012-01-01

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

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

    PubMed

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

    2008-09-01

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

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

    PubMed

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

    2016-06-01

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

  6. Density functional theory calculations and molecular dynamics simulations of the adsorption of biomolecules on graphene surfaces.

    PubMed

    Qin, Wu; Li, Xin; Bian, Wen-Wen; Fan, Xiu-Juan; Qi, Jing-Yao

    2010-02-01

    There is increasing attention in the unique biological and medical properties of graphene, and it is expected that biomaterials incorporating graphene will be developed for the graphene-based drug delivery systems and biomedical devices. Despite the importance of biomolecules-graphene interactions, a detailed understanding of the adsorption mechanism and features of biomolecules onto the surfaces of graphene is lacking. To address this, we have performed density functional theory (DFT) and molecular dynamics (MD) methods exploring the adsorption geometries, adsorption energies, electronic band structures, adsorption isotherms, and adsorption dynamics of l-leucine (model biomolecule)/graphene composite system. DFT calculations confirmed the energetic stability of adsorption model and revealed that electronic structure of graphene can be controlled by the adsorption direction of l-leucine. MD simulations further investigate the potential energy and van der Waals energy for the interaction processes of l-leucine/graphene system at different temperatures and pressures. We find that the van der Waals interaction between the l-leucine and the graphene play a dominant role in the adsorption process under a certain range of temperature and pressure, and the l-leucine molecule could be adsorbed onto graphene spontaneously in aqueous solution.

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

    SciTech Connect

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

    2011-01-07

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

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

    PubMed

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

    1979-01-01

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

  9. Effect of L-azetidine 2-carboxilic acid on the activity of the general amino-acid permease from Saccharomyces cerevisiae var. ellipsoideus.

    PubMed

    Iglesias, R; Ferreras, J M; Arias, F J; Muñoz, R; Rojo, M A; Girbés, T

    1991-01-01

    Addition of the L-proline analogue L-azetidine 2-carboxylic acid to growing cultures of Saccharomyces cerevisiae var. ellipsoideus promoted fast deactivation of the general aminoacid permease, measured as L-valine uptake, without an immediate decrease in the growth rate. Cells preincubated with the analogue for 3 h were unable to restore either growth ability or general aminoacid permease activity in analogue-free medium. Eadie-Hofstee plots of L-valine uptake in the presence of the analogue are consistent with a strong reduction in the number of active molecules of the general amino-acid permease located in the plasma membrane. Inhibitory effects on protein synthesis were seen after preincubations of the yeast with the analogue for 3 h although a 30 min preincubation had no effect.

  10. Fibroblast Activation Protein-Alpha, a Serine Protease that Facilitates Metastasis by Modification of Diverse Microenvironments

    DTIC Science & Technology

    2011-10-01

    lung tissue. We were not able to detect sufficient numbers of cells in this manner. We tried a different procedure for fixing the lungs after they...added after 24 hours. The films were fixed and evaluated microscopically. In four trials, 10 random microscopic fields were selected and... dosing by oral gavage once daily with 1.3 mg/kg L-valine-L-boroproline called talabostat (extracellular & intracellular DASH), 13.3 mg/kg L- glutamyl

  11. Enantioselective Friedel-Crafts reactions between phenols and N-tosylaldimines catalyzed by a leucine-derived bifunctional catalyst.

    PubMed

    Li, Guo-Xing; Qu, Jin

    2012-06-04

    Enantioselective Friedel-Crafts reactions between phenols and N-tosylaldimines were developed using a bifunctional catalyst readily prepared from L-leucine. The chiral benzylic amine products were obtained in high yields (up to 96% yield) and good to high enantiomeric excesses (up to 95% ee).

  12. Constituents of cultivated Agaricus blazei.

    PubMed

    Ueguchi, Yumi; Matsunami, Katsuyoshi; Otsuka, Hideaki; Kondo, Kazunari

    2011-04-01

    Two phenylhexane derivatives (1, 2), benzoylergostane (3), N-benzoyl-L-leucine methyl ester (4), two known ergostanes, and highly degraded incisterol were isolated from fruit bodies of Agaricus blazei. Compound 3 exhibited strong cytotoxicity toward HepG2 cells (IC(50) = 6.0 ± 0.33 μM).

  13. 21 CFR 172.829 - Neotame.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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 percent of the... basis. (c) The food additive neotame may be safely used as a sweetening agent and flavor enhancer...

  14. Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets.

    PubMed

    Hernández-Fisac, Inés; Fernández-Pascual, Sergio; Ortsäter, Henrik; Pizarro-Delgado, Javier; Martín del Río, Rafael; Bergsten, Peter; Tamarit-Rodriguez, Jorge

    2006-11-15

    OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.

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

    PubMed

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

    2015-10-01

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

  16. 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.

  17. Novel propanamide analogue and antiproliferative diketopiperazines from mangrove Streptomyces sp. Q24.

    PubMed

    Ye, Xuewei; Chai, Weiyun; Lian, Xiao-Yuan; Zhang, Zhizhen

    2017-06-01

    A new propanamide analogue (1), along with one known alkaloid (2) and four known diketopiperazines (3-6), was isolated from a cultured broth of the actinomycete Streptomyces sp. Q24 that was obtained from a sample of mangrove soil. The structures of these isolates were characterised as 3-acetylamino-N-2-thienyl-propanamide (1), N-acetyltryptamine (2), cyclo-(l-phenylalanine-l-4-hydroxyproline) (3), cyclo-(l-leucine-l-4-hydroxyproline) (4), cyclo-(l-phenylalanine-d-4-hydroxyproline) (5) and cyclo-(l-leucine-l-proline) (6) based on their NMR and HRESIMS data as well as optical rotation. Three diketopiperazines (3, 4, 6) showed activity in inhibiting the proliferation of human glioma U87-MG and U251 cells. This type of the new propanamide analogue (1) is first found from a nature source and the antiproliferative property of these three diketopiperazines against glioma cells is also reported herein for the first time.

  18. 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.

  19. Design of peptides with alpha,beta-dehydro residues: pseudo-tripeptide N-benzyloxycarbonyl-DeltaLeu-L-Ala-L-Leu-OCH3.

    PubMed

    Makker, Jyoti; Dey, Sharmistha; Kumar, Pravindra; Singh, Tej P

    2002-04-01

    The title peptide N-benzyloxycarbonyl-DeltaLeu-L-Ala-L-Leu-OCH(3) [methyl N-(benzyloxycarbonyl)-alpha,beta-dehydroleucyl-L-alanyl-L-leucinate], C(24)H(35)N(3)O(6), was synthesized in the solution phase. The peptide adopts a type II' beta-turn conformation which is stabilized by an intramolecular 4 --> 1 N-H* * *O hydrogen bond. The crystal packing is stabilized by two intermolecular N-H* * *O hydrogen bonds.

  20. Conversion into GABA (gamma-aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue.

    PubMed Central

    Fernández-Pascual, Sergio; Mukala-Nsengu-Tshibangu, André; Martín Del Río, Rafael; Tamarit-Rodríguez, Jorge

    2004-01-01

    We have carried out a detailed examination of L-glutamine metabolism in rat islets in order to elucidate the paradoxical failure of L-glutamine to stimulate insulin secretion. L-Glutamine was converted by isolated islets into GABA (gamma-aminobutyric acid), L-aspartate and L-glutamate. Saturation of the intracellular concentrations of all of these amino acids occurred at approx. 10 mmol/l L-glutamine, and their half-maximal values were attained at progressively increasing concentrations of L-glutamine (0.3 mmol/l for GABA; 0.5 and 1.0 mmol/l for Asp and Glu respectively). GABA accumulation accounted for most of the 14CO2 produced at various L-[U-14C]glutamine concentrations. Potentiation by L-glutamine of L-leucine-induced insulin secretion in perifused islets was suppressed by malonic acid dimethyl ester, was accompanied by a significant decrease in islet GABA accumulation, and was not modified in the presence of GABA receptor antagonists [50 micromol/l saclofen or 10 micromol/l (+)-bicuculline]. L-Leucine activated islet glutamate dehydrogenase activity, but had no effect on either glutamate decarboxylase or GABA transaminase activity, in islet homogenates. We conclude that (i) L-glutamine is metabolized preferentially to GABA and L-aspartate, which accumulate in islets, thus preventing its complete oxidation in the Krebs cycle, which accounts for its failure to stimulate insulin secretion; (ii) potentiation by L-glutamine of L-leucine-induced insulin secretion involves increased metabolism of L-glutamate and GABA via the Krebs cycle (glutamate dehydrogenase activation) and the GABA shunt (2-oxoglutarate availability for GABA transaminase) respectively, and (iii) islet release of GABA does not seem to play an important role in the modulation of the islet secretory response to the combination of L-leucine and L-glutamine. PMID:14763900

  1. Conversion into GABA (gamma-aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue.

    PubMed

    Fernández-Pascual, Sergio; Mukala-Nsengu-Tshibangu, André; Martín Del Río, Rafael; Tamarit-Rodríguez, Jorge

    2004-05-01

    We have carried out a detailed examination of L-glutamine metabolism in rat islets in order to elucidate the paradoxical failure of L-glutamine to stimulate insulin secretion. L-Glutamine was converted by isolated islets into GABA (gamma-aminobutyric acid), L-aspartate and L-glutamate. Saturation of the intracellular concentrations of all of these amino acids occurred at approx. 10 mmol/l L-glutamine, and their half-maximal values were attained at progressively increasing concentrations of L-glutamine (0.3 mmol/l for GABA; 0.5 and 1.0 mmol/l for Asp and Glu respectively). GABA accumulation accounted for most of the 14CO2 produced at various L-[U-14C]glutamine concentrations. Potentiation by L-glutamine of L-leucine-induced insulin secretion in perifused islets was suppressed by malonic acid dimethyl ester, was accompanied by a significant decrease in islet GABA accumulation, and was not modified in the presence of GABA receptor antagonists [50 micromol/l saclofen or 10 micromol/l (+)-bicuculline]. L-Leucine activated islet glutamate dehydrogenase activity, but had no effect on either glutamate decarboxylase or GABA transaminase activity, in islet homogenates. We conclude that (i) L-glutamine is metabolized preferentially to GABA and L-aspartate, which accumulate in islets, thus preventing its complete oxidation in the Krebs cycle, which accounts for its failure to stimulate insulin secretion; (ii) potentiation by L-glutamine of L-leucine-induced insulin secretion involves increased metabolism of L-glutamate and GABA via the Krebs cycle (glutamate dehydrogenase activation) and the GABA shunt (2-oxoglutarate availability for GABA transaminase) respectively, and (iii) islet release of GABA does not seem to play an important role in the modulation of the islet secretory response to the combination of L-leucine and L-glutamine.

  2. Nitric oxide (NO)--production and regulation of insulin secretion in islets of freely fed and fasted mice.

    PubMed

    Eckersten, Dag; Henningsson, Ragnar

    2012-02-10

    Production of nitric oxide through the action of nitric oxide synthase (NOS) has been detected in the islets of Langerhans. The inducible isoform of NOS (iNOS) is induced by cytokines and might contribute to the development of type-1 diabetes, while the constitutive isoform (cNOS) is thought to be implicated in the physiological regulation of insulin secretion. In the present study we have detected and quantified islet cNOS- and iNOS-derived NO production concomitant with measuring its influence on insulin secretion in the presence of different secretagogues: glucose, L-arginine, L-leucine and α-ketoisocaproic acid (KIC) both during fasting and freely fed conditions. In intact islets from freely fed mice both cNOS- and iNOS-activity was greatly increased by glucose (20 mmol/l). Fasting induced islet iNOS activity at both physiological (7 mmol/l) and high (20 mmol/l) glucose concentrations. NOS blockade increased insulin secretion both during freely fed conditions and after fasting. L-arginine stimulated islet cNOS activity and did not affect islet iNOS activity. l-leucine or KIC, known to enter the TCA cycle without affecting glycolysis, did not affect either islet cNOS- or iNOS activity. Accordingly, insulin secretion stimulated by L-leucine or KIC was unaffected by addition of L-NAME both during feeding and fasting. We conclude that both high glucose concentrations and fasting increase islet total NO production (mostly iNOS derived) which inhibit insulin secretion. The insulin secretagogues L-leucine and KIC, which do not affect glycolysis, do not interfere with the islet NO-NOS system.

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  4. Process Research and Development of Antibodies as Countermeasures for C. Botulinum

    DTIC Science & Technology

    2006-03-01

    Concentrations of the components in the control media. Components Composition (mg/L) CaCl2 (anhyd.) 99.1 CuSO4 *5H2O 0.00125 FeSO4*7H2O 0.415...18.37 L-Threonine 83.3 L-Tryptophan 17.8 L-Tyrosine*2Na* 2H2O 63 L-Valine 81.9 Biotin 0.02 D-Ca Pantothenate 4 Choline Chloride 18 Folic Acid 4 i

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

    PubMed

    Eikmanns, Bernhard J; Blombach, Bastian

    2014-12-20

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

  6. 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.

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

    PubMed

    Andrés, José M; Ceballos, Miriam; Maestro, Alicia; Sanz, Isabel; Pedrosa, Rafael

    2016-01-01

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

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

    PubMed Central

    Ceballos, Miriam; Maestro, Alicia; Sanz, Isabel

    2016-01-01

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

  9. Symmetrical drug-related intertriginous and flexural exanthema caused by valacyclovir.

    PubMed

    Daito, Junko; Hanada, Keiji; Katoh, Norito; Katoh, Sayoko; Sakamoto, Kakei; Asai, Jun; Takenaka, Hideya; Kishimoto, Saburo

    2009-01-01

    Drug-related eruptions that appear only on intertriginous or flexural folds and in gluteal areas have recently been termed symmetrical drug-related intertriginous and flexural exanthema (SDRIFE). We report a case of a 56-year-old woman with acute erythematous rash in the intertriginous areas after treatment with the L-valine ester of acyclovir, valacyclovir. Oral-challenge tests resulted in erythematous pruritic rash in the intertriginous area by valacyclovir. The patient was diagnosed as having SDRIFE due to valacyclovir.

  10. Asymmetric synthesis and biological evaluations of (+)- and (-)-6-dimethoxymethyl-1,4-dihydropyridine-3-carboxylic acid derivatives blocking N-type calcium channels.

    PubMed

    Yamamoto, Takashi; Ohno, Seiji; Niwa, Seiji; Tokumasu, Munetaka; Hagihara, Masako; Koganei, Hajime; Fujita, Shin-ichi; Takeda, Tomoko; Saitou, Yuki; Iwayama, Satoshi; Takahara, Akira; Iwata, Seinosuke; Shoji, Masataka

    2011-06-01

    An efficient asymmetric synthesis of 1,4-dihydropyridine derivatives is described. The key step is the stereoselective Michael addition using t-butyl ester of L-valine as a chiral auxiliary to achieve good ee (>95% for all the tested experiments) and moderate yield. With this method, (+)-4-(3-chlorophenyl)-6-dimethoxymethyl-2-methyl-1,4-dihydropyridine-3,5-dicarboxylic acid cinnamyl ester was obtained and was characterized as a promising N-type calcium channel blocker with improved selectivity over L-type compared to its (-)- and racemic isomers.

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

    PubMed Central

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

    2015-01-01

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

  12. Effect of the deformability of guest particles on the tensile strength of tablets from interactive mixtures.

    PubMed

    Mangal, Sharad; Lakio, Satu; Gengenbach, Thomas; Larson, Ian; Morton, David A V

    2016-12-05

    In this study, we investigated the influence of deformability of specifically-engineered guest particles on the tensile strength of tablets of interactive mixtures. The binder polyvinylpyrrolidone (PVP) of different molecular weights were spray dried with l-leucine to create guest particle formulations. The guest particle formulations were characterized by their particle size, surface l-leucine concentration and glass transition temperature (Tg). These spray-dried particles were then blended with paracetamol to form interactive mixtures, which were compacted into tablets and tablet tensile strength and elastic recovery were determined. The guest particles had particle diameters in the range of 1-10μm, and surfaces that were l-leucine enriched. The Tg of guest particle formulations increased with increasing molecular weight of the PVP. All the guest particle formulations formed an observed homogeneous interactive mixture with paracetamol. The tensile strength of the tablets of interactive mixtures increased with decreasing Tg of the guest particles. In these interactive mixtures, higher tensile strength was also associated with lower tablet elastic recovery. The elastic recovery of the tablets showed a correlation with the elastic recovery of the tablets of guest particles. Thus, our results indicated that the deformability of guest particles dictates the tensile strength of the tablets of these interactive mixtures.

  13. Low-damage milling of an amino acid thin film with cluster ion beam

    SciTech Connect

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

    2011-11-01

    In this work, we characterized the surface damage layer and sputtering yield of polycrystalline L-leucine films before and after irradiation with Ar cluster or monomer ion beams with x ray photoelectron spectroscopy and ellipsometry. Irradiation with Ar monomer ion beams induced heavy damage on the surface of L-leucine films, such as bond breaking and carbonization. In contrast, no significant surface damage was observed in the films irradiated with Ar cluster ion beams. The sputtering yield of L-leucine decreased dramatically with increasing fluence of monomer Ar ions and approached the value of the sputtering yield of graphite; but under irradiation with Ar cluster ion beams, the sputtering yield remained constant with fluence. The differences in sputtering yield behavior were explained in relation with the surface damage layer on organic materials. Thus, cluster ion beams could potentially be used to mill down biological materials without significant damage on the surface and could contribute to various applications in the analysis and processing of life matter.

  14. 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.

  15. Polypeptide Grafted Hyaluronan: Synthesis and Characterization

    SciTech Connect

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

    2010-01-01

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

  16. Synthesis of Pulcherriminic Acid by Bacillus subtilis

    PubMed Central

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

    1972-01-01

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

  17. 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.

  18. Investigation of the Changes in Aerosolization Behavior Between the Jet-Milled and Spray-Dried Colistin Powders Through Surface Energy Characterization.

    PubMed

    Jong, Teresa; Li, Jian; Morton, David A V; Zhou, Qi Tony; Larson, Ian

    2016-03-01

    This study aimed to investigate the surface energy factors behind improved aerosolization performance of spray-dried colistin powder formulations compared with those produced by jet milling. Inhalable colistin powder formulations were produced by jet milling or spray drying (with or without l-leucine). Scanning electron micrographs showed the jet-milled particles had irregularly angular shapes, whereas the spray-dried particles were more spherical. Significantly higher fine particle fractions were measured for the spray-dried (43.8%-49.6%) versus the jet-milled formulation (28.4%) from a Rotahaler at 60 L/min; albeit the size distribution of the jet-milled powder was smaller. Surprisingly, addition of l-leucine in the spray drying feed solution gave no significant improvement in fine particle fraction. As measured by inverse gas chromatography, spray-dried formulations had significantly (p < 0.001) lower dispersive, specific, and total surface energy values and more uniform surface energy distributions than the jet-milled powder. Interestingly, no significant difference was measured in the specific and total surface energy values between the spray-dried formulation with or without l-leucine. Based on our previous findings in the self-assembling behavior of colistin in aqueous solution and the surface energy data obtained here, we propose the self-assembly of colistin molecules during spray drying contributed significantly to the reduction of surface free energy and the superior aerosolization performance.

  19. Polypeptide Grafted Hyaluronan: Synthesis and Characterization

    PubMed Central

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

    2010-01-01

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

  20. Investigation of the changes in aerosolization behavior between the jet-milled and spray-dried colistin powders through surface energy characterization

    PubMed Central

    Jong, Teresa; Li, Jian; Mortonx, David A.V.; Zhou, Qi (Tony); Larson, Ian

    2016-01-01

    This study aimed to investigate the surface energy factors behind improved aerosolization performance of spray-dried colistin powder formulations compared to those produced by jet-milling. Inhalable colistin powder formulations were produced by jet-milling or spray-drying (with or without L-leucine). Scanning electron micrographs showed the jet-milled particles had irregularly angular shapes, while the spray-dried particles were more spherical. Significantly higher fine particle fractions (FPFs) were measured for the spray-dried (43.8-49.6%) vs. the jet-milled formulation (28.4 %) from a Rotahaler at 60L/min; albeit the size distribution of the jet-milled powder was smaller. Surprisingly, addition of L-leucine in the spray drying feed-solution gave no significant improvement in FPF. As measured by inverse gas chromatography, spray-dried formulations had significantly (p<0.001) lower dispersive, specific and total surface energy values and more uniform surface energy distributions than the jet-milled powder. Interestingly, no significant difference was measured in the specific and total surface energy values between the spray-dried formulation with or without L-leucine. Based upon our previous findings in the self-assembling behavior of colistin in aqueous solution and the surface energy data obtained here, we propose the self-assembly of colistin molecules during spray-drying, contributed significantly to the reduction of surface free energy and the superior aerosolization performance. PMID:26886330

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

    PubMed

    Sugimoto, Hiroshi; Kakehi, Masaaki; Jinno, Fumihiro

    2015-10-01

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

  2. Amino Acid Transport in Mycobacterium smegmatis

    PubMed Central

    Yabu, Kunihiko

    1970-01-01

    The transport of d-alanine, d-glutamic acid, and d-valine in Mycobacterium smegmatis was compared quantitatively with that of their l-isomers. It appeared that the uptake of d-alanine was mediated by an active process displaying saturation kinetics characteristic of enzyme function, whereas the uptake of d-glutamic acid was accomplished by a passive process showing diffusion kinetics. Both processes were involved in the uptake of l-alanine, l-glutamic acid, d-valine, and l-valine. d-Valine competed with l-valine for entry into the cell through a single active process. d-Alanine and l-alanine also utilized the same active process, but the d-isomer could not enter the cell through the passive process. The passive process exhibited characteristics of diffusion, but was sensitive to sulfhydryl-blocking reagents and showed competition among structurally related amino acids. These last findings suggested that the passive process is a facilitated diffusion. PMID:5437732

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

    PubMed Central

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

    2012-01-01

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

  4. Cationic bactericidal peptide 1018 does not specifically target the stringent response alarmone (p)ppGpp

    PubMed Central

    Andresen, Liis; Tenson, Tanel; Hauryliuk, Vasili

    2016-01-01

    The bacterial stringent response is a key regulator of bacterial virulence, biofilm formation and antibiotic tolerance, and is a promising target for the development of new antibacterial compounds. The intracellular nucleotide (p)ppGpp acts as a messenger orchestrating the stringent response. A synthetic peptide 1018 was recently proposed to specifically disrupt biofilms by inhibiting the stringent response via direct interaction with (p)ppGpp (de la Fuente-Núñez et al. (2014) PLoS Pathogens). We have interrogated the specificity of the proposed molecular mechanism. When inhibition of Pseudomonas aeruginosa planktonic and biofilm growth is tested simultaneously in the same assay, peptides 1018 and the control peptide 8101 generated by an inversion of the amino acid sequence of 1018 are equally potent, and, importantly, do not display a preferential activity against biofilm. 1018 inhibits planktonic growth of Escherichia coli equally efficiently either when the alleged target, (p)ppGpp, is essential (MOPS media lacking amino acid L-valine), or dispensable for growth (MOPS media supplemented with L-valine). Genetic disruption of the genes relA and spoT responsible for (p)ppGpp synthesis moderately sensitizes – rather than protects – E. coli to 1018. We suggest that the antimicrobial activity of 1018 does not rely on specific recognition of the stringent response messenger (p)ppGpp. PMID:27819280

  5. Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum.

    PubMed

    Marienhagen, Jan; Kennerknecht, Nicole; Sahm, Hermann; Eggeling, Lothar

    2005-11-01

    Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5'-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with L-glutamate, L-aspartate, and L-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) L-glutamate > 2-aminobutyrate > L-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes L-alanine to aminate 2-oxo-isovalerate, the L-valine precursor, and 2-oxo-butyrate. A second AT active with the L-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for L-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.

  6. Biosynthesis of tiglic, ethacrylic, and 2-methylbutyric acids in a carabid beetle, Pterostichus (Hypherpes) californicus.

    PubMed

    Attygalle, Athula B; Wu, Xiaogang; Will, Kipling W

    2007-05-01

    Tiglic, 2-methylbutyric, and ethacrylic acids are found in the pygidial gland defensive fluid of many carabid beetles. By injecting a deuterium-labeled precursor into the carabid beetle Pterostichus (Hypherpes) californicus, and analyzing the defensive fluid by gas chromatography/mass spectrometry, we were able to demonstrate that tiglic and ethacrylic acids are biosynthesized from isoleucine via 2-methylbutyric acid. Moreover, we observed that the injection of L-isoleucine induces an increased production of tiglic acid in P. californicus. A strong primary kinetic isotope effect was found to operate in the dehydrogenation step of 2-methylbutyric acid to tiglic and ethacrylic acids. Consequently, ethacrylic acid was found to preferentially accumulate the deuterium labeling from [2,3,4,4-(2)H(4)]isoleucine during our biosynthetic experiments.

  7. Jasmonate signaling and manipulation by pathogens and insects.

    PubMed

    Zhang, Li; Zhang, Feng; Melotto, Maeli; Yao, Jian; He, Sheng Yang

    2017-01-09

    Plants synthesize jasmonates (JAs) in response to developmental cues or environmental stresses, in order to coordinate plant growth, development or defense against pathogens and herbivores. Perception of pathogen or herbivore attack promotes synthesis of jasmonoyl-L-isoleucine (JA-Ile), which binds to the COI1-JAZ receptor, triggering the degradation of JAZ repressors and induction of transcriptional reprogramming associated with plant defense. Interestingly, some virulent pathogens have evolved various strategies to manipulate JA signaling to facilitate their exploitation of plant hosts. In this review, we focus on recent advances in understanding the mechanism underlying the enigmatic switch between transcriptional repression and hormone-dependent transcriptional activation of JA signaling. We also discuss various strategies used by pathogens and insects to manipulate JA signaling and how interfering with this could be used as a novel means of disease control.

  8. Whole cell biotransformation for reductive amination reactions.

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

  10. Effect of selectively introducing arginine and D-amino acids on the antimicrobial activity and salt sensitivity in analogs of human beta-defensins.

    PubMed

    Olli, Sudar; Rangaraj, Nandini; Nagaraj, Ramakrishnan

    2013-01-01

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

  11. A study regarding friction behaviour of lysine and isoleucine modified epoxy matrix

    NASA Astrophysics Data System (ADS)

    Bălan, I.; Bosoancă, R.; Căpăţină, A.; Graur, I.; Bria, V.; Ungureanu, C.

    2017-02-01

    The aim of this study is to point out the effect of L-lysine and L-isoleucine used as modifying agents for epoxy resins. The amino acids are largely used to turn the usual polymers in bio-compatible materials but they effect also other significant proprieties of formed materials. The general study developed in Polymer Composite Laboratory is focused on analysis of 14 amino acids used as modifying agents but the two above mentioned showed a special behaviour namely they re-crystalized during the polymerization of the matrix. The coefficient of friction was obtained through the calculation of friction torque measured with a loaded cell sensor. As far as we know, there is no report on the friction proprieties of amino acids modified epoxy resins.

  12. 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

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

    SciTech Connect

    Mallakpour, Shadpour; Dinari, Mohammad

    2012-09-15

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

  14. 1,25-Dihydroxyvitamin D3 Induces LL-37 and HBD-2 Production in Keratinocytes from Diabetic Foot Ulcers Promoting Wound Healing: An In Vitro Model

    PubMed Central

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

    2014-01-01

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

  15. 1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

    PubMed

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

    2014-01-01

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

  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

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

    PubMed

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

    2014-01-01

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

  18. Protective effects of leucine on redox status and mitochondrial-related gene abundance in the jejunum of intrauterine growth-retarded piglets during early weaning period.

    PubMed

    Huang, Qiang; Xu, Wen; Bai, Kai-Wen; He, Jin-Tian; Ahmad, Hussain; Zhou, Le; Zhang, Li-Li; Wang, Tian

    2017-04-01

    The aim of this study was to investigate the effects of dietary supplementation with 0.35% l-leucine on redox status and gene abundance relating to mitochondrial biogenesis and function in the jejunum of intrauterine growth-retarded (IUGR) piglets during early weaning period. According to a 2 × 2 factorial arrangement, 16 IUGR and 16 normal body weight (NBW) piglets were fed a basal diet without l-leucine supplementation or a basal diet plus 0.35% l-leucine supplementation from the age of 14 to 35 d. The results showed that compared with NBW piglets, IUGR piglets had a lower (p < 0.05) jejunal DNA concentration, a reduced (p < 0.05) manganese superoxide dismutase (MnSOD) and total antioxidant capability (T-AOC) activities and mitochondrial DNA content in the jejunum. Leucine supplementation increased (p < 0.05) MnSOD and T-AOC activities and decreased (p < 0.05) the malondialdehyde content in the jejunum of IUGR piglets. The mRNA gene abundance of nuclear respiratory factor-1 (NRF1), mitochondrial transcription factor A (TFAM), ATP synthase (ATPs), cytochrome c oxidase V (CcOX V), cytochrome c and glucokinase in the jejunum of IUGR piglets was reduced (p < 0.05) compared with NBW piglets. However, NRF1, peroxisome proliferation-activated receptor gamma coactivator-1 alpha, TFAM, ATPs and CcOX I mRNA gene abundance in the jejunum of IUGR piglets were increased (p < 0.05) by diets supplemented with leucine. These data indicate that leucine supplementation has therapeutic potential for attenuating intestinal oxidative stress and mitochondrial dysfunction in IUGR piglets during the early period of life via increasing enzyme activities and up-regulating mRNA gene abundance.

  19. 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.

  20. Study of the racemization observed in the amide bond forming reaction on silica gel.

    PubMed

    Norick, Amanda L; Li, Tingyu

    2005-01-01

    Racemization resulting from the coupling of N-(3,5-dinitrobenzoyl)-L-leucine and 3-aminopropyl silica gel with several amide-coupling reagents is further investigated in order to explain the much higher degree of racemization on silica gel, as compared with the similar reaction in solution. Based on experiments using different types of solid supports, limited pore access and surface microchemical environment are ruled out as the possible reason for the higher degree of racemization that occurred on silica gel. Steric hindrance of the solid support is thought to have caused the amino group to be more basic relative to its nucleophilicity, leading to a higher degree of racemization.

  1. 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

  2. Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin.

    PubMed

    Yamashita, Makiko; Harada, Gakuro; Matsumoto, Shin-ei; Aiba, Yoshihiro; Ichikawa, Akira; Fujiki, Tsukasa; Udono, Miyako; Kabayama, Shigeru; Yoshida, Tadashi; Zhang, Pingbo; Fujii, Hiroshi; Shirahata, Sanetaka; Katakura, Yoshinori

    2014-02-01

    In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14(+) monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14(+) monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.

  3. Effects of mercury on spermatogenesis studied by velocity sedimentation cell separation and serial mating.

    PubMed

    Lee, I P; Dixon, R L

    1975-07-01

    Recently the potential toxicity of environmental mercury has become a major concern. Human tissues contain mercury due to daily environmental exposure. Nearly all of the mercury contaminating food is in the form of methylmercury complexes, due to the biotransformation of inorganic mercury. This report compares the reproductive effects of methylmercury hydroxide and mercuric chloride in male mice. The mercuric compounds were administered intraperitoneally once at a dose of 1 mg/kg (based on Hg++ concentration), or spermatogenic cells were exposed in vitro to Hg+ concentrations ranging from 10(-3) to 10(-8)M. Spermatogenic cells were separated for biochemical studies using the velocity sedimentation technique, and in vivo serial mating was used to assess fertility. The effects of CH3Hg+ or Hg++ on the uptake of 3H-thymidine by spermatogonia, 3H-uridine by early elongated spermatids, and 3H-L-leucine by late elongated spermatids were studied. These in vitro experiments indicated that at 1-(-3) m CH2HgOH reduced thymidine incorporation by spermatogonia by 40%, uridine incorporation by elongated spermatids by 39% and L-leucine incorporation by late elongated spermatids by 40%. Results obtained with HgCl2 were similar but of lesser magnitude. In vivo administration of CH3HgOH and HgCl2 significantly inhibited the uptake of thymidine, uridine and L-leucine by their respective spermatogenic cells. Fertility profiles obtained from serial mating studies indicated that the primary effect of CH3HgOH was on spermatogonial cells, premeiotic spermatocytes and early elongated spermatids, with no apparent effect on spermatozoa in testis, peididymis or vas deferentia. HgCl2 also primarily affected spermatogonial and premeiotic cells, but the effect was less than that seen with CH3HgOH. Statistical analysis indicated significant antifertility effects. Inhibition of uptake of thymidine and uridine was well correlated with the functionality of these cells as reflected in the fertility

  4. ENZYMATIC FORMATION OF D-KYNURENINE

    DTIC Science & Technology

    x 10 to the -3 power M), and methylene blue (10 to the -4 power M) to stoichiometric amounts of D-kynurenine. The enzyme activity was evident only in...which distinguishes the D- and L-isomers of kynurenine. Methylene blue appeared essential for the reaction; FAD, FMN, DPN, TPN, or ferricyanide could...for kynurenine formation, whereas the presence of an H2O2 generating system (L amino acid oxidase and L-leucine) did not substitute for the methylene blue .

  5. Changing the substrate specificity of penicillin G acylase from Kluyvera citrophila through selective pressure.

    PubMed Central

    Roa, A; Garcia, J L; Salto, F; Cortes, E

    1994-01-01

    Escherichia coli (muT, mutD, Leu-) cells transformed with plasmid pYKD59 harbouring the pac gene encoding penicillin acylase (PA) from Kluyvera citrophila ATCC 21285 were exposed to environmental conditions that made expression of this enzyme essential for growth. Under these conditions, spontaneous mutants were isolated that used adipyl-L-leucine as the sole source of L-leucine. DNA sequencing of the mutant pac genes identified a transversion mutation of thymine to guanine at position 1163. This mutation was located in the beta-subunit of the enzyme and resulted in conversion of Phe-360 to valine. The assignment of this mutation to the shift in substrate specificity was further confirmed by site-directed mutagenesis. Secondary-structure prediction of the region surrounding Phe-360 suggests that this mutation should not produce any significant structural change. The purified mutant acylase was able to hydrolyse adipyl-, glutaryl-, valeryl-, caproyl-, heptanoyl- and phenoxyacetyl-L-leucine at pH 5 with greater efficiency than the wild-type enzyme. However, the mutant enzyme was not able to hydrolyse glutaryl-7-aminocephalosporanic acid and had lost 90% and 50% of activity on penicillin G and phenylacetyl-L-leucine respectively. Nevertheless, mutant PA retained its original activity on 6-nitro-3-phenylacetamidobenzoate and p-nitrophenylphenylacetate, suggesting that the binding specificity of PA by the acyl and amine moieties of the substrate are not independent phenomena. The small differences observed between the c.d. spectra of the mutant enzyme recorded at pH 5 and 8 suggest the existence of different conformational states at the two pH values, but these differences were indistinguishable from those observed in the native enzyme and cannot be correlated with the shift in substrate specificity. Our results demonstrate that it is possible to change the specificity of PA by laboratory evolution and use it to identify the amino acids involved in substrate recognition

  6. Targetable Polymer - Antiangiogenic Drug Conjugates for Systemic Breast Cancer Therapy

    DTIC Science & Technology

    2005-09-01

    1.7FL G-Fu_ Legends: HPMA= N-(2-hydroxypropyl) methacrylamide, G=Glycine, F=Phenylalanine, L= Leucine , Fu=Fumagillol, M,=weight average molecular...CM. The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase , MetAP-2. Proc NatlAcad Sci USA. 1997; 94(12...covalently modifies a conserved active-site histidine in the Escherichia coli methionine aminopeptidase . Proc Natl Acad Sci USA. 1998; 95(21): 12153-7

  7. Aminopeptidase from Brevibacterium linens: activation and inhibition.

    PubMed

    Foissy, H

    1978-04-18

    Activation and inhibition of a purified aminopeptidase from Brevibacterium linens was investigated using L-alpha-leucyl-4-nitroanilide and L-leucyl-L-leucine as substrates. The enzyme was activated by cobalt, provided that the enzyme was preincubated with the metal. Strong inhibitory effects were derived from heavy metals, metal-complexing compounds, reducing agents, the modification of aromatic amino acids, and the presence of hydrophobic substances or certain amino acids in the test mixtures. Supposing that this B. linens aminopeptidase plays a part during surface-ripening of cheeses, possible consequences of specific technological conditions for its activity are discussed.

  8. Liquid chromatographic determination of hippuric acid for the evaluation of ethacrynic acid as angiotensin converting enzyme inhibitor.

    PubMed

    Mehanna, A S; Dowling, M

    1999-05-01

    A rapid, simple and interference-free method is described to evaluate the inhibitory effects of organic compounds on the activity of angiotensin converting enzyme irrespective of their acid-base properties. The assay is based on the high performance liquid chromatographic separation of the synthetic substrate hippuryl-L-histidyl-L-leucine, the hydrolysis product hippuric acid and the test compound. Using the new method, the diuretic drug ethacrynic acid was found to act as an inhibitor for the enzyme in a non competitive mode.

  9. Photorelease of amino acids from novel thioxobenzo[f]benzopyran ester conjugates.

    PubMed

    Piloto, Ana M; Soares, Ana M S; Costa, Susana P G; Gonçalves, M Sameiro T

    2012-06-01

    Aiming at the enhancement of the performance of (9-methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl) methyl ester as photocleavable protecting group for the carboxylic acid function at long-wavelengths, 9-methoxy-3-thioxo-3H-benzo[f]benzopyran-L-valine and L-phenylalanine model conjugates were prepared through a thionation reaction of the corresponding oxo-benzobenzopyrans. These thioxobenzobenzopyran derivatives were subjected to photocleavage reactions in the same conditions as the parent oxo-benzobenzopyrans at different wavelengths of irradiation, and photocleavage data were obtained. It was found that the exchange of the carbonyl by a thiocarbonyl group enhanced the performance of the heterocyclic protecting group at 419 nm by improving the photolysis rates, making it an appropriate group for practical applications at long wavelengths.

  10. (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.

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

    SciTech Connect

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

    1994-12-01

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

  12. Crystal structure of catena-poly[silver(I)-μ-l-valinato-κ2 N:O

    PubMed Central

    Takagi, Yoshitaka; Okamoto, Youhei; Inoue, Chisato; Kasuga, Noriko Chikaraishi

    2017-01-01

    The reaction of Ag2O with l-valine (l-Hval, C5H11NO2) in a 1:2 molar ratio in water, followed by vapour diffusion, afforded a coordination polymer of the title compound, [Ag(C5H10NO2)]n, with N—Ag—O repeat units, which is classified as a type III silver(I) complex with amino acid ligands. The asymmetric unit consists of two independent units of [Ag(l-val)]. In the crystal, the polymeric chains run along [101], and neighbouring chains are linked via a weak Ag⋯Ag inter­action and N—H⋯O hydrogen bonds. The title complex exhibited anti­microbial activity against selected bacteria (Escherichia coli, Bacillus subtilis, Staphylococcous aureus and Psedomonas aeruginosa). PMID:28316807

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  15. Design of chiral monochloro-s-triazine reagents for the liquid chromatographic separation of amino acid enantiomers.

    PubMed

    Brückner, H; Wachsmann, M

    2003-05-23

    A series of chiral derivatizing reagents (CDRs) was synthesized by nucleophilic replacement of one chlorine atom in cyanuric chloride (2,4,6-trichloro-1,3,5-triazine; s-triazine) by alkoxy (methoxy, butoxy, 1,1,1-trifluoroethoxy) or aryloxy groups (phenoxy, nitrophenoxy, phenylphenoxy, 4-methylcoumaryloxy), and displacement of a second chlorine by L-alanine amide, L-phenylalanine amide, L-proline tert.-butyl ester, or Boc-L-lysine tert.-butyl ester. Further, CDRs were investigated in which two chlorine atoms in cyanuric chloride were substituted consecutively by L-valine amide and L-phenylalanine amide. The resulting CDRs having a remaining reactive chlorine were tested for their capability of derivatizing DL-amino acids followed by liquid chromatographic separation of the resulting diastereomers.

  16. Serum Metabolomics in Rats after Acute Paraquat Poisoning.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  18. 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.

  19. Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence of Corynebacterium glutamicum

    PubMed Central

    Marienhagen, Jan; Kennerknecht, Nicole; Sahm, Hermann; Eggeling, Lothar

    2005-01-01

    Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with l-glutamate, l-aspartate, and l-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) l-glutamate > 2-aminobutyrate > l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes l-alanine to aminate 2-oxo-isovalerate, the l-valine precursor, and 2-oxo-butyrate. A second AT active with the l-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for l-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far. PMID:16267288

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

    PubMed Central

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

    2012-01-01

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

  1. Transport of. cap alpha. -aminoisobutyric acid by Streptococcus pyogenes and its derived L-form

    SciTech Connect

    Reizer, J.; Panos, C.

    1982-01-01

    We studied the uptake of ..cap alpha..-aminoisobutyric acid (AIB) in Streptococcus pyogenes and its physiologically isotonic L-form. S. pyogenes cells starved for glucose or treated with carbonyl cyanide-m-chlorophenyl hydrazone accumulated limited amounts of AIB. A high apparent K/sub m/ value characterized the glucose-independent transport of AIB. The rate and extent of AIB accumulation significantly increased in the presence of glucose. Two saturable transport components with distinct apparent K/sub m/values characterized glycolysis-coupled transport of AIB. A biphasic Lineweaver-Burk plot was also obtained for L-alanine transport by glycolyzing S. pyogenes cells. AIB seems to share a common transport system(s) with glycine, L- and D-anine, L-serine, and L-valine. This was shown by the competitive exchange efflux of accumulated AIB. About 30% of the AIB uptake was not inhibited by a saturating amount of L-valine, indicating the existence of more than one system for AIB transport, p-Chloromercuribenzoate markedly inhibited the accumulation of AIB by both glycolyzing and glucose-starved cells. In contrast, carbonyl cyanide-m-chlorophenyl hydrazone affected only metabolism-dependent uptake of AIB, which was also sensitive to dinitrophenol, N-ethylmaleimide, iodoacetate, fluoride (NaF), arsenate, and N,N'-dicyclohexylcarbodiimide. These results are interpreted according to the chemiosmotic theory of Mitchell, whereby a proton motive force constitutes the driving force for AIB accumulation. AIB was not accumulated by the L-form. However, a temporary accumulation of AIB by a counterflow mechanism and a saturable system with a low apparent affinity were demonstrated for AIB transport by this organism. We suggest that a deficiency in the coupling of energy to AIB transport is responsible for the apparent lack of active AIB accumulation by the L-form.

  2. d-Amino Acid Position Influences the Anticancer Activity of Galaxamide Analogs: An Apoptotic Mechanism Study

    PubMed Central

    Bai, Defa; Yu, Siming; Zhong, Shenghui; Zhao, Bingxin; Qiu, Shaoling; Chen, Jianwei; Lunagariya, Jignesh; Liao, Xiaojian; Xu, Shihai

    2017-01-01

    Galaxamide, an extract from Galaxaura filamentosa, is a cyclic pentapeptide containing five l-leucines. Due to the particular cyclic structure and the excellent anticancer activity, synthesis of Galaxamide and its analogs and their subsequent bio-applications have attracted great attention. In the present work, we synthesized six Galaxamide analogs by replacing one of the l-leucines with phenylalanine and varying the d-amino acid position. The anticancer effect of the synthesized Galaxamide analogs was tested against four in vitro human cancer cell lines, human hepatocellular cells (HepG2), human breast cancer cell (MCF-7), human breast adenocarcinoma cells (MDA-MB-435) and a human cervical carcinoma cell line (Hela). Results showed that Galaxamide analogs with different d-amino acid positions displayed distinct anticancer potential. The Galaxamide analog containing d-amino acid at position 5 (Analog-6) presented the strongest anticancer activity. The mechanism study revealed that Analog-6 could cause the early apoptosis of HepG2 cells by inhibiting their growth in the sub-G1 stage of the cell cycle and induce the chromatin condensation and fragmentation, which can be seen as 68% of HepG2 cells inhibited in the sub-G1 stage. Moreover, a mitochondria-mediated pathway was found to be involved in the apoptotic process of Analog-6 on HepG2 cells. PMID:28287429

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

    PubMed

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

    2009-01-05

    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.

  4. [Formation Mechanism of the Disinfection By-product 1, 1-Dichloroacetone in Drinking Water].

    PubMed

    Ding, Chun-sheng; Meng, Zhuang; Xu, Yang-yang; Miao, Jia

    2015-05-01

    A novel method using methyl tertiary butyl ether (MTBE) as extractant and 1,2-dibromopropane as internal standard for the determination of the disinfection by-producs 1,1-dichloroacetone (DCAce) by gas chromatography mass spectrometry (GC-MS) was described. The formation process of DCAce and its influencing factors were discussed with L-leucine as the precursor during the chloramination process. The results indicated that the DCAce production increased with the increase of chloramine dosage when the chloramine addition was in the range of 5-30 mg · L(-1). The DCAce amount produced under alkaline condition was higher than those produced under the neutral and acidic conditions, and the DCAce amount reduced with the increase of pH value. Temperature was another important factor that affected the DCAce formation from methylamine especially in the range of 15-35°C , and the higher the temperature, the more the DCAce produced. The formation process of DCAce from L-leucine by chloramine consisted of a series of complicated reactions, including substitution, oxidation, bond breaking, amino diazotization, reduction and so on, and eventually DCAce was formed.

  5. d-Amino Acid Position Influences the Anticancer Activity of Galaxamide Analogs: An Apoptotic Mechanism Study.

    PubMed

    Bai, Defa; Yu, Siming; Zhong, Shenghui; Zhao, Bingxin; Qiu, Shaoling; Chen, Jianwei; Lunagariya, Jignesh; Liao, Xiaojian; Xu, Shihai

    2017-03-10

    Galaxamide, an extract from Galaxaura filamentosa, is a cyclic pentapeptide containing five l-leucines. Due to the particular cyclic structure and the excellent anticancer activity, synthesis of Galaxamide and its analogs and their subsequent bio-applications have attracted great attention. In the present work, we synthesized six Galaxamide analogs by replacing one of the l-leucines with phenylalanine and varying the d-amino acid position. The anticancer effect of the synthesized Galaxamide analogs was tested against four in vitro human cancer cell lines, human hepatocellular cells (HepG₂), human breast cancer cell (MCF-7), human breast adenocarcinoma cells (MDA-MB-435) and a human cervical carcinoma cell line (Hela). Results showed that Galaxamide analogs with different d-amino acid positions displayed distinct anticancer potential. The Galaxamide analog containing d-amino acid at position 5 (Analog-6) presented the strongest anticancer activity. The mechanism study revealed that Analog-6 could cause the early apoptosis of HepG₂ cells by inhibiting their growth in the sub-G1 stage of the cell cycle and induce the chromatin condensation and fragmentation, which can be seen as 68% of HepG₂ cells inhibited in the sub-G1 stage. Moreover, a mitochondria-mediated pathway was found to be involved in the apoptotic process of Analog-6 on HepG₂ cells.

  6. Mode of Action of the Toxin from Pseudomonas phaseolicola

    PubMed Central

    Patil, Suresh S.; Tam, Leslie Q.; Sakai, W. S.

    1972-01-01

    The specificity of the Pseudomonas phaseolicola toxin for enzyme inhibition and its relationship to toxin-induced chlorosis in bean leaves (Phaseolus vulgaris L.) was examined. The toxin showed no significant inhibitory activity against glutamine synthetase, glutamine transferase, carbamyl phosphate synthetase, aspartate carbamoyltransferase, or arginase at concentrations 100-fold higher than that needed to inhibit ornithine carbamoyltransferase by 50%. Protection from and reversal of toxin-induced chlorosis in bean leaves was attempted with several amino acids. Aside from protection with l-citrulline which was previously reported, only l-arginine-HCl and to a minor extent l-leucine and l-glutamine showed protection from chlorosis. l-Citrulline and l-arginine-HCl (but not l-glutamine and l-leucine) also reversed toxin-induced chlorosis. Ultrastructurally, cells from toxin-treated chlorotic tissues showed no observable changes as compared to nontreated tissues. This, together with the ability of the two amino acids to reverse chlorosis, indicated that the toxin causes a reversible biochemical lesion in treated tissue. While tissues from bean plants inoculated with P. phaseolicola showed a large accumulation of ornithine, toxin-treated tissues showed no accumulation of ornithine. The latter finding indicated that in addition to the ornithine carbamoyltransferase inhibitor, the pathogen may produce inhibitors of other ornithine metabolizing enzymes in inoculated tissues. Images PMID:16658052

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

    PubMed

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

    2016-05-01

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

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1980-03-01

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

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

    PubMed

    O'Dell, Luke A; Schurko, Robert W

    2009-08-28

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

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

    PubMed

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

    2012-02-01

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

  12. Effects of long chain fatty acids on solute absorption: perfusion studies in the human jejunum.

    PubMed Central

    Ammon, H V; Thomas, P J; Phillips, S F

    1977-01-01

    Perfusion studies were performed in healthy volunteers to test the hypothesis that net fluid secretion induced by fatty acids is accompanied by parallel reduction in solute transport. Ricinoleic acid provoked a marked net secretion of fluid and concomitantly inhibited the absorption of all solutes tested; these included glucose, xylose, L-leucine, L-lysine, Folic acid, and 2-mono-olein. Oleic acid also reduced net fluid and solute transport, but was less potent in reducing solute absorption than was ricinoleic acid. When fluid secretion was induced osmotically with mannitol, glucose and xylose absorption was not affected. The mechanism for this generalised effect of fatty acids on solute absorption is uncertain, possibly nonspecific, and might be related to mucosal damage and altered mucosal permeability induced by these agents. PMID:590838

  13. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  15. 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.

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

    PubMed

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

    2013-10-01

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

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

    SciTech Connect

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

    2008-02-05

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

  18. Amperometric biosensor based on diamond paste for the enantioanalysis of L-lysine.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Nejem, R'afat Mahmoud; van Staden, Jacobus Frederick; Aboul-Enein, Hassan Y

    2012-05-15

    An amperometric biosensor was proposed for the enantioanalysis of L-lysine. The biosensor is based on the impregnation of L-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650 mV. The biosensor exhibited a linear concentration range between 1 and 100 nmol/L with a limit of detection of 4 pmol/L. The selectivity of the biosensor is high over other amino acids, such as L-serine, L-leucine, L-aspartic acid, L-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of L-lysine in serum samples and pharmaceutical compounds.

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

    NASA Technical Reports Server (NTRS)

    Holstege, Gert; Blok, Bertil F.

    1989-01-01

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

  20. Purification and partial characterization of an L-amino acid oxidase from bushmaster snake (Surucucu Pico de Jaca) Lachesis muta muta venom.

    PubMed

    Sánchez, E O; Magalhães, A

    1991-01-01

    1. L-amino acid oxidase (L-AO) from the venom of Lachesis muta muta was purified 72 times (38%) by gel filtration on Sephadex G-100, followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-300. 2. The protein was shown to be homogeneous by polyacrylamide gel electrophoresis at pH 8.5, immunoelectrophoresis, immunodiffusion and isoelectric focusing. Its specific activity was 44.4 units/mg protein, using 7.5 mM L-leucine as substrate and O-dianisidine as electron donor, at pH 7.6 and 25 degrees C. The increase in absorbance at 436 nm was recorded. 3. The enzyme was characterized as a glycoprotein with an S20,w = 6.72, MW = 138,000 and pI = 5.2. It presented maxima at 389 and 460 nm and contained 2 mol of FAD per mole protein.

  1. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  3. alpha-Aminoalkylphosphonates as a tool in experimental optimisation of P1 side chain shape of potential inhibitors in S1 pocket of leucine- and neutral aminopeptidases.

    PubMed

    Drag, Marcin; Grembecka, Jolanta; Pawełczak, Małgorzata; Kafarski, Paweł

    2005-08-01

    The synthesis and biological activity studies of the series of structurally different alpha-aminoalkylphosphonates were performed in order to optimise the shape of the side chain of the potential inhibitors in S1 pocket of leucine aminopeptidase [E.C.3.4.11.1]. Analysis of a series of compounds with aromatic, aliphatic and alicyclic P1 side chains enabled to find out the structural features, optimal for that fragment of inhibitors of LAP. The most active among all investigated compounds were the phosphonic analogues of homo-tyrosine (K(i)=120 nM) and homo-phenylalanine (K(i)=140 nM), which even as racemic mixtures were better inhibitors in comparison with the best till now-phosphonic analogue of l-leucine (230 nM). Additional comparison of the inhibitory activity obtained for aminopeptidase N (APN, E.C.3.4.11.2) give insight into structural preferences of both enzymes.

  4. Evaluation of selected micronized poloxamers as tablet lubricants.

    PubMed

    Desai, D; Zia, H; Quadir, A

    2007-10-01

    The primary objective of this study was to compare the lubrication properties of micronized poloxamer 188 (Lmicrotrol micro 68) and micronized poloxamer 407 (Lmicrotrol micro 127) with certain conventional lubricants such as magnesium stearate and stearic acid. The secondary objective was to use these micronized poloxamers as water-soluble tablet lubricants in preparation of effervecsent tablets. The results showed that these micronized poloxamers have superior lubrication properties compared with stearic acid, with no negative effect on tablet hardness, friability, disintegration, or dissolution. Moreover, lubricant mixing time had no significant effect on tablet properties when poloxamers were used as lubricants. Effervescent tablets also were produced successfully using micronized poloxamers as lubricants. The micronized poloxamers had a better lubrication effect in comparison with that of water-soluble lubricant l-leucine.

  5. High cell density cultivation of Brevibacterium linens and formation of proteinases and lipase.

    PubMed

    Adamitsch, Bernhard F; Karner, Ferdinand; Hampel, Werner A

    2003-05-01

    Brevibacterium linens forms hydrolytic enzymes which can be used to accelerate the ripening of cheese without causing bitterness. B. linens ATCC 9172 was grown to a high cell density (50 g dry wt l-1 after 60 h) in a mineral medium containing lactic acid, soy-peptone and ammonium sulphate by applying a continuous feed of nutrients. The maximal activities of L-leucine aminopeptidase and cell-associated proteinase were 286 U l-1 and 202 U l-1, respectively. The cell-associated lipolytic activity exhibited a strong and sudden increase at 46 h, resulting in a maximum of 9.5 U g-1 dry wt; thus the volumetric productivity of proteolytic and lipolytic activity was 4220 U l-1 h-1 and 7.3 U l-1 h-1, respectively.

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

    PubMed

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

    2011-04-20

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

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2009-01-01

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

  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

    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. 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:

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

    PubMed

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

    2015-01-01

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

  12. Metabolic pathways and fermentative production of L-aspartate family amino acids.

    PubMed

    Park, Jin Hwan; Lee, Sang Yup

    2010-06-01

    The L-aspartate family amino acids (AFAAs), L-threonine, L-lysine, L-methionine and L-isoleucine have recently been of much interest due to their wide spectrum of applications including food additives, components of cosmetics and therapeutic agents, and animal feed additives. Among them, L-threonine, L-lysine and L-methionine are three major amino acids produced currently throughout the world. Recent advances in systems metabolic engineering, which combine various high-throughput omics technologies and computational analysis, are now facilitating development of microbial strains efficiently producing AFAAs. Thus, a thorough understanding of the metabolic and regulatory mechanisms of the biosynthesis of these amino acids is urgently needed for designing system-wide metabolic engineering strategies. Here we review the details of AFAA biosynthetic pathways, regulations involved, and export and transport systems, and provide general strategies for successful metabolic engineering along with relevant examples. Finally, perspectives of systems metabolic engineering for developing AFAA overproducers are suggested with selected exemplary studies.

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

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

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

    1991-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-08-01

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

  16. Detection of a new piperideine alkaloid in the pygidial glands of some Stenus beetles.

    PubMed

    Wittmann, Isabel; Schierling, Andreas; Dettner, Konrad; Göhl, Matthias; Schmidt, Jürgen; Seifert, Karlheinz

    2015-09-01

    Rove beetles of the genus Stenus produce and store bioactive alkaloids like stenusine (3), 3-(2-methylbut-1-enyl)pyridine (4), and cicindeloine (5) in their pygidial glands to protect themselves from predation and microorganismic infestation. The biosynthesis of stenusine (3), 3-(2-methylbut-1-enyl)pyridine (4), and cicindeloine (5) was previously investigated in Stenus bimaculatus, Stenus similis, and Stenus solutus, respectively. The piperideine alkaloid cicindeloine (5) occurs also as a major compound in the pygidial gland secretion of Stenus cicindeloides. The three metabolites follow the same biosynthetic pathway, where the N-heterocyclic ring is derived from L-lysine and the side chain from L-isoleucine. The different alkaloids are finally obtained by few modifications of shared precursor molecules, such as 2,3,4,5-tetrahydro-5-(2-methylbutylidene)pyridine (1). This piperideine alkaloid was synthesized and detected by GC/MS and GC at a chiral phase in the pygidial glands of Stenus similis, Stenus tarsalis, and Stenus cicindeloides.

  17. 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.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    Dave, Anuja; Graham, Ian A.

    2012-01-01

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

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

    PubMed

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

    2016-09-02

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

  2. Plant Volatiles Increase Sex Pheromone Attraction of Holotrichia parallela (Coleoptera: Scarabaeoidea).

    PubMed

    Ju, Qian; Guo, Xiao-Qiang; Li, Xiao; Jiang, Xiao-Jing; Jiang, Xiang-Guo; Ni, Wan-Li; Qu, Ming-Jing

    2017-03-01

    Holotrichia parallela (Coleoptera: Scarabaeoidea) is a notorious pest of many crops. To improve the effectiveness of its female-produced sex pheromone (L-isoleucine methyl ester:(R)-(-)-linalool = 6:1), 14 plant volatiles, including dodecanoic acid, dodecanal, farnesol, α-farnesene, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, (Z)-3-hexenyl acetate, (E)-2-hexenyl acetate, (R)-(+)-limonene, α-phellandrene, α-pinene, ocimene, methyl benzoate, and benzaldehyde, were individually evaluated using electroantennography and olfactometer assays. (E)-2-Hexenyl acetate and (Z)-3-hexenyl acetate were found to elicit the strongest responses in both males and females. Further testing of these two compounds in mixtures with the sex pheromone indicated that (E)-2-hexenyl acetate had a stronger synergistic effect than (Z)-3-hexenyl acetate. Field evaluations showed that mixtures of (E)-2-hexenyl acetate and the sex pheromone resulted in significantly higher catches than the sex pheromone alone. Using a 5:1 mixture of the sex pheromone and (E)-2-hexenyl acetate, the maximum number of females per trap per day was 14, showing a synergistic effect of a factor of four. For males, a 3:1 mixture of the sex pheromone and (E)-2-hexenyl acetate yielded a maximum number of 310 individuals per trap per day, equivalent to a synergistic effect of 175%. These results may provide the basis for the development of efficient pest management systems against H. parallela using plant volatiles and insect sex pheromones.

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

    PubMed

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

    2015-06-07

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

  4. Synthesis and spectroscopic DNA binding investigations of dibutyltin N-(5-chlorosalicylidene)-leucinate

    NASA Astrophysics Data System (ADS)

    Zhang, Shufang; Yuan, Hongyu; Tian, Laijin

    2017-02-01

    A new dibutyltin N-(5-Chlorosalicylidene)-leucinate (DNCL) was synthesized by the reaction of dibutyltin dichloride with in situ formed potassium N-(5-chlorosalicylidene)-L-isoleucinate 3 characterized by elemental analysis, IR, 1H NMR (1H, 13C and 119Sn) spectra. The interaction between DNCL and calf thymus deoxyribonucleic acid (ctDNA) in a pH 7.4 Tris-HCl buffer solution was investigated by UV-Vis spectroscopy, fluorescence spectroscopy, and viscosity measurements. It was found that DNCL molecules could intercalate into the base pairs of DNA, forming a DNCL-DNA complex with a binding constant of Kf = 5.75 × 105 L mol-1 (310 K). The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) and Gibbs free energy (ΔG) were calculated to be 1.16 × 105 J mol-1, 486.5 J K-1 mol -1 and -3.48 × 104 J mol-1 at 310 K, respectively. Hydrophobic interaction was the predominant intermolecular force in stabilizing the DNCL-DNA complex.

  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. 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.

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    1974-01-01

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

  9. Chronic leucine exposure results in reduced but reversible glucose-stimulated insulin secretion in INS-1 cells.

    PubMed

    Zhang, Xiujuan; Han, Wenxia; Jiang, Xiuyun; Li, Min; Gao, Ling; Zhao, Jia Jun

    2014-06-01

    Previous studies have demonstrated that sustained high leucine exposure decreases glucose-stimulated insulin secretion (GSIS). However, whether this effect is recoverable following the removal of leucine is unclear. Pancreatic/duodenal homeobox-1 (PDX-1) and its downstream target, glucose transporter 2 (GLUT2), are reported to be positively associated with insulin secretion. However, it also remains unclear whether the effect of leucine on GSIS is accompanied by alterations in PDX-1 and GLUT2. In the present study, insulin secretion, insulin content, PDX-1 and GLUT2 protein expression in INS-1 (rat insulinoma cell line) cells were assessed following a 24-h incubation in 40 mmol/l leucine. Half of the cells were incubated in leucine-free media for a further 24 h to observe the abovementioned effects. In contrast to the control, 40 mmol/l leucine for 24 or 48 h diminished GSIS at high glucose concentrations by 11% (P=0.026) or 22% (P=0.003), insulin content by 14% (P=0.008) or 20% (P=0.002), as well as decreasing PDX-1 and GLUT2 expression. When leucine was removed from the media for a further 24-h incubation, in comparison with those cells that were maintained in leucine treatment for 24 and 48 h, the high GSIS increased by 13% (P=0.032) and 27% (P=0.002), insulin content was augmented by 10% (P=0.014) and 20% (P=0.003), and the protein expression of PDX-1 and GLUT2 also increased. The present study demonstrates that sustained high concentrations of leucine induce a reversible impairment of GSIS and alter insulin content, which is mediated by PDX-1 and GLUT2, in INS-1 cells.

  10. Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.

    PubMed

    Ihara, Kohei; Sato, Kazuki; Hori, Hatsuhiro; Makino, Yumiko; Shigenobu, Shuji; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2017-04-01

    The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less β-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of β-galactosidase activity (approximately 17-fold) in the presence of 6 mM l-alanine, l-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp-deficient cells showed almost no β-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide -161 to -83 with a physiologically relevant affinity (apparent KD, 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by l-alanine and l-leucine, but not by Ala-Ala and d-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l-alanine, which eventually leads to intracellular homeostasis of l-alanine concentrations.

  11. Mechanism of peptide hydrolysis by co-catalytic metal centers containing leucine aminopeptidase enzyme: a DFT approach.

    PubMed

    Zhu, Xiaoxia; Barman, Arghya; Ozbil, Mehmet; Zhang, Tingting; Li, Shanghao; Prabhakar, Rajeev

    2012-02-01

    In this density functional theory study, reaction mechanisms of a co-catalytic binuclear metal center (Zn1-Zn2) containing enzyme leucine aminopeptidase for two different metal bridging nucleophiles (H(2)O and -OH) have been investigated. In addition, the effects of the substrate (L-leucine-p-nitroanilide → L-leucyl-p-anisidine) and metal (Zn1 → Mg and Zn2 → Co, i.e., Mg1-Zn2 and Mg1-Co2 variants) substitutions on the energetics of the mechanism have been investigated. The general acid/base mechanism utilizing a bicarbonate ion followed by this enzyme is divided into two steps: (1) the formation of the gem-diolate intermediate, and (2) the cleavage of the peptide bond. With the computed barrier of 17.8 kcal/mol, the mechanism utilizing a hydroxyl nucleophile was found to be in excellent agreement with the experimentally measured barrier of 18.7 kcal/mol. The rate-limiting step for reaction with L-leucine-p-nitroanilide is the cleavage of the peptide bond with a barrier of 17.8 kcal/mol. However, for L-leucyl-p-anisidine all steps of the mechanism were found to occur with similar barriers (18.0-19.0 kcal/mol). For the metallovariants, cleavage of the peptide bond occurs in the rate-limiting step with barriers of 17.8, 18.0, and 24.2 kcal/mol for the Zn1-Zn2, Mg1-Zn2, and Mg1-Co2 enzymes, respectively. The nature of the metal ion was found to affect only the creation of the gem-diolate intermediate, and after that all three enzymes follow essentially the same energetics. The results reported in this study have elucidated specific roles of both metal centers, the nucleophile, indirect ligands, and substrates in the catalytic functioning of this important class of binuclear metallopeptidases.

  12. Protease activities of rumen protozoa.

    PubMed Central

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

    1984-01-01

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

  13. Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

    PubMed Central

    Lobaton, C D; Moreno, A; Oxender, D L

    1984-01-01

    We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20. Images PMID:6717430

  14. Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

    PubMed

    Lobaton, C D; Moreno, A; Oxender, D L

    1984-03-01

    We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.

  15. 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.

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

    PubMed Central

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

    1990-01-01

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

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

    SciTech Connect

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

    2012-12-07

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

  18. Molecular interactions of α-amino acids insight into aqueous β-cyclodextrin systems.

    PubMed

    Ekka, Deepak; Roy, Mahendra Nath

    2013-10-01

    Qualitative and quantitative analysis of molecular interaction prevailing in glycine, L-alanine, L-valine and aqueous solution of β-cyclodextrin (β-CD) have been probed by thermophysical properties. Density (ρ), viscosity (η), and ultrasonic speed (u) measurements have been reported at different temperatures. The extent of interaction (solute-solvent interaction) is expressed in terms of the limiting apparent molar volume ([Formula: see text]), viscosity B-coefficient and limiting apparent molar adiabatic compressibility ([Formula: see text]). The changes on the enthalpy ([Formula: see text]) and entropy ([Formula: see text]) of the encapsulation analysis give information about the driving forces governing the inclusion. The temperature dependence behaviour of partial molar quantities and group contributions to partial molar volumes has been determined for the amino acids. The trends in transfer volumes, [Formula: see text], have been interpreted in terms of solute-cosolute interactions based on a cosphere overlap model. The role of the solvent (aqueous solution of β-CD) and the contribution of solute-solute and solute-solvent interactions to the solution complexes have also been analyzed through the derived properties.

  19. Correlation analysis of urine metabolites and clinical staging in patients with ovarian cancer

    PubMed Central

    Jiang, Ting; Lin, Yunliang; Yin, Haiqin; Wang, Shanshan; Sun, Qinglei; Zhang, Peihai; Bi, Wenxiang

    2015-01-01

    This study is to investigate the correlation between urine metabolites and clinical staging in patients with ovarian cancer. The urina sanguinis from 56 cases of primary epithelial ovarian cancer patients and 15 healthy volunteers was collected and the urine metabolites were extracted. Ultra high performance liquid chromatography/time-of-flight mass spectrometry (UPLC-Q-TOF-MS) analysis was performed. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to analyze the mass spectrometry data. Database retrieval and comparison of the screened metabolites were performed and one-way ANOVA and least significant difference (LSD) t test were carried out. PCA analysis of UPLC-Q-TOF-MS results showed that the score plots of samples from healthy people and patients with ovarian cancer at different clinical stages were separated. Further PLS-DA analysis significantly improved the classification results. The R2X was 0.757, the R2Y was 0.977 and the Q2Y was 0.87, indicating that the model stability and predictability were good. Eight metabolites, including N-acetylneuraminic acid-9-phosphate, 5’-methioadenosine, uric acid-3-nucleoside, pseudouridine, L-valine, succinic acid, L-proline and β-nicotinamide mononucleotide were identified. The contents of these metabolites increased with the development of the disease. There was correlation between urine metabolites and clinical staging in patients with ovarian cancer. PMID:26770415

  20. L-tyrosine potentiates the anorexia induced by mixed-acting sympathomimetic drugs in hyperphagic rats.

    PubMed

    Hull, K M; Maher, T J

    1990-11-01

    The effects of L-tyrosine (L-TYR) on the anorectic activity of several mixed-acting sympathomimetics were determined during the dark cycle in rats made hyperphagic by food deprivation. L-TYR (200 mg/kg) significantly potentiated the anorectic activity of phenylpropanolamine, (-)-ephedrine and (+)-amphetamine by 48, 50 and 37%, respectively. When the dose of L-TYR was varied (25-400 mg/kg), a significant dose-dependent relationship was noted. The observed potentiation was positively correlated with increases in brain TYR concentrations; blockade of L-TYR uptake into the brain by the coadministration of L-valine prevented this potentiation. Various other L-amino acids, as well as D-TYR, failed to mimic the potentiating action of L-TYR. As determined by alpha-methyl-p-TYR pretreatment, the L-TYR-induced potentiation was dependent upon increased catecholamine synthesis. Although various other mixed-acting sympathomimetic anorexiants were similarly potentiated by L-TYR, the direct-acting beta-2 adrenoceptor anorexiants, salbutamol and methoxyphenamine, were not. These results indicate that L-TYR specifically potentiates the anorectic activity of the studied mixed-acting sympathomimetics and are consistent with the requirement of the central conversion of L-TYR to catecholamines via TYR hydroxylase for this response. The possibility that the effect of mixed-acting sympathomimetics is normally limited by the availability of L-TYR is suggested.

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

    PubMed

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

    2016-03-01

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

  2. Enantioselective conjugate addition of nitro compounds to α,β-unsaturated ketones: an experimental and computational study.

    PubMed

    Manzano, Rubén; Andrés, José M; Álvarez, Rosana; Muruzábal, María D; de Lera, Ángel R; Pedrosa, Rafael

    2011-05-16

    A series of chiral thioureas derived from easily available diamines, prepared from α-amino acids, have been tested as catalysts in the enantioselective Michael additions of nitroalkanes to α,β-unsaturated ketones. The best results are obtained with the bifunctional catalyst prepared from L-valine. This thiourea promotes the reaction with high enantioselectivities and chemical yields for aryl/vinyl ketones, but the enantiomeric ratio for alkyl/vinyl derivatives is very modest. The addition of substituted nitromethanes led to the corresponding adducts with excellent enantioselectivity but very poor diastereoselectivity. Evidence for the isomerization of the addition products has been obtained from the reaction of chalcone with [D(3)]nitromethane, which shows that the final addition products epimerize under the reaction conditions. The epimerization explains the low diastereoselectivity observed in the formation of adducts with two adjacent tertiary stereocenters. Density functional studies of the transition structures corresponding to two alternative activation modes of the nitroalkanes and α,β-unsaturated ketones by the bifunctional organocatalyst have been carried out at the B3LYP/3-21G* level. The computations are consistent with a reaction model involving the Michael addition of the thiourea-activated nitronate to the ketone activated by the protonated amine of the organocatalyst. The enantioselectivities predicted by the computations are consistent with the experimental values obtained for aryl- and alkyl-substituted α,β-unsaturated ketones.

  3. Development and validation of a systematic UPLC-MS/MS method for simultaneous determination of three phenol impurities in ritonavir.

    PubMed

    Venugopal, N; Vijaya Bhaskar Reddy, A; Madhavi, G

    2014-03-01

    A stability indicating gradient reverse phase UPLC-MS/MS method was developed and validated for the simultaneous determination of three phenol impurities in ritonavir drug substance. The chromatographic separation was performed on Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution of 0.05% ammonia in methanol and 5.0 mM ammonium acetate buffer (30:70, v/v) at a flow rate of 0.2 mL/min. Both negative and positive electrospray ionization (ESI) modes were operated simultaneously for the quantification of three phenol impurities. The total run time was 11 min, within which ritonavir and its three impurities were well separated. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. The calibration curves showed a good linearity over the concentration range of 0.3-1.5 ppm for phenol and 0.1-1.5 ppm for both 4-nitrophenol and N-phenoxycarbonyl-L-valine (NPV). The determination coefficient obtained was >0.9998 in each case. The method had very low limit of detection (LOD) and limit of quantification (LOQ) and the accuracy lies between 97.8% and 103.2% for all the three phenol impurities. The developed method was successfully applied for five formulation batches of ritonavir to determine its phenol impurities.

  4. Identification of two novel pigment precursors and a reddish-purple pigment involved in the blue-green discoloration of onion and garlic.

    PubMed

    Imai, Shinsuke; Akita, Kaori; Tomotake, Muneaki; Sawada, Hiroshi

    2006-02-08

    By using a model reaction system representing blue-green discoloration that occurs when purees of onion (Allium cepa L.) and garlic (Allium sativum L.) are mixed, we isolated two pigment precursors (PPs) and a reddish-purple pigment (PUR-1) and determined their chemical structures. PPs were isolated from a heat-treated solution containing color developer (CD) and either l-valine or l-alanine, and their structures were determined as 2-(3,4-dimethylpyrrolyl)-3-methylbutanoic acid (PP-Val), and 2-(3,4-dimethyl-1H-pyrrolyl) propanoic acid (PP-Ala), respectively. Next, PUR-1 was isolated from a heat-treated solution containing PP-Val and allicin, and its structure was determined as (1E)-1-(1-((1S)-1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-yl)-prop-1-enylene-3-(1-((1S)-1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-ylidenium). The structure of PUR-1 suggested that PP molecules containing a 3,4-dimethyl pyrrole ring had been cross-linked by an allyl group of allicin to form conjugated pigments. While PUR-1 is a dipyrrole compound exhibiting a reddish-purple color, a color shift toward blue to green can be expected as the cross-linking reaction continues to form, for example, tri- or tetrapyrrole compounds.

  5. Formulation of yeast-leavened bread with reduced salt content by using a Lactobacillus plantarum fermentation product.

    PubMed

    Valerio, Francesca; Conte, Amalia; Di Biase, Mariaelena; Lattanzio, Veronica M T; Lonigro, S Lisa; Padalino, Lucia; Pontonio, Erica; Lavermicocca, Paola

    2017-04-15

    A Lactobacillus plantarum fermentation product (Bio21B), obtained after strain growth (14h) in a wheat flour-based medium, was applied in the bread-making process as taste enhancer, in order to obtain a yeast-leavened bread with reduced salt content (20% and 50%) with respect to a reference bread (REF) not containing the fermentation product. Sensory analysis indicated that the Bio21B bread with salt reduced by 50% had a pleasant taste similar to the salt-containing bread (REF). l-Glutamate and total free amino acid content did not differ between REF and Bio21B breads, while the acids lactic, acetic, phenyllactic, 4-OH-phenyllactic and indole-3-lactic were present only in Bio21B breads. Moreover, the presence of several umami (uridine monophosphate, inosine monophosphate, adenosine, and guanosine) and kokumi (γ-l-glutamyl-l-valine) taste-related molecules was ascertained both in REF and in Bio21B breads. Therefore, a possible role of the acidic molecules in compensating the negative perception of salt reduction can be hypothesized.

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

    PubMed

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

    2015-11-25

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

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

    PubMed

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

    2009-06-01

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

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

    PubMed

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

    2014-10-27

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

  9. Supramolecular catalysis with extended aggregates and gels: inversion of stereoselectivity caused by self-assembly.

    PubMed

    Rodríguez-Llansola, Francisco; Miravet, Juan F; Escuder, Beatriu

    2010-07-26

    L-Proline-L-valine dipeptide derivatives, which self-assemble in toluene, have been studied as stereoselective catalysts in the conjugate addition of cyclohexanone to trans-beta-nitrostyrene. Remarkable effects on the stereoselectivity are observed associated to the aggregation of the catalyst. Outstanding differences were observed between the catalytic activity of compound 1, which forms supramolecular gels in toluene, and compound 2, which is not a gelator. In the former case, the enantioselectivity of the reaction was almost insensitive to changes in catalyst concentration and temperature, but in the case of compound 2, the catalytic activity was very much affected by those variables. Structural studies indicate that the results can be rationalized by taking into account significant conformational changes experienced by the catalytic L-proline derivatives associated with the aggregation process. The results highlight that catalyst self-assembly is a very important issue to consider in the stereoselective outcome of organocatalytic reactions. Especially relevant is the fact that the use of supramolecular gels as organocatalysts emerges as a technique that affords reliable and constant stereoselectivity in different conditions with the added value of easy catalyst recovery.

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

    PubMed

    Bhushan, Ravi; Nagar, Hariom

    2015-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed

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

    1993-10-15

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

  13. Simultaneous determination of atenolol and amiloride in pharmaceutical preparations by capillary zone electrophoresis with capacitively coupled contactless conductivity detection.

    PubMed

    Al Azzam, Khaldun M; Saad, Bahruddin; Aboul-Enein, Hassan Y

    2010-09-01

    Capillary zone electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE-C(4)D) has been employed for the determination of atenolol and amiloride in pharmaceutical formulations. Acetic acid (150 mm) was used as background electrolyte. The influence of several factors (detector excitation voltage and frequency, buffer concentration, applied voltage, capillary temperature and injection time) was studied. Non-UV-absorbing L-valine was used as internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28 degrees C, 25 kV and using hydrodynamic injection (25 s). The separation was effected in an uncoated fused-silica capillary (75 microm, i.d. x 52 cm). The CE-C(4)D method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 5-250 microg/mL for the studied analytes. The relative standard deviations of intra- and inter-day migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of atenolol and amiloride in different pharmaceutical tablet formulations.

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

    PubMed Central

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

    2008-01-01

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

  15. Supramolecular effects and molecular discrimination by macrocyclic hosts embedded in synthetic bilayer membranes.

    PubMed Central

    Murakami, Y; Hayashida, O

    1993-01-01

    Hybrid assemblies were prepared in combinations of a synthetic lipid, involving an L-alanine residue interposed between an anionic head group and a hydrophobic double-chain segment, with cage-type cyclophanes having a three-dimensionally extended hydrophobic cavity. Incorporation behavior of the cage-type hosts into the multi- and single-walled vesicules was characterized by highly sensitive differential scanning calorimetry and dynamic light-scattering measurements as well as by gel-filtration chromatography and electronic absorption spectroscopy. Both hosts were found to be completely incorporated into a relatively polar domain close to the vesicular surface. Anionic guest molecules, such as naphthol yellow S, dimethylsulfonazo III, bromopyrogallol red, and orange G, were effectively incorporated into the hydrophobic cavities provided by the cage-type cyclophanes embedded in the single-walled bilayer membrane, as confirmed by circular dichroism spectroscopy; the formation constants for 1:1 host-guest complexes were in the order of 10(4)-10(5) mol.dm-3. This means that the binding affinities of the cage-type hosts are largely retained even when the hosts are incorporated into the vesicle. Moreover, the pyridinium moieties bound to the chiral L-valine residues in the bridging components of the hosts underwent conformational changes upon inclusion of the guest, as confirmed by circular dichroism spectroscopy and molecular mechanics and dynamics calculations. Images PMID:8433975

  16. Membrane permeability as a cause of transport defects in experimental Fanconi syndrome. A new hypothesis.

    PubMed Central

    Bergeron, M; Dubord, L; Hausser, C; Schwab, C

    1976-01-01

    The injection of sodium maleate (200-400 mg/kg) into rats produces aminoaciduria along with glycosuria and phosphaturia, resembling the Fanconi syndrome. This experimental model was studied by means of microinjections into proximal convoluted tubules of the kidney, stop-flow diuresis, and microperfusion of single nephrons. Our results show that, in maleate-treated rats, competition between amino acids or related structures (L-proline, L-OH-proline, and glycine) possesses the same characteristics, and net influx of amino acids appear normal at the proximal nephron. Data obtained by classical stop-flow techniques and single nephron microperfusions also indicate a normal entry of labeled amino acids (L-lysine, glycine, L-valine, L-proline, L-cystine), and 3-0-methyl-D-[3H]glucose and [32P]phosphate from the luminal side of the proximal tubule cell. However, the efflux of molecules from the cell appears enhanced throughout the proximal and distal tubule; molecules that exit at this site are excreted directly into the urine. Our results suggest that the phosphaturia, aminoaciduria, and glycosuria of the experimental Fanconi syndrome can be explained by a modification of the cell membrane permeability (increased efflux) at distal sites of the nephron rather than by a modification of the membrane transport (decreased influx) at the proximal sites, as is currently accepted. Our data also stress the importance of efflux phenomena in membrane transport. PMID:1262464

  17. Characterization of alanine catabolism in Pseudomonas aeruginosa and its importance for proliferation in vivo.

    PubMed

    Boulette, Megan L; Baynham, Patricia J; Jorth, Peter A; Kukavica-Ibrulj, Irena; Longoria, Aissa; Barrera, Karla; Levesque, Roger C; Whiteley, Marvin

    2009-10-01

    The opportunistic pathogen Pseudomonas aeruginosa causes a variety of infections in immunocompromised individuals, including individuals with the heritable disease cystic fibrosis. Like the carbon sources metabolized by many disease-causing bacteria, the carbon sources metabolized by P. aeruginosa at the host infection site are unknown. We recently reported that l-alanine is a preferred carbon source for P. aeruginosa and that two genes potentially involved in alanine catabolism (dadA and dadX) are induced during in vivo growth in the rat peritoneum and during in vitro growth in sputum (mucus) collected from the lungs of individuals with cystic fibrosis. The goals of this study were to characterize factors required for alanine catabolism in P. aeruginosa and to assess the importance of these factors for in vivo growth. Our results reveal that dadA and dadX are arranged in an operon and are required for catabolism of l-alanine. The dad operon is inducible by l-alanine, d-alanine, and l-valine, and induction is dependent on the transcriptional regulator Lrp. Finally, we show that a mutant unable to catabolize dl-alanine displays decreased competitiveness in a rat lung model of infection.

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

    PubMed

    Eisele, T A

    1996-01-01

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

  19. An evaluation of acute hydrogen sulfide poisoning in rats through serum metabolomics based on gas chromatography-mass spectrometry.

    PubMed

    Zhang, Meiling; Deng, Mingjie; Ma, Jianshe; Wang, Xianqin

    2014-01-01

    Hydrogen sulfide (H2S) is the second leading cause of toxin-related deaths in the operational site. Its main target organs of toxic effects are the central nervous system and respiratory system. In this study, we developed a serum metabonomic method, based on gas chromatography-mass spectrometry (GC/MS), to evaluate the effect of acute poisoning by hydrogen sulfide on rats. Pattern recognition analysis, including both principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA), revealed that acute hydrogen sulfide poisoning induced metabolic perturbations. Compared to the control group, the level of urea, glucose, glyceryl stearate in rat serum of the poisoning group increased after two hours, and the level of glucose, docosahexaenoic acid, glyceryl stearate and arachidonic acid in rat serum of the poisoning group increased after 48 h, while the L-valine, galactose, L-tyrosine levels decreased. Our results indicate that metabonomic methods based on GC/MS may be useful to elucidate acute hydrogen sulfide poisoning through the exploration of biomarkers.

  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.

  1. The Origin of Amino Acids in Lunar Regolith Samples

    NASA Technical Reports Server (NTRS)

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

    2016-01-01

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

  2. Catabolism of L-methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit.

    PubMed

    Gonda, Itay; Lev, Shery; Bar, Einat; Sikron, Noga; Portnoy, Vitaly; Davidovich-Rikanati, Rachel; Burger, Joseph; Schaffer, Arthur A; Tadmor, Ya'akov; Giovannonni, James J; Huang, Mingyun; Fei, Zhangjun; Katzir, Nurit; Fait, Aaron; Lewinsohn, Efraim

    2013-05-01

    Sulfur-containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur-containing and other volatiles. L-methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C- and ²H-labeled L-methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an L-methionine aminotransferase and preserves the main carbon skeleton of L-methionine. The second route apparently involves the action of an L-methionine-γ-lyase activity, releasing methanethiol, a backbone for formation of thiol-derived aroma volatiles. Exogenous L-methionine also generated non-sulfur volatiles by further metabolism of α-ketobutyrate, a product of L-methionine-γ-lyase activity. α-Ketobutyrate was further metabolized into L-isoleucine and other important melon volatiles, including non-sulfur branched and straight-chain esters. Cell-free extracts derived from ripe melon fruit exhibited L-methionine-γ-lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing L-methionine-γ-lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co-segregated with the levels of sulfur-containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that L-methionine is a precursor of both sulfur and non-sulfur aroma volatiles in melon fruit.

  3. Aminostratigraphy of Middle and Late Pleistocene deposits in The Netherlands and the southern part of the North Sea Basin

    NASA Astrophysics Data System (ADS)

    Meijer, T.; Cleveringa, P.

    2009-09-01

    A review of all available amino acid racemization D (alloisoleucine)/L (isoleucine) data from the whole shell of four molluscan species from Late and late Middle Pleistocene deposits of the Netherlands is presented. The data allow the distinction of 5 aminostratigraphical units, NAZ (Netherlands Amino Zone) A-E, each representing a temperate stage. The zones are correlated with marine isotope stages 1, 5e, 7, 9, and 11 respectively. Apart from NAZ-D (MIS 9), in all aminozones the marine transgression reached the present-day onshore area of the Netherlands. The transgression during NAZ-C (Oostermeer Interglacial: MIS 7) seems to be at least as widespread as its counterpart during NAZ-B (Eemian: MIS 5e) in the southern bight of the North Sea Basin. The stratigraphic position of the Oostermeer Interglacial is just below deposits of the Drente phase of the Saalian and because of this position the interglacial marine deposits have formerly erroneously considered to be of Holsteinian age. Neede, the 'classic' Dutch Holsteinian site, is dated in NAZ-E (MIS 11), like Noordbergum. Although the validity of these zones has been checked with independent data, some overlap between succeeding zones may occur. The relation between amino acid data from elsewhere in the North Sea Basin and the Netherlands amino zonation is discussed. The deposits at the Holsteinian stratotype Hummelsbüttel in North West Germany are dated in NAZ-D. This interglacial correlates with MIS 9. The Belvédère Interglacial, which is of importance for its archaeology, is in NAZ-D (MIS 9) and therefore of Holsteinian age as well. The lacustroglacial 'pottery clays' in the Noordbergum area are deposits from two glacial stages, which can be correlated with MIS 8 and 10 (the Elsterian). The pottery clay that is considered equivalent to the German 'Lauenburger Ton' correlates with MIS 10.

  4. Analysis of Arabidopsis JAZ gene expression during Pseudomonas syringae pathogenesis.

    PubMed

    Demianski, Agnes J; Chung, Kwi Mi; Kunkel, Barbara N

    2012-01-01

    The jasmonates (JAs) comprise a family of plant hormones that regulate several developmental processes and mediate responses to various abiotic and biotic stresses, including pathogens. JA signalling is manipulated by several strains of the bacterial pathogen Pseudomonas syringae, including P. syringae strain DC3000, using the virulence factor coronatine (COR) as a mimic of jasmonyl-L-isoleucine (JA-Ile). To better understand the JA-Ile-mediated processes contributing to P. syringae disease susceptibility, it is important to investigate the regulation of JA signalling during infection. In Arabidopsis thaliana, JASMONATE ZIM-DOMAIN (JAZ) proteins are negative regulators of JA signalling. The transcription factor JASMONATE INSENSITIVE1 (JIN1/ATMYC2) has been implicated in the regulation of JAZ gene expression. To investigate the regulation of JAZ genes during P. syringae pathogenesis, we examined JAZ gene expression during infection of Arabidopsis by DC3000. We found that eight of the 12 JAZ genes are induced during infection in a COR-dependent manner. Unexpectedly, the induction of the majority of JAZ genes during infection was not dependent on JIN1, indicating that JIN1 is not the only transcription factor regulating JAZ genes. A T-DNA insertion mutant and an RNA interference line disrupted for the expression of JAZ10, one of the few JAZ genes regulated by JIN1 during infection, exhibited enhanced JA sensitivity and increased susceptibility to DC3000, with the primary effect being increased disease symptom severity. Thus, JAZ10 is a negative regulator of both JA signalling and disease symptom development.

  5. Effects of Light and Wounding on Jasmonates in Rice phyAphyC Mutants

    PubMed Central

    Brendel, Rita; Svyatyna, Katharina; Jikumaru, Yusuke; Reichelt, Michael; Mithöfer, Axel; Takano, Makoto; Kamiya, Yuji; Nick, Peter; Riemann, Michael

    2014-01-01

    Jasmonates (JA) are lipid-derived plant hormones. They have been shown to be important regulators of photomorphogenesis, a developmental program in plants, which is activated by light through different red and blue light sensitive photoreceptors. In rice, inhibition of coleoptile growth by light is a central event in photomorphogenesis. This growth inhibition is impaired, when jasmonate biosynthesis is knocked out. Previously, we found that JASMONATE RESISTANT 1 (OsJAR1) transcripts were not induced in the phytochrome (phy) mutant phyAphyC. Therefore, in the current study we investigated the regulation of JA and its highly bioactive derivative (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), as well as the transcriptional regulation of several JA-dependent genes both in wild type and phyAphyC mutant. JA and JA-Ile levels increased in the mutant seedlings in response to blue light. However, in phyAphyC mutant leaves, which were continuously wounded, JA and JA-Ile levels were lower compared to those in the wild type. Hence, the mutation of phyA and phyC has differential effects on jasmonate levels depending on the tissue and developmental stage. Our results suggest that the contribution of JA-Ile to signaling during photomorphogenesis of rice is minor, as coleoptile phenotypes of phyAphyC mutants resemble those of jasmonate-deficient mutants despite the fact that induction by blue light leads to higher levels of JA-Ile compared to the wild type. We postulate that phyA and phyC could control the activity of specific enzymes metabolizing JA to active derivatives. PMID:27135497

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

    SciTech Connect

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

    2014-09-21

    Application of sets of {sup 13}C-{sup 13}C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ({sup 13}C′) and aliphatic ({sup 13}C{sub aliphatic}) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly {sup 13}C,{sup 15}N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of {sup 13}C′-{sup 13}C{sub aliphatic} distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform {sup 13}C,{sup 15}N-labeling on the FGAIL fragment.

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

    USGS Publications Warehouse

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

    1989-01-01

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

  8. Pregabalin reduces the release of synaptic vesicles from cultured hippocampal neurons.

    PubMed

    Micheva, Kristina D; Taylor, Charles P; Smith, Stephen J

    2006-08-01

    Pregabalin [S-[+]-3-isobutylGABA or (S)-3-(aminomethyl)-5-methylhexanoic acid, Lyrica] is an anticonvulsant and analgesic medication that is both structurally and pharmacologically related to gabapentin (Neurontin; Pfizer Inc., New York, NY). Previous studies have shown that pregabalin reduces the release of neurotransmitters in several in vitro preparations, although the molecular details of these effects are less clear. The present study was performed using living cultured rat hippocampal neurons with the synaptic vesicle fluorescent dye probe FM4-64 to determine details of the action of pregabalin to reduce neurotransmitter release. Our results indicate that pregabalin treatment, at concentrations that are therapeutically relevant, slightly but significantly reduces the emptying of neurotransmitter vesicles from presynaptic sites in living neurons. Dye release is reduced in both glutamic acid decarboxylase (GAD)-immunoreactive and GAD-negative (presumed glutamatergic) synaptic terminals. Furthermore, both calcium-dependent release and hyperosmotic (calcium-independent) dye release are reduced by pregabalin. The effects of pregabalin on dye release are masked in the presence of l-isoleucine, consistent with the fact that both of these compounds have a high binding affinity to the calcium channel alpha(2)-delta protein. The effect of pregabalin is not apparent in the presence of an N-methyl-d-aspartate (NMDA) antagonist [D(-)-2-amino-5-phosphonopentanoic acid], suggesting that pregabalin action depends on NMDA receptor activation. Finally, the action of pregabalin on dye release is most apparent before and early during a train of electrical stimuli when vesicle release preferentially involves the readily releasable pool.

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

    NASA Astrophysics Data System (ADS)

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

    2004-05-01

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

  10. A one-pot procedure for the preparation of N-9-fluorenylmethyloxycarbonyl-α-amino diazoketones from α-amino acids.

    PubMed

    Siciliano, Carlo; De Marco, Rosaria; Guidi, Ludovica Evelin; Spinella, Mariagiovanna; Liguori, Angelo

    2012-12-07

    The study describes a new "one-pot" route to the synthesis of N-9-fluorenylmethyloxycarbonyl (Fmoc) α-amino diazoketones. The procedure was tested on a series of commercially available free or side-chain protected α-amino acids employed as precursors. The conversion into the title compounds was achieved by masking and activating the α-amino acids with a single reagent, namely, 9-fluorenylmethyl chloroformate (Fmoc-Cl). The resulting N-protected mixed anhydrides were reacted with diazomethane to lead to the α-amino diazoketones, which were isolated by flash column chromatography in very good to excellent overall yields. The versatility of the procedure was verified on lipophilic α-amino acids and further demonstrated by the preparation of N-Fmoc-α-amino diazoketones also from α-amino acids containing side-chain masking groups, which are orthogonal to the Fmoc one. The results confirmed that tert-butyloxycarbonyl (Boc), tert-butyl ((t)Bu), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), three acid-labile protecting groups mostly adopted in the solution and solid-phase peptide synthesis, are compatible to the adopted reaction conditions. In all cases, the formation of the corresponding C-methyl ester of the starting amino acid was not observed. Moreover, the proposed method respects the chirality of the starting α-amino acids. No racemization occurred when the procedure was applied to the synthesis of the respective N-Fmoc-protected α-amino diazoketones from L-isoleucine and L-threonine and to the preparation of a diastereomeric pair of N-Fmoc-protected dipeptidyl diazoketones.

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

    PubMed

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

    2016-07-01

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

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

    SciTech Connect

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

    2006-03-01

    S. typhimurium biodegradative threonine deaminase (TdcB), a member of the β-family of PLP-dependent enzymes, has been overexpressed, purified and crystallized in three different crystal forms using the hanging-drop vapour-diffusion method. Biodegradative threonine deaminase (TdcB) catalyzes the deamination of l-threonine to α-ketobutyrate, the first reaction in the anaerobic breakdown of l-threonine to propionate. Unlike the biosynthetic threonine deaminase, TdcB is insensitive to l-isoleucine and is activated by AMP. Here, the cloning of TdcB (molecular weight 36 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli is reported. TdcB was purified to homogeneity using Ni–NTA affinity column chromatography and crystallized using the hanging-drop vapour-diffusion technique in three different crystal forms. Crystal forms I (unit-cell parameters a = 46.32, b = 55.30, c = 67.24 Å, α = 103.09, β = 94.70, γ = 112.94°) and II (a = 56.68, b = 76.83, c = 78.50 Å, α = 66.12, β = 89.16, γ = 77.08°) belong to space group P1 and contain two and four molecules of TdcB, respectively, in the asymmetric unit. Poorly diffracting form III crystals were obtained in space group C2 and based on the unit-cell volume are most likely to contain one molecule per asymmetric unit. Two complete data sets of resolutions 2.2 Å (crystal form I) and 1.7 Å (crystal form II) were collected at 100 K using an in-house X-ray source.

  13. 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.

  14. Motifs in the C-terminal region of the Penicillium chrysogenum ACV synthetase are essential for valine epimerization and processivity of tripeptide formation.

    PubMed

    Wu, Xiaobin; García-Estrada, Carlos; Vaca, Inmaculada; Martín, Juan-Francisco

    2012-02-01

    The first step in the penicillin biosynthetic pathway is the non-ribosomal condensation of L-α-aminoadipic acid, L-cysteine and L-valine into the tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV). This reaction is catalysed by the multienzyme ACV synthetase (ACVS), which is encoded in the filamentous fungus Penicillium chrysogenum by the pcbAB gene. This enzyme contains at least ten catalytic domains. The precise role of the C-terminal domain of this multidomain NRPS still remains obscure. The C-terminal region of ACVS bears the epimerase and the thioesterase domains and may be involved in the epimerization of LLL-ACV to LLD-ACV and in the hydrolysis of the thioester bond. In this work, the conserved motifs (3371)EGHGRE(3376) (located in the putative epimerase domain) and (3629)GWSFG(3633) (located in the thioesterase domain) were changed by site-directed-mutagenesis to LGFGLL and GWAFG, respectively. In addition, the whole thioesterase domain (230 amino acids) and the different parts of this domain were deleted. The activity of these mutant enzymes was assessed in vivo by two different procedures: i) through the quantification of bisACV produced by the fungus and ii) by quantifying the benzylpenicillin production using tailored strains of P. chrysogenum, which lack the pcbAB gene, as host strains. All indicated mutant enzymes showed lower or null activity than the control strain confirming that E3371, H3373, R3375 and E3376 belong to the epimerase active centre. Different fragments included in the C-terminal region of ACVS control thioester hydrolysis. Overexpression of the sequence encoding the ACVS integrated thioesterase domain as a separate (stand-alone) transcriptional unit complemented mutants lacking the integrated thioesterase domain, although with low ACV releasing activity, suggesting that the stand-alone thioesterease interacts with the other ACVS domains.

  15. 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.

  16. Organometallic complexes of bulky, optically active, C3-symmetric tris(4S-isopropyl-5,5-dimethyl-2-oxazolinyl)phenylborate (ToP*)

    DOE PAGES

    Xu, Songchen; Magoon, Yitzhak; Reinig, Regina R.; ...

    2015-07-16

    A bulky, optically active monoanionic scorpionate ligand, tris(4S-isopropyl-5,5-dimethyl-2-oxazolinyl)phenylborate (ToP*), is synthesized from the naturally occurring amino acid l-valine as its lithium salt, Li[ToP*] (1). That compound is readily converted to the thallium complex Tl[ToP*] (2) and to the acid derivative H[ToP*] (3). Group 7 tricarbonyl complexes ToP*M(CO)3 (M = Mn (4), Re (5)) are synthesized by the reaction of MBr(CO)5 and Li[ToP*] and are crystallographically characterized. The νCO bands in their infrared spectra indicate that π back-donation in the rhenium compounds is greater with ToP* than with non-methylated tris(4S-isopropyl-2-oxazolinyl)phenylborate (ToP). The reaction of H[ToP*] and ZnEt2 gives ToP*ZnEt (6), whilemore » ToP*ZnCl (7) is synthesized from Li[ToP*] and ZnCl2. The reaction of ToP*ZnCl and KOtBu followed by addition of PhSiH3 provides the zinc hydride complex ToP*ZnH (8). In this study, compound 8 is the first example of a crystallographically characterized optically active zinc hydride. We tested its catalytic reactivity in the cross-dehydrocoupling of silanes and alcohols, which provided Si-chiral silanes with moderate enantioselectivity.« less

  17. Organometallic complexes of bulky, optically active, C3-symmetric tris(4S-isopropyl-5,5-dimethyl-2-oxazolinyl)phenylborate (ToP*)

    SciTech Connect

    Xu, Songchen; Magoon, Yitzhak; Reinig, Regina R.; Schmidt, Bradley M.; Ellern, Arkady; Sadow, Aaron D.

    2015-07-16

    A bulky, optically active monoanionic scorpionate ligand, tris(4S-isopropyl-5,5-dimethyl-2-oxazolinyl)phenylborate (ToP*), is synthesized from the naturally occurring amino acid l-valine as its lithium salt, Li[ToP*] (1). That compound is readily converted to the thallium complex Tl[ToP*] (2) and to the acid derivative H[ToP*] (3). Group 7 tricarbonyl complexes ToP*M(CO)3 (M = Mn (4), Re (5)) are synthesized by the reaction of MBr(CO)5 and Li[ToP*] and are crystallographically characterized. The νCO bands in their infrared spectra indicate that π back-donation in the rhenium compounds is greater with ToP* than with non-methylated tris(4S-isopropyl-2-oxazolinyl)phenylborate (ToP). The reaction of H[ToP*] and ZnEt2 gives ToP*ZnEt (6), while ToP*ZnCl (7) is synthesized from Li[ToP*] and ZnCl2. The reaction of ToP*ZnCl and KOtBu followed by addition of PhSiH3 provides the zinc hydride complex ToP*ZnH (8). In this study, compound 8 is the first example of a crystallographically characterized optically active zinc hydride. We tested its catalytic reactivity in the cross-dehydrocoupling of silanes and alcohols, which provided Si-chiral silanes with moderate enantioselectivity.

  18. Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

    PubMed Central

    Wang, Shiwei; Yu, Jing; Suvira, Milomir; Setlow, Peter; Li, Yong-qing

    2015-01-01

    Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis). PMID:26636757

  19. Enzymatic activation of double-targeted 5'-O-L-valyl-decitabine prodrug by biphenyl hydrolase-like protein and its molecular design basis.

    PubMed

    Tao, Wenhui; Zhao, Dongyang; Sun, Mengchi; Li, Meng; Zhang, Xiangyu; He, Zhonggui; Sun, Yinghua; Sun, Jin

    2017-04-01

    A primary focus of this research was to explore the activation process and mechanism of decitabine (5-aza-2'-deoxycytidine, DAC) prodrug. Recently, it has been reported that biphenyl hydrolase-like protein (BPHL) can play an important role in the activation of some amino acid nucleoside prodrugs with a general preference for hydrophobic amino acids and 5'-esters. Therefore, we put forward a bold hypothesis that this novel enzyme may be primarily responsible for the activation process of DAC prodrug as well. 5'-O-L-valyl-decitabine (L-val-DAC) was synthesized before and can be transported across biological membranes by the oligopeptide transporter (PEPT1), granting it much greater utility in vivo. In this report, L-val-DAC was found to be a good substrate of BPHL protein (K m 0.59 mM; k cat/K m 553.69 mM(-1) s(-1)). After intestinal absorption, L-val-DAC was rapidly and almost completely hydrolyzed to DAC and L-valine. The catalysis was mainly mediated by the BPHL hydrolase and resulted in the intestinal first-pass effect of L-val-DAC after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The structural insights using computational molecular docking showed that BPHL had a unique binding mode for L-val-DAC. As a fundamental basis, the simulation was employed to explain the catalytic mechanism in molecular level. In conclusion, BPHL was at least one of the primary candidate enzymes for L-val-DAC prodrug activation. This promising double-targeted prodrug approach have more advantages than the traditional targeted designs due to its higher transport and more predictable activation, thereby leading to a favorable property for oral delivery.

  20. Integrative analysis of transcriptomic and metabolomic profiling of ascites syndrome in broiler chickens induced by low temperature.

    PubMed

    Shi, Shourong; Shen, Yiru; Zhao, Zhenhua; Hou, Zhuocheng; Yang, Ying; Zhou, Huaijun; Zou, Jianmin; Guo, Yuming

    2014-11-01

    Ascites syndrome (AS) still has an unacceptably high incidence rate in both humans and animals although there have been many studies on AS. To continue our previous pathological and biochemical investigation on the underlying mechanisms of AS incidence in broiler chickens, cutting-edge technologies including RNA-seq and metabolimics were used by directly comparing AS chickens and healthy controls. The RNA-seq analysis in the liver identified 390 differentially expressed genes (DEGs), among which 212 genes were up-regulated and 178 genes were down-regulated in the AS group compared to the control. For the down-regulated DEGs, further gene ontology (GO) analysis suggested that lipid metabolism, cell differentiation, enzyme linked receptor protein signaling pathway and steroid biosynthesis pathway were significantly enriched. For up-regulated DEGs, the cholesterol metabolic process has the lowest p value (0.000966) of fold enrichment while the cholesterol biosynthetic process has the highest fold enrichment (46.67). The metabolomic analysis of serum revealed statistically significant changes in the concentrations of LysoPC(20 : 4), LysoPC(16 : 0), LysoPC(18 : 0), LysoPC(18 : 1), LysoPC(18 : 2), PC(14 : 1/20 : 1), PC(20 : 4/18 : 0), PC(14 : 1/22 : 1), dihydroxyacetone, indoleacrylic acid, ursodeoxycholic acid, l-valine, and l-tryptophan. The integrative analysis of transcriptome and metabolome indicated that two biological pathways of tryptophan biosynthesis and metabolism, and glycerophospholipid metabolism may contribute to the induction of AS in broilers. These findings have provided novel insights into our understanding of molecular mechanisms of AS incidence in broilers.

  1. Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

    PubMed

    Hayashi, Masaya; Okada, Akane; Yamamoto, Kumiko; Okugochi, Tomomi; Kusaka, Chika; Kudou, Daizou; Nemoto, Michiko; Inagaki, Junko; Hirose, Yuu; Okajima, Toshihide; Tamura, Takashi; Soda, Kenji; Inagaki, Kenji

    2016-12-21

    l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

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

    PubMed Central

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

    2012-01-01

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

  3. Improvement of Aspergillus nidulans penicillin production by targeting AcvA to peroxisomes.

    PubMed

    Herr, Andreas; Fischer, Reinhard

    2014-09-01

    Aspergillus nidulans is able to synthesize penicillin and serves as a model to study the regulation of its biosynthesis. Only three enzymes are required to form the beta lactam ring tripeptide, which is comprised of l-cysteine, l-valine and l-aminoadipic acid. Whereas two enzymes, AcvA and IpnA localize to the cytoplasm, AatA resides in peroxisomes. Here, we tested a novel strategy to improve penicillin production, namely the change of the residence of the enzymes involved in the biosynthesis. We tested if targeting of AcvA or IpnA (or both) to peroxisomes would increase the penicillin yield. Indeed, AcvA peroxisomal targeting led to a 3.2-fold increase. In contrast, targeting IpnA to peroxisomes caused a complete loss of penicillin production. Overexpression of acvA, ipnA or aatA resulted in 1.4, 2.8 and 3.1-fold more penicillin, respectively in comparison to wildtype. Simultaneous overexpression of all three enzymes resulted even in 6-fold more penicillin. Combination of acvA peroxisomal targeting and overexpression of the gene led to 5-fold increase of the penicillin titer. At last, the number of peroxisomes was increased through overexpression of pexK. A strain with the double number of peroxisomes produced 2.3 times more penicillin. These results show that penicillin production can be triggered at several levels of regulation, one of which is the subcellular localization of the enzymes.

  4. Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy.

    PubMed

    Wang, Shiwei; Yu, Jing; Suvira, Milomir; Setlow, Peter; Li, Yong-qing

    2015-01-01

    Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis).

  5. Identification and quantification of metabolites of AB-CHMINACA in a urine specimen of an abuser.

    PubMed

    Wurita, Amin; Hasegawa, Koutaro; Minakata, Kayoko; Gonmori, Kunio; Nozawa, Hideki; Yamagishi, Itaru; Suzuki, Osamu; Watanabe, Kanako

    2016-03-01

    We experienced an autopsy case in which the cause of death was judged as poisoning by multiple new psychoactive substances, including AB-CHMINACA, 5-fluoro-AMB and diphenidine [Forensic Toxicol. 33 (2015): 45-53]. Although unchanged AB-CHMINACA could be detected from 8 solid tissues, it could neither be detected from blood nor urine specimens. In this article, we obtained eight kinds of reference standards of AB-CHMINACA metabolites from a commercial source. The AB-CHMINACA metabolites from the urine specimen of the abuser were extracted by a modified QuEChERS method and analyzed by liquid chromatography-tandem mass spectrometry before and after hydrolysis with β-glucuronidase. Among the eight AB-CHMINACA metabolites tested, only 2 metabolites could be identified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, the concentrations of the two metabolites were not increased, suggesting that the metabolites were not in the conjugated forms. The metabolites detected were 4-hydroxycyclohexylmethyl AB-CHMINACA (M1), followed by N-[[1-(cyclohexylmethyl)-1H-indazol-3-yl]carbonyl]-l-valine (M3). Their concentrations were 52.8 ± 3.44 and 41.3 ± 5.04 ng/ml (n=10) for M1 and M3, respectively. Although there is one preceding report showing the estimations of metabolism of AB-CHMINACA without reference standards, this is the first report dealing with exact identification using reference standards, and quantification of M1 and M3 in an authentic urine specimen.

  6. The impact of chronic imipramine treatment on amino acid concentrations in the hippocampus of mice.

    PubMed

    Nagasawa, Mao; Murakami, Tatsuro; Tomonaga, Shozo; Furuse, Mitsuhiro

    2012-09-01

    The relationship between antidepressants and monoamine concentrations in the brain has been well investigated, but few studies have investigated the relationship between antidepressants and amino acid concentrations in the brain. The purpose of the present study was therefore to investigate the effect of the chronic antidepressant imipramine on amino acid and monoamine concentrations in the mouse brain and plasma. Chronic imipramine treatment decreased the concentration of 5-hydroxyindoleaceticacid/5-hydroxytryptamine in the cerebral cortex and increased that of norepinephrine (NE) in the hippocampus. Since these changes were conspicuous effects of the antidepressant, we concluded that imipramine acts on the central nervous system. No change in amino acid concentrations in plasma was induced by chronic imipramine treatment, but several changes were confirmed in the cerebral cortex, the hypothalamus and the hippocampus. Chronic imipramine treatment caused increases in L-methionine, L-tyrosine, and L-lysine in the cerebral cortex, and an increase in L-aspartate in the hypothalamus. Contrary to this, the concentrations of L-aspartate, L-serine, L-asparagine, glycine, L-glutamine, gamma-aminobutyric acid, L-threonine, L-arginine, L-proline, L-valine, and L-methionine in the hippocampus were decreased by chronic imipramine treatment. The present results demonstrate that the metabolism of several amino acids in the brain, but not of those in plasma, was altered by chronic imipramine treatment. The findings in the present study may help to further elucidate the relationship between amino acids and the effects and side effects of antidepressants.

  7. Investigation of Isovaline Enantiomeric Excesses and Other C5 Amino Acids in Carbonaceous Meteorites

    NASA Technical Reports Server (NTRS)

    Dworkin, Jason P.; Glavin, Daniel P.

    2009-01-01

    The origin of biological homochirality is one of the most perplexing puzzles to understanding the emergence of life on Earth. While many models have been proposed, the only reported non-biologically generated. compounds that show a significant enantiomeric excess are a few amino acids in the CM2 Murchison and Murray meteorites (e.g. Pizzarello and Cronin 2000; Pizzarello et al, 2008). Of these isovaline (alpha-ethyl-alanine) is of particular interest since it is typically abundant in CM2 meteorites, is exceedingly rare in biology, and due to its chemical structure is likely to maintain its primordial D/L ratio. Instead of the gas chromatography-mass spectrometry (GC-MS) technique employed by Pizzarello et al., we have used liquid chromatography-fluorescence detection/time of flight-mass spectrometry (LC-FD/ToF-MS) to study the enantiomeric ratio of isovaline in the CM2 meteorites Murchison and LEW90500 and the CR2 QUE99177. We have placed particular emphasis on understanding the suite of C5 amino acids in these meteorites. In doing so, we have determined that D and L 3-aminopentanoic acid co-elutes with Lisovaline and L-valine under common chromatographic conditions (Glavin and Dworkin 2006) for omicron-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). We have devised a method to separate these compounds and we will report the actual D/ L ratios of isovaline in these meteorites and how they compare to the GC-MS measurements of Pizzarello and co-workers.

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

    PubMed Central

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

    2015-01-01

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

  9. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    SciTech Connect

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

  10. Electron spin-lattice relaxation in radicals containing two methyl groups, generated by /γ-irradiation of polycrystalline solids

    NASA Astrophysics Data System (ADS)

    Harbridge, James R.; Eaton, Sandra S.; Eaton, Gareth R.

    2002-12-01

    The effects of methyl rotation on electron spin-lattice relaxation times were examined by pulsed electron paramagnetic resonance for the major radicals in γ-irradiated polycrystalline α-amino isobutyric acid, dimethyl-malonic acid, and L-valine. The dominant radical is the same in irradiated dimethyl-malonic acid and α-amino isobutyric acid. Continuous wave saturation recovery was measured between 10 and 295 K at S-band and X-band. Inversion recovery, echo-detected saturation recovery, and pulsed electron-electron double resonance (ELDOR) data were obtained between 77 and 295 K. For the radicals in the three solids, recovery time constants measured by the various techniques were not the same, because spectral diffusion processes contribute differently for each measurement. Hyperfine splitting due to the protons of two methyl groups is resolved in the EPR spectra for each of the samples. Pulsed ELDOR data were obtained to characterize the spectral diffusion processes that transfer magnetization between hyperfine lines. Time constants were obtained for electron spin-lattice relaxation ( T1e), nuclear spin relaxation ( T1n), cross-relaxation ( Tx1), and spin diffusion ( Ts). Between 77 and 295 K rapid cross-relaxation (Δ Ms=±1, Δ MI=∓1) was observed for each sample, which is attributed to methyl rotation at a rate that is approximately equal to the electron Larmor frequency. The large temperature range over which cross-relaxation was observed suggests that methyl groups in the radical and in the lattice, with different activation energies for rotation, contribute to the rapid cross-relaxation. Activation energies for methyl and amino group rotation between 160 and 1900 K (1.3-16 kJ/mol) were obtained by analysis of the temperature dependence of 1/ T1e at S-band and X-band in the temperature intervals where the dynamic process dominates T1e.

  11. Simultaneous modulation of transport and metabolism of acyclovir prodrugs across rabbit cornea: An approach involving enzyme inhibitors.

    PubMed

    Katragadda, Suresh; Talluri, Ravi S; Mitra, Ashim K

    2006-08-31

    The aim of this study is to identify the class of enzymes responsible for the hydrolysis of amino acid and dipeptide prodrugs of acyclovir (ACV) and to modulate transport and metabolism of amino acid and dipeptide prodrugs of acyclovir by enzyme inhibitors across rabbit cornea. l-Valine ester of acyclovir, valacyclovir (VACV) and l-glycine-valine ester of acyclovir, gly-val-acyclovir (GVACV) were used as model compounds. Hydrolysis studies of VACV and GVACV in corneal homogenate were conducted in presence of various enzyme inhibitors. IC(50) values were determined for the enzyme inhibitors. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Complete inhibition of VACV hydrolysis was observed in the presence of Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl-fluoride) and PCMB (p-chloromercuribenzoic acid). Similar trend was also observed with GVACV in the presence of bestatin. IC(50) values of PCMB and bestatin for VACV and GVACV were found to be 3.81+/-0.94 and 0.34+/-0.08muM respectively. Eserine, tetraethyl pyrophosphate (TEPP) and diisopropyl fluorophosphate (DFP) also produced significant inhibition of VACV hydrolysis. Transport of VACV and GVACV across cornea showed decreased metabolic rate and modulation of transport in presence of PCMB and bestain respectively. The principle enzyme classes responsible for the hydrolysis of VACV and GVACV were carboxylesterases and aminopeptidases respectively. Enzyme inhibitors modulated the transport and metabolism of prodrugs simultaneously even though their affinity towards prodrugs was distinct. In conclusion, utility of enzyme inhibitors to modulate transport and metabolism of prodrugs appears to be promising strategy for enhancing drug transport across cornea.

  12. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    DOE PAGES

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; ...

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel)more » initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.« less

  13. Synthesis, structural elucidation, biological, antioxidant and nuclease activities of some 5-Fluorouracil-amino acid mixed ligand complexes

    NASA Astrophysics Data System (ADS)

    Shobana, Sutha; Subramaniam, Perumal; Mitu, Liviu; Dharmaraja, Jeyaprakash; Arvind Narayan, Sundaram

    2015-01-01

    Some biologically active mixed ligand complexes (1-9) have been synthesized from 5-Fluorouracil (5-FU; A) and amino acids (B) such as glycine (gly), L-alanine (ala) and L-valine (val) with Ni(II), Cu(II) and Zn(II) ions. The synthesized mixed ligand complexes (1-9) were characterized by various physico-chemical, spectral, thermal and morphological studies. 5-Fluorouracil and its mixed ligand complexes have been tested for their in vitro biological activities against some pathogenic bacterial and fungal species by the agar well diffusion method. The in vitro antioxidant activities of 5-Fluorouracil and its complexes have also been investigated by using the DPPH assay method. The results demonstrate that Cu(II) mixed ligand complexes (4-6) exhibit potent biological as well as antioxidant activities compared to 5-Fluorouracil and Ni(II) (1-3) and Zn(II) (7-9) mixed ligand complexes. Further, the cleaving activities of CT DNA under aerobic conditions show moderate activity with the synthesized Cu(II) and Ni(II) mixed ligand complexes (1-6) while no activity is seen with Zn(II) complexes (7-9). Binding studies of CT DNA with these complexes show a decrease in intensity of the charge transfer band to the extent of 5-15% along with a minor red shift. The free energy change values (Δ‡G) calculated from intrinsic binding constants indicate that the interaction between mixed ligand complex and DNA is spontaneous.

  14. Synthesis, structural elucidation, biological, antioxidant and nuclease activities of some 5-Fluorouracil-amino acid mixed ligand complexes.

    PubMed

    Shobana, Sutha; Subramaniam, Perumal; Mitu, Liviu; Dharmaraja, Jeyaprakash; Arvind Narayan, Sundaram

    2015-01-05

    Some biologically active mixed ligand complexes (1-9) have been synthesized from 5-Fluorouracil (5-FU; A) and amino acids (B) such as glycine (gly), L-alanine (ala) and L-valine (val) with Ni(II), Cu(II) and Zn(II) ions. The synthesized mixed ligand complexes (1-9) were characterized by various physico-chemical, spectral, thermal and morphological studies. 5-Fluorouracil and its mixed ligand complexes have been tested for their in vitro biological activities against some pathogenic bacterial and fungal species by the agar well diffusion method. The in vitro antioxidant activities of 5-Fluorouracil and its complexes have also been investigated by using the DPPH assay method. The results demonstrate that Cu(II) mixed ligand complexes (4-6) exhibit potent biological as well as antioxidant activities compared to 5-Fluorouracil and Ni(II) (1-3) and Zn(II) (7-9) mixed ligand complexes. Further, the cleaving activities of CT DNA under aerobic conditions show moderate activity with the synthesized Cu(II) and Ni(II) mixed ligand complexes (1-6) while no activity is seen with Zn(II) complexes (7-9). Binding studies of CT DNA with these complexes show a decrease in intensity of the charge transfer band to the extent of 5-15% along with a minor red shift. The free energy change values (Δ(‡)G) calculated from intrinsic binding constants indicate that the interaction between mixed ligand complex and DNA is spontaneous.

  15. Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement

    PubMed Central

    Haack, Tobias B; Jackson, Christopher B; Murayama, Kei; Kremer, Laura S; Schaller, André; Kotzaeridou, Urania; de Vries, Maaike C; Schottmann, Gudrun; Santra, Saikat; Büchner, Boriana; Wieland, Thomas; Graf, Elisabeth; Freisinger, Peter; Eggimann, Sandra; Ohtake, Akira; Okazaki, Yasushi; Kohda, Masakazu; Kishita, Yoshihito; Tokuzawa, Yoshimi; Sauer, Sascha; Memari, Yasin; Kolb-Kokocinski, Anja; Durbin, Richard; Hasselmann, Oswald; Cremer, Kirsten; Albrecht, Beate; Wieczorek, Dagmar; Engels, Hartmut; Hahn, Dagmar; Zink, Alexander M; Alston, Charlotte L; Taylor, Robert W; Rodenburg, Richard J; Trollmann, Regina; Sperl, Wolfgang; Strom, Tim M; Hoffmann, Georg F; Mayr, Johannes A; Meitinger, Thomas; Bolognini, Ramona; Schuelke, Markus; Nuoffer, Jean-Marc; Kölker, Stefan; Prokisch, Holger; Klopstock, Thomas

    2015-01-01

    Objective Short-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme that is involved in the oxidation of fatty acids and essential amino acids such as valine. Here, we describe the broad phenotypic spectrum and pathobiochemistry of individuals with autosomal-recessive ECHS1 deficiency. Methods Using exome sequencing, we identified ten unrelated individuals carrying compound heterozygous or homozygous mutations in ECHS1. Functional investigations in patient-derived fibroblast cell lines included immunoblotting, enzyme activity measurement, and a palmitate loading assay. Results Patients showed a heterogeneous phenotype with disease onset in the first year of life and course ranging from neonatal death to survival into adulthood. The most prominent clinical features were encephalopathy (10/10), deafness (9/9), epilepsy (6/9), optic atrophy (6/10), and cardiomyopathy (4/10). Serum lactate was elevated and brain magnetic resonance imaging showed white matter changes or a Leigh-like pattern resembling disorders of mitochondrial energy metabolism. Analysis of patients’ fibroblast cell lines (6/10) provided further evidence for the pathogenicity of the respective mutations by showing reduced ECHS1 protein levels and reduced 2-enoyl-CoA hydratase activity. While serum acylcarnitine profiles were largely normal, in vitro palmitate loading of patient fibroblasts revealed increased butyrylcarnitine, unmasking the functional defect in mitochondrial β-oxidation of short-chain fatty acids. Urinary excretion of 2-methyl-2,3-dihydroxybutyrate – a potential derivative of acryloyl-CoA in the valine catabolic pathway – was significantly increased, indicating impaired valine oxidation. Interpretation In conclusion, we define the phenotypic spectrum of a new syndrome caused by ECHS1 deficiency. We speculate that both the β-oxidation defect and the block in l-valine metabolism, with accumulation of toxic methacrylyl-CoA and acryloyl

  16. The effects of heat activation on Bacillus spore germination, with nutrients or under high pressure, with or without various germination proteins.

    PubMed

    Luu, Stephanie; Cruz-Mora, Jose; Setlow, Barbara; Feeherry, Florence E; Doona, Christopher J; Setlow, Peter

    2015-04-01

    Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75 °C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the L-asparagine-glucose-fructose-K(+) mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation.

  17. Characterization of CeO{sub 2} crystals synthesized with different amino acids

    SciTech Connect

    Atla, Shashi B.; Wu, Min-Nan; Pan, Wei; Hsiao, Yu Tang; Sun, An-Cheng; Tseng, Min-Jen; Chen, Yen-Ju; Chen, Chien-Yen

    2014-12-15

    We investigated the relationship between the structures of the CeO{sub 2} products (particle size, morphology and their characteristics) prepared using different amino acids. Cerium hydroxide carbonate precursors were initially prepared by a hydrothermal method and were subsequently converted to CeO{sub 2} by its thermal decomposition. Various amino acids were used as structure-directing agents in the presence of cerium nitrate and urea as precursors. The results indicate morphology selectivity using different amino acids; CeO{sub 2} structures, such as quasi-prism-sphere, straw-bundle, urchin-flower like and polyhedron prisms, indeed could be produced. Raman and photoluminescence studies indicate the presence of oxygen vacancies in the CeO{sub 2} samples. Photoluminescence spectra of CeO{sub 2} with L-Valine exhibit stronger emission compared with other amino acids utilized under this study, indicating the higher degree of defects in these particles. This study clearly indicates that the degree of defects varied in the presence of different amino acids. Improved precision to control the crystal morphology is important in various material applications and our study provides a novel method to achieve this specificity. - Highlights: • We used urea hydrolysis of process for synthesis of CeO{sub 2}. • Structures have been directed using various amino acids. • We obtained straw bundle-like, quasi prism-sphere, polyhedron prisms and urchin flower-like based on amino acids. • We have found that amino acids could achieve the specificity of different degrees of defects. • This could provide the “tailor-make” of cerium crystals.

  18. Delivery of DNAzyme targeting aurora kinase A to inhibit the proliferation and migration of human prostate cancer.

    PubMed

    Xing, Zhen; Gao, Sai; Duan, Yan; Han, Haobo; Li, Li; Yang, Yan; Li, Quanshun

    2015-01-01

    Herein, a polyethylenimine derivative N-acetyl-L-leucine-polyethylenimine (N-Ac-L-Leu-PEI) was employed as a carrier to achieve the delivery of DNAzyme targeting aurora kinase A using PC-3 cell as a model. Flow cytometry and confocal laser scanning microscopy demonstrated that the derivative could realize the cellular uptake of nanoparticles in an energy-dependent and clathrin-mediated pathway and obtain a high DNAzyme concentration in the cytoplasm through further endosomal escape. After DNAzyme transfection, expression level of aurora kinase A would be downregulated at the protein level. Meanwhile, the inhibition of cell proliferation was observed through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell colony formation assay, attributing to the activation of apoptosis and cell cycle arrest. Through flow cytometric analysis, an early apoptotic ratio of 25.93% and G2 phase of 22.58% has been detected after N-Ac-L-Leu-PEI-mediated DNAzyme transfection. Finally, wound healing and Transwell migration assay showed that DNAzyme transfection could efficiently inhibit the cell migration. These results demonstrated that N-Ac-L-Leu-PEI could successfully mediate the DNAzyme delivery and downregulate the expression level of aurora kinase A, triggering a significant inhibitory effect of excessive proliferation and migration of tumor cells.

  19. An efficient biosurfactant-producing bacterium Selenomonas ruminantium CT2, isolated from mangrove sediment in south of Thailand.

    PubMed

    Saimmai, Atipan; Onlamool, Theerawat; Sobhon, Vorasan; Maneerat, Suppasil

    2013-01-01

    Biosurfactant-producing bacteria, isolate CT2, was isolated from mangrove sediment in the south of Thailand. The sequence of the 16S rRNA gene from isolate CT2 showed 100 % similarity with Selenomonas ruminantium. The highest biosurfactant production (5.02 g/l) was obtained when the cells were grown on minimal salt medium containing 15 g/l molasses and 1 g/l commercial monosodium glutamate supplemented with 1 g/l NaCl, 0.1 g/l leucine, 5 % (v/v) inoculum size at 30 °C and 150 rpm after 54 h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5 mN/m), a small CMC value (8 mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test, FT-IR, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility.

  20. Response of Bacillus Spores to Combinations of Germinative Compounds

    PubMed Central

    Foerster, Harold F.; Foster, J. W.

    1966-01-01

    Foerster, Harold F. (University of Texas, Austin), and J. W. Foster. Response of Bacillus spores to combinations of germinative compounds. J. Bacteriol. 91:1168–1177. 1966.—Spores of 21 strains of Bacillus megaterium and 25 other strains representing 13 species of Bacillus were produced under standardized conditions. The germination of a washed spore suspension of each strain was measured as a response to various combinations of 30 different germinative compounds. The strains were first typed with respect to their response to “primary” germination compounds, i.e., glucose, l-alanine, inosine, and l-alanine-inosine mixture, and also Na+ and K+. The second stage was the determination of the response to various organic and inorganic anions and cations, each strain being supplied with the “primary” compounds best for it. Marked differences in germination patterns were observed among species and strains of the same species. No relation to established taxonomic lines was evident. A nonspecific requirement for ions was found for all strains, but not all ions were effective. A striking degree of interchangeability of germinative chemicals was found. “Fractional germination” was very common. A mixture of l-alanine and inosine and various ions was the best germinative solution for most strains. Some anomalous germination patterns were encountered. Those studied included a strain whose cells lysed spontaneously upon germination and other strains for which l-leucine had striking germinative powers. PMID:4956331

  1. Cold Osmotic Shock in Saccharomyces cerevisiae

    PubMed Central

    Patching, J. W.; Rose, A. H.

    1971-01-01

    Saccharomyces cerevisiae NCYC 366 is susceptible to cold osmotic shock. Exponentially growing cells from batch cultures grown in defined medium at 30 C, after being suspended in 0.8 m mannitol containing 10 mm ethylenedia-minetetraacetic acid and then resuspended in ice-cold 0.5 mm MgCl2, accumulated the nonmetabolizable solutes d-glucosamine-hydrochloride and 2-aminoisobutyrate at slower rates than unshocked cells; shocked cells retained their viability. Storage of unshocked batch-grown cells in buffer at 10 C led to an increase in ability to accumulate glucosamine, and further experiments were confined to cells grown in a chemostat under conditions of glucose limitation, thereby obviating the need for storing cells before use. A study was made of the effect of the different stages in the cold osmotic shock procedure, including the osmotic stress, the chelating agent, and the cold Mg2+-containing diluent, on viability and solute-accumulating ability. Growth of shocked cells in defined medium resembled that of unshocked cells; however, in malt extract-yeast extract-glucose-peptone medium, the shocked cells had a longer lag phase of growth and initially grew at a slower rate. Cold osmotic shock caused the release of low-molecular-weight compounds and about 6 to 8% of the cell protein. Neither the cell envelope enzymes, invertase, acid phosphatase and l-leucine-β-naphthylamidase, nor the cytoplasmic enzyme, alkaline phosphatase, were released when yeast cells were subjected to cold osmotic shock. PMID:5001201

  2. 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.

  3. Para-position derivatives of fungal anthelmintic cyclodepsipeptides engineered with Streptomyces venezuelae antibiotic biosynthetic genes.

    PubMed

    Yanai, Koji; Sumida, Naomi; Okakura, Kaoru; Moriya, Tatsuki; Watanabe, Manabu; Murakami, Takeshi

    2004-07-01

    PF1022A, a cyclooctadepsipeptide possessing strong anthelmintic properties and produced by the filamentous fungus Rosellinia sp. PF1022, consists of four alternating residues of N-methyl-L-leucine and four residues of D-lactate or D-phenyllactate. PF1022A derivatives obtained through modification of their benzene ring at the para-position with nitro or amino groups act as valuable starting materials for the synthesis of compounds with improved anthelmintic activities. Here we describe the production of such derivatives by fermentation through metabolic engineering of the PF1022A biosynthetic pathway in Rosellinia sp. PF1022. Three genes cloned from Streptomyces venezuelae, and required for the biosynthesis of p-aminophenylpyruvate from chorismate in the chloramphenicol biosynthetic pathway, were expressed in a chorismate mutase-deficient strain derived from Rosellinia sp. PF1022. Liquid chromatography-mass spectrometry and NMR analyses confirmed that this approach facilitated the production of PF1022A derivatives specifically modified at the para-position. This fermentation method is environmentally safe and can be used for the industrial scale production of PF1022A derivatives.

  4. New hydroxamate inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme active-site recognition.

    PubMed

    Bourdel, E; Doulut, S; Jarretou, G; Labbe-Jullie, C; Fehrentz, J A; Doumbia, O; Kitabgi, P; Martinez, J

    1996-08-01

    Selective and mixed inhibitors of the three zinc metallopeptidases that degrade neurotensin (NT), e.g. endopeptidase 24-16 (EC 3.4.24.16), endopeptidase 24-11 (EC 3.4.24.11 or neutral endopeptidase, NEP) and endopeptidase 24-15 (EC 3.4.24.15), and leucine-aminopeptidase (type IV-S), that degrades the NT-related peptides, Neuromedin N (NN), are of great interest. On the structural basis of compound JMV 390-1 (N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)-benzylpropyl]-L- isoleucyl-L-leucine), which was a full inhibitor of the major NT degrading enzymes, several hydroxamate inhibitors corresponding to the general formula HONHCO-CH2-CH(CH2-C6H5)CO-X-Y-OH (with X-Y = dipeptide) have been synthesized. Compound 7a (X-Y = Ile-Ala) was nearly 40-times more potent in inhibiting EC 24-16 than NEP and more than 800-times more potent than EC 24-15, with an IC50 (12 nM) almost equivalent to that of compound JMV 390-1. Therefore, this compound is an interesting selective inhibitor of EC 24-16, and should be an interesting probe to explore the physiological involvement of EC 24-16 in the metabolism of neurotensin.

  5. Treating psoriasis by targeting its susceptibility gene Rel.

    PubMed

    Fan, Tingting; Wang, Shaowen; Yu, Linjiang; Yi, Huqiang; Liu, Ruiling; Geng, Wenwen; Wan, Xiaochun; Ma, Yifan; Cai, Lintao; Chen, Youhai H; Ruan, Qingguo

    2016-04-01

    Psoriasis is a chronic inflammatory disorder of the skin. Accumulating evidence indicates that the Rel gene, a member of the NF-κB family, is a risk factor for the disease. We sought to investigate whether psoriasis can be prevented by directly targeting the Rel gene transcript, i.e., the c-Rel mRNA. Using chemically-modified c-Rel specific siRNA (siRel) and poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) micelles, we successfully knocked down the expression of c-Rel, and showed that the expression of cytokine IL-23, a direct target of c-Rel that can drive the development of IL-17-producing T cells, was markedly inhibited. More importantly, treating mice with siRel not only prevented but also ameliorated imiquimod (IMQ)-induced psoriasis. Mechanistic studies showed that siRel treatment down-regulated the expression of multiple inflammatory cytokines. Taken together, these results indicate that the susceptibility gene Rel can be targeted to treat and prevent psoriasis.

  6. Effects of taxifolin on the activity of angiotensin-converting enzyme and reactive oxygen and nitrogen species in the aorta of aging rats and rats treated with the nitric oxide synthase inhibitor and dexamethasone.

    PubMed

    Arutyunyan, Tamara V; Korystova, Antonina F; Kublik, Ludmila N; Levitman, Maria Kh; Shaposhnikova, Vera V; Korystov, Yuri N

    2013-12-01

    The action of taxifolin on the angiotensin-converting enzyme (ACE) and the formation of reactive oxygen and nitrogen species (ROS/RNS) in the aorta of aging rats and rats treated with nitric oxide synthase inhibitor (N ω-nitro-L-arginine methyl ester (L-NAME)) or dexamethasone have been studied. The ACE activity in aorta sections was determined by measuring the hydrolysis of hippuryl-L-histidyl-L-leucine, and the ROS/RNS production was measured by oxidation of dichlorodihydrofluorescein. It was shown that taxifolin at a dose of 30-100 μg/kg/day decreases the ACE activity in the aorta of aging rats and of rats treated with L-NAME or dexamethasone to the level of the ACE activity in young control rats. Taxifolin (100 μg/kg/day) was found to also reduce the amount of ROS/RNS in the aorta that increased as a result of L-NAME intake. L-NAME treatment increases the contribution of 5-lipoxygenase and NADPH oxidase to ROS/RNS production in the aorta, while taxifolin (100 μg/kg/day) decreases the contribution of these enzymes to the normal level.

  7. Identification of human pulmonary alkaline phosphatase isoenzymes.

    PubMed

    Capelli, A; Cerutti, C G; Lusuardi, M; Donner, C F

    1997-04-01

    An increase of alkaline phosphatase (ALP) activity has been observed in the bronchoalveolar lavage fluid (BALF) of patients affected by pulmonary fibrosis in chronic interstitial lung disorders. To characterize the ALP isoenzymes in such cases, we used gel filtration, agarose gel electrophoresis, heat and amino acid inhibition assays, wheat-germ agglutinin (WGA) precipitation, and an immunoassay specific for the bone-isoform of ALP. Only one anodic band representing a high-molecular-weight isoform of ALP (Mr approximately 2,000 kDa) was observed on electrophoresis of BALF. The inhibition assay results were consistent for a tissue-nonspecific isoenzyme sensitive to a temperature of 56 degrees C (71.9 +/- 2.5% inhibition) and to homoarginine (65.7 +/- 1.9%), and resistant to L-phenylalanine and L-leucine. Less than 13% of ALP activity was heat-stable. After incubation of BALF specimens with glycosyl-phosphatidylinositol-phospholipase D plus Nonidet P-40, or with phosphatidylinositol-phospholipase C alone, an electrophoretic cathodic band (Mr approximately 220 kDa) appeared near the bone band of a standard serum. With the WGA assay, 84.4 +/- 3.3% of ALP precipitated and the band disappeared. After immunoassay for the bone isoform, a mean of less than 5% enzyme activity was measured. We conclude that the ALP found in BALF is a pulmonary isoform of a tissue nonspecific isoenzyme.

  8. Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

    SciTech Connect

    Raub, T.J.; Denny, J.B.; Roberts, R.M.

    1986-01-01

    The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4/sup 0/C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodiumdodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with (/sup 3/H)D-glucosamine or (/sup 3/H)L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecule haptenized with trinitrophenol (TNP) groups were followed radio-chemically by means of (/sup 125/I)anti-DNP antibodies. Within 15 min at 37/sup 0/C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t/sub 1/2/ approx. 5 min). Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.

  9. Aspartate-specific peptidases in Salmonella typhimurium: mutants deficient in peptidase E.

    PubMed Central

    Carter, T H; Miller, C G

    1984-01-01

    The only dipeptide found to serve as a leucine source for a Salmonella strain lacking peptidases N, A, B, D, P, and Q was alpha-L-aspartyl-L-leucine. A peptidase (peptidase E) that specifically hydrolyzes Asp-X peptides was identified and partially purified from cell extracts. The enzyme (molecular weight, 35,000) is inactive toward dipeptides with N-terminal asparagine or glutamic acid. Mutants (pepE) lacking this enzyme were isolated by screening extracts for loss of the activity. Genetic mapping placed the pepE locus at 91.5 map units and established the gene order metA pepE zja-861::Tn5 malB. Duplications of the pepE locus showed a gene dosage effect on levels of peptidase E, suggesting that pepE is the structural gene for this enzyme. Mutations in pepE resulted in the loss of the ability to grow on Asp-Pro as a proline source but did not affect utilization of other dipeptides with N-terminal aspartic acid. Loss of peptidase E did not cause a detectable impairment in protein degradation. Two other peptidases present in cell extracts of mutants lacking peptidases N, A, B, D, P, Q, and E also hydrolyze many Asp-X dipeptides. Images PMID:6086568

  10. Identification of Immunopotentiating Lactic Acid Bacteria that Induce Antibody Production by in vitro Stimulated Human Peripheral Blood Mononuclear Cells.

    PubMed

    Yamashita, Makiko; Hitaka, Akira; Fujino, Himiko; Matsumoto, Takashi; Hasegawa, Takanori; Morimatsu, Fumiki; Fujiki, Tsukasa; Katakura, Yoshinori

    2012-01-01

    L-leucyl-L-leucine methyl ester (LLME) is known to remove lysosome-rich cells from human peripheral blood mononuclear cells (PBMCs). To evaluate the immunopotentiating ability of lactic acid bacteria (LAB), we adopted the in vitro stimulation protocol of LLME-treated PBMCs as a model assay system and monitored the level of antibody produced by stimulated PBMCs. The results indicated that several LAB strains have immunopotentiating ability against PBMCs, as evidenced by the enhanced antibody production and increased number of antigen-specific B cells. Next, we identified T cells as the direct target cells of the immunopotentiating LAB strain L32, suggesting that L32 induced antibody production by PBMCs through T-cell activation. Finally, we tested the immunopotentiating ability of ligands for Toll-like receptor 2 (TLR2), which is known to mediate the LAB signal, and observed that both L32 and one of the TLR2 ligands, LTA-BS, induced antigen-specific antibody production by in vitro stimulated PBMC. This suggests that L32 and LTA-BS can be used as an adjuvant for stimulating immune reaction in PBMCs.

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

    PubMed

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

    2009-08-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

    Zahmatkesh, Saeed

    2011-02-01

    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 poly (amide-imide)s containing L-amino acids were prepared from the synthesized dicarboxylic acids with two synthetic aromatic diamines in an ionic liquid (IL) as a green, safe and eco-friendly medium and also reactions catalysis agent. Evaluation of data shows that IL is the better polyamidation medium than the reported method and the catalysis stand on the higher inherent viscosities of the obtained PAIs and the rate of polymerizations beyond the greener reaction conditions and deletion of some essential reagents in conventional manners. Characterization were performs by means of IR, MS and (1)H NMR spectroscopy, elemental analysis, specific rotation, thermogravimetric analysis and differential scanning calorimetric techniques. Molecular weights of the obtained polymers were evaluated viscometrically, and the measured inherent viscosities were in the range 0.43-0.85 dL/g. These polymers were readily soluble in many organic solvents. These polymers still kept good thermal stability with glass transition temperatures in the range of 94-154°C, and the decomposition temperature under the nitrogen atmosphere for 10% weight-loss temperatures in excess of 308°C.

  15. Molecular Dynamics Simulation and NMR Investigation of the Association of the β-Blockers Atenolol and Propranolol with a Chiral Molecular Micelle

    PubMed Central

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

    2015-01-01

    Molecular dynamics simulations and NMR spectroscopy were used to compare the binding of two β-blocker drugs to the chiral molecular micelle poly-(sodium undecyl-(L)-leucine-valine). The molecular micelle is used as a chiral selector in capillary electrophoresis. This study is part of a larger effort to understand the mechanism of chiral recognition in capillary electrophoresis by characterizing the molecular micelle binding of chiral compounds with different geometries and charges. Propranolol and atenolol were chosen because their structures are similar, but their chiral interactions with the molecular micelle are different. Molecular dynamics simulations showed both propranolol enantiomers inserted their aromatic rings into the molecular micelle core and that (S)-propranolol associated more strongly with the molecular micelle than (R)-propranolol. This difference was attributed to stronger molecular micelle hydrogen bonding interactions experienced by (S)-propranolol. Atenolol enantiomers were found to bind near the molecular micelle surface and to have similar molecular micelle binding free energies. PMID:26257464

  16. 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

    2014-10-06

    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.

  17. Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light

    SciTech Connect

    Aryal, Uma K.; Stockel, Jana; Welsh, Eric A.; Gritsenko, Marina A.; Nicora, Carrie D.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2012-02-03

    Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a 13C15N-L-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 422 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly and degradation showed higher levels of isotope incorporation suggesting that these biochemical pathways are important for growth under non-diazotrophic conditions. Calculation of relative isotope abundances (RIA) values allowed to measure actual active protein synthesis over time for different biochemical pathways under non-diazotrophic conditions. Overall results demonstrated the utility of 'non-steady state' pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

  18. [B-, H- and L-cathepsin-like activity in blood plasma of patients with diseases of the thyroid, parathyroid and adrenal glands].

    PubMed

    Kalinichenko, O V; Myshunina, T M; Tron'ko, M D

    2010-01-01

    B-, H- and L-catepsine-like activity regarding Na-benzoyl-D,L-arginine-4-nitroanilide, L-leucine-4-nitroanilide and azocasein was studied in the blood plasma of patients with different diseases of thyroid, parathyroid and adrenal glands. It has been shown that the high H- catepsine-like activity confirming the activation of blood catepsin H secretion accompanied by the tissue growth under any pathology took place in the blood plasma of patients with all the above diseases. High B- catepsine-like activity in the blood plasma of patients with thyroid diseases was exceptionally fixed under the nodular forms of thyroid pathology, while the character of changes in L catepsine-like activity under these diseases was not so natural. In diaseses of the adrenal glands the changes in the B- and L-catepsine-like activity were only shown in the blood plasma of patients with cerebral layer tumors but not the gland cortex: B-catepsine-like activity increased in the blood plasma of patients with benign or malignant tumors, and L-catepsine-like activity decreased under benign tumor from chromaffin tissue. The established picture of changes in enzymatic activity in the blood plasma of patients does not allow to think that the determination of blood B- and L- catepsine-like activity can be recommended for obtaining additional information at diagnosis thyroid diseases.

  19. 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

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

    NASA Astrophysics Data System (ADS)

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

    2002-02-01

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

  1. A levodopa dry powder inhaler for the treatment of Parkinson's disease patients in off periods.

    PubMed

    Luinstra, Marianne; Grasmeijer, Floris; Hagedoorn, Paul; Moes, Jan Reindert; Frijlink, Henderik W; de Boer, Anne H

    2015-11-01

    Adequate treatment of Parkinson's patients in off periods with orally administered levodopa is hindered by a poor bioavailability and a slow onset of action. Hence, there is a need for a fast and reliable alternative as for instance via pulmonary administration of the drug. We developed a levodopa containing powder formulation for pulmonary delivery by a recently presented high dose dry powder inhaler (Cyclops). The objective was to produce the drug formulation by means of simple techniques such as micronization, either as pure active substance or with a minimum amount of excipients. After an initial screening on dispersion behaviour, the most promising formulation in the Cyclops was characterized in vitro over a range of pressure drops (2-6 kPa) and doses (20, 30 and 40 mg), representative of those to be expected in practice. A co-micronized levodopa formulation with 2% L-leucine appeared to yield the best aerosol properties for inhalation and highest delivered dose reproducibility. The combination of this particular formulation and the Cyclops inhaler seems to meet the basic requirements for satisfactory deposition in the airways. This formulation is therefore expected to be a promising candidate for the treatment of Parkinson's patients in an off period.

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

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

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

  3. Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying

    PubMed Central

    Okuda, Tomoyuki; Suzuki, Yumiko; Kobayashi, Yuko; Ishii, Takehiko; Uchida, Satoshi; Itaka, Keiji; Kataoka, Kazunori; Okamoto, Hirokazu

    2015-01-01

    In this study, two types of biodegradable polycation (PAsp(DET) homopolymer and PEG-PAsp(DET) copolymer) were applied as vectors for inhalable dry gene powders prepared by spray freeze drying (SFD). The prepared dry gene powders had spherical and porous structures with a 5~10-μm diameter, and the integrity of plasmid DNA could be maintained during powder production. Furthermore, it was clarified that PEG-PAsp(DET)-based dry gene powder could more sufficiently maintain both the physicochemical properties and in vitro gene transfection efficiencies of polyplexes reconstituted after powder production than PAsp(DET)-based dry gene powder. From an in vitro inhalation study using an Andersen cascade impactor, it was demonstrated that the addition of l-leucine could markedly improve the inhalation performance of dry powders prepared by SFD. Following pulmonary delivery to mice, both PAsp(DET)- and PEG-PAsp(DET)-based dry gene powders could achieve higher gene transfection efficiencies in the lungs compared with a chitosan-based dry gene powder previously reported by us. PMID:26343708

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

    PubMed Central

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

    1996-01-01

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

  5. 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.

  6. Proton-detected 3D (15)N/(1)H/(1)H isotropic/anisotropic/isotropic chemical shift correlation solid-state NMR at 70kHz MAS.

    PubMed

    Pandey, Manoj Kumar; Yarava, Jayasubba Reddy; Zhang, Rongchun; Ramamoorthy, Ayyalusamy; Nishiyama, Yusuke

    2016-01-01

    Chemical shift anisotropy (CSA) tensors offer a wealth of information for structural and dynamics studies of a variety of chemical and biological systems. In particular, CSA of amide protons can provide piercing insights into hydrogen-bonding interactions that vary with the backbone conformation of a protein and dynamics. However, the narrow span of amide proton resonances makes it very difficult to measure (1)H CSAs of proteins even by using the recently proposed 2D (1)H/(1)H anisotropic/isotropic chemical shift (CSA/CS) correlation technique. Such difficulties due to overlapping proton resonances can in general be overcome by utilizing the broad span of isotropic chemical shifts of low-gamma nuclei like (15)N. In this context, we demonstrate a proton-detected 3D (15)N/(1)H/(1)H CS/CSA/CS correlation experiment at fast MAS frequency (70kHz) to measure (1)H CSA values of unresolved amide protons of N-acetyl-(15)N-l-valyl-(15)N-l-leucine (NAVL).

  7. Lycopene production from synthetic medium by Blakeslea trispora NRRL 2895 (+) and 2896 (-) in a stirred-tank fermenter.

    PubMed

    Liu, Xiu-Ji; Liu, Rui-Sang; Li, Hong-Mei; Tang, Ya-Jie

    2012-06-01

    The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V(tip) = 0.926 - 2.161 m/s) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (-) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 μmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.

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

    PubMed Central

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

    2007-01-01

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

  9. Stimulation of L-asparate beta-decarboxylase formation by L-glutamate in Pseudomonas dacunhae and Improved production of L-alanine.

    PubMed

    Shibatani, T; Kakimoto, T; Chibata, I

    1979-09-01

    The formation of L-asparate beta-decarboxylase by Pseudomonas dacunhae was compared on media containing a variety of organic acids and amino acids as a carbon source. Although the enzyme was formed constitutively when the organism was grown on basal medium or on that containing tricarboxylic acid cycle intermediates, it was induced twofold by L-glutamate and repressed one-tenth by L-serine. L-Glutamine, L-proline, L-leucine, glycine, and L-threonine also showed induction effects lower than that of L-glutamate. L-Glutamate derepressed the serine effect. This glutamate effect was observed effect was observed with other microoganisms, e.g., Achromobacter pestifer and Achromobacter liquidum. Since the intermediates from L-glutamate metabolism had no effect, this induction effect was specific to L-glutamate. The formation of some glutamate-related enzymes was measured and is discussed in relation to the formation of L-asparate beta-decarboxylase. L-Asparate beta-decarboxylase was purified to an electrophoretically homogenous state from L-glutamate-grown cells of P. dacunhae, and some properties were compared with those of the enzyme from fumarate-grown cells. The two enzymes were identical in disc electrophoresis, molecular weight, and some enzymatic properties. The industrial production of L-alanine from L-aspartic acid acid was improved by using the culture broth with highly induced L-asparate beta-decarboxylase (9.4 U/ml of broth).

  10. Human Aging Is a Metabolome-related Matter of Gender.

    PubMed

    Jové, Mariona; Maté, Ianire; Naudí, Alba; Mota-Martorell, Natalia; Portero-Otín, Manuel; De la Fuente, Mónica; Pamplona, Reinald

    2016-05-01

    A molecular description of the mechanisms by which aging is produced is still very limited. Here, we have determined the plasma metabolite profile by using high-throughput metabolome profiling technologies of 150 healthy humans ranging from 30 to 100 years of age. Using a nontargeted approach, we detected 2,678 metabolite species in plasma, and the multivariate analyses separated perfectly two groups indicating a specific signature for each gender. In addition, there is a set of gender-shared metabolites, which change significantly during aging with a similar tendency. Among the identified molecules, we found vitamin D2-related compound, phosphoserine (40:5), monoacylglyceride (22:1), diacylglyceride (33:2), and resolvin D6, all of them decreasing with the aging process. Finally, we found three molecules that directly correlate with age and seven that inversely correlate with age, independently of gender. Among the identified molecules (6 of 10 according to exact mass and retention time), we found a proteolytic product (l-γ-glutamyl-l-leucine), which increased with age. On the contrary, a hydroxyl fatty acid (25-hydroxy-hexacosanoic), a polyunsaturated fatty acid (eicosapentaenoic acid), two phospholipids (phosphocholine [42:9]and phosphoserine [42:3]) and a prostaglandin (15-keto-prostaglandin F2α) decreased with aging. These results suggest that lipid species and their metabolism are closely linked to the aging process.

  11. Electrochemical Aptatoxisensor Responses on Nanocomposites Containing Electro-Deposited Silver Nanoparticles on Poly(Propyleneimine) Dendrimer for the Detection of Microcystin-LR in Freshwater

    PubMed Central

    Bilibana, Mawethu P.; Williams, Avril R.; Rassie, Candice; Sunday, Christopher E.; Makelane, Hlamulo; Wilson, Lindsay; Ntshongontshi, Nomaphelo; Jijana, Abongile N.; Masikini, Milua; Baker, Priscilla G. L.; Iwuoha, Emmanuel I.

    2016-01-01

    A sensitive and reagentless electrochemical aptatoxisensor was developed on cobalt (II) salicylaldiimine metallodendrimer (SDD–Co(II)) doped with electro-synthesized silver nanoparticles (AgNPs) for microcystin-LR (L, l-leucine; R, l-arginine), or MC-LR, detection in the nanomolar range. The GCE|SDD–Co(II)|AgNPs aptatoxisensor was fabricated with 5’ thiolated aptamer through self-assembly on the modified surface of the glassy carbon electrode (GCE) and the electronic response was measured using cyclic voltammetry (CV). Specific binding of MC-LR with the aptamer on GCE|SDD–Co(II)|AgNPs aptatoxisensor caused the formation of a complex that resulted in steric hindrance and electrostatic repulsion culminating in variation of the corresponding peak current of the electrochemical probe. The aptatoxisensor showed a linear response for MC-LR between 0.1 and 1.1 µg·L−1 and the calculated limit of detection (LOD) was 0.04 µg·L−1. In the detection of MC-LR in water samples, the aptatoxisensor proved to be highly sensitive and stable, performed well in the presence of interfering analog and was comparable to the conventional analytical techniques. The results demonstrate that the constructed MC-LR aptatoxisensor is a suitable device for routine quantification of MC-LR in freshwater and environmental samples. PMID:27845719

  12. Transport of the aromatic amino acids into isolated rat liver cells. Properties of uptake by two distinct systems.

    PubMed Central

    Salter, M; Knowles, R G; Pogson, C I

    1986-01-01

    The transport of the aromatic amino acids into isolated rat liver cells was studied. There was a rapid and substantial binding of the aromatic amino acids, L-alanine and L-leucine to the plasma membrane. This has important consequences for the determination of rates of transport and intracellular concentrations of the amino acids. Inhibition studies with a variety of substrates of various transport systems gave results consistent with aromatic amino acid transport being catalysed by two systems: a 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH)-insensitive aromatic D- and L-amino acid-specific system, and the L-type system (BCH-sensitive). The BCH-insensitive component of transport was Na+-independent and facilitated non-concentrative transport of the aromatic amino acids; it was unaffected by culture of liver cells for 24 h, by 48 h starvation, dexamethasone phosphate or glucagon. Kinetic properties of the BCH-inhibitable component were similar to those previously reported for the L2-system in liver cells. The BCH-insensitive component was a comparatively low-Km low-Vmax. transport system that we suggest is similar to the T-transport system previously seen only in human red blood cells. The results are discussed with reference to the importance of the T- and L-systems in the control of aromatic L-amino acid degradation in the liver. PMID:3954748

  13. Optimization of the mated fermentation process for the production of lycopene by Blakeslea trispora NRRL 2895 (+) and NRRL 2896 (-).

    PubMed

    Wang, Jin-Feng; Liu, Xiu-Ji; Liu, Rui-Sang; Li, Hong-Mei; Tang, Ya-Jie

    2012-05-01

    The mated fermentation process for the production of lycopene by Blakeslea trispora NRRL 2895 (+) and NRRL 2896 (-) was systematically optimized in shake flasks. The ratio of the (+) to (-) strains, the lycopene cyclase inhibitors piperidine and creatinine, the trisporic acid structural analog abscisic acid, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) precursor leucine, and the mevalonate kinase enhancer penicillin were all identified as key factors affecting lycopene biosynthesis. With an optimal ratio of 5:1 for the (+) to (-) strains and the addition of 6 g/L creatinine on day 3, the highest lycopene production was 98.1 ± 15.5 mg/L. Based on the above result, the addition of 0.1 g/L penicillin on day 4, 150 μmol/L abscisic acid on day 3 or 0.5 g/L leucine on day 4 enhanced lycopene production to 119.7 ± 17.2, 120.6 ± 12.3 and 135.2 ± 7.0 mg/L, respectively. Finally, an integrated strategy by combining the above key factors was developed, and the highest lycopene production of 156.2 ± 15.4 mg/L was obtained, which was enhanced by 134.9% comparing with its production of 66.5 ± 3.6 mg/L before the optimization process of this work. The results obtained in this study may be useful for large-scale industrial lycopene production.

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

    SciTech Connect

    Hu,G.; Lin, G.; Wang, M.; Dick, L.; Xu, R.; Nathan, C.; Li, H.

    2006-01-01

    Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 Angstroms resolution reveals a substrate-binding pocket with composite features of the distinct {beta}1, {beta}2 and {beta}5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the a-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapeptides of the a-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-{beta}-(1-naphthyl)-l-alanine-l-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

  15. Electrochemical Imprinted Polycrystalline Nickel-Nickel Oxide Half-Nanotube-Modified Boron-Doped Diamond Electrode for the Detection of L-Serine.

    PubMed

    Dai, Wei; Li, Hongji; Li, Mingji; Li, Cuiping; Wu, Xiaoguo; Yang, Baohe

    2015-10-21

    This paper presents a novel and versatile method for the fabrication of half nanotubes (HNTs) using a flexible template-based nanofabrication method denoted as electrochemical imprinting. With use of this method, polycrystalline nickel and nickel(II) oxide (Ni-NiO) HNTs were synthesized using pulsed electrodeposition to transfer Ni, deposited by radio frequency magnetron sputtering on a porous polytetrafluoroethylene template, onto a boron-doped diamond (BDD) film. The Ni-NiO HNTs exhibited semicircular profiles along their entire lengths, with outer diameters of 50-120 nm and inner diameters of 20-50 nm. The HNT walls were formed of Ni and NiO nanoparticles. A biosensor for the detection of L-serine was fabricated using a BDD electrode modified with Ni-NiO HNTs, and the device demonstrated satisfactory analytical performance with high sensitivity (0.33 μA μM(-1)) and a low limit of detection (0.1 μM). The biosensor also exhibited very good reproducibility and stability, as well as a high anti-interference ability against amino acids such as L-leucine, L-tryptophan, L-cysteine, L-phenylalanine, L-arginine, and L-lysine.

  16. [Responses of peptide hydrolases of the small and large intestines in rats on the administration of antibiotics].

    PubMed

    Borshchëv, Iu Iu; Gromova, L V; Ermolenko, E I; Grefner, N M; Borshchëva, I Iu; Gruzdkov, A A

    2012-06-01

    Effects of antibiotics on the structure and functional state of the intestine are not clear. We investigated some structural parameters of the small and large intestine, and activities of two intestinal peptide hydrolases in rats after administration of ampicillin and metronidazole during 3 and 5 days. After 3 days of antibiotic administration a decrease in the weight of mucosa in the small intestine, accompanied with a reduction in the villous height and width in this part of the intestine, and in the weight ofmucosa in the colon occured. At the same time the number of goblet cells in the small intestinal epithelium was increased. Specific activities of aminopeptidase M, and glycyl-L-leucine dipeptidase (micromol/min per g) in the mucosa of the small intestine were increased, and the total activities (micromol/min calculated per a part of the intestine) of the same enzymes did not change. The administration of antibiotics for 5 days resulted in increase of specific activity ofaminopeptidase M in the mucosa of the proximal part of the small intestine. In the chyme of the small intestine and colon, activities of the same enzymes (micromol/min calculated per a part of the intestine) were increased on the third and fifth days of the antibiotic administration. Thus, the application ofampicillin and metronidazole within 3-5 days causes a disturbance of the structural and functional parameters in the small and large intestines, which is most pronounced on the third day of the drug administration.

  17. Taste responses of bullfrog to pungent stimuli.

    PubMed

    Yoshii, K; Matui, T

    1994-02-21

    Taste responses of bullfrogs to various pungent compounds and taste substances were electrophysiologically recorded from glossopharyngeal nerves. The threshold concentrations were approximately 10(-7) M for piperine, approximately 10(-6) M for capsaicin and approximately 10(-4) M for allyl isothiocyanate. At any concentration examined, piperine was more potent than capsaicin. Both piperine and capsaicin elicited desensitizing responses, but the taste receptors recovered from the desensitization within 10 min after washing with deionized water. Cross-adaptation experiments revealed that capsaicin only partially desensitizes receptors for piperine, L-leucine, HCl or quinine. Perfusion of the lingual artery with a solution containing no added Ca decreased the responses to capsaicin. Such a solution has been shown to suppress the taste nerve responses by blocking synaptic transmissions between taste cells and taste nerves [8]. These results suggest that the gustatory effects of capsaicin are different from its pharmacological effects on sensory neurons. It is likely that capsaicin and other pungent compounds, when they act as seasonings, stimulate taste cells rather than the free nerve endings of the sensory neurons.

  18. Helical Oligopeptides of a Quaternized Amino Acid with Tunable Chiral-Induction Ability and an Anomalous pH Response.

    PubMed

    Cho, Joonil; Ishida, Yasuhiro; Aida, Takuzo

    2017-04-06

    A series of octamer (8-mer) and hexadecamer (16-mer) oligopeptides of 4-aminopiperidine-4-carboxylic acid (Api) with l-leucine as a chiral auxiliary at their N or C termini were synthesized. By using circular dichroism spectroscopy, the conformational profiles of the peptides were systematically studied, which revealed that the α-helix-formation ability of the peptides is determined by the combination of parameters, which includes peptide length, state of the piperidine groups in the Api units, and position of the chiral auxiliary. When the piperidines were in the free-base state, the peptides showed a low propensity to form helical structures. However, the protonation and acylation of the piperidines enhanced the formation of helical structures, such that the order for helix-formation ability was protonated>acylated>free base. In terms of peptide length, the 16-mers generally showed higher helix-formation ability than the corresponding 8-mers, and one of the 16-mers showed helicity at the highest level reported thus far for oligopeptides of a similar length. It was also found that the sensitivity of the helical structure towards the state of the piperidine groups changed drastically depending on the chiral auxiliary position; the N-terminal chiral peptides were more sensitive than the C-terminal chiral analogues.

  19. 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.

  20. Quantifying the relative contributions of different solute carriers to aggregate substrate transport

    PubMed Central

    Taslimifar, Mehdi; Oparija, Lalita; Verrey, Francois; Kurtcuoglu, Vartan; Olgac, Ufuk; Makrides, Victoria

    2017-01-01

    Determining the contributions of different transporter species to overall cellular transport is fundamental for understanding the physiological regulation of solutes. We calculated the relative activities of Solute Carrier (SLC) transporters using the Michaelis-Menten equation and global fitting to estimate the normalized maximum transport rate for each transporter (Vmax). Data input were the normalized measured uptake of the essential neutral amino acid (AA) L-leucine (Leu) from concentration-dependence assays performed using Xenopus laevis oocytes. Our methodology was verified by calculating Leu and L-phenylalanine (Phe) data in the presence of competitive substrates and/or inhibitors. Among 9 potentially expressed endogenous X. laevis oocyte Leu transporter species, activities of only the uniporters SLC43A2/LAT4 (and/or SLC43A1/LAT3) and the sodium symporter SLC6A19/B0AT1 were required to account for total uptake. Furthermore, Leu and Phe uptake by heterologously expressed human SLC6A14/ATB0,+ and SLC43A2/LAT4 was accurately calculated. This versatile systems biology approach is useful for analyses where the kinetics of each active protein species can be represented by the Hill equation. Furthermore, its applicable even in the absence of protein expression data. It could potentially be applied, for example, to quantify drug transporter activities in target cells to improve specificity. PMID:28091567

  1. 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.

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

    PubMed

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

    2005-09-01

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

  3. A molecular dynamics simulation study of the association of 1,1";-binaphthyl-2,2";-diyl hydrogenphosphate enantiomers with a chiral molecular micelle

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

    Molecular dynamics (MD) simulations were used to investigate the binding of 1,1";-binaphthyl-2,2";-diyl hydrogenphosphate (BNP) enantiomers to the molecular micelle poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)). Poly(SULV) is used as a chiral selector in capillary electrophoresis separations. Four poly(SULV) binding pockets were identified and either (R)-BNP or (S)-BNP were docked into each pocket. MD simulations were then used to identify the preferred BNP binding site. Within the preferred site, both enantiomers formed hydrogen bonds with poly(SULV) and penetrated into the poly(SULV) core. Comparisons of BNP enantiomer binding to the preferred poly(SULV) pocket showed that (S)-BNP formed stronger hydrogen bonds, moved deeper into the binding site, and had a lower poly(SULV) binding free energy than the (R) enantiomer. Finally, MD simulation results were in agreement with capillary electrophoresis and NMR experiments. Each technique showed (S)-BNP interacted more strongly with poly(SULV) than (R)-BNP and that the site of chiral recognition was near the poly(SULV) leucine chiral center.

  4. Mutations in TBCK, Encoding TBC1-Domain-Containing Kinase, Lead to a Recognizable Syndrome of Intellectual Disability and Hypotonia

    PubMed Central

    Bhoj, Elizabeth J.; Li, Dong; Harr, Margaret; Edvardson, Shimon; Elpeleg, Orly; Chisholm, Elizabeth; Juusola, Jane; Douglas, Ganka; Guillen Sacoto, Maria J.; Siquier-Pernet, Karine; Saadi, Abdelkrim; Bole-Feysot, Christine; Nitschke, Patrick; Narravula, Alekhya; Walke, Maria; Horner, Michele B.; Day-Salvatore, Debra-Lynn; Jayakar, Parul; Vergano, Samantha A. Schrier; Tarnopolsky, Mark A.; Hegde, Madhuri; Colleaux, Laurence; Crino, Peter; Hakonarson, Hakon

    2016-01-01

    Through an international multi-center collaboration, 13 individuals from nine unrelated families and affected by likely pathogenic biallelic variants in TBC1-domain-containing kinase (TBCK) were identified through whole-exome sequencing. All affected individuals were found to share a core phenotype of intellectual disability and hypotonia, and many had seizures and showed brain atrophy and white-matter changes on neuroimaging. Minor non-specific facial dysmorphism was also noted in some individuals, including multiple older children who developed coarse features similar to those of storage disorders. TBCK has been shown to regulate the mammalian target of rapamycin (mTOR) signaling pathway, which is also stimulated by exogenous leucine supplementation. TBCK was absent in cells from affected individuals, and decreased phosphorylation of phospho-ribosomal protein S6 was also observed, a finding suggestive of downregulation of mTOR signaling. Lastly, we demonstrated that activation of the mTOR pathway in response to L-leucine supplementation was retained, suggesting a possible avenue for directed therapies for this condition. PMID:27040691

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

    PubMed

    Suzuki, Yumiko; Okuda, Tomoyuki; Okamoto, Hirokazu

    2016-01-01

    Cationic polymers are being studied as non-viral gene delivery vectors. Poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)) and their block copolymers with poly(ethylene glycol), PEG-PAsp(DET), have been reported as efficient biodegradable non-viral vectors which form a polyplex with plasmid DNA (pDNA). However, the polyplexes are not stable because PAsp(DET) and PEG-PAsp(DET) are easily subjected to hydrolysis; therefore, they need to be prepared on site. In this study, using the biodegradable polycations as non-viral vectors, PAsp(DET) and PEG-PAsp(DET), we investigated the effects of L-leucine (Leu) on the polyplex. We prepared solutions and dry powders with and without Leu. Both dry powders had large and porous particles and Leu acted as a dispersing agent. The transfection activity of the sample solutions decreased within a month. However, the decrease in the transfection activity was partially suppressed by the dry powder with Leu at 5 and 25°C at 3 months. Furthermore, transfection experiments revealed that Leu exhibited a pDNA-stabilizing effect in the solution and dry powder. Similar results were observed for pDNA integrity, where a polyplex was formed in the dry powder. The results suggest that Leu is a candidate stabilizer to protect pDNA from degradation.

  6. Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases.

    PubMed

    Dutoit, Raphaël; Brandt, Nathalie; Legrain, Christianne; Bauvois, Cédric

    2012-01-01

    Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.

  7. Rapid screening of aminopeptidase N inhibitors by capillary electrophoresis with electrophoretically mediated microanalysis.

    PubMed

    Wang, Hairong; Xu, Wenfang; Cao, Jiangying; Wang, Weihong

    2015-01-01

    In this work, an electrophoretically mediated microanalysis (EMMA) method with a partial-filling technique was setup to evaluate the inhibitory potency of novel compounds toward aminopeptidase N (APN). It was necessary to optimize the electrophoretic conditions with respect to the kinetic constraints and for attaining high sensitivity. In our setup, a part of the capillary was filled with the incubation buffer for the enzyme reaction, whereas the rest was filled with a suitable BGE for the separation of substrates and products. To monitor the performance of the newly developed method, the kinetic constants (Km and Vmax) for the catalyzed dissociation of L-Leucine-p-nitroanilide in the presence of APN as well as the inhibition constant (IC50 ) of a known competitive inhibitor, that is bestatin, were determined and these results were compared with those obtained by a classical spectrophotometric assay. The developed EMMA method was subsequently applied to the screening of 30 APN inhibitors. Whereas the inhibition potency of these inhibitors (expressed in IC50 values) were significantly underestimated by the EMMA method, the order of the inhibitory potential of these various compounds was found in agreement with the literature.

  8. 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.

  9. Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations.

    PubMed

    de Souza, Anderson Proust Gonçalves; Vicente, Maristela de Araújo; Klein, Raphael Contelli; Fietto, Luciano Gomes; Coutrim, Maurício Xavier; de Cássia Franco Afonso, Robson José; Araújo, Leandro Dias; da Silva, Paulo Henrique Alves; Bouillet, Leoneide Erica Maduro; Castro, Ieso Miranda; Brandão, Rogelio Lopes

    2012-02-01

    In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5',5″-trifluor-D: ,L: -leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. "Cachaça" produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in "cachaça" production.

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. Intracellular fates of cell-penetrating block copolypeptide vesicles.

    PubMed

    Sun, Victor Z; Li, Zhibo; Deming, Timothy J; Kamei, Daniel T

    2011-01-10

    The block copolypeptide poly(l-homoarginine)(60)-b-poly(l-leucine)(20) (R(60)L(20)) was previously found to self-assemble into versatile vesicles with controllable size and encapsulate hydrophilic cargo. These R(60)L(20) vesicles also demonstrated the ability to cross the cell membrane and transport encapsulated cargo into different cell lines. To assess the potential for using the R(60)L(20) vesicles as drug delivery vehicles further, we have investigated their endocytosis and intracellular trafficking behavior. Using drugs that inhibit different endocytosis pathways, we identified macropinocytosis to be a major process by which the R(60)L(20) vesicles enter HeLa cells. Subsequent immunostaining experiments demonstrated that the vesicles entered the early endosomes but not the lysosomes, suggesting that they recycle back to the cell surface. Overall, our studies indicate that the R(60)L(20) vesicles are able to enter cells intact with their cargos, and although some manage to escape from early endosomes, most are trapped within these intracellular compartments.

  13. Microscopical examination of the localisation patterns of two novel rhodamine derivatives in normal and neoplastic colonic mucosa.

    PubMed

    Atlamazoglou, V; Yova, D; Kavantzas, N; Loukas, S

    2001-01-01

    Tissue characterisation by fluorescence imaging, using exogenous fluorophores, is a promising method for cancer detection. Histochemical alterations in the composition of mucins, when neoplastic transformations occur, could be exploited to derive more selective fluoroprobes indicative of early malignant transformation. The aim of this work was to develop and examine tumour selective fluoroprobes for colon cancer diagnosis, as well as to determine the morphological components where selective dye accumulation has occurred. Two novel fluoroprobes: rhodamine B-L-leucine amide and rhodamine B-phenylboronic acid were synthesised and examined together with Mayer's mucicarmine, alexa 350-wheat germ agglutinin (WGA) and tetramethyl rhodamine-concanavalin A (ConA). Fluorescence microscopy studies were performed with deparaffinised human colon sections, using an epifluorescence microscope equipped with a colour CCD camera. The intense accumulation of the novel fluoroprobes was localised in the amorphous material in the lumen of neoplastic crypts. To gain insight into the localisation patterns, mucicarmine, alexa 350-WGA and tetramethyl rhodamine-ConA were used. Alexa 350-WGA reacted primarily with mucin secreted in the malignant crypt lumen suggesting that this material is rich in sialic acid and N-acetylglucosaminyl residues. These derivatives clearly and consistently distinguished non-neoplastic from neoplastic human colon tissue sections. The intense accumulation at the altered mucins indicates that they could be used as fluoroprobes of biochemical alterations for carcinoma detection.

  14. Investigating on the Correlation Between Some Biological Activities of Marine Sponge-Associated Bacteria Extracts and Isolated Diketopiperazines.

    PubMed

    Abd El-Hady, Faten K; Fayad, Walid; Iodice, Carmine; El-Shahid, Zeinab A; Abdel-Aziz, Mohamed S; Crudele, Egle; Tommonaro, Giuseppina

    2017-01-01

    Marine organisms have been considered as the richest sources of novel bioactive metabolites, which can be used for pharmaceutical purposes. In the last years, the interest for marine microorganisms has grown for their enormous biodiversity and for the evidence that many novel compounds isolated from marine invertebrates are really synthesized by their associated bacteria. Nevertheless, the discovery of a chemical communication Quorum sensing (QS) between bacterial cells and between bacteria and host has gained the researchers to expand the aim of their study toward the role of bacteria associated with marine invertebrates, such as marine sponge. In the present paper, we report the evaluation of biological activities of different extracts of bacteria Vibrio sp. and Bacillus sp. associated with marine sponges Dysidea avara and Ircinia variabilis, respectively. Moreover, we evaluated the biological activities of some diketopiperazines (DKPs), previously isolated, and able to activate QS mechanism. The results showed that all extracts, fractions, and DKPs showed low scavenging activity against DPPH and superoxide anion, low cytotoxic and anti-tyrosinase activities, but no antimicrobial and acetylcholinesterase inhibitory activities. One DKP [cyclo-(trans-4-hydroxy-L-prolyl-L-leucine)] has the highest α-glucosidase inhibitory activity even than the standard acarbose.

  15. AFRRI (Armed Forces Radiobiology Research Institute) reports, July, August, September 1989. Technical report

    SciTech Connect

    Not Available

    1989-11-01

    This volume contains AFRRI Scientific Reports SR 89-26 through SR89-39 and Technical Report TR89-1 for Jul-Sep 1989. Partial contents include: Induction of marrow hypoxia by radioprotective agents; Cell-cycle radiation response: Role of intracellular factors; Characteristics of radiation-induced performance changes in bar-press avoidance with and without a preshock warning cue; Norepinephrine-induced phosphorylation of a 25 kd phosphoprotein in rat aorta is altered in intraperitoneal sepsis; Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry; Tropism of canine neutrophils to xanthine oxidase; Effects of acute sublethal gamma radiation exposure on aggressive behavior in male mice: A dose-response study; Progressive behavioral changes during the maturation of rats with early radiation-induced hypoplasia of fascia dentata granule cells; Stomach nodules in pigeons; An assessment of the behavioral toxicity of high-energy iron particles compared to other qualities of radiation; L-leucyl-L-leucine methyl ester treatment of canine marrow and peripheral blood cells; Localization of cyclo-oxygenase and prostaglandin E2 in the secretory granule of the mast cell; Radioprotection of mice with interleukin-1: Relationship to the number of spleen colony-forming units; Survival after total-body irradiation. I. Effects of partial small-bowel shielding; Laboratory x-ray irradiator for cellular radiobiology research studies: Dosimetry report.

  16. Inhibitory properties of nerve-specific human glutamate dehydrogenase isozyme by chloroquine.

    PubMed

    Choi, Myung-Min; Kim, Eun-A; Choi, Soo Young; Kim, Tae Ue; Cho, Sung-Woo; Yang, Seung-Ju

    2007-11-30

    Human glutamate dehydrogenase exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. In the nervous system, GDH is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter during neurotransmission. Chloroquine has been known to be a potent inhibitor of house-keeping GDH1 in permeabilized liver and kidney-cortex of rabbit. However, the effects of chloroquine on nerve-specific GDH2 have not been reported yet. In the present study, we have investigated the effects of chloroquine on hGDH2 at various conditions and showed that chloroquine could inhibit the activity of hGDH2 at dose-dependent manner. Studies of the chloroquine inhibition on enzyme activity revealed that hGDH2 was relatively less sensitive to chloroquine inhibition than house-keeping hGDH1. Incubation of hGDH2 was uncompetitive with respect of NADH and non-competitive with respect of 2-oxoglutarate. The inhibitory effect of chloroquine on hGDH2 was abolished, although in part, by the presence of ADP and L-leucine, whereas GTP did not change the sensitivity to chloroquine inhibition. Our results show a possibility that chloroquine may be used in regulating GDH activity and subsequently glutamate concentration in the central nervous system.

  17. Somatostatin and the intestinal transport of glucose and other nutrients in the anaesthetised rat.

    PubMed Central

    Daumerie, C; Henquin, J C

    1982-01-01

    The effects of somatostatin on oral glucose tolerance and on intestinal absorption of glucose and other nutrients have been studied in anaesthetised rats. Intravenous somatostatin (0.1-0.6 nmol/min) increased the rate of gastric emptying. After intraduodenal administration of glucose, the rise in peripheral plasma levels of the sugar was delayed, but finally exaggerated by somatostatin, which inhibited the insulin response. Absorption was evaluated by measuring the disappearance of radioactive nutrients from the lumen of a 'tied duodenojejunal loop'. At a luminal concentration of 4 mmol/l of 3-0-methylglucose, neither disappearance of the sugar from the lumen nor its appearance in plasma was affected by somatostatin. Passive transport of 3-0-methylglucose (100 mmol/l) was not significantly modified by somatostatin, although the appearance of the labelled tracer in plasma was delayed. Somatostatin had no significant effect on absorption of galactose (4 mmol/l), sucrose (40 mmol/l), leucine (4 mmol/l) or palmitate (0.1 and 0.4 mmol/l). These results show that somatostatin delays appearance of ingested sugars in peripheral plasma without direct effect on the absorption sites; this delay may result from changes in intestinal motility, enzyme secretion and splanchnic blood flow. PMID:6121743

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

    PubMed

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

    2000-03-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  1. A Combination of Leucine, Metformin, and Sildenafil Treats Nonalcoholic Fatty Liver Disease and Steatohepatitis in Mice

    PubMed Central

    Fu, Lizhi; Li, Fenfen; Cao, Qiang; Cui, Xin; Wu, Rui; Shi, Hang

    2016-01-01

    Sirt1, AMPK, and eNOS modulate hepatic energy metabolism and inflammation and are key players in the development of NASH. L-leucine, an allosteric Sirt1 activator, synergizes with low doses of metformin or sildenafil on the AMPK-eNOS-Sirt1 pathway to reverse mild NAFLD in preclinical mouse models. Here we tested a possible multicomponent synergy to yield greater therapeutic efficacy in NAFLD/NASH. Liver cells and macrophages or an atherogenic diet induced NASH mouse model was treated with two-way and three-way combinations. The three-way combination Sild-Met-Leu increased hepatic fatty acid oxidation and reduced lipogenic gene expression and inflammatory marker in vitro. In mice, Sild-Met-Leu reduced the diet induced increases of ALT, TGFβ, PAI-1, IL1β, and TNFα, hepatic collagen expression, and nearly completely reversed hepatocyte ballooning and triglyceride accumulation, while all two-way combinations had only modest effects. Therefore, these data provide preclinical evidence for therapeutic efficacy of Sild-Met-Leu in the treatment of NAFLD and NASH. PMID:28042486

  2. Impact of epiphytic and endogenous enzyme activities of senescent maize leaves and roots on the soil biodegradation process.

    PubMed

    Zafar Amin, Bilal Ahmad; Beaugrand, Johnny; Debeire, Philippe; Chabbert, Brigitte; Bertrand, Isabelle

    2011-11-01

    This study was focused on investigating the role of the initial residue community, i.e. microorganisms and enzymes from the epiphytic and endophytic compartments, in soil decomposition processes. Aerial and underground parts (leaves and roots) of maize (Zea mays L.) plants were γ-irradiated, surface-sterilized with sodium hypochlorite (NaOCl)/ethanol or non-sterilized (controls), while the outer surface morphology of maize leaves and roots was examined by scanning electron microscopy (SEM). Non-sterilized and sterilized maize leaves and roots were incubated in soil to study carbon (C) mineralization kinetics and enzyme dynamics (L-leucine aminopeptidase, CBH-1, xylanase, cellulase and laccase). SEM results showed that initial microbial colonization was more pronounced on non-sterilized leaf and root surfaces than on sterilized samples. The hypochlorite treatment removed a part of the soluble components of leaves by washing and no specific effect of any type of colonizing microorganisms was observed on C mineralization. In contrast, γ irradiation and hypochlorite treatments did not affect root chemical characteristics and the quantitative effect of initial residue-colonizing microorganisms on C mineralization was demonstrated. The variations in C mineralization and enzyme dynamics between non-sterilized and sterilized roots suggested that activities of epiphytic and endogenic microorganisms were of the same order of magnitude.

  3. Amino-acid transporters in T-cell activation and differentiation.

    PubMed

    Ren, W; Liu, G; Yin, J; Tan, B; Wu, G; Bazer, F W; Peng, Y; Yin, Y

    2017-03-02

    T-cell-mediated immune responses aim to protect mammals against cancers and infections, and are also involved in the pathogenesis of various inflammatory or autoimmune diseases. Cellular uptake and the utilization of nutrients is closely related to the T-cell fate decision and function. Research in this area has yielded surprising findings in the importance of amino-acid transporters for T-cell development, homeostasis, activation, differentiation and memory. In this review, we present current information on amino-acid transporters, such as LAT1 (l-leucine transporter), ASCT2 (l-glutamine transporter) and GAT-1 (γ-aminobutyric acid transporter-1), which are critically important for mediating peripheral naive T-cell homeostasis, activation and differentiation, especially for Th1 and Th17 cells, and even memory T cells. Mechanically, the influence of amino-acid transporters on T-cell fate decision may largely depend on the mechanistic target of rapamycin complex 1 (mTORC1) signaling. These discoveries remarkably demonstrate the role of amino-acid transporters in T-cell fate determination, and strongly indicate that manipulation of the amino-acid transporter-mTORC1 axis could ameliorate many inflammatory or autoimmune diseases associated with T-cell-based immune responses.

  4. Molecularly designed surfaces for blood deheparinization using an immobilized heparin-binding peptide.

    PubMed

    Martins, M Cristina L; Curtin, Scott A; Freitas, Sidónio C; Salgueiro, Pedro; Ratner, Buddy D; Barbosa, Mário A

    2009-01-01

    Systemic heparinization, used during haemodialysis to prevent blood clotting on the extracorporeal circuit, leads to a high incidence of hemorrhagic complications. The adverse reactions associated with heparin neutralization using protamine sulphate justify the development of an alternative system for blood deheparinization. The main objective of this work is to design nanostructured surfaces with the capacity to bind heparin from blood in a selective way. A heparin-binding polypeptide, composed of L-lysine and L-leucine (pKL), was synthesized and immobilized, in different concentrations, onto self-assembled monolayers (SAMs) terminated with tetra(ethylene-glycol) (EG4 SAMs). Immobilization was performed using a fixed concentration of pKL after surface activation to different degrees using a range of CDI (N,N'-carbonyldiimidazole) concentrations. Results demonstrated that the presence of pKL increases heparin adsorption to EG4-SAMs, independently of the pKL concentration and the way of immobilization (adsorption or covalent bound). Selectivity towards heparin was successfully achieved on SAMs with low concentrations of immobilized pKL (9-17% of pKL). Surfaces were characterized using ellipsometry, contact angle measurements, Fourier transform infrared reflection absorption spectroscopy (IRAS), atomic force microscopy, and X-ray photoelectron spectroscopy. Heparin adsorption was assessed using IRAS and N-sulphonate-(35)S-heparin. Therefore, this study could give a good contribution for the design of blood deheparinization devices.

  5. Interactions of leukocytes and platelets with poly(lysine/leucine) immobilized on tetraethylene glycol-terminated self-assembled monolayers.

    PubMed

    Martins, M Cristina L; Ochoa-Mendes, Vanessa; Ferreira, Gisela; Barbosa, Judite N; Curtin, Scott A; Ratner, Buddy D; Barbosa, Mário A

    2011-05-01

    Surfaces that bind heparin are important for biomaterials for blood deheparinization. In our recent work it was demonstrated that a polypeptide composed of L-lysine and L-leucine (pKL), after immobilization onto tetra(ethylene glycol) terminated self-assembled monolayers (EG4-SAMs), can bind heparin from blood plasma in a selective, concentration-dependent way. During this work the effect of this peptide on platelet adhesion and activation and leukocyte adhesion was studied. The surface charge of these nanostructured surfaces was evaluated in order to correlate the effect of positively charged amine groups and hydrophobic methyl groups on the behavior of platelets and leukocyte adhesion. The results demonstrated that the presence of pKL decreased leukocyte adhesion to EG4-SAMs at all concentrations used. This effect is even more pronounced when surfaces were pre-immersed in heparinized plasma. In contrast, there is an increase in platelet adhesion and activation with increased percentage immobilized pKL. This effect is enhanced when surfaces were pre-immersed in heparinized plasma. However, adsorbed pKL in very low amounts does not induce platelet adhesion and activation compared with EG4, even when pre-immersed in plasma. Since only low pKL amounts are necessary to induce heparin selectivity, these results are promising for the development of heparin-binding biomaterials for blood deheparinization.

  6. Funastrain c II: a cysteine endopeptidase purified from the latex of Funastrum clausum.

    PubMed

    Morcelle, Susana R; Trejo, Sebastián A; Canals, Francesc; Avilés, Francesc X; Priolo, Nora S

    2004-04-01

    A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2009-02-01

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

  9. Calcium triggers beta-defensin (hBD-2 and hBD-3) and chemokine macrophage inflammatory protein-3 alpha (MIP-3alpha/CCL20) expression in monolayers of activated human keratinocytes.

    PubMed

    Pernet, I; Reymermier, C; Guezennec, A; Branka, J-E; Guesnet, J; Perrier, E; Dezutter-Dambuyant, C; Schmitt, D; Viac, J

    2003-12-01

    The inducible epidermal beta-defensins and the chemokine macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20) are important mediators involved in innate and adaptive immunity and in the recruitment of immune cells. The aim of our study was to determine whether calcium could trigger the induction of beta-defensins (hBD-2 and hBD-3) mRNA and the release of MIP-3alpha by normal human keratinocyte monolayers. Epidermal cells derived from foreskin were cultured in defined medium supplemented with different calcium levels (0.09, 0.8 and 1.7 mM) and were stimulated or not with the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha 1-500 ng/ml) or interferon-gamma (INF-gamma 1-100 ng/ml). A high calcium concentration (1.7 mM) alone applied in culture medium for 4 days was sufficient to induce hBD-2 and hBD-3 mRNA expression. Whatever interindividual variability in the expression of hBD-2 and hBD-3 mRNA and MIP-3alpha secretion, the addition of TNF-alpha for a short duration (26h), initiated a dose-dependent and coordinated up-regulation of hBD-2 and hBD-3 mRNA and MIP-3alpha release in keratinocyte cultures. Unlike hBD-2 and hBD-3 mRNA was preferentially stimulated by IFN-gamma rather than TNF-alpha. In our experimental conditions, L-isoleucine, described to stimulate beta-defensin in bovine epithelial cells, did not exert any effect either on hBD-2 and hBD-3 transcripts or MIP-3alpha protein. Taken together, these results confirm the major role of the maturation/differentiation process of normal human keratinocytes in the induction of inducible beta-defensins and MIP-3alpha chemokine, which contribute in vivo to the immunosurveillance of the skin barrier function.

  10. Endocannabinoid-mediated long-term depression of afferent excitatory synapses in hippocampal pyramidal cells and GABAergic interneurons.

    PubMed

    Péterfi, Zoltán; Urbán, Gabriella M; Papp, Orsolya I; Németh, Beáta; Monyer, Hannah; Szabó, Gábor; Erdélyi, Ferenc; Mackie, Ken; Freund, Tamás F; Hájos, Norbert; Katona, István

    2012-10-10

    Although endocannabinoids have emerged as essential retrograde messengers in several forms of synaptic plasticity, it remains controversial whether they mediate long-term depression (LTD) of glutamatergic synapses onto excitatory and inhibitory neurons in the hippocampus. Here, we show that parvalbumin- and somatostatin/metabotropic glutamate receptor 1(a) (mGlu(1a))-positive GABAergic interneurons express diacylglycerol lipase-α (DGL-α), a synthesizing enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), albeit at lower levels than principal cells. Moreover, this lipase accumulates postsynaptically around afferent excitatory synapses in all three cell types. To address the role of retrograde 2-AG signaling in LTD, we investigated two forms: (1) produced by postsynaptic spiking paired with subsequent presynaptic stimulation or (2) induced by group I mGlu activation by (S)-3,5-dihydroxyphenylglycine (DHPG). Neither form of LTD was evoked in the presence of the mGlu(5) antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], the DGL inhibitor THL [N-formyl-l-leucine (1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]dodecyl ester], or the intracellularly applied Ca(2+) chelator BAPTA in CA1 pyramidal cells, fast-spiking interneurons (representing parvalbumin-containing cells) and interneurons projecting to stratum lacunosum-moleculare (representing somatostatin/mGlu(1a)-expressing interneurons). Both forms of LTD were completely absent in CB(1) cannabinoid receptor knock-out mice, whereas pharmacological blockade of CB(1) led to inconsistent results. Notably, in accordance with their lower DGL-α level, a higher stimulation frequency or higher DHPG concentration was required for LTD induction in interneurons compared with pyramidal cells. These findings demonstrate that hippocampal principal cells and interneurons produce endocannabinoids to mediate LTD in a qualitatively similar, but quantitatively different manner. The shifted induction threshold implies that

  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

    2015-01-01

    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 < 0.01) between the two groups of liposomal OP dry powders and OP solution with t-test, which indicated that the drug released slowly from liposomal OP dry powders in the lung. To sum up, dry powders formulation of liposome-encapsulated OP for inhalation was suitable for pulmonary administration, which offering the opportunity to reduce dosing frequency.

  12. Improved narrowband dipolar recoupling for homonuclear distance measurements in rotating solids.

    PubMed

    Goobes, G; Vega, S

    2002-02-01

    Recovery of the magnetic dipolar interaction between nuclei bearing the same gyromagnetic ratio in rotating solids can be promoted by synchronous rf irradiation. Determination of the dipolar interaction strength can serve as a tool for structural elucidation in polycrystalline powders. Spinning frequency dependent narrow-band (nb) RFDR and SEDRA experiments are utilized as simple techniques for the determination of dipolar interactions between the nuclei in coupled homonuclear spin pairs. The magnetization exchange and coherence dephasing due to a fixed number of rotor-synchronously applied pi-pulses is monitored at spinning frequencies in the vicinity of the rotational resonance (R(2)) conditions. The powder nbRFDR and nbSEDRA decay curves of spin magnetizations and coherences, respectively, as a function of the spinning frequency can be measured and analyzed using simple rate equations providing a quantitative measure of the dipolar coupling. The effects of the phenomenological relaxation parameters in these rate equations are discussed and an improved methodology is suggested for analyzing nbRFDR data for small dipolar couplings. The distance between the labeled nuclei in the 1,3-(13)C(2)-hydroxybutyric acid molecule is rederived using existing nbRFDR results and the new simulation procedure. A nbSEDRA experiment has been performed successfully on a powder sample of singly labeled 1-(13)C-L-leucine measuring the dipolar interaction between the labeled carboxyl carbon and the natural abundant beta-carbon. Both narrowband techniques are employed for the determination of the nuclear distances between the side-chain carbons of leucine and its carbonyl carbon in a tripeptide Leu-Gly-Phe that is singly (13)C-labeled at the leucine carbonyl carbon position.

  13. Improved Narrowband Dipolar Recoupling for Homonuclear Distance Measurements in Rotating Solids

    NASA Astrophysics Data System (ADS)

    Goobes, G.; Vega, S.

    2002-02-01

    Recovery of the magnetic dipolar interaction between nuclei bearing the same gyromagnetic ratio in rotating solids can be promoted by synchronous rf irradiation. Determination of the dipolar interaction strength can serve as a tool for structural elucidation in polycrystalline powders. Spinning frequency dependent narrow-band (nb) RFDR and SEDRA experiments are utilized as simple techniques for the determination of dipolar interactions between the nuclei in coupled homonuclear spin pairs. The magnetization exchange and coherence dephasing due to a fixed number of rotor-synchronously applied π-pulses is monitored at spinning frequencies in the vicinity of the rotational resonance (R2) conditions. The powder nbRFDR and nbSEDRA decay curves of spin magnetizations and coherences, respectively, as a function of the spinning frequency can be measured and analyzed using simple rate equations providing a quantitative measure of the dipolar coupling. The effects of the phenomenological relaxation parameters in these rate equations are discussed and an improved methodology is suggested for analyzing nbRFDR data for small dipolar couplings. The distance between the labeled nuclei in the 1,3-13C2-hydroxybutyric acid molecule is rederived using existing nbRFDR results and the new simulation procedure. A nbSEDRA experiment has been performed successfully on a powder sample of singly labeled 1-13C-L-leucine measuring the dipolar interaction between the labeled carboxyl carbon and the natural abundant β-carbon. Both narrowband techniques are employed for the determination of the nuclear distances between the side-chain carbons of leucine and its carbonyl carbon in a tripeptide Leu-Gly-Phe that is singly 13C-labeled at the leucine carbonyl carbon position.

  14. Dietary Leucine Supplementation Improves the Mucin Production in the Jejunal Mucosa of the Weaned Pigs Challenged by Porcine Rotavirus.

    PubMed

    Mao, Xiangbing; Liu, Minghui; Tang, Jun; Chen, Hao; Chen, Daiwen; Yu, Bing; He, Jun; Yu, Jie; Zheng, Ping

    2015-01-01

    The present study was mainly conducted to determine whether dietary leucine supplementation could attenuate the decrease of the mucin production in the jejunal mucosa of weaned pigs infected by porcine rotavirus (PRV). A total of 24 crossbred barrows weaned at 21 d of age were assigned randomly to 1 of 2 diets supplemented with 1.00% L-leucine or 0.68% L-alanine (isonitrogenous control) for 17 d. On day 11, all pigs were orally infused PRV or the sterile essential medium. During the first 10 d of trial, dietary leucine supplementation could improve the feed efficiency (P = 0.09). The ADG and feed efficiency were impaired by PRV infusion (P<0.05). PRV infusion also increased mean cumulative score of diarrhea, serum rotavirus antibody concentration and crypt depth of the jejunal mucosa (P<0.05), and decreased villus height: crypt depth (P = 0.07), goblet cell numbers (P<0.05), mucin 1 and 2 concentrations (P<0.05) and phosphorylated mTOR level (P<0.05) of the jejunal mucosa in weaned pigs. Dietary leucine supplementation could attenuate the effects of PRV infusion on feed efficiency (P = 0.09) and mean cumulative score of diarrhea (P = 0.09), and improve the effects of PRV infusion on villus height: crypt depth (P = 0.06), goblet cell numbers (P<0.05), mucin 1 (P = 0.08) and 2 (P = 0.07) concentrations and phosphorylated mTOR level (P = 0.08) of the jejunal mucosa in weaned pigs. These results suggest that dietary 1% leucine supplementation alleviated the decrease of mucin production and goblet cell numbers in the jejunal mucosa of weaned pigs challenged by PRV possibly via activation of the mTOR signaling.

  15. Recombinant expression and characterization of a L-amino acid oxidase from the fungus Rhizoctonia solani.

    PubMed

    Hahn, Katharina; Neumeister, Katrin; Mix, Andreas; Kottke, Tilman; Gröger, Harald; Fischer von Mollard, Gabriele

    2017-04-01

    L-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of L-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. L-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant L-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active L-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic L-amino acids as well as methyl esters of these L-amino acids. The progress of the conversion of L-phenylalanine and L-leucine into the corresponding α-keto acids was determined by HPLC and (1)H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50-100-fold by SDS treatment. K m values ranging from 0.9-10 mM and v max values from 3 to 10 U mg(-1) were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant L-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.

  16. Nutritional profile of phytococktail from trans-Himalayan plants.

    PubMed

    Dhar, Priyanka; Tayade, Amol B; Kumar, Jatinder; Chaurasia, Om P; Srivastava, Ravi B; Singh, Shashi B

    2013-01-01

    We estimated the nutritive value, vitamin content, amino acid composition, fatty acid content, and mineral profile of a phytococktail comprising sea buckthorn (Hippophae rhamnoides), apricot (Prunus armeniaca), and roseroot (Rhodiola imbricata) from trans-Himalaya. The free vitamin forms in the phytococktail were determined by rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS). Vitamin E and B-complex vitamins were detected as the principle vitamins. Reversed-phase high performance liquid chromatography (RP-HPLC) with pre-column derivatization was used for identification and quantification of amino acids. Eight essential and eleven non-essential amino acids were quantified, and the content ranged between 76.33 and 9485.67 µg/g. Among the essential amino acids, L-methionine, L-phenylalanine, L-lysine, L-leucine, and L-histidine were found to be the dominant contributors. We also quantified the fatty acids in the phytococktail by using gas chromatography coupled with a flame ionization detector (GC-FID) with fatty acid methyl esters (FAMEs) derivatization. The analysis revealed the presence of 4 major fatty acids contributing to the total lipid content. Palmitic acid was found to be the rich source of saturated fatty acid (SFA) and constituted ∼31% of the total lipid content. Among the unsaturated fatty acids (UFAs), palmitoleic acid (43.47%), oleic acid (20.89%), and linoleic acid (4.31%) were prominent. The mineral profiling was carried out by inductively coupled plasma optical emission spectrometer (ICP-OES), and it was found to contain a number of important dietary mineral elements. The harsh climatic conditions, difficult terrain, and logistic constraints at high altitude regions of Indian trans-Himalayan cold desert lead to the scarcity of fresh fruits and vegetables. Therefore, the source of multiple vitamins, essential amino acids, fatty acids, and dietary minerals from the phytococktail would provide great health benefit in the

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

    SciTech Connect

    Ledee, Dolena R.; Kajimoto, Masaki; O'Kelly-Priddy, Colleen M.; Olson, Aaron; Isern, Nancy G.; 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 closely examined the role of prolonged systemic pyruvate supplementation in modifying metabolic parameters during the unique conditions of ventricular unloading provided by ECMO. Twelve male mixed breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (Group C) or pyruvate (Group P) during ECMO for 8 hours. Over the final hour piglets received [2-13C] pyruvate, and [13C6]-L-leucine, as an indicator for oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of all measured CAC intermediates. Group P showed greater anaplerotic flux through pyruvate carboxylation although pyruvate oxidation relative to citrate synthase flux was similar to Group C. The groups demonstrated similar leucine fractional contributions to acetyl-CoA and fractional protein synthesis rates. Pyruvate also promoted an increase in the phosphorylation state of several nutrient sensitive enzymes, such as AMPK and ACC, and promoted O-GlcNAcylation through the hexosamine biosynthetic pathway (HBP). In conclusion, prolonged pyruvate supplementation during ECMO modified anaplerotic pyruvate flux and elicited changes in important nutrient and energy sensitive pathways, while preserving protein synthesis. Therefore, the observed results support the further study of nutritional supplementation and its downstream effects on cardiac adaptation during ventricular unloading.

  18. 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.

  19. Light-Activated Amino Acid Transport Systems in Halobacterium halobium Envelope Vesicles: Role of Chemical and Electrical Gradients

    NASA Technical Reports Server (NTRS)

    MacDonald, Russell E.; Greene, Richard V.; Lanyi, Janos K.

    1977-01-01

    The accumulation of 20 commonly occurring L-amino acids by cell envelope vesicles of Halobacterium halobium, in response to light-induced membrane potential and an artificially created sodium gradient, has been studied. Nineteen of these amino acids are actively accumulated under either or both of these conditions. Glutamate is unique in that its uptake is driven only by a chemical gradient for sodium. Amino acid concentrations at half-maximal uptake rates (Km) and maximal transport rates (V(sub max) have been determined for the uptake of all 19 amino acids. The transport systems have been partially characterized with respect to groups of amino acids transported by common carriers, cation effects, and relative response to the electrical and chemical components of the sodium gradient, the driving forces for uptake. The data presented clearly show that the carrier systems, which are responsible for uptake of individual amino acids, are as variable in their properties as those found in other organisms, i. e., some are highly specific for individual amino acids, some transport several amino acids competitively, some are activated by a chemical gradient of sodium only, and some function also in the complete absence of such a gradient. For all amino acids, Na(+) and K(+) are both required for maximal rate of uptake. The carriers for L-leucine and L-histidine are symmetrical in that these amino acids are transported in both directions across the vesicle membrane. It is suggested that coupling of substrate transport to metabolic energy via transient ionic gradients may be a general phenomenon in procaryotes.

  20. Proteasome inhibition induces hsp30 and hsp70 gene expression as well as the acquisition of thermotolerance in Xenopus laevis A6 cells

    PubMed Central

    Young, Jordan T. F.

    2009-01-01

    Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge. PMID:19838833

  1. Effects of single-walled carbon nanotubes on soil microorganisms

    NASA Astrophysics Data System (ADS)

    Jin, L.; Chung, H.; Son, Y.

    2011-12-01

    Single-walled carbon nanotubes (SWCNTs) are novel materials that have the potential to be used in various commercial fields due to their unique physicochemical properties. As a result of commercial development of nanotechnology, SWCNTs may be discharged to the soil environment with unknown consequences. However, there are as yet no data in the scientific literature that demonstrate the effects of SWCNTs on microbial function in soils. Therefore, we aimed to determine the effects of SWCNTs on soil microbial activity through a 2-week incubation study on urban soils supplemented with different concentrations of SWCNTs ranging from 0 to 1000 μg CNT/g soil. Fluorometric test using fluorogenic substrates were employed for the measurement of several enzyme activities in soil samples. More specifically, we determined the changes in the activities of cellobiohydrolase, β-1,4-glucosidase, β-1,4-xylosidase, β-1,4-N-acetylglucosaminidase, L-leucine aminopeptidase and acid phosphatase which play important roles in the carbon, nitrogen, and phosphorus cycles in response to the addition of SWCNTs. We found that microbial enzyme activities decreased as the concentrations of SWCNT added increased. The lowest enzyme activities were observed under 1000 μg CNT/g soil. The overall pattern shows that enzyme activities decreased slightly in the first 2-3 days and increased in the later stage of the incubation. Our results suggest that relatively high concentrations of SWCNTs can inhibit microbial activities, and this may be due to microbial cell membrane damage caused by SWCNTs. However, further study needs to be conducted to determine the mechanism responsible for inhibitory effect of SWCNTs on soil microbial activity. It can be concluded that changes in the activities of extracellular enzymes can indicate the effect of SWCNTs on soil microorganisms and nutrient cycling.

  2. In Vitro Evaluation of Inhalable Verapamil-Rifapentine Particles for Tuberculosis Therapy.

    PubMed

    Parumasivam, T; Chan, J G Y; Pang, A; Quan, D H; Triccas, J A; Britton, W J; Chan, H K

    2016-03-07

    Recent studies have demonstrated that efflux pumps of Mycobacterium tuberculosis (M. tb) provide a crucial mechanism in the development of drug resistant to antimycobacterial drugs. Drugs that inhibit these efflux pumps, such as verapamil, have shown the potential in enhancing the treatment success. We therefore hypothesized that the combined inhaled administration of verapamil and a first-line rifamycin antibiotic will further improve the treatment efficacy. An inhalable dry powder consisting of amorphous verapamil and crystalline rifapentine with l-leucine as an excipient was produced by spray drying. The in vitro aerosol characteristic of the powder, its microbiological activity and stability were assessed. When the powder was dispersed by an Osmohaler, the total fine particle fraction (FPFtotal, wt % of particles in aerosol <5 μm) of verapamil and rifapentine was 77.4 ± 1.1% and 71.5 ± 2.0%, respectively. The combination drug formulation showed a minimum inhibitory concentration (MIC90) similar to that of rifapentine alone when tested against both M. tb H37Ra and M. tb H37Rv strains. Importantly, the combination resulted in increased killing of M. tb H37Ra within the infected macrophage cells compared to either verapamil or rifapentine alone. In assessing cellular toxicity, the combination exhibited an acceptable half maximal inhibitory concentration (IC50) values (62.5 μg/mL) on both human monocytic (THP-1) and lung alveolar basal epithelial (A549) cell lines. Finally, the powder was stable after 3 months storage in 0% relative humidity at 20 ± 3 °C.

  3. Binding of cationic peptides (KX)4K to DPPG bilayers. Increasing the hydrophobicity of the uncharged amino acid X drives formation of membrane bound β-sheets: A DSC and FT-IR study.

    PubMed

    Hädicke, André; Blume, Alfred

    2016-06-01

    The binding of cationic peptides of the sequence (KX)4K to lipid vesicles of negatively charged dipalmitoyl-phosphatidylglycerol (DPPG) was investigated by differential scanning calorimetry (DSC) and temperature dependent Fourier-transformed infrared (FT-IR) spectroscopy. The hydrophobicity of the uncharged amino acid X was changed from G (glycine) over A (alanine), Abu (α-aminobutyric acid), V (valine) to L (leucine). The binding of the peptides caused an increase of the phase transition temperature (Tm) of DPPG by up to 20°C. The shift depended on the charge ratio and on the hydrophobicity of the amino acid X. Unexpectedly, the upward shift of Tm increased with increasing hydrophobicity of X. FT-IR spectroscopy showed a shift of the CH2 stretching vibrations of DPPG to lower frequency, particularly for bilayers in the liquid-crystalline phase, indicating an ordering of the hydrocarbon chains when the peptides were bound. Changes in the lipid C=O vibrational band indicated a dehydration of the lipid headgroup region after peptide binding. (KG)4K was bound in an unordered structure at all temperatures. All other peptides formed intermolecular antiparallel β-sheets, when bound to gel phase DPPG. However, for (KA)4K and (KAbu)4K, the β-sheets converted into an unordered structure above Tm. In contrast, the β-sheet structures of (KV)4K and (KL)4K remained stable even at 80°C when bound to the liquid-crystalline phase of DPPG. Strong aggregation of DPPG vesicles occurred after peptide binding. For the aggregates, we suggest a structure, where aggregated single β-sheets are sandwiched between opposing DPPG bilayers with a dehydrated interfacial region.

  4. [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.

  5. Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae).

    PubMed

    Fialho, Maria C Q; Moreira, Nathalia R; Zanuncio, José C; Ribeiro, Alberto F; Terra, Walter R; Serrão, José E

    2012-06-01

    Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 μM); CAL2 (17 kDa, pHo 5.5, km 11 μM Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM l-Leucine-p-nitroanilide), α-glucosidase (90 kDa, pHo 5.0, km 5mM with p-nitrophenyl α-d-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans.

  6. Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N.

    PubMed

    Bernkop-Schnürch, A; Zarti, H; Walker, G F

    2001-11-01

    The purpose of this study was to evaluate the potential of polycarbophil-cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 microM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP-Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP-Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 +/- 4 and 60 +/- 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP-Cys (0.25% w/v, pH 6) 11 +/- 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides.

  7. Pulmonary immunization of guinea pigs with diphtheria CRM-197 antigen as nanoparticle aggregate dry powders enhance local and systemic immune responses.

    PubMed

    Muttil, Pavan; Pulliam, Brian; Garcia-Contreras, Lucila; Fallon, John Kevin; Wang, Chenchen; Hickey, Anthony James; Edwards, David A

    2010-12-01

    This study establishes the immune response elicited in guinea pigs after pulmonary and parenteral immunizations with diphtheria CRM-197 antigen (CrmAg). Several spray-dried powders of formalin-treated/untreated CrmAg nanoaggregates with L-leucine were delivered to the lungs of guinea pigs. A control group consisting of alum with adsorbed CrmAg in saline was administered by intramuscular injection. Animals received three doses of powder vaccines containing 20 or 40 μg of CrmAg. The serum IgG titers were measured for 16 weeks after the initial immunization; IgA titers were measured at the time of sacrifice in the broncho-alveolar lavage fluid. Further, toxin neutralization tests in naïve guinea pigs were performed for a few select serum samples. Histopathology of the lung tissues was conducted to evaluate inflammation or injury to the lung tissues. While the highest titer of serum IgG antibody was observed in guinea pigs immunized by the intramuscular route, those animals immunized with dry powder formulation by the pulmonary route, and without the adjuvant alum, exhibited high IgA titers. A pulmonary administered dry powder, compared to parenteral immunization, conferred complete protection in the toxin neutralization test. Mild inflammation was observed in lung tissues of animals receiving dry powder vaccines by the pulmonary route. Thus, administering novel CrmAg as dry powders to the lungs may be able to overcome some of the disadvantages observed with the existing diphtheria vaccine which is administered by the parenteral route. In addition, these powders will have the advantage of eliciting a high mucosal immune response in the lungs without using traditional adjuvants.

  8. Β-alanine and l-histidine transport across the inner blood-retinal barrier: potential involvement in L-carnosine supply.

    PubMed

    Usui, Takuya; Kubo, Yoshiyuki; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi

    2013-08-01

    The supply of L-carnosine, a bioactive dipeptide of β-alanine and l-histidine, to the retina across the blood-retinal barrier (BRB) was studied. The in vivo and in vitro studies revealed low uptake activities for [(3)H]Gly-Sar, a representative dipeptide, suggesting that l-carnosine transport plays only a minor role at the BRB. The in vivo study using rats showed approximately 18- and 23-fold greater retinal uptake indexes (RUI) for [(3)H]β-alanine and [(3)H]l-histidine compared with that of a paracellular marker, respectively. The RUI of [(3)H]β-alanine was taurine- and γ-aminobutyric acid-sensitive, and the in vitro uptake by TR-iBRB2 cells showed time- concentration- and temperature-dependent [(3)H]β-alanine uptake, suggesting that a carrier-mediated process was involved in β-alanine transport across the inner BRB. [(3)H]β-Alanine uptake was inhibited by taurine and β-guanidinopropionic acid, suggesting that taurine transporter (TAUT/SLC6A6) is responsible for the influx transport of β-alanine across the inner BRB. Regarding l-histidine, the l-leucine-sensitive RUI of [(3)H]l-histidine was identified, and the in vitro [(3)H]l-histidine uptake by TR-iBRB2 cells suggested that a carrier-mediated process was involved in l-histidine transport across the inner BRB. The inhibition profile suggested that L-type amino acid transporter (LAT1/SLC7A5) is responsible for the influx transport of l-histidine across the inner BRB. These results show that the influx transports of β-alanine and l-histidine across the inner BRB is carried out by TAUT and LAT1, respectively, suggesting that the retinal l-carnosine is supplied by enzymatic synthesis from two kinds of amino acids transported across the inner BRB.

  9. The molecular mechanism of intestinal levodopa absorption and its possible implications for the treatment of Parkinson's disease.

    PubMed

    Camargo, Simone M R; Vuille-dit-Bille, Raphael N; Mariotta, Luca; Ramadan, Tamara; Huggel, Katja; Singer, Dustin; Götze, Oliver; Verrey, François

    2014-10-01

    Levodopa (L-DOPA) is the naturally occurring precursor amino acid for dopamine and the main therapeutic agent for neurologic disorders due to dopamine depletion, such as Parkinson's disease. l-DOPA absorption in small intestine has been suggested to be mediated by the large neutral amino acids transport machinery, but the identity of the involved transporters is unknown. Clinically, coadministration of l-DOPA and dietary amino acids is avoided to decrease competition for transport in intestine and at the blood-brain barrier. l-DOPA is routinely coadministered with levodopa metabolism inhibitors (dopa-decarboxylase and cathechol-O-methyl transferase inhibitors) that share structural similarity with levodopa. In this systematic study involving Xenopus laevis oocytes and Madin-Darby canine kidney epithelia expression systems and ex vivo preparations from wild-type and knockout mice, we identified the neutral and dibasic amino acids exchanger (antiporter) b(0,+)AT-rBAT (SLC7A9-SLC3A1) as the luminal intestinal l-DOPA transporter. The major luminal cotransporter (symporter) B(0)AT1 (SLC6A19) was not involved in levodopa transport. L-Leucine and L-arginine competed with levodopa across the luminal enterocyte membrane as expected for b(0,+)AT-rBAT substrates, whereas dopa-decarboxylase and cathechol-O-methyl transferase inhibitors had no effect. The presence of amino acids in the basolateral compartment mimicking the postprandial phase increased transepithelial levodopa transport by stimulating basolateral efflux via the antiporter LAT2-4F2 (SLC7A8-SLC3A2). Additionally, the aromatic amino acid uniporter TAT1 (SLC16A10) was shown to play a major role in l-DOPA efflux from intestinal enterocytes. These results identify the molecular mechanisms mediating small intestinal levodopa absorption and suggest strategies for optimization of delivery and absorption of this important prodrug.

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

    PubMed Central

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

    2015-01-01

    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

  11. Dry powder inhalation of macromolecules using novel PEG-co-polyester microparticle carriers.

    PubMed

    Tawfeek, Hesham M; Evans, Andrew R; Iftikhar, Abid; Mohammed, Afzal R; Shabir, Anjum; Somavarapu, Satyanarayana; Hutcheon, Gillian A; Saleem, Imran Y

    2013-01-30

    This study investigated optimizing the formulation parameters for encapsulation of a model mucinolytic enzyme, α-chymotrypsin (α-CH), within a novel polymer; poly(ethylene glycol)-co-poly(glycerol adipate-co-ω-pentadecalactone), PEG-co-(PGA-co-PDL) which were then applied to the formulation of DNase I. α-CH or DNase I loaded microparticles were prepared via spray drying from double emulsion (w(1)/o/w(2)) utilizing chloroform (CHF) as the organic solvent, L-leucine as a dispersibility enhancer and an internal aqueous phase (w(1)) containing PEG4500 or Pluronic(®) F-68 (PLF68). α-CH released from microparticles was investigated for bioactivity using the azocasein assay and the mucinolytic activity was assessed utilizing the degradation of mucin suspension assay. The chemical structure of PEG-co-(PGA-co-PDL) was characterized by (1)H NMR and FT-IR with both analyses confirming PEG incorporated into the polymer backbone, and any unreacted units removed. Optimum formulation α-CH-CHF/PLF68, 1% produced the highest bioactivity, enzyme encapsulation (20.08±3.91%), loading (22.31±4.34 μg/mg), FPF (fine particle fraction) (37.63±0.97%); FPD (fine particle dose) (179.88±9.43 μg), MMAD (mass median aerodynamic diameter) (2.95±1.61 μm), and the mucinolytic activity was equal to the native non-encapsulated enzyme up to 5h. DNase I-CHF/PLF68, 1% resulted in enzyme encapsulation (17.44±3.11%), loading (19.31±3.27 μg/mg) and activity (81.9±2.7%). The results indicate PEG-co-(PGA-co-PDL) can be considered as a potential biodegradable polymer carrier for dry powder inhalation of macromolecules for treatment of local pulmonary diseases.

  12. Human interleukin-1 beta induced stimulation of insulin release from rat pancreatic islets is accompanied by an increase in mitochondrial oxidative events.

    PubMed

    Eizirik, D L; Sandler, S

    1989-11-01

    Acute exposure of pancreatic islets to interleukin-1 beta results in an increase in insulin release, while an extension of the exposure time induces a functional suppression and eventually, destruction of the B-cells. We have recently suggested that the interleukin-1 beta induced inhibition of islet function is mediated through an impairment in oxidative metabolism. The aim of the current study was to investigate if the acute, stimulatory effects of interleukin-1 beta on islet function could also be related to changes in the substrate metabolism. For this purpose, rat islets were exposed for 90-120 min to 30 pmol/l human recombinant interleukin-1 beta (biological activity of 2.5 U/ml) and their function and metabolism characterized during this period. The cytokine did not increase insulin release in the presence of 1.7 or 5.5 mmol/l glucose but in both the presence of 16.7 mmol/l glucose or 10 mmol/l leucine + 2 mmol/l glutamine there was a 50% increase in insulin release. Interleukin-1 beta exposure increased the oxidation of D-[U-14C]glucose at 5.5 mmol/l glucose by 25% and at 16.7 mmol/l glucose by 60%. Carbohydrate and amino acid metabolism were further examined in the presence of D-[5-3H]glucose, D-[6-14C]glucose, [1-14C]pyruvate, L-[U-14C]glutamine, L-[U-14C]leucine and L-[1-14C]leucine. There was no difference between control islets and interleukin-1 beta exposed islets in terms of D-[5-3H]glucose utilization or [1-14C]pyruvate decarboxylation, but the oxidation of D-[6-14C]glucose was increased by 64% in the interleukin-1 beta exposed islets.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed Central

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

    2015-01-01

    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 [2-13C] pyruvate, as a reference substrate for oxidation, and [13C6]-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

  14. 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.

  15. Jejunal microvilli atrophy and reduced nutrient transport in rats with advanced liver cirrhosis: improvement by Insulin-like Growth Factor I

    PubMed Central

    Castilla-Cortázar, Inma; Pascual, María; Urdaneta, Elena; Pardo, Javier; Puche, Juan Enrique; Vivas, Bárbara; Díaz-Casares, Amelia; García, María; Díaz-Sánchez, Matías; Varela-Nieto, Isabel; Castilla, Alberto; González-Barón, Salvador

    2004-01-01

    Background Previous results have shown that in rats with non-ascitic cirrhosis there is an altered transport of sugars and amino acids associated with elongated microvilli. These alterations returned to normal with the administration of Insulin-Like Growth Factor-I (IGF-I). The aims of this study were to explore the evolution of these alterations and analyse the effect of IGF-I in rats with advanced cirrhosis and ascites. Thus, jejunal structure and nutrient transport (D-galactose, L-leucine, L-proline, L-glutamic acid and L-cystine) were studied in rats with ascitic cirrhosis. Methods Advanced cirrhosis was induced by CCl4 inhalation and Phenobarbital administration for 30 weeks. Cirrhotic animals were divided into two groups which received IGF-I or saline during two weeks. Control group was studied in parallel. Jejunal microvilli were studied by electron microscopy. Nutrient transport was assessed in brush border membrane vesicles using 14C or 35S-labelled subtracts in the three experimental groups. Results Intestinal active Na+-dependent transport was significantly reduced in untreated cirrhotic rats. Kinetic studies showed a decreased Vmax and a reduced affinity for sugar and four amino acids transporters (expressed as an increased Kt) in the brush border membrane vesicles from untreated cirrhotic rats as compared with controls. Both parameters were normalised in the IGF-I-treated cirrhotic group. Electron microscopy showed elongation and fusion of microvilli with degenerative membrane lesions and/or notable atrophy. Conclusions The initial microvilli elongation reported in non ascitic cirrhosis develops into atrophy in rats with advanced cirrhosis and nutrient transports (monosaccharides and amino acids) are progressively reduced. Both morphological and functional alterations improved significantly with low doses of IGF-I. PMID:15196310

  16. Degradation capability of the coastal environment adjacent to the Itata River in central Chile (36.5° S)

    NASA Astrophysics Data System (ADS)

    Pantoja, S.; Gutiérrez, M. H.; Ampuero, P.; Tejos, E.

    2011-08-01

    The response of the coastal ocean influenced by both river discharges and inputs of photosynthetically derived organic carbon product of upwelling, was evaluated by estimating rates of microbial hydrolysis of macromolecules with the goal of estimating the potential degradation capability of the coastal ecosystem off central Chile. Extracellular enzymatic activity (EEA) in seawater was dominated by aminopeptidase activity on substrate L-leucine-4-methyl-7-coumarinylamide (MCA-leu) (1.2 to 182 nmol l-1 h-1) followed by 4-methylumbelliferyl-ß-D-glucoside (MUF-glu) (0.08-61 nmol l-1 h-1) and 4-methylumbelliferyl-ß-D-cellobiose (MUF-cel) (0.15-7 nmol l-1 h-1), with the highest rates measured during spring-summer. In riverine waters, extracellular enzymatic hydrolysis remained within the range of 45 to 131 nmol l-1 h-1 for MCA-leu and ca. 20 nmol l-1 h-1 for glucosidic substrates, year-round. Contrary to the EEA observed for the marine water column, surface sediment extracellular enzymatic hydrolysis of MCA-leu (0.04 to 6.13 nmol g-1 dw h-1) was in the same order of magnitude as the rates observed for MUF-cel (0.004 to 5.1 nmol g-1 dw h-1) and MUF-glu (0.007 to 10.5 nmol g-1 dw h-1). Moreover, hydrolysis in sediments was characterized by higher rates during winter compared with spring-summer in the coastal and estuarine zone. The five years of data allowed us to evaluate the potential capability of microbial processing of organic carbon in the coastal area adjacent to the Itata river discharge where the increase in primary production in the productive seasons is accompanied by the increase in hydrolysis of macromolecules.

  17. Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis.

    PubMed

    Acosta, Daniel; Cancela, Martín; Piacenza, Lucia; Roche, Leda; Carmona, Carlos; Tort, José F

    2008-03-01

    Leucyl aminopeptidases (LAP) from different parasitic organisms are attracting attention as relevant players in parasite biology, and consequently being considered as candidates for drug and vaccine design. In fact, the highest protection level achieved in ruminant immunization by a native antigen was previously reported by us, using a purified LAP as immunogen in a sheep trial against fasciolosis. Here, we report the cloning of a full-length cDNA from adult F. hepatica encoding a member of the M17 family of LAP (FhLAP) and functional expression and characterization of the corresponding enzyme. FhLAP was closely related to Schistosoma LAPs, but interestingly distant from their mammalian host's homologues, and was expressed in all stages of the parasite life cycle. The recombinant enzyme, functionally expressed in Escherichia coli, showed a marked amidolytic preference against the synthetic aminopeptidase substrate l-leucine-7-amino-4-methylcoumarin (Leu-AMC) and was also active against Cys-AMC and Met-AMC. Both native and recombinant enzyme were stimulated by the addition of divalent cations predominantly Mn(2+), and strongly inhibited by bestatin and cysteine. Physico-chemical properties, localization by immunoelectron microscopy, MALDI-TOF analysis, and cross-reactivity of anti-rFhLAP immune serum demonstrated that the recombinant enzyme was identical to the previously purified gut-associated LAP from adult F. hepatica. Vaccination trials using rFhLAP for rabbit immunization showed a strong IgG response and a highly significant level of protection after experimental infection with F. hepatica metacercariae, confirming that FhLAP is a relevant candidate for vaccine development.

  18. A comparative study on the rectal aminopeptidase enzymatic activities of different species.

    PubMed

    Acartürk, F; Parlatan, Z I

    2003-03-01

    The aim of the present study was to compare the enzymatic activity of four different aminopeptidases (aminopeptidase N, leucine aminopeptidase, aminopeptidase A, aminopeptidase B) in rectal homogenates from different species: rabbit, rat, guinea-pig, sheep and human. Different substrates were used as the relative specific substrates for the determination of aminopeptidase enzymatic activity. For this purpose, 4-methoxy-2-naphthylamide of L-alanine for aminopeptidase N, 4-methoxy-2-naphthylamide of L-leucine for leucine aminopeptidase, 4-methoxy-2-naphthylamide of L-glutamic acid for aminopeptidase A and 4-methoxy-2-naphthylamide of L-arginine for aminopeptidase B were employed. The rectal aminopeptidase enzymatic activity was determined spectrofluorometrically. The inhibition of activity of aminopeptidase in the presence of bestatin and puromycin inhibitors was also investigated. The results showed the presence of aminopeptidase enzymatic activity in all rectal homogenates. Sheep and guinea-pig had the greatest aminopeptidase activity. The four aminopeptidase activities of rat and rabbit were not significantly different from each other. Human data was not evaluated statistically, due to insufficient sample. But the values of human data was close to those of the rabbit and rat values except for aminopeptidase A. Based on the data of the hydrolysis and inhibition of the 4-methoxy-2-naphthylamide substrates, it was rather difficult to determine the aminopeptidase type in the rectal homogenates of the species studied. It has been found that the aminopeptidase activities of rat and rabbit were not statistically different from each other and the human data were close to them.

  19. Kinetics and conformational stability studies of recombinant leucine aminopeptidase.

    PubMed

    Hernández-Moreno, Ana V; Villaseñor, Francisco; Medina-Rivero, Emilio; Pérez, Néstor O; Flores-Ortiz, Luis F; Saab-Rincón, Gloria; Luna-Bárcenas, Gabriel

    2014-03-01

    Leucine aminopeptidase from Vibrio proteolyticus is a broad specificity N-terminal aminopeptidase that is widely used in pharmaceutical processes where the removal of N-terminal residues in recombinant proteins is required. We previously reported the expression of a heterologous construction of the mature protein fused to a 6-histidine tag that presents a reasonable refolding rate for its use at industrial level. Here, we investigate this recombinant leucine aminopeptidase (rLAP) to explain the gain of activity observed when incubated at 37 °C after its production. Unfolding transitions of rLAP as a function of urea concentration were monitored by circular dichroism (CD) and fluorescence (FL) spectroscopy exhibiting single transitions by both techniques. Free energy change for unfolding measured by CD and FL spectroscopy are 2.8 ± 0.4 and 3.7 ± 0.4 kcal mol(-1), respectively. Thermal stability conformation of rLAP is 2.6 ± 0.1 and 6.1 kcal mol(-1) for CD and Nano-Differential Scanning Calorimetry (Nano-DSC), respectively. Enzyme activity was assessed with L-leucine-p-nitroanilide (L-pNA) as substrate. The catalytic efficiency was 3.87 ± 0.10 min(-1) μM(-1) at 37 °C and pH 8.0. Kinetic and conformation studies show differences between the enzyme native and rLAP; however rLAP is selective and specific to remove N-terminal groups from amino acids.

  20. RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs.

    PubMed

    Rinaldi, Gabriel; Morales, Maria E; Alrefaei, Yousef N; Cancela, Martín; Castillo, Estela; Dalton, John P; Tort, José F; Brindley, Paul J

    2009-10-01

    Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the S. mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP1 and SmLAP2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate l-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by >or=80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases.

  1. In Situ Ratiometric Quantitative Tracing of Intracellular Leucine Aminopeptidase Activity via an Activatable Near-Infrared Fluorescent Probe.

    PubMed

    Gu, Kaizhi; Liu, Yajing; Guo, Zhiqian; Lian, Cheng; Yan, Chenxu; Shi, Ping; Tian, He; Zhu, Wei-Hong

    2016-10-03

    Leucine aminopeptidase (LAP), one of the important proteolytic enzymes, is intertwined with the progress of many pathological disorders as a well-defined biomarker. To explore fluorescent aminopeptidase probe for quantitative detection of LAP distribution and dynamic changes, herein we report a LAP-targeting near-infrared (NIR) fluorescent probe (DCM-Leu) for ratiometric quantitative trapping of LAP activity in different kinds of living cells. DCM-Leu is composed of a NIR-emitting fluorophore (DCM) as a reporter and l-leucine as a triggered moiety, which are linked together by an amide bond specific for LAP cleavage. High contrast on the ratiometric NIR fluorescence signal can be achieved in response to LAP activity, thus enabling quantification of endogenous LAP with "build-in calibration" as well as minimal background interference. Its ratiometric NIR signal can be blocked in a dose-dependent manner by bestatin, an LAP inhibitor, indicating that the alteration of endogenous LAP activity results in these obviously fluorescent signal responses. It is worth noting that DCM-Leu features striking characteristics such as a large Stokes shift (∼205 nm), superior selectivity, and strong photostability responding to LAP. Impressively, not only did we successfully exemplify DCM-Leu in situ ratiometric trapping and quantification of endogenous LAP activity in various types of living cells, but also, with the aid of three-dimensional confocal imaging, the intracellular LAP distribution is clearly observed from different perspectives for the first time, owing to the high signal-to-noise of ratiometric NIR fluorescent response. Collectively, these results demonstrate preclinical potential value of DCM-Leu serving as a useful NIR fluorescent probe for early detection of LAP-associated disease and screening inhibitor.

  2. Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: the two YxxL segments within a single CD3zeta-ITAM are functionally distinct.

    PubMed

    Sunder-Plassmann, R; Lialios, F; Madsen, M; Koyasu, S; Reinherz, E L

    1997-08-01

    Functional analysis of the immunoreceptor tyrosine-based activation motif (ITAM) derived from the membrane-proximal ITAM of CD3zeta demonstrates that mutations at either the tyrosine or leucine residues in the N-terminal YxxL segment of the ITAM abolish all signal transduction functions of this ITAM. In contrast, mutations at the tyrosine or leucine residues in the C-terminal YxxL segment abrogate signals for interleukin (IL)-2 production but do not prevent tyrosine phosphorylation of the N-terminal tyrosine of the ITAM, lck association with the ITAM, activation of phospholipase C-gamma1 or calcium mobilization. Cross-linking of chimeric receptors containing a C-terminal YxxL leucine mutation induces tyrosine phosphorylation of ZAP70 but without stable binding to the phosphorylated ITAM. These results indicate that the two YxxL segments in an ITAM are functionally distinct and that both are essential for ZAP70 binding and IL-2 production. Furthermore, tyrosine phosphorylation of ZAP70 per se is not sufficient to trigger the downstream events leading to IL-2 production. Substitution of an alanine for the bulky side chain at the Y+1 position of the N-terminal YxxL segment reduces the receptor cross-linking requirement necessary to achieve cellular activation and the absolute dependence on lck in this process. Our results reveal that both the number of ITAM as well as the specific amino acid residues within a single ITAM determine the extent of chimeric receptor cross-linking required to trigger tyrosine phosphorylation-dependent signaling events.

  3. Biochar affects soil organic matter cycling and microbial functions but does not alter microbial community structure in a paddy soil.

    PubMed

    Tian, Jing; Wang, Jingyuan; Dippold, Michaela; Gao, Yang; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2016-06-15

    The application of biochar (BC) in conjunction with mineral fertilizers is one of the most promising management practices recommended to improve soil quality. However, the interactive mechanisms of BC and mineral fertilizer addition affecting microbial communities and functions associated with soil organic matter (SOM) cycling are poorly understood. We investigated the SOM in physical and chemical fractions, microbial community structure (using phospholipid fatty acid analysis, PLFA) and functions (by analyzing enzymes involved in C and N cycling and Biolog) in a 6-year field experiment with BC and NPK amendment. BC application increased total soil C and particulate organic C for 47.4-50.4% and 63.7-74.6%, respectively. The effects of BC on the microbial community and C-cycling enzymes were dependent on fertilization. Addition of BC alone did not change the microbial community compared with the control, but altered the microbial community structure in conjunction with NPK fertilization. SOM fractions accounted for 55% of the variance in the PLFA-related microbial community structure. The particulate organic N explained the largest variation in the microbial community structure. Microbial metabolic activity strongly increased after BC addition, particularly the utilization of amino acids and amines due to an increase in the activity of proteolytic (l-leucine aminopeptidase) enzymes. These results indicate that microorganisms start to mine N from the SOM to compensate for high C:N ratios after BC application, which consequently accelerate cycling of stable N. Concluding, BC in combination with NPK fertilizer application strongly affected microbial community composition and functions, which consequently influenced SOM cycling.

  4. A highly sensitive and simply operated protease sensor toward point-of-care testing.

    PubMed

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

    2016-04-21

    Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.

  5. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

    SciTech Connect

    Kamolrat, Torkamol; Gray, Stuart R.

    2013-03-22

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using {sup 3}H-labelled phenylalanine. Protein breakdown was measured using {sup 3}H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion.

  6. 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 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/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.

  7. Inhalable Ipratropium Bromide Particle Engineering with Multicriteria Optimization.

    PubMed

    Vinjamuri, Bhavani Prasad; Haware, Rahul V; Stagner, William C

    2016-11-21

    Spray-dried ipratropium bromide (IPB) microspheres for oral inhalation were engineered using Quality by Design. The interrogation of material properties, process parameters, and critical product quality attributes interplay enabled rational product design. A 2(7-3) screening design exhibited the Maillard reaction between L-leucine (LL) and lactose at studied outlet temperatures (OT) >130°C. A response surface custom design was used in conjunction with multicriteria optimization to determine the operating design space to achieve inhalable microparticles. Statistically significant predictive models were developed for volume median diameter (p = 0.0001, adjusted R (2)  = 0.9938), span (p = 0.0278, adjusted R (2)  = 0.7912), yield (p = 0.0020, adjusted R (2)  = 0.9320), and OT (p = 0.0082, adjusted R (2)  = 0.8768). An independent verification batch confirmed the model's predictive capability. The prediction and actual values were in good agreement. Particle size and span were 3.32 ± 0.09 μm and 1.71 ± 0.18, which were 4.7 and 5.3% higher than the predicted values. The process yield was 50.3%, compared to the predicted value of 65.3%. The OT was 100°C versus the predicted value of 105°C. The label strength of IPB microparticles was 99.0 to 105.9% w/w suggesting that enrichment occurred during the spray-drying process. The present study can be utilized to initiate the design of the first commercial IPB dry powder inhaler.

  8. Molecular Imaging of Urogenital Diseases

    PubMed Central

    Cho, Steve Y.; Szabo, Zsolt; Morgan, Russell H.

    2013-01-01

    There is an expanding and exciting repertoire of PET imaging radiotracers for urogenital diseases, particularly in prostate cancer, renal cell cancer, and renal function. Prostate cancer is the most commonly diagnosed cancer in men. With growing therapeutics options for the treatment of metastatic and advanced prostate cancer, improved functional imaging of prostate cancer beyond the limitations of conventional computed tomography (CT) and bone scan (BS) is becoming increasingly important for both clinical management and drug development. PET radiotracers beyond 18F-Fluorodeoxyglucose (FDG) for prostate cancer include 18F-Sodium Fluoride, 11C-Choline and 18F-Fluorocholine and 11C-Acetate. Other emerging and promising PET radiotracers include a synthetic L-leucine amino acid analog (anti-18F-FACBC), dihydrotestosterone analog (18F-FDHT) and prostate specific membrane antigen (PSMA) based PET radiotracers (ex. 18F-DCFBC, 89Zr-DFO-J591, 68Ga(HBED-CC)). Larger prospective and comparison trials of these PET radiotracers are needed to establish the role of PET/CT in prostate cancer. Renal cell cancer imaging with FDG PET/CT although available can be limited, especially for detection of the primary tumor. Improved renal cell cancer detection with carbonic anhydrase IX (CAIX) based antibody (124I-girentuximab) and radioimmunotherapy targeting with 177Lu-cG250 appear promising. Evaluation of renal injury by imaging renal perfusion and function with novel PET radiotracers include p-18F-fluorohippurate (18F-PFH) and hippurate m-cyano-p-18F-fluorohippurate (18F-CNPFH) and Rubidium-82 chloride (typically used for myocardial perfusion imaging). Renal receptor imaging of the renal renin angiotensin system with a variety of selective PET radioligands are also becoming available for clinical translation. PMID:24484747

  9. Transpulmonary flux of S-nitrosothiols and pulmonary vasodilation during nitric oxide inhalation: role of transport.

    PubMed

    Torok, Jordan A; Brahmajothi, Mulugu V; Zhu, Hongmei; Tinch, Brian T; Auten, Richard L; McMahon, Timothy J

    2012-07-01

    Inhaled nitric oxide (iNO) is used to treat pulmonary hypertension and is being investigated for prevention of bronchopulmonary dysplasia in neonates. Extrapulmonary effects of iNO are widely recognized, but the underlying chemistry and pharmacology are poorly understood. Growing evidence suggests that, in addition to acting via diffusion, NO can be converted into nitrosants capable of reacting with endogenous L-cysteine (L-Cys) in the alveolar lining fluid, forming S-nitrosothiol (SNO)-L-cysteine (CSNO). CSNO can then enter cells via the type L amino acid transporter (LAT). To determine the influence of LAT and supplemental L-Cys on the functional activity of iNO and transpulmonary movement of SNOs or other related species, we exposed C57Bl6 mice to nebulized L-Cys or D-cysteine (D-Cys) and/or LAT competitors. Isolated lungs were then perfused with physiologic buffer while effluent was collected to assay perfusate SNOs. Nebulized L-Cys, but not D-Cys, augmented the iNO-induced increase in circulating SNOs in the effluent without altering iNO-induced pulmonary vasodilation. Addition to the perfusate of either L-leucine (L-Leu) or 2-amino-2-norborane carboxylic acid, two distinct LAT competitors, inhibited appearance in the perfusate of SNOs in L-Cys-exposed lungs; a higher concentration of L-Leu significantly inhibited the iNO-induced pulmonary vasodilation as well as SNO accumulation. We conclude that iNO-induced pulmonary vasodilation and the transpulmonary movement of iNO-derived SNOs are mediated in part by formation of extracellular CSNO, uptake by alveolar epithelial LAT, and/or export by LAT from the pulmonary endothelium into the circulation. Therapies that exploit and optimize LAT-dependent SNO transport might improve the efficacy of and clinical outcomes with NO-based therapy by improving systemic SNO delivery.

  10. Leptin and leucine synergistically regulate protein metabolism in C2C12 myotubes and mouse skeletal muscles.

    PubMed

    Mao, Xiangbing; Zeng, Xiangfang; Huang, Zhimin; Wang, Junjun; Qiao, Shiyan

    2013-07-28

    Leucine and leptin play important roles in regulating protein synthesis and degradation in skeletal muscles in vitro and in vivo. However, the objective of the present study was to determine whether leptin and leucine function synergistically in regulating protein metabolism of skeletal muscles. In the in vitro experiment, C2C12 myotubes were cultured for 2 h in the presence of 5 mm-leucine and/or 50 ng/ml of leptin. In the in vivo experiment, C57BL/6 and ob/ob mice were randomly assigned to be fed a non-purified diet supplemented with 3 % L-leucine or 2·04 % L-alanine (isonitrogenous control) for 14 d. Ob/ob mice were injected intraperitoneally with sterile PBS or recombinant mouse leptin (0·1 μg/g body weight) for 14 d. In C57BL/6 mice, dietary leucine supplementation increased (P< 0·05) plasma leptin, leptin receptor expression and protein synthesis in skeletal muscles, but reduced (P< 0·05) plasma urea and protein degradation in skeletal muscles. Dietary leucine supplementation and leptin injection increased the relative weight of the gastrocnemius and soleus muscles in ob/ob mice. Moreover, leucine and leptin treatments stimulated (P< 0·05) protein synthesis and inhibited (P< 0·05) protein degradation in C2C12 myotubes and skeletal muscles of ob/ob mice. There were interactions (P< 0·05) between the leucine and leptin treatments with regard to protein metabolism in C2C12 myotubes and soleus muscles of ob/ob mice but not in the gastrocnemius muscles of ob/ob mice. Collectively, these results suggest that leptin and leucine synergistically regulate protein metabolism in skeletal muscles both in vitro and in vivo.

  11. 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-β-lactone structure with an N-formyl-l-leucine group attached as an ester to the 5-hydroxy group. It has been suggested that the α-branched 3,5-dihydroxy fatty acid β-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 β-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 α-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 α-branched 3,5-dihydroxy fatty acid chain with an N-formylated leucine residue. Finally, the 3β-hydroxysteroid dehydrogenase-homologous gene lstD might be responsible for the reduction of the β-keto group of the biosynthetic intermediate, thereby facilitating the formation of the unique β-lactone ring. PMID:25239907

  12. a Single Crystal Study of Deuterons in a Dipeptide, and, Intercavity Dynamics of Xenon in Zeolite a.

    NASA Astrophysics Data System (ADS)

    Gerald, Rex Ervin, II

    In part I of this thesis, the goal is to determine for the first time the complete chemical shift (CS) tensor for an amide proton in the biologically important model peptide N-Acetyl-D, L-valine (NAV). The method of single crystal deuterium nuclear magnetic resonance (NMR) is used. By measuring deuterium NMR spectra for different orientations of a single crystal of NAV in an external magnetic field we are able to extract both an electric field gradient (EFG) tensor and a chemical shift tensor for the amide deuteron site. Both tensors are electronic molecular properties and intimately connected. The EFG tensor is easily measured and its connection to chemical bonding allows us to remove the ambiguity of assigning the amide CS tensor to one of the two chemically equivalent but magnetically inequivalent amide deuteron sites. A knowledge of amide shielding tensors has implications in NMR relaxation measurements and solid -state structure elucidation of important bio-molecules. The EFG and CS tensors for the carboxyl proton in NAV were also determined, and the results together with the amide proton findings were compared to ab initio calculations in the literature, and correlations with hydrogen bonding discussed. In Part II of this thesis the first systematic study of xenon occluded in zeolite type A is accomplished. The distributions and chemical shifts of xenon clusters of one to eight xenon atoms trapped in the alpha -cavities of NaA are measured at two different temperatures. Also, the temperature dependence of the chemical shifts for all the clusters is recorded. The measured chemical shifts and distributions are used to predict the chemical shift of the observed ^{129}Xe NMR resonances in zeolite CaA, thereby providing a paradigm for the qualitative understanding of the observed ^{129}Xe chemical shifts in medium pore zeolites. Finally, a second paradigm which describes the effect of the chemical shift anisotropy and xenon mobility on the observed ^{129}Xe NMR

  13. Global Regulation of Food Supply by Pseudomonas putida DOT-T1E▿ †

    PubMed Central

    Daniels, Craig; Godoy, Patricia; Duque, Estrella; Molina-Henares, M. Antonia; de la Torre, Jesús; del Arco, José María; Herrera, Carmen; Segura, Ana; Guazzaroni, M. Eugenia; Ferrer, Manuel; Ramos, Juan Luis

    2010-01-01

    Pseudomonas putida DOT-T1E was used as a model to develop a “phenomics” platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Δcrc, Δcrp, ΔcyoB, and ΔptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d-fructose is influenced by CyoB, and l-arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine

  14. Use of Raman Spectroscopy and Phase-Contrast Microscopy To Characterize Cold Atmospheric Plasma Inactivation of Individual Bacterial Spores

    PubMed Central

    Wang, Shiwei; Doona, Christopher J.; Setlow, Peter

    2016-01-01

    ABSTRACT Raman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and nonnutrient germination of multiple individual Bacillus subtilis spores treated with cold atmospheric plasma (CAP). Major results for this work include the following: (i) >5 logs of spores deposited on glass surfaces were inactivated by CAP treatment for 3 min, while deposited spores placed inside an impermeable plastic bag were inactivated only ∼2 logs in 30 min; (ii) >80% of the spores treated for 1 to 3 min with CAP were nonculturable and retained CaDPA in their core, while >95% of spores treated with CAP for 5 to 10 min lost all CaDPA; (iii) Raman measurements of individual CAP-treated spores without CaDPA showed differences from spores that germinated with l-valine in terms of nucleic acids, lipids, and proteins; and (iv) 1 to 2 min of CAP treatment killed 99% of spores, but these spores still germinated with nutrients or exogenous CaDPA, albeit more slowly and to a lesser extent than untreated spores, while spores CAP treated for >3 min that retained CaDPA did not germinate via nutrients or CaDPA. However, even after 1 to 3 min of CAP treatment, spores germinated normally with dodecylamine. These results suggest that exposure to the present CAP configuration severely damages a spore's inner membrane and key germination proteins, such that the treated spores either lose CaDPA or can neither initiate nor complete germination with nutrients or CaDPA. Analysis of the various CAP components indicated that UV photons contributed minimally to spore inactivation, while charged particles and reactive oxygen species contributed significantly. IMPORTANCE Much research has shown that cold atmospheric plasma (CAP) is a promising tool for the inactivation of spores in the medical and food industries. However, knowledge about the effects of plasma treatment on spore properties is limited, especially at the single-cell level. In this

  15. New Insights into Amino Acid Preservation in the Early Oceans using Modern Analytical Techniques

    NASA Astrophysics Data System (ADS)

    Parker, E. T.; Brinton, K. L.; Burton, A. S.; Glavin, D. P.; Dworkin, J. P.; Bada, J.

    2015-12-01

    Protein- and non-protein-amino acids likely occupied the oceans at the time of the origin and evolution of life. Primordial soup-, hydrothermal vent-, and meteoritic-processes likely contributed to this early chemical inventory. Prebiotic synthesis and carbonaceous meteorite studies suggest that non-protein amino acids were likely more abundant than their protein-counterparts. Amino acid preservation before abiotic and biotic destruction is key to biomarker availability in paleoenvironments and remains an important uncertainty. To constrain primitive amino acid lifetimes, a 1992 archived seawater/beach sand mixture was spiked with D,L-alanine, D,L-valine (Val), α-aminoisobutyric acid (α-AIB), D,L-isovaline (Iva), and glycine (Gly). Analysis by high performance liquid chromatography with fluorescence detection (HPLC-FD) showed that only D-Val and non-protein amino acids were abundant after 2250 days. The mixture was re-analyzed in 2012 using HPLC-FD and a triple quadrupole mass spectrometer (QqQ-MS). The analytical results 20 years after the inception of the experiment were strikingly similar to those after 2250 days. To confirm that viable microorganisms were still present, the mixture was re-spiked with Gly in 2012. Aliquots were collected immediately after spiking, and at 5- and 9-month intervals thereafter. Final HPLC-FD/QqQ-MS analyses were performed in 2014. The 2014 analyses revealed that only α-AIB, D,L-Iva, and D-Val remained abundant. The disappearance of Gly indicated that microorganisms still lived in the mixture and were capable of consuming protein amino acids. These findings demonstrate that non-protein amino acids are minimally impacted by biological degradation and thus have very long