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Conversion of ammonia or urea into essential amino acids, L -leucine, L -valine, and L -isoleucine using artificial cells containing an immobilized multienzyme system and dextran-NAD +  

Microsoft Academic Search

A multienzyme system consisting of leucine dehydrogenase (EC, L-lactic dehydrogenase (EC, urease (EC,\\u000a and dextran-NAD+ was microencapsulated within artificial cells. This system could convert ammonia and urea into essential amino acids,L-leucine,L-valine, andL-isoleucine.L-lactate acted as a cosubstrate for the regeneration of dextran-NADH. Greater concentrations of L-lactate favored the higher\\u000a conversion ratios. The effects of ammonium salts and urea

Kang Fu Gu; Thomas Ming Swi Chang



Diagnosis of maple syrup urine disease by determination of l-valine, l-isoleucine, l-leucine and l-phenylalanine in neonatal blood spots by gas chromatography–mass spectrometry  

Microsoft Academic Search

A novel method was developed for the diagnosis of maple syrup urine disease (MSUD) by the determination of l-valine, l-leucine, l-isoleucine and l-phenylalanine in dried blood spots of newborns by gas chromatography–mass spectrometry (GC–MS). The four amino acids were extracted from blood samples by methanol and derivatized by n-butanol and trifluroacetic anhydride under optimum reaction conditions. The corresponding single derivatives

Chunhui Deng; Yonghui Deng



Diagnosis of maple syrup urine disease by determination of L-valine, L-isoleucine, L-leucine and L-phenylalanine in neonatal blood spots by gas chromatography-mass spectrometry.  


A novel method was developed for the diagnosis of maple syrup urine disease (MSUD) by the determination of L-valine, L-leucine, L-isoleucine and L-phenylalanine in dried blood spots of newborns by gas chromatography-mass spectrometry (GC-MS). The four amino acids were extracted from blood samples by methanol and derivatized by n-butanol and trifluroacetic anhydride under optimum reaction conditions. The corresponding single derivatives of the four amino acids were obtained under the optimum conditions. Their contents in blood samples were analyzed quantitatively by measurement of their derivatives by GC-MS in selected ion monitoring mode. MSUD can be diagnosed on the basis of the ratio of the total content of L-leucine and L-isoleucine to that of L-phenylalanine. The present method only took a short time to perform and required minimal sample preparation, which provided low detection limits and a relative standard deviation of less than 5.0%. The derivatization reactions of the four amino acids, L-valine, L-isoleucine, L-leucine and L-phenylalanine, with n-butanol and trifluroacetic anhydride were investigated and the optimum reaction conditions, including reaction time and temperature, were obtained and used for the determination of the amino acids in plasma samples. PMID:12860033

Deng, Chunhui; Deng, Yonghui



Influence of l -isoleucine and pantothenate auxotrophy for l -valine formation in Corynebacterium glutamicum revisited by metabolome analyses  

Microsoft Academic Search

The effect of different amounts of supplemented l-isoleucine and pantothenate has been analysed with the auxotrophic strain Corynebacterium glutamicum\\u000a ?ilvA\\u000a ?panB, showing that the final biomass concentration of this preliminary l-valine production strain can be controlled by the amount of added l-isoleucine. One gramme cell dry weight is formed from 48 ?mol l-isoleucine. Different amounts of available pantothenate affect the intracellular

Tobias Bartek; Pia Makus; Bianca Klein; Siegmund Lang; Marco Oldiges



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

Microsoft Academic Search

The 400-MHz ¹H 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

Daisuke Kohda; Gota Kawai; Shigeyuki Yokoyama; Makoto Kawakami; Shoji Mizushima; Tatsuo Miyazawa



Chemo-enzymatic synthesis of isotopically labelled L-Valine, L-Isoleucine and allo-isoleucine  

Microsoft Academic Search

Syntheses of (3R)-[4,4,4-D3]-L-valine, [15N]-L-isoleucine and [15N]-allo-isoleucine from homochiral 2-alkylated carboxylic acids are described. The approach involves a one-carbon homologation of the carboxylic acid to give the corrresponding ?-substituted ?-keto ester which is converted directly to the ?-amino acid in a one-pot procedure involving two enzyme catalysed reactions (Candida cylindracea lipase to hydroluse the ester and leucine dehydrogenase to catalyse the

Nicholas M. Kelly; R. Gordon Reid; Christine L. Wellis; Peter L. Winton



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


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

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



Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis  

Microsoft Academic Search

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain a-keto acids which are precursors of

Norbert Obermeier; Karl Poralla



Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation.  


The previously constructed strain Corynebacterium glutamicumilvNM13 with acetohydroxy acid synthase, resistant to inhibition by all three branched-chain amino acids (L-valine, L-isoleucine and L-leucine), was used as a basis to develop a new type of valine producer by genetic engineering. The main strategy was to modulate expression of the genes involved in the biosynthesis of branched-chain amino acids. The activity of the promoters P-ilvD (dihydroxyacid dehydratase) and P-ilvE (transaminase) was up-modulated and the activity of the promoters P-ilvA (threonine deaminase) and P-leuA (isopropylmalate synthase) was down-modulated by site-directed mutagenesis. A constructed weak promoter of ilvA (or leuA), which was introduced into the C. glutamicum chromosome via a gene-replacement technique reduced the biosynthetic rate of isoleucine (or leucine), which lowered the mutant growth rate and increased valine production. Overexpression of ilvD and ilvE driven by the strong mutant promoters P-ilvDM7 and P-ilvEM6 resulted in an even higher level of valine production. Thus, the strain C. glutamicum ilvNM13 DeltapanB P-ilvAM1CG P-ilvDM7 P-ilvEM6, having all mutations constructed within the chromosome, produced 136 mM valine in a 48-h cultivation. PMID:19121344

Holátko, Jirí; Elisáková, Veronika; Prouza, Marek; Sobotka, Miroslav; Nesvera, Jan; Pátek, Miroslav



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

PubMed Central

Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of Propionibacterium freudenreichii, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of l-leucine, ?-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced ?-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal nor?-hydroxyisocaproic acid was detected. l-Isoleucine and l-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24°C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening.

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



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


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

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



Molecular Structure of L-Isoleucine  

NSDL National Science Digital Library

L-Isoleucine is an essential, branched-chain, aliphatic amino acid that is found in many proteins. It is an important compound for hemoglobin synthesis and regulates energy and blood sugar levels. Together with leucine and valine, it metabolizes in muscle tissue and promotes muscle recovery, wound healing, including the growth of new tissue and increases growth hormone production. Only the L-form occurs in mammalian protein.



A novel l-isoleucine-4?-dioxygenase and l-isoleucine dihydroxylation cascade in Pantoea ananatis  

PubMed Central

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.

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



(L)-Valine production with minimization of by-products' synthesis in Corynebacterium glutamicum and Brevibacterium flavum.  


Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for L-valine production by over-expressing ilvEBN ( r ) C genes at 31 °C in 72 h fermentation. Different strategies were carried out to reduce the by-products' accumulation in L-valine fermentation and also to increase the availability of precursor for L-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of L-isoleucine. Effect of different relative dissolved oxygen on L-valine production and by-products' formation was recorded, indicating that 15 % saturation may be the most appropriate relative dissolved oxygen for L-valine fermentation with almost no L-lactic acid and L-glutamate formed. To minimize L-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of L-alanine accumulated by alaT inactivated strains harboring ilvEBN ( r ) C genes, L-alanine concentration was reduced to 0.18 g/L by C. glutamicum ATCC13032MPilvA?avtA pDXW-8-ilvEBN ( r ) C, and 0.22 g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. Meanwhile, L-valine production and conversion efficiency were enhanced to 31.15 g/L and 0.173 g/g by C. glutamicum ATCC13032MPilvA?avtA pDXW-8-ilvEBN ( r ) C, 38.82 g/L and 0.252 g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. This study provides combined strategies to improve L-valine yield by minimization of by-products' production. PMID:22552525

Hou, Xiaohu; Chen, Xinde; Zhang, Yue; Qian, He; Zhang, Weiguo



Construction of l-Isoleucine Overproducing Strains of Corynebacterium glutamicum  

NASA Astrophysics Data System (ADS)

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

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


Rational design of Escherichia coli for L-isoleucine production.  


Metabolic engineering of Escherichia coli was performed to construct a 100% rationally engineered strain capable of overproducing L-isoleucine, an important branched-chain amino acid. The thrABC (encoding L-threonine biosynthetic enzymes), ilvA (encoding feedback-resistant threonine dehydratase), ilvIH (encoding feedback-resistant acetohydroxy acid synthase III), and ygaZH (encoding branched-chain amino acid exporter) genes were amplified by plasmid-based overexpression. The ilvCED (encoding L-isoleucine biosynthetic enzymes) and lrp (encoding global regulator Lrp) genes were also amplified by chromosomal promoter replacement in order to further increase the flux toward L-isoleucine. The final engineered E. coli strain was able to produce 9.46 g/L of L-isoleucine with a yield of 0.14 g/g of glucose by fed-batch culture. The overall design principles described here for the production of highly regulated product should be useful in designing strains for the production of other similar bioproducts. PMID:23656230

Park, Jin Hwan; Oh, Jae Eun; Lee, Kwang Ho; Kim, Ji Young; Lee, Sang Yup



L-leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/?-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst.  


L-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/?-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of L-leucine and L-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of L-methionine and L-ethionine in the same manner as previously described L-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids. PMID:22584432

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



Synthesis, structure, DNA binding and oxidative cleavage activity of ternary ( l-leucine\\/isoleucine) copper(II) complexes of heterocyclic bases  

Microsoft Academic Search

Six ternary ?-amino acid copper(II) complexes of the general formula [Cu(AA)(B)(H2O)](X) (1–6), where AA is l-leu=l-leucine (1–3) or l-ile=l-isoleucine (4–6), B is a N,N-donor heterocyclic base, viz. 2,2?-bipyridine (bpy, 1, 4), 1,10-phenanthroline (phen, 2, 5) and dipyrido[3,2:2?,3?-f]quinoxaline (dpq, 3, 6) and X=ClO4-\\/NO3- have been synthesized, characterized, and their DNA binding and cleavage activity studied. The bpy and dpq complexes of

Ramakrishna Rao; Ashis K. Patra; P. R. Chetana



Construction of an L-isoleucine overproducing strain of Escherichia coli K-12.  


The genes for a threonine deaminase that is resistant to feedback inhibition by L-isoleucine and for an active acetohydroxyacid synthase II were introduced by a plasmid into a L-threonine-producing recombinant strain of Escherichia coli K-12. Analysis of culture broth of the strain using 13C nuclear magnetic resonance suggested that alpha, beta-dihydroxy-beta-methylvalerate (DHMV) and alpha-keto-beta-methylvalerate (KMV), the third and the fourth intermediates in the L-isoleucine biosynthetic pathway from L-threonine, respectively, accumulated in the medium in amounts comparable to that of L-isoleucine. The ratio of accumulated L-isoleucine:DHMV:KMV were approximately 2:1:1. The concentration of accumulated L-isoleucine increased by twofold after the additional introduction of the genes for dihyroxyacid dehydratase (DH) and transaminase-B (TA-B), and the intermediates no longer accumulated. The resultant strain TVD5 accumulated 10 g/l of L-isoleucine from 40 g/l of glucose. PMID:10361680

Hashiguchi, K; Takesada, H; Suzuki, E; Matsui, H



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


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

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



Possibility of wound dressing using poly( l-leucine)\\/poly(ethylene glycol)\\/poly( l-leucine) triblock copolymer  

Microsoft Academic Search

ABA-type block copolymers (abbreviated as LEL) composed of poly(l-leucine) (PLL) as the A component and poly(ethylene glycol) (PEG) as the B component were synthesized by ring-opening polymerization of l-leucine N-carboxyanhydride initiated by primary amino group located at both ends of PEG chain. A silver sulfadiazine (AgSD)-impregnated wound dressing of sponge type was prepared by the lyophilization method. Morphological structure of

Hyun-Jung Kim; Eun-Young Choi; Jong-Suk Oh; Hyun-Chul Lee; Sung-Sik Park; Chong-Su Cho



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

PubMed Central

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.

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




Microsoft Academic Search

We have investigated the preequilibrium kinetics of the L-isoleucine activation reaction catalyzed by Ile-tRNA synthetase in the presence of a fluorescent reporter group, 2-p-toluidinylnaphthalene-6-sulfonate, using the stopped-flow technique. It is found that of all the reactants involved, L-isoleucine binds slowest to the enzyme, apparently in a two-step process. The kinetics of the reaction are invariant in the presence of co-reactants,

E. Holler; Melvin Calvin



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


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 study is warranted to substantiate the therapeutic effect of L-isoleucine. PMID:21766553

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



Freezing capture of polymorphic aggregates of bolaamphiphilic (L)-valine-based molecular hydrogelators.  


Nanostructured xerogels have been prepared by the freeze-drying of hydrogels and aggregates formed by bolaamphiphilic L-valine derivatives after aging under different environmental conditions. A wide variety of shapes and sizes has been achieved by a simple methodology. These nanostructures have been studied by SEM and WAXD and a dramatic influence of structural flexibility on the kinetics of aggregation has been observed. Such flexibility and a modulation of the hydrophobic effect have shown a profound influence in the packing of these compounds and revealed a high degree of polymorphism. PMID:24668870

Nebot, Vicent J; Díaz-Oltra, Santiago; Smets, Johan; Fernández Prieto, Susana; Miravet, Juan F; Escuder, Beatriu



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

NASA Astrophysics Data System (ADS)

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.

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



Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum.  


Threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the L-isoleucine biosynthesis pathway of Corynebacterium glutamicum, but their activities are usually feedback-inhibited. In this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an L-isoleucine producing strain C. glutamicum JHI3-156. Sequence analysis showed that there was only a single amino acid substitution (Phe383Val) in the feedback-resistant threonine dehydratase, and there were three mutated amino acids (Pro176Ser, Asp426Glu, and Leu575Trp) in the big subunit of feedback-resistant acetohydroxy acid synthase. The mutated threonine dehydratase over-expressed in E. coli not only showed completely resistance to L-isoleucine inhibition, but also showed enhanced activity. The mutated acetohydroxy acid synthase over-expressed in E. coli showed more resistance to L-isoleucine inhibition than the wild type. Over-expression of the feedback-resistant threonine dehydratase or acetohydroxy acid synthase in C. glutamicum JHI3-156 led to increase of L-isoleucine production; co-expression of them in C. glutamicum JHI3-156 led to 131.7% increase in flask cultivation, and could produce 30.7g/L L-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance L-isoleucine production in C. glutamicum. PMID:22771937

Yin, Lianghong; Hu, Xiaoqing; Xu, Daqing; Ning, Jianfei; Chen, Jian; Wang, Xiaoyuan



Fluorescence biosensing system with a UV-LED excitation for l-leucine detection  

Microsoft Academic Search

A high-sensitive l-leucine biosensors consisting of l-leucine dehydrogenase (LeuDH) immobilized membrane on the edge of a fiber was developed and demonstrated using a high-intensity ultra violet light-emitting diodes (UV-LED) excitation system (?=340nm), an optical fiber probe and a photomultiplier tube. The biosensors were fabricated by attaching l-leucine dehydrogenase immobilized polytetrafluoroethylene (PTFE) membrane with 2-methacryloyloxyethyl phosphorylcholine polymer (PMEH) polymer to the

Tomoyuki Koshida; Takahiro Arakawa; Tomoko Gessei; Daishi Takahashi; Hiroyuki Kudo; Hirokazu Saito; Kazuyoshi Yano; Kohji Mitsubayashi



Effects of an Escherichia coli ilvA mutant gene encoding feedback-resistant threonine deaminase on L-isoleucine production by Brevibacterium flavum.  


A mutated ilvA gene of Escherichia coli encoding a threonine deaminase that is resistant to feedback inhibition by L-isoleucine was obtained. It was functional in Brevibacterium flavum, and a wild strain of B. flavum into which it was introduced became able to convert exogeneous L-homoserine and L-threonine to L-isoleucine. When it was introduced into a L-threonine-producing B. flavum strain, the transformant accumulated 20 g/liter L-isoleucine from 100 g/liter glucose. PMID:9028039

Hashiguchi, K; Kojima, H; Sato, K; Sano, K



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


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

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



Metabolic Function of Corynebacterium glutamicum Aminotransferases AlaT and AvtA and Impact on L-Valine Production  

Microsoft Academic Search

Aminotransferases (ATs) interacting with L-alanine are the least studied bacterial ATs. Whereas AlaT converts pyruvate to L-alanine in a glutamate-dependent reaction, AvtA is able to convert pyruvate to L-alanine in an L-valine-dependent manner. We show here that the wild type of Corynebacterium glutamicum with a deletion of either of the corresponding genes does not exhibit an explicit growth deficiency. However,

Jan Marienhagen; Lothar Eggeling



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


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

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



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

PubMed Central

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

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



Postcolumn co-immobilized leucine dehydrogenase-NADH oxidase reactor for the determination of branched-chain amino acids by high-performance liquid chromatography with chemiluminescence detection  

Microsoft Academic Search

A liquid chromatographic system with a co-immobilized leucine dehydrogenase-NADH oxidase reactor is described for the determination of branched-chain amino acids such as l-leucine, l-isoleucine and l-valine. The enzymes were simultaneously immobilized on tresylate-containing poly(vinyl alcohol) beads. The separation was achieved by means of an ODS column with elution with phosphate buffer (pH 7.5). The hydrogen peroxide produced was detected chemiluminometrically

Nobutoshi Kiba; Yukio Oyama; Akira Kato; Motohisa Furusawa



Induction of oxidative stress in rat brain by the metabolites accumulating in maple syrup urine disease  

Microsoft Academic Search

Maple syrup urine disease (MSUD) is an inherited disorder caused by deficiency of branched-chain l-2-keto acid dehydrogenase complex activity. Affected patients present severe brain dysfunction manifested as convulsions, coma, psychomotor delay and mental retardation. However, the underlying mechanisms of these neurological findings are virtually unknown. In this study, we tested the in vitro effect of l-leucine, l-isoleucine and l-valine, the

Raquel Bridi; Jana??na Araldi; Miriam B. Sgarbi; Carla G. Testa; Karina Durigon; Moacir Wajner; Carlos Severo Dutra-Filho



Synthesis of the Quinoline-Linked Triazolopyrimidine Analogues and Their Interactions with the Recombinant Tobacco Acetolactate Synthase  

Microsoft Academic Search

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. Triazolopyrimidine sulfonamide (TP) is a mixed-type inhibitor of ALS with respect to both pyruvate and thiamine pyrophosphate. In this study, we synthesized new substituted quinoline-linked TP analogues and several TP analogues which contained either unsubstituted aminoquinolines or amino isoquinolines. In addition, we examined the

Sung Keon Namgoong; Hyun Jung Lee; Young Sook Kim; Jung-Hyu Shin; Jong-Khn Che; Do Young Jang; Gun Sung Kim; Jae Won Yoo; Moon-Kyeong Kang; Mee-Wha Kil; Jung-Do Choi; Soo-Ik Chang



Inhibition ofEscherichia coli Isoleucine Biosynthesis by Isoleucine Tetrazole  

Microsoft Academic Search

orglycyl-L- threonine, and,ina valine-resistant mutant,byL-valine. Partial reversal of growth inhibition was effected byL-leucine, L-methionine, orL-homoserine. The tetrazole inhibited theactivity ofthebiosynthetic threonine deaminase (EC L-threonine hydrolyase (deaminating)), theinhibition being relieved by L-valine. Thetetrazole also inhibited isoleucyl-transfer ribonucleic acid(tRNA) synthetase (EC6.1.1.5 L-isoleucine: tRNAligase (adenosine monophosphate)), butwas without effect on theactivities ofa-isopropylmalate synthetase or acetohydroxy acidsynthetase. Oneclass ofisoleucine tetrazole-resistant mutants produced biosynthetic




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

NASA Astrophysics Data System (ADS)

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

Yoshiura, Hiromu; Nagano, Hiroshi; Hirasawa, Izumi



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


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

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



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

NASA Astrophysics Data System (ADS)

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

Puhal Raj, A.; Ramachandra Raja, C.



A combined 17 O RAPT and MQ-MAS NMR study of l-leucine  

Microsoft Academic Search

We report the application of rotor-assisted population transfer (RAPT) to measure the quadrupolar coupling constant (Cq) for spin 52 nuclei. Results from numerical simulations are presented on the magnitude of enhancement factor as a function of frequency offsets, i.e. the RAPT profile. Experimental O17 RAPT profile is traced for the amino acid l-leucine. In addition, results from MQ-MAS experiments are

Subramanian Prasad; Ted M. Clark; Ramesh Sharma; Hyung-Tae Kwak; Philip J. Grandinetti; Herbert Zimmermann



L-leucine and a nonmetabolized analogue activate pancreatic islet glutamate dehydrogenase  

Microsoft Academic Search

The release of insulin evoked by nutrients in the pancreatic beta-cell is attributed to either the activation of a stereospecific receptor by the nutrient molecule itself or the generation of one or more signal(s) through the intracellular metabolism of the nutrient secretagogue1. The first of these hypotheses is apparently supported by the fact that nonmetabolized amino acids, especially the L-leucine

Abdullah Sener; Willy J. Malaisse



l-Leucine Supplementation Worsens the Adiposity of Already Obese Rats by Promoting a Hypothalamic Pattern of Gene Expression that Favors Fat Accumulation  

PubMed Central

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.

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



Slow-onset feedback inhibition: inhibition of Mycobacterium tuberculosis alpha-isopropylmalate synthase by L-leucine.  


This report describes the first demonstration of slow-onset feedback inhibition of an enzyme that catalyzes the first committed step in a biosynthetic pathway. alpha-Isopropylmalate synthase (IPMS) catalyzes the first committed step of the l-leucine biosynthetic pathway and is feedback-inhibited by l-leucine. Initial velocity experiments on the Mycobacterium tuberculosis IPMS indicate that inhibition by l-leucine is linearly noncompetitive versus alpha-ketoisovalerate. Time-courses displayed a burst of product formation followed by a linear steady-state rate when reactions were initiated by the addition of enzyme. The burst rate showed a hyperbolic dependence on the concentration of l-leucine indicating that inhibition proceeds in two steps, an initial rapid binding step followed by slow isomerization to a more tightly bound complex. PMID:16011356

de Carvalho, Luiz P S; Argyrou, Argyrides; Blanchard, John S



l-Leucine transport in human breast cancer cells (MCF7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter  

Microsoft Academic Search

The transport of l-leucine by two human breast cancer cell lines has been examined. l-Leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na+-independent pathway. l-Leucine uptake by both cell lines was inhibited by l-alanine, d-leucine and to a lesser extent by l-lysine but not by l-proline. Estrogen (17?-estradiol) stimulated l-leucine uptake by MCF-7 but not by MDA-MB-231

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



Synthesis, crystal structure and DNA-binding properties of a new copper(II) complex with l-valine Schiff base and 1,10-phenanthroline  

Microsoft Academic Search

A new copper(II) complex, [Cu(sal-l-val)phen] (sal-l-val=a Schiff base derived from salicylaldehyde and l-valine, phen=1,10-phenanthroline), has been synthesized and characterized by elemental analysis, IR spectra and X-ray single crystal diffraction. Results show that this crystal structure of the copper(II) complex belongs to triclinic crystal system, P1 space group with crystallographic data: a=10.3710(16)Å, b=13.9350(19)Å, c=16.670(2)Å, ?=79.970(2)°, ?=75.623(2)°, ?=84.160(3)° and Z=4. The copper(II)

Jianfang Dong; Lianzhi Li; Guihua Liu; Tao Xu; Daqi Wang



Total structures of colistin minor components.  


Structural characterization of the colistin (CL) components were carried out using Frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS), tandem mass spectrometry (MS/MS) and the amino acid analysis proposed by MARFEY, and the total structures of 4 minor components including the absolute configuration of the constituent amino acids were proposed. The structures of the minor components were the same as those of the main component colistin A or B except that L-leucine is replaced by L-valine or L-isoleucine. PMID:9666178

Ikai, Y; Oka, H; Hayakawa, J; Kawamura, N; Mayumi, T; Suzuki, M; Harada, K



Development of a high-performance liquid chromatographic system with enzyme reactors for the determination of N-acetyl branched-chain amino acids  

Microsoft Academic Search

Immobilized enzymes were used as column reactors in a high-performance liquid chromatographic system for the specific detection of N-acetyl branched-chain amino acids (AcBCAs) such as N-acetyl-l-valine (AcVal), N-acetyl-l-leucine (AcLeu) and N-acetyl-l-isoleucine (AcIle). Aminoacylase and leucine dehydrogenase were immobilized onto poly(vinyl alcohol) beads. The AcBCAs were separated as three peaks on a Capcell C1 SG120 column with 0.03M phosphate buffer (pH

Nobutoshi Kiba; Yukio Oyama; Motohisa Furusawa



Chemiluminometric branched chain amino acids determination with immobilized enzymes by flow-injection analysis  

Microsoft Academic Search

A tri-enzyme sensor was developed for the flow-injection determination of branched chain amino acids (L-valine, L-leucine and L-isoleucine). Leucine dehydrogenase, NADH oxidase and peroxidase were coimmobilized covalently on tresylate-hydrophilic vinyl polymer beads and packed into transparent PTFE tube (20cm×1.0 i.d.), which was used as flow cell. The calibration graph was linear for 30nM–5?M; the detection limit (signal-to-noise=3) was 10nM. The

Nobutoshi Kiba; Masaki Tachibana; Kazue Tani; Takao Miwa



Soluble Overexpression in Escherichia coli,and Purification and Characterization of Wild-Type Recombinant Tobacco Acetolactate Synthase  

Microsoft Academic Search

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. The wild-type ALS gene fromNicotiana tabacumwas cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS2 was used to transformEscherichia colistrain XL1-Blue, and the wild-type tobacco ALS (wALS) was expressed in the bacteria as a protein fused with glutathioneS-transferase (GST). The fusion

Soo-Ik Chang; Moon-Kyeong Kang; Jung-Do Choi; Sung Keon Namgoong



ZnCl2-mediated practical protocol for the synthesis of Amadori ketoses.  


An efficient and practical protocol for the synthesis of Amadori ketoses N-(1-deoxy-D-fructose-1-yl) amino acid (amino acid=L-valine (1), L-leucine (2), L-isoleucine (3), L-tryptophan (4), L-phenylalanine (5), L-arginine (6) has been accomplished by employing ZnCl2 as a catalyst. The developed method circumvents protection and deprotection steps as well as tedious ion-exchange and column chromatographic techniques. The accomplished Amadori ketoses showed moderate to weak angiotensin I converting enzyme (ACE) inhibitory activity. PMID:24731352

Harohally, Nanishankar V; Srinivas, Sudhanva M; Umesh, Sushma



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


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

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



Thermal, Dielectric Studies on Pure and Amino Acid L-Glutamic Acid, L-Histidine L-Valine Doped Potassium Dihydrogen Phosphate Single Crystals  

NASA Astrophysics Data System (ADS)

Amino acids (L-Glutamic acid, L-Histidine, L-Valine) doped potassium dihydrogen phosphate crystals were grown by the solution growth technique. Slow cooling as well as slow evaporation methods were employed to grow these crystals. The concentration of dopants in the mother solution was varied from 0.1 mole % to 10 mole %. The solubility data for all dopant concentrations were determined. The variation in pH and the corresponding habit modification of the grown crystals were characterized with UV - VIS, FT-IR and SHG trace elements, and dielectric studies reveal slight distortion of lattice parameter for the heavily doped KDP crystals. TGA-DTA studies reveal good thermal stability. The dopants increase the hardness value of the material, which also depends on the concentration of the dopants. Amino acids doping improved the NLO properties. The detailed results on the spectral parameters, habit modifications and constant values will be presented.

Kumaresan, P.; Babu, S. Moorthy; Anbarasan, P. M.


A platinum(II) terpyridine metallogel with an L-valine-modified alkynyl ligand: interplay of Pt???Pt, ?-? and hydrogen-bonding interactions.  


A series of platinum(II) terpyridine complexes with L-valine-modified alkynyl ligands has been synthesized. A complex with an unsubstituted terpyridine and one valine unit on the alkynyl is shown to be capable of gel formation, which is in sharp contrast to the gelation properties of the corresponding organic counterparts. Upon sol-gel transition, a drastic color change from yellow to red is observed, which is indicative of the involvement of Pt???Pt interactions. Through the concentration- and temperature-dependent UV/Vis absorption, emission, circular dichroism, and (1) H?NMR studies, the contribution of hydrogen bonding, Pt???Pt and ?-? stacking interactions as driving forces for gelation have been established, and the importance of maintaining a delicate balance between different intermolecular forces has also been illustrated. PMID:24123486

Po, Charlotte; Ke, Zhihai; Tam, Anthony Yiu-Yan; Chow, Hak-Fun; Yam, Vivian Wing-Wah



Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains  

PubMed Central

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ?aceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ?aceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.

Mahr, Regina; Helfrich, Stefan; Noh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia



Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.  


The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ?aceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ?aceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains. PMID:24465669

Mustafi, Nurije; Grünberger, Alexander; Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia



The effect of L-leucine on the absorption of levodopa, studied by regional jejunal perfusion in man.  

PubMed Central

1. A new method for perfusing a 10 cm segment of jejunum in humans has been used in seven subjects to study the effect of the amino acid L-leucine (40 mM) on the intestinal absorption of levodopa (2.5 mM). The tube contains six channels and has two inflatable balloons, which enable a perfusion of a closed and defined segment of the proximal small intestine. 2. L-leucine decreased the intestinal absorption of levodopa from 40 +/- 19 to 21 +/- 15% but was without effect on the absorption of antipyrine, benserazide and D-glucose. 3. We confirm that levodopa is absorbed by the active transport system normally responsible for the absorption of large neutral amino acids (LNAA) in humans. Oral absorption by passive diffusion, probably by the paracellular route, might also occur for levodopa in the proximal part of the small intestine.

Lennernas, H; Nilsson, D; Aquilonius, S M; Ahrenstedt, O; Knutson, L; Paalzow, L K



Methods for the Synthesis of L-Leucine Selectively Labelled with Carbon13 or Deuterium in either Diastereotopic Methyl Group  

Microsoft Academic Search

A versatile approach is described for the enantioselective synthesis of isotopically labelled L-leucine involving the preparation of 2-oxo-4-methylpentanoic acid labelled selectively with carbon-13 or deuterium in either the pro-R or pro-S methyl group followed by a reductive amination of the ketone catalysed by leucine dehydrogenase. This strategy is applied to the total synthesis of (2S.4R)-[5.5.5-D3]-leucine using CD3I as the source

Nicholas M. Kelly; R. Gordon Reid; Christine L. Willis; Peter L. Winton



Size-Selective Encapsulation Property of Unimolecular Reverse Micelle Consisting of Hyperbranched D-Glucan Core and L-Leucine Ethyl Ether Shell  

Microsoft Academic Search

Summary: The synthesis of a unimolecular reverse micelle (3) consisting of hyperbranched D-glucan as the core and L-leucine ethyl ester as the shell was accomplished through the carbamation reaction of the hyperbranched D-glucan (1) with the N-carbonyl L-leucine ethyl ester (2) in pyridine at 100 ºC. The polymer 3 was soluble in a large variety of organic solvents, such as

Toshifumi Satoh; Yoshikazu Kitajyo; Ryosuke Sakai; Atsushi Narumi; Kenji Takahashi; Harumi Kaga; Noriaki Kaneko; Toyoji Kakuchi



Thermal, dielectric studies on pure and amino acid ( L-glutamic acid, L-histidine, L-valine) doped KDP single crystals  

NASA Astrophysics Data System (ADS)

Amino acids ( L-glutamic acid, L-histidine, L-valine) doped potassium dihydrogen phospate crystals are grown by solution growth technique. Slow cooling as well as slow evaporation methods were employed to grow these crystals. The concentration of dopants in the mother solution was varied from 0.1 mol% to 10 mol%. The solubility data for all dopants concentration were determined. There is variation in pH value and hence, there is habit modification of the grown crystals were characterized with UV-VIS, FT-IR studies, SHG trace elements and dielectric studies reveal slight distortion of lattice parameter for the heavily doped KDP crystals. UV-Visible spectra confirm the improvement in the transparency of these crystals on doping metal ions. FT-IR spectra reveal strong absorption band between 1400 and 1600 cm -1 for metal ion doped crystals. TGA-DTA studies reveal good thermal stability. The dopants increase the hardness value of the material and it also depends on the concentration of the dopants. Amino acids doping improved the NLO properties. The detailed results on the spectral parameters, habit modifications and constant values will be presented.

Kumaresan, P.; Moorthy Babu, S.; Anbarasan, P. M.



Synthesis, crystal structure and DNA-binding properties of a new copper(II) complex with L-valine Schiff base and 1,10-phenanthroline  

NASA Astrophysics Data System (ADS)

A new copper(II) complex, [Cu(sal- L-val)phen] (sal- L-val = a Schiff base derived from salicylaldehyde and L-valine, phen = 1,10-phenanthroline), has been synthesized and characterized by elemental analysis, IR spectra and X-ray single crystal diffraction. Results show that this crystal structure of the copper(II) complex belongs to triclinic crystal system, P1 space group with crystallographic data: a = 10.3710(16) Å, b = 13.9350(19) Å, c = 16.670(2) Å, ? = 79.970(2)°, ? = 75.623(2)°, ? = 84.160(3)° and Z = 4. The copper(II) complex has four independent structures in the crystallographic asymmetric unit, three of them show distorted square-pyramidal and the fourth displays distorted trigonal bipyramid CuN 3O 2 coordination geometry. In the crystal, ?-? stacking and the intermolecular hydrogen bonds form two-dimensional network. The interactions between the copper(II) complex and calf thymus DNA(CT-DNA) have been studied by using UV, fluorescence and CD spectra, as well as viscosity and thermal denaturation measurements. Experiment results confirm that the copper(II) complex binds to CT-DNA in an intercalative mode.

Dong, Jianfang; Li, Lianzhi; Liu, Guihua; Xu, Tao; Wang, Daqi



Decreased pancreatic islet response to L-leucine in the spontaneously diabetic GK rat: enzymatic, metabolic and secretory data  

Microsoft Academic Search

\\u000a \\u000a Abstract\\u000a \\u000a   \\u000a \\u000a Aims\\/hypothesis. Pancreatic islets from hereditarily non-insulin-dependent diabetic Goto-Kakizaki (GK) rats have a deficient insulin response\\u000a not only to d-glucose but also to l-leucine. Our aim was to explain the cellular mechanism(s) underlying the beta-cell unresponsiveness to this amino acid. Methods. Freshly collagenase isolated islets from GK rats and healthy Wistar control rats matched with them for sex and

M.-H. Giroix; C. Saulnier; B. Portha



Preparation and Characterization of New Optically Active Poly(amide-imide)s from N,N?-(bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetra carboxylic)-bis-L-isoleucine and Aromatic Diamines  

Microsoft Academic Search

Five new optically active aromatic poly(amide-imide)s (PAIs) 5a–e were prepared from a direct polycondensation reaction of a new diacid of N,N?-(bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetra carboxylic)-bis-L-isoleucine 3 with various aromatic diamines 4a–e in a medium consisting of triphenyl phosphite (TPP), calcium chloride (CaCl2), pyridine (Py) and N-methyl-2-pyrrolidone (NMP). The polycondensation reaction produced a series of novel poly(amid-imide)s 5a–e in quantitative yields with inherent viscosities

Khalil Faghihi; Mohsen Hajibeygi; Samira Bayat



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

NASA Astrophysics Data System (ADS)

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

Tamasi, Gabriella; Casolaro, Mario; Cini, Renzo



Experimental and theoretical studies on physicochemical properties of L-leucine nitrate—a probable nonlinear optical material  

NASA Astrophysics Data System (ADS)

An amino acid salt L-leucine nitrate (LN) has been synthesized and single crystals were grown by the controlled evaporation of the aqueous solution at constant temperature (30±0.01)°C. The structural and physicochemical properties of the grown crystals were characterized by X-ray powder diffraction and thermal analysis. The optical transmission spectra and second harmonic generation (SHG) were investigated to study its linear and nonlinear optical properties. SHG efficiency is comparable to that of KDP. The dipole moment, polarizability, first order hyperpolarizability and the HOMO-LUMO energy gap of LN were calculated at the framework of Hartree-Fock (HF) and density functional theory (DFT) to confirm the suitability of the crystal for nonlinear optical applications. First hyperpolarizability was found to be 9.4 times of that of urea.

Adhikari, Soma; Kar, Tanusree



Investigating expression systems for the stable large-scale production of recombinant L -leucine-dehydrogenase from Bacillus cereus in Escherichia coli  

Microsoft Academic Search

The established Escherichia coli expression vectors ptrc99a, pKK223-3, pPL?, pAsk75, pRA95, and pRA96, which differ in copy number, mode of induction, selection\\u000a marker, and use of par sequences for stabilization, were investigated for the stable expression of recombinant L-leucine dehydrogenase from Bacillus cereus with a view to large-scale production. Best results were achieved with pIET98, a runaway-replication system derived from

M. B. Ansorge; M. R. Kula



Design, synthesis, photochemical properties and cytotoxic activities of water-Soluble caged l-Leucyl- l-leucine methyl esters that control apoptosis of immune cells  

Microsoft Academic Search

l-Leucyl-l-leucine methyl esters (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. Glucose-carrying 2-nitrobenzyl (2-NB) and 2-nitrophenethyl (2-NPE) caged LeuLeuOMe, 1a and b, were synthesized and their photochemical and immunological properties were studied. Caged glycine methyl esters (GlyOMe), 2a,b, were also prepared to examine the cytotoxic activity of the photolytic byproducts from 1a,b. All the caged compounds

Hironori Mizuta; Soichiro Watanabe; Yuji Sakurai; Keiko Nishiyama; Toshiaki Furuta; Yoshiro Kobayashi; Michiko Iwamura



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


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

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



Formation of amino acid (L-leucine, L-phenylalanine) derived volatile flavour compounds by Moraxella phenylpyruvica and Staphylococcus xylosus in cured meat model systems.  


A bacterial strain isolated from Danish immersion curing brine, Moraxella phenylpyruvica 0100, and a commercial meat starter culture, Staphylococcus xylosus DD34, were tested for their ability to form characteristic volatile compounds in minimal medium with the added amino acid L-leucine or L-phenylalanine under different environmental conditions (pH 5.5 and 6.0; 0 and 210 ppm nitrate; pre-incubation with and without agitation) and compared with respect to their ability to form volatile compounds in cured meat extracts and vacuum-packed cured meat cuts. The characteristic cured meat aroma precursors/compounds 3-methylbutanal and 3-methylbutanol were found to be formed in cured meat extracts and vacuum-packed cured meat cuts inoculated with M. phenylpyruvica. These volatiles are most probably formed by metabolic conversion of the amino acid L-leucine by M. phenylpyruvica, as they were also produced in minimal media with added L-leucine inoculated with this organism. The characteristic L-phenylalanine derived compound, benzaldehyde, formed by M. phenylpyruvica in minimal medium in the presence of nitrate (210 ppm), was not produced in any noticeable amount in cured meat extracts or vacuum-packed cured meat inoculated with M. phenylpyruvica. In contrast, benzacetaldehyde, which has been described as a possible metabolic product of the microbial conversion of L-phenylalanine, was found to be a characteristic volatile compound formed in cured meat extracts and vacuum-packed cured meat inoculated with M. phenylpyruvica, indicating an alternative metabolic pathway for L-phenylalanine by this organism in a cured meat environment. Even though S. xylosus was able to form volatile compounds characteristic of cured meats (3-methylbutanal, 3-methylbutanol) in minimal media with added L-leucine, this bacterial strain seemed not to be able to produce these characteristic volatiles in the studied cured meat systems. The present data imply that M. phenylpyruvica, in particular, is a potential meat starter for ensuring superior flavour development in cured meat. PMID:9706803

Møller, J K; Hinrichsen, L L; Andersen, H J



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


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. PMID:16741983

Rozwadowski, Z



NRPSs and amide ligases producing homopoly(amino acid)s and homooligo(amino acid)s.  


Microorganisms are capable of producing a wide variety of biopolymers. Homopoly(amino acid)s and homooligo(amino acid)s, which are made up of only a single type of amino acid, are relatively rare; in fact, only two homopoly(amino acid)s have been known to occur in nature: poly(?-L-lysine) (?-PL) and poly(?-glutamic acid) (?-PGA). Bacterial enzymes that produce homooligo(amino acid)s, such as L-?-lysine-, L-valine-, L-leucine-, L-isoleucine-, L-methionine-, and L-glutamic acid-oligopeptides and poly(?-l-glutamic acid) (?-PGA) have recently been identified, as well as ?-PL synthetase and ?-PGA synthetase. This article reviews the current knowledge about these unique enzymes producing homopoly(amino acid)s and homooligo(amino acid)s. PMID:23817633

Hamano, Yoshimitsu; Arai, Toshinobu; Ashiuchi, Makoto; Kino, Kuniki



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

Microsoft Academic Search

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

Khalil Faghihi; Mohsen Hajibeygi; Meisam Shabanian



Synthesis and properties of novel flame-retardant and thermally stable poly(amideimide) s from N,N? -(bicyclo[2,2,2]oct-7-ene-tetracarboxylic)-bis-L-amino acids and phosphine oxide moiety by two different methods  

Microsoft Academic Search

N,N?-(bicyclo[2,2,2]oct-7-ene-tetracarboxylic)-bis-L-amino acids3a-g were synthesized by the condensation reaction of bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic dianhydride1 with two equimolars of Lalanine2a, L-valine2b, L-leucine2c, L-isoleucine2d, L-phenyl alanine2e, L-2-aminobutyric acid2f and L-histidine2g in an acetic acid solution. Seven new poly(amide-imide)s PAIs5a-g were synthesized through the direct polycondensation reaction of seven chiralN,N?-(bicyclo[2,2,2]oct-7-ene-tetracarboxylic)-bis-L-amino acids3a-g with bis(3-amino phenyl) phenyl phosphine oxide4 by two different methods: direct polycondensation in a medium

Khalil Faghihi; Mohsen Hajibeygi; Meisam Shabanian



The leucine regulon of Escherichia coli K-12: a mutation in rblA alters expression of L-leucine-dependent metabolic operons.  


We have isolated and characterized a highly pleiotropic Escherichia coli mutant affected in the activity of a number of enzymes involved in different metabolic pathways, all of which are regulated by leucine. Selected for its ability to grow with L-serine as sole carbon source, the rbl-1::Tn10 mutant had high levels of L-serine deaminase activity (due to increased transcription of the structural gene) and of another amino acid-degrading enzyme, L-threonine dehydrogenase, and decreased transcription of the operons serA and ilvIH, coding for biosynthetic enzymes. The rbl mutation suppressed the slow growth of a metK mutant, deficient in S-adenosylmethionine synthetase. Furthermore, metK mutants spontaneously accumulated faster-growing rbl-like derivatives, and a commonly used metK strain, RG62, carries such a mutation. The rbl gene is located near 20 min on the E. coli genetic map. All phenotypes of the rbl mutant could be observed in rbl+ strains cultivated in the presence of L-leucine, and exogenous L-leucine had little further effect on the rbl strains. We propose that the rbl gene product is the regulator of a global response to leucine. PMID:2165479

Tuan, L R; D'Ari, R; Newman, E B



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

SciTech Connect

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

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



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


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

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



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

SciTech Connect

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.

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



Determination of Isotope Enrichment in Cor H-Labelled BranchedChain l-Amino Acids From Physiological Fluids by Gas Chromatography-Mass Spectrometry: Use of l-Leucine Dehydrogenase for Specific Preparation of the Quinoxalinol Derivatives  

Microsoft Academic Search

A procedure is described which allows the specific and sensitive estimation of C- or H-label enrichment in branched-chain l-amino acids from physiological fluids: Amino acids are isolated from deproteinized samples by cation exchange chromatography. Specific conversion of branched-chain l-amino acids to their respective 2-oxo acids is achieved by treatment with l-leucine dehydrogenase from Bacillus sp. Reaction of the 2-oxo acids

U. Matthiesen; P. Schadewaldt



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

Microsoft Academic Search

This study concerns the synthesis of novel multi block polyurethane (PU) copolymers containing cyclodipeptide, taking the\\u000a advantage of ionic liquids (ILs) under microwave irradiation. For this, l-leucine anhydride cyclodipeptide (LACP) was prepared and then a new class of poly(ether-urethane-urea)s (PEUUs) was synthesized\\u000a in molten ammonium type ILs. ILs were used as reaction media and PUs were prepared via two-step polymerization

Fatemeh Rafiemanzelat; Elahe Abdollahi


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


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

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



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

NASA Technical Reports Server (NTRS)

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.

Chipot, C.; Pohorille, A.



Solvent Effects on the Protonation Constants of Some ? Amino Acid Esters in 1,4Dioxane–Water Mixtures  

Microsoft Academic Search

The stoichiometric protonation constants of some ?-amino acid esters (glycine methyl ester, glycine t-butyl ester, l-valine methyl ester, l-valine ethyl ester, l-valine t-butyl ester, l-serine methyl ester, l-serine ethyl ester, l-leucine methyl ester, l-leucine ethyl ester, l-leucine t-butyl ester, l-alanine methyl ester, l-alanine benzyl ester, l-phenylalanine methyl ester, l-phenylalanine ethyl ester, and l-phenylalanine t-butyl ester) in water and 20%, 40%,

Alev Do?an; Nazife Aslan; Esin Canel; Esma K?l?ç



Synthesis and allosteric modulation of the dopamine receptor by peptide analogs of L-prolyl-L-leucyl-glycinamide (PLG) modified in the L-proline or L-proline and L-leucine scaffolds.  


Novel analogs of L-prolyl-L-leucylglycinamide (PLG) were synthesized wherein the prolyl residue was replaced with other amino acids based on a 3,5-disubstituted proline scaffold. In some examples, the L-leucyl residue was also replaced by L-valine. These analogs were tested for their ability to enhance the binding of [(3)H]-N-propylnorapomorphine to short isoform of human dopamine D? receptors. Compounds 18b and 19b, increased [(3)H] NPA binding at concentrations between 10(-12) and 10(-9) M, which is similar to the effect of PLG in this assay and, provides evidences that these compounds are acting as allosteric modulators of dopamine D? receptors. PMID:24013414

Ferreira da Costa, Joana; Caamaño, Olga; Fernández, Franco; García-Mera, Xerardo; Sampaio-Dias, Ivo E; Brea, José Manuel; Cadavid, María Isabel



Threonine deaminase from Salmonella typhimurium. Relationship between regulatory sites.  


Kinetic analysis of the biosynthetic threonine deaminase, EC, from Samonella typhimurium yields hyperbolic substrate saturation curves in the absence of, and higher order substrate saturation curves in the presence of, L-isoleucine. L-Valine reverses this effect of L-isoleucine by restoring the hyperbolic substrate saturation curves. The inhibition of enzyme activity and the reversal of valine stimulation is a function of a second order concentration of L-isoleucine, whereas antagonism of inhibition is a function of first order concentration of valine. The antagonistic effects on enzyme activity of L-isoleucine and of L-valine appear as competitive in diagnostic plots. Threonine deaminase possesses two L-isoleucine binding sites (Kd equals 3.6 muM) and one L-valine binding site (Kd equals 26 muM); the binding of these ligands appear competitive. Exclusion of L-valine requires the binding of 2 molecules of L-isoleucine whereas binding of a single L-valine molecule prevents the binding of 2 L-isoleucine molecules. Cooperative binding of L-isoleucine is not observed under any of the conditions tested. Two cases, expressed in terms of modified Adair equations and based upon the assumption that L-threonine also serves as an activator ligand which binds to the L-valine site, are presented. Case I states that liganding of the activator sites must percede substrate-binding at the active site, and Case II states that the activator site liganding is required solely for reactivation of the L-isoleucine-inhibited enzyme. Analysis of kinetic data by a curve-fitting process suggests that Case II described the relationship between the activator site and the L-isoleucine sites. An enzymatically inactive derivative of threonine deaminase, prepared by reduction with borohydride, binds isoleucine and valine in a manner similar to native holoenzyme. Binding of L-threonine and L-valine to the derivatized enzyme is competitive. The Kd for threonine binding is 3 mM, which is in excellent agreement with the Kd determined by the curve fitting process. It is concluded that the modulation of threonine deaminase activity is wrought by interaction between inhibitor sites and an activator site rather than inhibitor and active sites and that induced transitions rather than concerted transitions more adequately describe the underlying regulatory principle. PMID:1089662

Decedue, C J; Hofler, J G; Burns, R O



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

PubMed Central

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.

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



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


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

Blombach, Bastian; Eikmanns, Bernhard J



Sequence diversity of the peptaibol antibiotic suzukacillin-A from the mold Trichoderma viride.  


From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C-terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence:Ac-Aib-Ala-Aib-Ala-Aib-Ala(6)-Gln-Aib-Lx(9)-Aib-Gly-Aib(12)-Aib-Pro-Vx(15)-Aib-Vx(17)-Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (alpha-aminoisobutyric acid); Iva (D-isovaline); Lx (L-leucine or L-isoleucine); Vx (L-valine or D-isovaline); Fol (L-phenylalaninol)). PMID:16245259

Krause, Corina; Kirschbaum, Jochen; Jung, Günther; Brückner, Hans



A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli.  

PubMed Central

A novel L-threonine transport system is induced in Escherichia coli cells when incubated in amino acid-rich medium under anaerobic conditions. Genetic and biochemical analyses with plasmids harboring mutations in the anaerobically expressed tdcABC operon indicated that the tdcC gene product was responsible for L-threonine uptake. Competition experiments revealed that the L-threonine transport system is also involved in L-serine uptake and is partially shared for L-leucine transport; L-alanine, L-valine, and L-isoleucine did not affect L-threonine uptake. Transport of L-threonine was inhibited by the respiratory chain inhibitors KCN and carbonyl cyanide m-chlorophenylhydrazone and was Na+ independent. These results identify for the first time an E. coli gene encoding a permease specific for L-threonine-L-serine transport that is distinct from the previously described threonine-serine transport systems. A two-dimensional topological model predicted from the amino acid composition and hydropathy plot showed that the TdcC polypeptide appears to be an integral membrane protein with several membrane-spanning domains exhibiting a striking similarity with other bacterial permeases.

Sumantran, V N; Schweizer, H P; Datta, P



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


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

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



Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters.  


A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters. PMID:12930836

Babu, Ellappan; Kanai, Yoshikatsu; Chairoungdua, Arthit; Kim, Do Kyung; Iribe, Yuji; Tangtrongsup, Sahatchai; Jutabha, Promsuk; Li, Yuewei; Ahmed, Nesar; Sakamoto, Shinichi; Anzai, Naohiko; Nagamori, Seishi; Endou, Hitoshi



l-leucine dehydrogenase from Bacillus cereus  

Microsoft Academic Search

An improved method for the production ofl-leucine dehydrogenase is described employing a mutant with a constitutive enzyme and a fed-batch cultivation technique yielding high cell concentrations. Purification ofl-leucine dehydrogenase to homogeneity was carried out starting with 30 kgBacillus cereus cells by heat treatment at 63°C, followed by two liquid-liquid extraction steps and three conventional column chromatographies. Crystals have been obtained

Horst Schütte; Werner Hummel; Hsin Tsai; Maria-Regina Kula



Study of the aminolysis of the activated esters of N-substituted amino acids by trimethylsilyl derivatives of the amino components  

Microsoft Academic Search

1.Study has been made of the kinetics of the aminolysis of the N-hydroxysuccinimide esters of the Ntert-butyloxycarbonyl derivatives of L-proline and L-leucine by the ethyl esters of L-isoleucine, tert-butylamine and their trimethylsilyt-substituted derivatives, and by the trimethylsilyl ester of N-trimethyl L-isoleucine.2.Aminolysis by these trimethylsilyl derivatives proceeds through elimination of the N-trimethylsilyl group of the amino component, the reaction rate being

A. I. Yurtanov; Yu. A. Davidovich; S. V. Rogozhin



Actinoplanes teichomyceticus ATCC 31121 as a cell factory for producing teicoplanin  

PubMed Central

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



Aminoacylation of rat liver transfer RNA with L-penicillamine. On the specificity of the aminoacylation reaction.  


L-]14C]Penicillamine is bound to RNA from rat liver in an in vitro reaction catalyzed by rat liver aminoacyl-tRNA synthetases. Addition of certain naturally occuring amino acids results in a significant decrease of L-penicillamine binding. The most potent inhibitor of this binding is L-valine, followed by L-isoleucine and L-threonine. Amino acids without structural relationship to L-penicillamine in the non-functional part of the molecule, such as L-phenylalanine, are ineffective. Studies on the competition of L-penicillamine and L-isoleucine, respectively, with L-valine demonstrate the high specificity of the aminoacylation reaction. They show that the change of L-penicillamine binding to tRNA Val is considerably lower than that of L-valine. PMID:318863

Lodemann, E; Ulrich, P; Wacker, A



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


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

Yoshii, K; Kobatake, Y; Kurihara, K



Selective enhancement and suppression of frog gustatory responses to amino acids  

PubMed Central

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.



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

PubMed Central

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

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



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


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

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



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


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

Mourad, G; Williams, D; King, J



A mathematical model for the branched chain amino acid biosynthetic pathways of Escherichia coli K12.  


As a first step toward the elucidation of the systems biology of the model organism Escherichia coli, it was our goal to mathematically model a metabolic system of intermediate complexity, namely the well studied end product-regulated pathways for the biosynthesis of the branched chain amino acids L-isoleucine, L-valine, and L-leucine. This has been accomplished with the use of kMech (Yang, C.-R., Shapiro, B. E., Mjolsness, E. D., and Hatfield, G. W. (2005) Bioinformatics 21, in press), a Cellerator (Shapiro, B. E., Levchenko, A., Meyerowitz, E. M., Wold, B. J., and Mjolsness, E. D. (2003) Bioinformatics 19, 677-678) language extension that describes a suite of enzyme reaction mechanisms. Each enzyme mechanism is parsed by kMech into a set of fundamental association-dissociation reactions that are translated by Cellerator into ordinary differential equations. These ordinary differential equations are numerically solved by Mathematica. Any metabolic pathway can be simulated by stringing together appropriate kMech models and providing the physical and kinetic parameters for each enzyme in the pathway. Writing differential equations is not required. The mathematical model of branched chain amino acid biosynthesis in E. coli K12 presented here incorporates all of the forward and reverse enzyme reactions and regulatory circuits of the branched chain amino acid biosynthetic pathways, including single and multiple substrate (Ping Pong and Bi Bi) enzyme kinetic reactions, feedback inhibition (allosteric, competitive, and non-competitive) mechanisms, the channeling of metabolic flow through isozymes, the channeling of metabolic flow via transamination reactions, and active transport mechanisms. This model simulates the results of experimental measurements. PMID:15657047

Yang, Chin-Rang; Shapiro, Bruce E; Hung, She-Pin; Mjolsness, Eric D; Hatfield, G Wesley



Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.  

PubMed Central

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.

Heyne, R I; de Vrij, W; Crielaard, W; Konings, W N



The role of JAR1 in Jasmonoyl- l -isoleucine production during Arabidopsis wound response  

Microsoft Academic Search

The Arabidopsis thaliana (L.) Heynh. JASMONATE RESISTANT 1( JAR1) locus is essential for pathogen defense, but its role in wound response has not been investigated. JAR1 encodes an enzyme that conjugates jasmonic acid (JA) to isoleucine, which was recently shown to function directly in CORONATINE INSENSITIVE 1 (COI1)-mediated signal transduction. Leaf wounding rapidly increased the level of JA–Ile by about

Walter P. Suza; Paul E. Staswick



Determination of R- and S-3-methyl-2-oxopentanoate enantiomers in human plasma: suitable method for label enrichment analysis  

Microsoft Academic Search

A sensitive method for the determination of S- and R-3-methyl-2-oxopentanoate enantiomers (KMV, (?-keto-?-methylval-erate) in physiological fluids suitable for isotope enrichment analysis is described: after extraction with acid, 2-oxo acids are separated from interfering amino acids by cation-exchange chromatography. Reductive amination of the branched-chain 2-oxo acids by use of l- leucine dehydrogenase yields the corresponding l-amino acids. l-Isoleucine and l-alloisoleucine which

Peter Schadewaldt; Udo Wendel; Hans-Werner Hammen



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


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

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



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

Microsoft Academic Search

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

Shadpour Mallakpour; Fatemeh Zeraatpisheh



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

Microsoft Academic Search

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

Shadpour Mallakpour; Samaneh Soltanian; Mohammad R. Sabzalian



Development of Competence of Haemophilus influenzae  

PubMed Central

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

Spencer, Hugh T.; Herriott, Roger M.



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

PubMed Central

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

Liesenfeld, Andrea



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

NASA Technical Reports Server (NTRS)

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.

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



Supramolecular gelators based on benzenetricarboxamides for ionic liquids.  


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. PMID:24652194

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



Age-Related Increase in Alanine Aminotransferase Correlates with Elevated Levels of Plasma Amino Acids, Decanoylcarnitine, Lp-PLA2 Activity, Oxidative Stress, and Arterial Stiffness.  


We investigated plasma metabolite profiles that correlated with age-related serum alanine aminotransferase (ALT) levels. The study included 602 healthy, nondiabetic subjects (aged 30-65 years); 393 individuals had normal ALT levels at baseline. Fifty-three (13.5%) individuals developed elevated ALT levels after 3 years. The remaining 340 subjects with normal ALT were matched to the elevated-ALT group (n = 53) for age, gender, BMI, fasting glucose, and ALT to form the control group (n = 53). At the 3-year follow-up, the elevated-ALT group exhibited greater increases in waist circumference, serum free fatty acid, ALT, aspartate aminotransferase (AST), ?-glutamyltransferase (GGT), bilirubin, plasma oxidized LDL, Lp-PLA2 activity, urinary 8-epi-prostaglandin F2? (8-epi-PGF2?), and brachial-ankle pulse-wave velocity (ba-PWV) compared to the control group after baseline adjustment. The elevated-ALT group exhibited greater increases in plasma l-valine (q = 0.036), l-leucine (q = 0.012), l-phenylalanine (q = 0.012), and decanoylcarnitine (q = 0.002). Mean ALT levels positively correlated with changes in these four metabolites, which correlated with changes in AST, GGT, Lp-PLA2 activity, urinary 8-epi-PGF2?, and ba-PWV. Mean ALT changes did not significantly correlate with HOMA-insulin resistance. These results suggest that increased plasma levels of l-valine, l-leucine, l-phenylalanine, and decanoylcarnitine precede insulin resistance during periods of elevated ALT. This metabolic disturbance coincides with enhanced risk factors for cardiovascular disease. PMID:24874467

Jung, Saem; Kim, Oh Yoen; Kim, Minjoo; Song, Juheui; Lee, Sang-Hyun; Lee, Jong Ho



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)

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.

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



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

NASA Astrophysics Data System (ADS)

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

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



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

Microsoft Academic Search

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

Jihoe Kim; Daniel Darley; Thorsten Selmer; Wolfgang Buckel



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

PubMed Central

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.

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



Industrial production of amino acids by coryneform bacteria.  


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

Hermann, Thomas



Taxonomy of Phytopathogenic Pseudomonads1  

PubMed Central

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

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



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


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 ?-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 ?mol/g wet wt. l-Valine and leucine ethyl, benzyl, and tert-butyl esters inhibit the Leu?Glu pathway without decreasing tissue total glutamate. PMID:24699330

Schachter, David; Buteau, Jean



Polymeric alkenoxy amino acid surfactants: II. Chiral separations of beta-blockers with multiple stereogenic centers.  


Two amino acid-based (leucine and isoleucine) alkenoxy micelle polymers were employed in this study for the separation of multichiral center-bearing beta-blockers, nadolol and labetalol. These polymers include polysodium N-undecenoxy carbonyl-L-leucinate (poly-L-SUCL) and polysodium N-undecenoxy carbonyl-L-isoleucinate (poly-L-SUCIL). Detailed synthesis and characterization were reported in our previous paper [26]. It was found that poly-L-SUCIL gives better chiral separation than poly-L-SUCL for both nadolol and labetalol isomers. The use of 50-100 mM poly-L-SUCIL as a single chiral selector provided separation of four and three isomers of labetalol and nadolol, respectively. Further optimization in separation of both enantiomeric pairs of nadolol and labetalol was achieved by evaluation of type and concentration of organic solvents, capillary temperature as well type and concentration of cyclodextrins. A synergistic approach, using a combination of poly-L-SUCIL and sulfated beta-CD (S-beta-CD) was evaluated and it showed dramatic separation for enantiomeric pairs of nadolol. On the other hand for labetalol enantiomers, separation was slightly decreased or remain unaffected using the dual chiral selector system. Finally, simultaneous separation of both nadolol and labetalol enantiomers was achieved in a single run using 25 mM poly-L-SUCIL and 5% w/v of S-beta-CD in less then 35 min highlighting the importance of high-throughput chiral analysis. PMID:15004846

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



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


A series of new ternary zinc(II) complexes [Zn(L(1-10))(phen)], where phen is 1,10-phenanthroline and H(2)L(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, (1)H NMR, and (13)C NMR spectra. The IR spectra of the complexes showed large differences between nu(as)(COO) and nu(s)(COO), Deltanu (nu(as)(COO)-nu(s)(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. PMID:17049913

Boghaei, Davar M; Gharagozlou, Mehrnaz



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

PubMed Central

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

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



Molecular dynamics simulation of aqueous solutions of 26-unit segments of p(NIPAAm) and of p(NIPAAm) "doped" with amino acid based comonomers.  


We have performed 75-ns molecular dynamics (MD) simulations of aqueous solutions of a 26-unit NIPAAm oligomer at two temperatures, 302 and 315 K, below and above the experimentally determined lower critical solution temperature (LCST) of p(NIPAAm). We have been able to show that at 315 K the oligomer assumes a compact form, while it keeps a more extended form at 302 K. A similar behavior has been demonstrated for a similar NIPAAm oligomer, where two units had been substituted by methacryloyl-l-valine (MAVA) comonomers, one of them being charged and one neutral. For another analogous oligomer, where the same units had been substituted by methacryloyl-l-leucine (MALEU) comonomers, no transition from the extended to the more compact conformation has been found within the same simulation time. Statistical analysis of the trajectories indicates that this transition is related to the dynamics of the oligomer backbone, and to the formation of intramolecular hydrogen bonds and water-bridges between distant units of the solute. In the MAVA case, we have also evidenced an important role of the neutral MAVA comonomer in stabilizing the compact coiled structure. In the MALEU case, the corresponding comonomer is not equally efficacious and, possibly, is even hindering the readjustment of the oligomer backbone. Finally the self-diffusion coefficient of water molecules surrounding the oligomers at the two temperatures for selected relevant times is observed to characteristically depend on the distance from the solute molecules. PMID:18759405

Gangemi, Fabrizio; Longhi, Giovanna; Abbate, Sergio; Lebon, France; Cordone, Roberto; Ghilardi, Gian Paolo; Fornili, Sandro L



Raman spectra of amino acids and their aqueous solutions  

NASA Astrophysics Data System (ADS)

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

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



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


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

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



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

PubMed Central

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), ?-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 (?z). SACV and VACV exhibited approximately five fold increase in area under the curve (AUC) values relative to ACV (p<0.05). Cmax(T) (maximum concentration) of SACV was observed to be 39 ± 22 µM in plasma which is 2 times better than VACV and 15 times better than ACV. Clast(T) (concentration at the last time point) of SACV was observed to be 0.18 ± 0.06 µM in plasma which is 2 times better than VACV and 3 times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (Cmax) and eliminated at varying rates (?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.

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



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

PubMed Central

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

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



Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli.  


Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme. PMID:2005118

Eisenstein, E



Preferential Treatment: Interaction Between Amino Acids and Minerals  

NASA Astrophysics Data System (ADS)

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.

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



Diversity of l-leucine catabolism in various microorganisms involved in dairy fermentations, and identification of the rate-controlling step in the formation of the potent flavour component 3-methylbutanal  

Microsoft Academic Search

Various microorganisms, belonging to the genera Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Propionibacterium, Brevibacterium, Corynebacterium and Arthrobacter, used in dairy fermentations such as cheese making, were analysed for their potential to convert leucine into flavour components, most notably 3-methylbutanal. A large variation between and within species was observed for various enzyme activities involved in the conversion pathway, e.g. transaminases, a-hydroxy acid

B. A. Smit; W. J. M. Engels; J. T. M. Wouters; G. Smit



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


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

Anantharaj, S; Jayakannan, M



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

PubMed Central

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.

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



Ocular Sustained Release Nanoparticles Containing Stereoisomeric Dipeptide Prodrugs of Acyclovir  

PubMed Central

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

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



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.  


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. PMID:16966812

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



Synthesis and characterization of new polyamides derived from alanine and valine derivatives  

PubMed Central

Background Many efforts have been recently devoted to design, investigate and synthesize biocompatible, biodegradable polymers for applications in medicine for either the fabrication of biodegradable devices or as drug delivery systems. Many of them consist of condensation of polymers having incorporated peptide linkages susceptible to enzymatic cleavage. Polyamides (PAs) containing ?-amino acid residues such as L-leucine, L-alanine and L-phenylalanine have been reported as biodegradable materials. Furthermore, polyamides (PAs) derived from C10 and C14 dicarboxylic acids and amide-diamines derived from 1,6-hexanediamine or 1,12-dodecanediamine and L-phenylalanine, L-valyl-L-phenylalanine or L-phenylalanyl-L-valine residues have been reported as biocompatible polymers. We have previously described the synthesis and thermal properties of a new type of polyamides-containing amino acids based on eight new symmetric meta-oriented protected diamines derived from coupling of amino acids namely; Fomc-glycine, Fmoc-alanine, Fomc-valine and Fomc-leucine with m-phenylene diamine or 2,6-diaminopyridine. Results revealed that incorporation of pyridine onto the polymeric backbone of all series decreases the thermal stability. Here we describe another family of polyamides based on benzene dicarboxylic acid, pyridine dicarboxylic acid, and ?-amino acid linked to benzidine and 4,4?-oxydianiline to study the effect of the dicarboxylic acid as well as the amino acids on the nature and thermal stability of the polymers. Results We report here the preparation of a new type of polyamides based on benzene dicarboxylic acid, pyridine dicarboxylic acid, and ?-amino acid linked to benzidine and 4,4?-oxydianiline to study the effect of the dicarboxylic acid as well as the amino acids on the nature and thermal stability of polymers. The thermal properties of the polymers were evaluated by different techniques. Results revealed that structure-thermal property correlation based on changing the dicarboxylic acid monomer or the diamine monomer demonstrated an interesting connection between a single change (changing the dicarboxylic acids in each series while the diamine is fixed) and thermal properties. The newly prepared polymers may possess biodegradability and thus may find some applications as novel biomaterials. Conclusions The thermal properties of the new type of polyamides based on benzene dicarboxylic acid, pyridine dicarboxylic acid, and ?-amino acid (alanine and valine) linked to benzidine and 4,4?-oxydianiline were evaluated by thermal gravimetric (TG), differential thermal gravimetric (DTG) and differential thermal analysis (DTA) techniques. Results revealed that the structure-thermal property correlation based on changing the dicarboxylic acid monomer or the diamine monomer demonstrated an interesting connection between a single change (changing the dicarboxylic acids in each series while the diamine is fixed) and thermal properties. In addition, pyridine-containing polymers exhibited semicrystalline characteristic with melting temperature, Tm. where none of the valine-containing polymers showed a melting and crystallization peak indicating that the polymers were amorphous. This is expected since L-valine side chain can inhibit close packing and eliminate crystallization. The newly prepared polymers may possess biodegradability and thus may find some applications as novel biomaterials.



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

PubMed Central

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

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



Cloning and functional characterization of a system ASC-like Na+-dependent neutral amino acid transporter.  


A cDNA was isolated from mouse testis which encodes a Na+-dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40-44% identity), Na+-dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G.(1993) J. Biol. Chem. 268, 15329-15332; Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. O., Guadano-Ferraz, A., and Fremeau, T., Jr. (1993) J. Biol. Chem. 268, 15351-15355) and a mouse adipocyte differentiation-associated gene product AAAT (94% identity; Liao, K., and Lane, D.(1995) Biochem. Biophys. Res. Commun. 208, 1008-1015). When expressed in Xenopus laevis oocytes, ASCT2 exhibited Na+-dependent uptakes of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine at high affinity with Km values around 20 microM. L-Methionine, L-leucine, L-glycine, and L-valine were also transported by ASCT2 but with lower affinity. The substrate selectivity of ASCT2 was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (Km = 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na+ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na+ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na+. Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na+-dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family. PMID:8662767

Utsunomiya-Tate, N; Endou, H; Kanai, Y



Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes  

Microsoft Academic Search

.   When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1\\u000a is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8,

S. Vincenti; M. Castagna; A. Peres; V. F. Sacchi



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

NASA Technical Reports Server (NTRS)

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.

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




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...


A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases  

Microsoft Academic Search

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of l-leucine with ?-ketoglutarate results in formation of ?-ketoisocaproate, which is reductively aminated back to l-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340nm due to NADH oxidation is measured continuously. The specific activities obtained by

Arthur J. L. Cooper; Myra Conway; Susan M. Hutson



Reduction of the off-flavor volatile generated by the yogurt starter culture including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in soymilk.  


Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus establish a symbiotic relationship in milk; however, S. thermophilus predominantly grows in soymilk. This study determined that excess diacetyl was notably generated mainly by S. thermophilus in soymilk, and this flavor compound created an unpleasant odor in fermented soymilk. The addition of l-valine to soymilk reduced the amount of diacetyl and increased the levels of acetoin during fermentation by S. thermophilus . In addition, it was found that the expression of the ilvC gene was repressed and that of the als and aldB genes was stimulated in S. thermophilus by l-valine. Sensory evaluations with the triangle difference test and a preference test showed that the soymilk fermented with l-valine was significantly preferred compared with that without l-valine. In this study, we successfully controlled the metabolic flux of S. thermophilus in soymilk and produced more favorable fermented soymilk without the use of genetically modified lactic acid bacteria strains. PMID:24495115

Kaneko, Daisuke; Igarashi, Toshinori; Aoyama, Kenji



Kinetic Evidence for Inter-Domain Communication in the Allosteric Regulation of ?-Isopropylmalate Synthase from M. tuberculosis†  

PubMed Central

The enzyme ?-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been identified as a possible target for design of new anti-tubercular therapeutics. Recently it was shown that MtIPMS is subject to slow-onset, feedback inhibition by L-leucine, the first instance of an allosteric regulator utilizing this mechanism. Structural studies are inconsistent with allosteric mechanisms including changes to the quarternary structure or large, rigid-body conformational changes to the enzyme upon L-leucine binding. Thus, the allosteric regulation may result from a discrete inhibitory signal transmitted to the active site upon L-leucine binding in the regulatory domain, a distance of over 50 Å In order to test this mechanism, site-directed mutagenesis was employed to construct enzymes with substitutions at phylogenetically conserved active site residues near the interface of the catalytic and linker domains. The substitutions had wide ranging effects on the kinetics of L-leucine inhibition, with some modest effects on the kinetic parameters of catalysis. The most dramatic result was the finding that the Y410F mutant form of MtIPMS is insensitive to L-leucine inhibition, suggesting that this residue has completely uncoupled the inhibitory signal to the active site. Overall, the data are consistent with a mechanism of allosteric regulation described by the inter-domain communication of the inhibitory signal from the regulatory to catalytic domain and implicate the interaction between the linker and catalytic domains as critical determinants of inhibitory signal transmission.

de Carvalho, Luiz Pedro S.; Frantom, Patrick A.; Argyrou, Argyrides; Blanchard, John S.



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


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

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




Microsoft Academic Search

Fluorescence properties of purified isoleucyl-tRNA-synthetase isolated from E. coli B have been studied. No changes in the quantum yield, energy or polarization of the emission were detected in the presence (either individually or in combinations) of the substrates and cofactors required for activation of L-isoleucine. In 2.5 M urea enzyme activity and intrinsic fluorescence intensity (at 340 nm) each decrease

Geoffrey R. Penzer; Edward L. Bennett; Melvin. Calvin



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

SciTech Connect

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.

Hada, Masaki; Hontani, Yusaku; Ichiki, Kazuya; Seki, Toshio [Department of Nuclear Engineering, Kyoto University, Sakyo, Kyoto, 606-8501 (Japan); Ibuki, Sachi; Ninomiya, Satoshi; Matsuo, Jiro [Quantum Science and Engineering Center, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Aoki, Takaaki [Department of Electronic Science and Engineering, Kyoto University, Nishigyo, Kyoto, 615-8530 (Japan)



Synthesis, Characterization, and Catalytic Activity of Polymer Anchored Amino Acid Mn(II) Complexes  

Microsoft Academic Search

Chloromethylated styrene?divinylbenzene copolymer beads with 6% and 8% crosslink were chemically modified by reaction with L?valine leading to the incorporation of nitrogen and oxygen as ligating sites on the surface of the polymer. These polymeric ligands on treatment with a solution of manganese(II) acetate gave the corresponding metal complex. The polymer supported Mn(II) complexes were characterized by elemental analyses, FT?IR,

V. B. Valodkar; G. L. Tembe; M. Ravindranathan; R. N. Ram; H. S. Rama



Absorption of neutral and basic amino acids across the body surface of two annelid species  

Microsoft Academic Search

Kurzfassung In Versuchen zur Aufnahme verschiedener Aminosäuren durch die Körperoberfläche bei dem OligochaetenEnchytraeus albidus und dem PolychaetenNereis diversicolor betrug die Absorptionsrate von14C-L-Arginin aus einer Umgebungskonzentration von 10 µM nur wenige Prozent der für Glycin und L-Valin bestimmten Werte. Nach einer halbstündigen Versuchsdauer waren neutrale14C-Aminosäuren im Organismus 9- bis 15fach gegenüber dem Medium angereichert, die L-Argininverteilung blieb dagegen unterhalb des Äquilibriums.

D. Siebers



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

Microsoft Academic Search

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

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



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

Microsoft Academic Search

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

M. Sivasankaran Nair; C. Ravi Samuel Raj



Quantitative determination of small amounts of L-penicillamine in D-penicillamine after desulfurization with Raney nickel.  


Commercially available D-penicillamine is desulfurized with Raney nickel at room temperature in aqueous solution. L-Valine derived from contaminating L-penicillamine is determined colorimetrically by the amino acid oxidase/peroxidase reaction, using 2,2'-azino-di-[3-ethyl-benz-thiazoline-sulfonate(6)] (ABTS). The procedure assures the quantitation of L-penicillamine contaminating D-penicillamine down to the level of 0.1%. PMID:7387748

Lodemann, E; El-Kirdassy, Z H; Wacker, A



Indium-copper-mediated barbier-grignard-type alkylation reaction of imines in aqueous media.  


An efficient system of In/CuI/InCl3 was developed for Barbier-Grignard-type alkylation reactions of simple imines, using a one-pot condensation of various aldehydes, amines (including aliphatic and chiral version), and alkyl iodides in water or aqueous media. The reactions proceeded efficiently at room temperature to give the desired products in moderate to good yields. Good diastereoselectivities were obtained when using L-valine methyl ester as substrate. PMID:18052071

Shen, Zhi-Liang; Loh, Teck-Peng



Secondary structures of peptides  

Microsoft Academic Search

75.46 MHz 13C-NMR CP\\/MAS spectra of solid poly(1-alanines) with various degrees of polymerization were measured. The signals of all three carbons are split into two peaks. One peak presents the a-helix structure and one the pleated sheet form. Equimolar mixtures of helical poly(L-alanine) with poly(glycine) and poly(L-valine) were measured under various conditions. It was found that the peak areas are

Detlef Müller; Hans R. Kricheldorf



Continuous Enzymatic Synthesis with Coenzyme Regeneration.  

National Technical Information Service (NTIS)

NAD(H) dependent enzymatic production of L-alanine and L-leucine was performed in an enzyme membrane reactor (EMR). An EMR is a continuous stirred tank reactor which is equipped with an ultrafiltration (UF) membrane at the exit of the reactor to retain en...

R. Wichmann



Constituents of cultivated Agaricus blazei.  


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 ?M). PMID:21240679

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



Studies on Human Antibodies. IV. Purification and Properties of Anti-a and Anti-B Obtained by Absorption and Elution from Insoluble Blood Group Substances.  

National Technical Information Service (NTIS)

Insoluble blood group substances prepared by copolymerization of soluble blood group substances with N-carboxy-L-leucine anhydride were used to absorb blood group antibodies from two human, high-titered anti-A sera. After the absorbants were washed free o...

M. E. Kaplan E. A. Kabat



Role of L-threonine dehydrogenase in the catabolism of threonine and synthesis of glycine by Escherichia coli.  

PubMed Central

The enzyme L-threonine dehydrogenase was demonstrated in extracts of Escherichia coli K-12, and was shown to be the first enzyme of the pathway converting threonine to glycine. The enzyme was induced by L-leucine, but not by its substrate, L-threonine. The metabolic significance of leucine as a catabolic signal for amino acid degradation is considered.

Newman, E B; Kapoor, V; Potter, R



Acetone Formation in the Vibrio Family: a New Pathway for Bacterial Leucine Catabolism  

PubMed Central

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of l-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. l-Leucine, but not d-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of l-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only ?-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d7)-l-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.

Nemecek-Marshall, Michele; Wojciechowski, Cheryl; Wagner, William P.; Fall, Ray



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

PubMed Central

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

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



Synthesis and characterization of new optically active and organosoluble poly(ester-imide)s based on bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic diimide by direct polycondensation  

Microsoft Academic Search

New optically active poly(ester-imide)s PEIs were prepared from newly synthesized N,N?-(bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic)-bis-l-isoleucine diacid 4 via direct polycondensation with various aromatic diols in a system of tosyl chloride (TsCl), pyridine (Py), and N,N-dimethylformamide (DMF). The reactions with bicyclo TsCl were significantly promoted by controlling alcoholysis with diols,\\u000a in the presence of catalytic amounts of DMF, to give a series of optically active

Khalil Faghihi; Meisam Shabanian; Mohsen Hajibeygi



Conquering isoleucine auxotrophy of Escherichia coli BLR(DE3) to recombinantly produce spider silk proteins in minimal media.  


Large-scale production of recombinant spider silk proteins is a long-term goal for their industrial use. Therefore, we have recently developed a process for bacterial production. Due to a highly repetitive gene sequence of spider silks, the host strain E. coli BLR(DE3) was employed since it shows no homologue recombination. Although perfectly suited for production in full media, the BLR strain does not grow in cost-effective minimal media, indicating a previously not reported L: -isoleucine auxotrophy. We provide evidence that mutated threonine deaminase is likely responsible for the detected auxotrophy of BLR. PMID:17611722

Schmidt, Martin; Römer, Lin; Strehle, Martin; Scheibel, Thomas



Isoleucine as a possible bridge between exogenous delivery and terrestrial enhancement of homochirality.  


We report a highly enantioselective oligomerization of isoleucine stereomers in the salt-induced peptide formation reaction under plausibly prebiotic earth conditions. Up to 6.5-fold superiority in reactivity of L-isoleucine was observed, compared to its D-enantiomer, after 14 evaporation cycles in the presence of Cu(2+) and NaCl. Since isoleucine is among the proteinogenic amino acids that were found enantioenriched in meteorites, this present work may further correlate the extraterrestrial delivery and endogenous production of biological homochirality by virtue of a protein constituent rather than the rarely occurring ?-methylated amino acids. PMID:22968664

Li, Feng; Fitz, Daniel; Rode, Bernd M



Amino Acid Transport by the Filamentous Fungus Arthrobotrys conoides1  

PubMed Central

Uptake of l-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. l-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 × 10?5 to 7.5 × 10?5m. Modification of the amino or carboxyl group abolishes l-valine uptake. The process is competitively inhibited by d-valine, glycine, and other neutral amino acids (Ki = 1.5 × 10?5 to 4.0 × 10?5m), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of Ki values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge.

Gupta, Rishab K.; Pramer, David



A New Peptide Isolated from a Marine Derived Streptomyces bacillaris  

PubMed Central

A new peptide, l-O-Lac-l-Val-d-O-Hiv-d-Val (1), consisting of d-valine, l-valine, l-lactic acid, and 3-d-hydroxyisovaleric acid, was isolated from the culture of the marine sediment derived Streptomyces bacillaris. The planar structure of compound 1 was assigned by 1D, 2D NMR and mass spectroscopic analyses. Following acid and base hydrolysis, the absolute configuration of the valine residues in 1 were determined by application of the advanced Marfey’s method and the absolute configurations of hydroxy acids units were determined by a HPLC method based on Mosher’s reagents.

Hu, Youcai



Molar extinction coefficients in aqueous solutions of some amino acids  

Microsoft Academic Search

Mass attenuation coefficients of amino acids viz. glycine (C2H5NO2), l-Serine (C3H7NO3), l-Theronine (C4H9NO3), l-Proline (C5H9NO2), l-Valine (C5H11NO2) and l-Phenylalanine (C9H11NO2) in aqueous solutions have been determined at 81, 356, 511, 662, 1173 and 1332 keV by the gamma-ray transmission method in a narrow beam good geometry setup. Precisely measured densities of these solutions were used for the determination of these

Kulwant Singh; G. K. Sandhu; Gagandeep Kaur; B. S. Lark



Indium-silver- and zinc-silver-mediated barbier-grignard-type alkylation reactions of imines by using unactivated alkyl halides in aqueous media.  


In the presence of In or Zn/AgI/InCl(3), an efficient and practical method for the Barbier-Grignard-type alkylation reactions of simple imines by using a one-pot condensation of various aldehydes, amines (including the aliphatic and chiral version), and secondary alkyl iodides has been developed. The reaction proceeded more efficiently in water than in organic solvents. Without the use of CuI, it mainly gave the imine self-reductive coupling product, which was not the alkylated product. Good diastereoselectivities (up to 92:8 dr) were obtained when L-valine methyl ester was used as the substrate. PMID:18058963

Shen, Zhi-Liang; Cheong, Hao-Lun; Loh, Teck-Peng



Valine requirement of nursery pigs.  


Six experiments were conducted to determine the true digestible valine requirement of 5- to 20-kg pigs. In Exp. 1, a valine-deficient diet for 5- to 10-kg pigs was developed and validated in terms of growth performance in response to supplemental L-valine. A different basal diet was validated for 10- to 20-kg pigs in Exp. 2. Both diets were demonstrated to be deficient in valine and to support performance equivalent to typical nursery diets when fortified with L-valine. In Exp. 3, true ileal digestibility of valine in the two basal diets was determined in eight pigs fitted with a simple T-cannula at the terminal ileum. Another four pigs received an enzymatically hydrolyzed casein-based diet to determine endogenous contributions to collected ileal digesta. The two diets were found to have true valine digestibilities of 82% (5- to 10-kg pigs) and 86% (10- to 20-kg pigs). In Exp. 4, 80 weaned pigs (5.8 kg) were offered the basal diet fortified with five incremental doses (0.08%) of L-valine. Weight gain increased quadratically (P < 0.05) with increasing levels of valine. Broken-line analysis revealed a true digestible valine requirement of 0.86 +/- 0.03%. In Exp. 5, the true digestible valine requirement of 10- to 20-kg pigs was estimated with 120 pigs (10.9 kg) using the second basal diet fortified with six incremental doses (0.05%) of L-valine. The data suggested a digestible valine requirement level of about 0.775%, which was reevaluated in Exp. 6, wherein pigs did not respond to levels of digestible valine higher than 0.775%. In conclusion, requirement estimates were 2.50 and 2.22 g of true digestible valine per megacalorie of ME for 5- to 10- and 10- to 20-kg pigs, respectively. These empirical estimates are in close agreement with recent estimates of the National Research Council Subcommittee on Swine Nutrition of 2.48 and 2.11 g of true digestible valine per megacalorie of ME, respectively. PMID:11374542

Mavromichalis, I; Kerr, B J; Parr, T M; Albin, D M; Gabert, V M; Baker, D H



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

Microsoft Academic Search

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

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



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


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

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



The binding site of zinc and indium metal to amino acid derivatized squarate complexes - Implications in inhibitor and mediator designs.  


Three novel metal squaric acid-peptide complexes, SQI-SQIII were prepared by addition of indium triflate or zinc chloride to the previously reported compounds [1], 3-(hydroxymethylamino)-4-(l-isoleucine methyl ester)-3-cyclobutene-1,2-dione (squarate 1), and 3-(hydroxymethylamino)-2-(l-isoleucine methyl ester)-4-thioxo-2-cyclobuten-1-one (squarate 2). The structures of SQI-SQIII were elucidated using NMR analysis. The electrochemical applications of two of these metal-squaric acid systems (SQI and SQII) were also investigated. Incorporation of SQII as a mediator, in the previously optimized Pt/p(HEMA)/p(pyrrole)/GOx electrode using the ionic liquid [bmim][BF(4)] as the solvent medium, produced a biosensor with enhanced properties, namely a sensitivity of 175.9mA/M d-glucose, working potential of +200mV, large linear range (0-12mM) and a detection limit of 1x10(-6)M. PMID:20598337

Ramroop-Singh, Natasha; Narinesingh, Dyer; Singh, Gurdial; Seto, Christopher T; Comeau, Anthony B



Effects of insulin secretagogues on phospholipid metabolism in pancreatic beta-cells.  


The effect of insulin secretagogues on the incorporation of [32-P] orthophosphate into phospholipids was studied in microdissected islets from obese-hyperglycemic mice. Increased 32-P-labelling was observed after incubation for 60 min with 10 mM L-leucine, 10 mM L-arginine or 20 mM D-glucose. Most of the label occurred in the phosphatidyl inositol fraction. The effect of L-leucine was additive to that induced by D-glucose while the effect of L-arginine was not. Glibenclamide (0.05 mM) was ineffective whether or not D-glucose was present. The results suggest that there is no direct correlation between the releasing actions of insulin secretagogues and changes in the metabolism of certain phospholipids and that phospholipid metabolism may be stimulated through more than one mechanism. PMID:804934

Fex, G; Lernmark, A



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

PubMed Central

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

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



The barbamide biosynthetic gene cluster: a novel marine cyanobacterial system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) origin involving an unusual trichloroleucyl starter unit  

Microsoft Academic Search

Barbamide was extracted from the marine cyanobacterium Lyngbya majuscula strain 19L as a chlorinated lipopeptide for its potent molluscicidal activity. Precursor incorporation studies indicated that it is derived from acetate, l-phenylalanine, l-leucine and l-cysteine. The gene cluster responsible for biosynthesis of barbamide (bar) was cloned and characterized in this study. DNA sequence analysis of cosmid pLM49 revealed a cluster of

Zunxue Chang; Patricia Flatt; William H. Gerwick; Viet-Anh Nguyen; Christine L. Willis; David H. Sherman



Purification and characterization of leucine dehydrogenase from an alkaliphilic halophile, Natronobacterium magadii MS3  

Microsoft Academic Search

Leucine dehydrogenase (l-leucine: NAD+ oxidoreductase, deaminating, EC was purified to homogeneity from the crude extract of an alkaliphilic halophile, Natronobacterium magadii MS-3, with a yield of 16%. The enzyme had a molecular mass of about 330kDa and consisted of six subunits identical in molecular mass (55kDa). The enzyme required a high concentration of salt for stability and activity. It

Reina Katoh; Shinji Ngata; Akira Ozawa; Toshihisa Ohshima; Masahiro Kamekura; Haruo Misono



Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus  

Microsoft Academic Search

Leucine dehydrogenase (l-leucine: NAD+ oxidoreductase, deaminating, EC has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its

Toshihisa Ohshima; Shinji Nagata; Kenji Soda



Cloning, sequencing and overexpression of the leucine dehydrogenase gene from Bacillus cereus 1 The sequence data reported in this paper have been submitted to the EMBL\\/GenBank Data Libraries under the accession number BankIt27708U51099 1  

Microsoft Academic Search

The l-leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced. The open reading frame has 1101 bp and codes for a protein of 39.9 kDa. The deduced amino acid sequence of the LeuDH from B. cereus shares 70–80% identity with LeuDH's from the thermophilic strains B. stearothermophilus and Thermoactinomyces intermedius. The active

Tanja Stoyan; Achim Recktenwald; Maria-Regina Kula



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

Microsoft Academic Search

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

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



Anti-angiogenic activity of ursodeoxycholic acid and its derivatives  

Microsoft Academic Search

Ursodeoxycholic acid (UDCA) and its derivatives were examined for anti-angiogenic activities by using the chick embryo chorioallantoic membrane (CAM) assay. The presence of UDCA or its derivatives inhibited angiogenesis in a dose-dependent manner; the dose required for half-maximal inhibition (ID50) was 4 ?g per CAM for UDCA. The conjugate forms of UDCA with glycine methyl ester (HS-1030), l-leucine benzyl ester

Hongsuk Suh; Eun-Jin Jung; Tae-Hyong Kim; Ho-Young Lee; Yoo-Hoi Park; Kyu-Won Kim



Microbial uptake of dissolved organic matter in Mcmurdo Sound, Antarctica  

Microsoft Academic Search

The distribution and activity of bacterioplankton, and the turnover of dissolved organic matter (DOM) were examined in McMurdo Sound, Antarctica. On the eastern side of the Sound, bacteria averaged 6.5×108 l-1, and turnover rates of dissolved adenosine triphosphate, D-glucose and l-leucine averaged 16, 116 and 124 h, respecitvely. These molecules as well as thymidine were taken up maximally from 0°

R. E. Hodson; F. Azam; A. F. Carlucci; J. A. Fuhrman; D. M. Karl; O. Holm-Hansen



Polyamino Acids as Man-Made Catalysts  

Microsoft Academic Search

Polyamino acids are easy to prepare by nucleophile-initiated polymerisation of amino acid N-carboxyanhydrides. Polymers such as poly-(l)-leucine act as robust catalysts for the epoxidation of a wide range of electron-poor alkenes, such as ?-substituted ?,?-unsaturated ketones. The optically active epoxides so formed may be transformed into heterocyclic compounds, polyhydroxylated\\u000a materials and biologically active compounds such as diltiazem and taxol side

Joanne V. Allen; Stanley M. Roberts; Natalie M. Williamson


Neuroglobin is up-regulated in the cerebellum of pups exposed to maternal epileptic seizures  

Microsoft Academic Search

To evaluate a potential insult in the cerebellum of pups exposed to maternal epileptic seizures during intrauterine life, female rats were subjected to pilocarpine-induced epilepsy. Pups from different litters were sacrificed at 1, 3, 7 and 14 post-natal days (PN) and neuroglobin (Ngb) and gliosis were analyzed in the cerebellum by Western blotting (WB) and RT-PCR. 14C-l-leucine-[14C-Leu] incorporation was used

Daiana Correia Lima; Ana Carolina Cossa; Sandra Regina Perosa; Elaine Menezes de Oliveira; José Antonio da Silva; Maria José da Silva Fernandes; Iara Ribeiro da Silva; Elisa Mieko Suemitsu Higa; Maria da Graça Naffah-Mazzacoratti; Esper Abrão Cavalheiro; Débora Amado



Enzymatic activities of activated sludge  

Microsoft Academic Search

Summary As a potential measure of active biomass in an automated and continuous system, enzyme activities of activated sludge were sought that would allow a rapid and accurate determination. Using p-nitrophenylphosphate, bis-p-nitrophenylphosphate, p-nitrophenyl-a- and ß-D-gluco- or galactopyranosides, and the p-nitroanilides of L-alanine, L-leucine, L-lysine, L-glutamic acid and L-phenylalanine, we could establish that the corresponding phosphatase, cyclic phosphodiesterase, glycosidase and aminopeptidase

M. Teuber; K. E. U. Brodisch



Digestive-absorptive function of the intestinal brush border in uremia1  

Microsoft Academic Search

Amino acid absorption was studied in chronic uremic rats. Intestinal transport of L-leucine appears to be inhibited with mild uremic intoxication, whereas severe uremia enhances absorption. Brush border activity of intestinal maltase and disaccharidases is higher in rats with chronic renal insufficiency. The same holds for -y-glutamyl-transpeptidase activity. Am. J. Clin. Nut,. 31: 1642-1646, 1978. Although morphological disorders of the

V. Wizemann; D. Ludwig; R. Kuhi; I. Burgmann


New observations on peptide bond formation using CDMT  

Microsoft Academic Search

The optimized formation of the peptide bond by means of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) has been found to occur rapidly and essentially quantitatively in a one-pot, one-step procedure. This new method is effective for the coupling of a variety of reactive partners, including chiral amino acids (e.g. N-acetyl-l-leucine) without significant loss of configuration. Significant racemization was observed when the typical literature conditions

Christine E. Garrett; Xinglong Jiang; Kapa Prasad; Oljan Repi?



Encapsulation–release property of amphiphilic hyperbranched d-glucan as a unimolecular reverse micelle  

Microsoft Academic Search

The synthesis of a novel unimolecular reverse micelle, the hyperbranched d-glucan carbamate (3), was accomplished through the carbamation reaction of the hyperbranched d-glucan (1) with the N-carbonyl l-leucine ethyl ester (2) in pyridine at 100°C. Polymer 3 was soluble in a large variety of organic solvents, such as methanol, acetone, chloroform, and ethyl acetate, and insoluble in water, which remarkably

Yoshikazu Kitajyo; Tomoko Imai; Yoko Sakai; Masaki Tamaki; Hirofumi Tani; Kenji Takahashi; Atsushi Narumi; Harumi Kaga; Noriaki Kaneko; Toshifumi Satoh; Toyoji Kakuchi



The hydrophobicity of bacteria — An important factor in their initial adhesion at the air-water inteface  

Microsoft Academic Search

Bacteria isolated from the surface and the subsurface water at four stations along the Swedish west coast were assessed for their hydrophobicity with hydrophobic interaction chromatography (HIC). The surface bacteria were sampled by the Teflon sheet technique. [3H]-l-leucine metabolically labeled isolates were run on a column packed with Octyl-Sepharose CL-4B gel. The relative hydrophobicity of the bacteria was expressed as

Björn Dahlbäck; Malte Hermansson; Staffan Kjelleberg; Birgitta Norkrans



A facile and efficient method for preparation of chiral supported poly(styrene–divinylbenzene) copolymers  

Microsoft Academic Search

A new and efficient method for preparation of optically active poly(styrene–divinylbenzene) copolymers (PS-DVB) is presented here. This is carried out by Friedel–Crafts acylation reaction of chiral N-phthaloyl l-leucine acid chloride with PS-DVB beads in the presence of aluminum chloride as Lewis acid catalyst and 1,2-dichloroethane as the solvent at ambient temperature. Reagents’ amounts and reaction conditions are mentioned and four

Ali-Reza Mahdavian; Sepideh Khoee



Activity of protease extracted from rice-rhizosphere soils under double cropping of rice and wheat  

Microsoft Academic Search

Protease-active extracts were prepared from 2 rice-rhizosphere and non-rhizosphere soil samples 3 proteolytic isolates from the soil samples which were taken from water logged paddy fields (Gray Lowland Soils) under double cropping of rice and wheat that had been treated with chemical fertilizer (CF) or organic manure (OM). The activities were assayed and characterized using an artificial substrate, benzyloxycarbonyl-L-phenylalanyl-L-tyrosyl-L-leucine (ZFTL).

Koichi Hayano; Katsuji Watanabe; Susumu Asakawa



Proteasome inhibition induces both pro- and anti-cell death pathways in prostate cancer cells  

Microsoft Academic Search

The proteasome-mediated protein degradation is critical for regulation of a variety of cellular processes, including cell cycle, cell death, differentiation and immune response. Proteasome inhibitors have recently been shown to be potent anti-cancer agents against a variety of cancer cells. Our study demonstrated that proteasome inhibitor MG132 (carbobenzoxy-l-leucyle-l-leucyl-l-leucinal) was a potent death-inducing agent for PC3 prostate cancer cells. MG132-induced cell

Wending Yang; Jason Monroe; Yonghong Zhang; David George; Eric Bremer; Honglin Li



Copper(II) and Zinc(II) Complexes of Schiff Bases Derived from Amino Acids and O-Vanillin  

Microsoft Academic Search

Some new Cu(II) and Zn(II) complexes of Schiff bases derived from o-vanillin and glycine, DL-alanine, DL-valine, DL-methionine, L-leucine, and L-phenylalanine have been synthesized and characterized by elemental analyses, molar electrical conductivity, electronic and infrared spectra, and thermogravimetric analyses. The results suggested that the Schiff bases are bivalent anions with tridentate ONO donors derived from the carboxylate oxygen, imino nitrogen, and

Guangbin Wang; James C. Chang



Primary Tissue Culture of Normal Adult Human Decapsulated Adrenal Cortex: Radioautographic Studies on the Metabolic Effects of ACTH1-24  

Microsoft Academic Search

ACTH-deprived primary normal adult human adrenocortical cells on the 23rd day in vitro were found to lack any DNA synthetic and proliferative activity, but incorporated uridine-3H and l-leucine-3H actively. In cortical cells of the same age treated for 2 and 7 days with ACTH1-24 (3 X 10-6M) the incorporation of the precursors for RNA and DNA synthesis and the cell

Ubaldo Armato; Paola G. Andreis; Enrica Draghi; Virgilio Meneghelli



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

PubMed Central

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



LAT1 overexpression and function compensates downregulation of ASCT2 in an in vitro model of renal proximal tubule cell ageing.  


Amino acid transporters provide cells neutral amino acids indispensable for growth and proliferation-dependent protein synthesis. This study evaluates whether prolonged serial cell passaging during 6 months (over 50 passages) may induce changes in amino acid transporters properties in Opossum kidney (OK) proximal tubular cells. High passage OK cells exhibit polyploidy, but no difference in the proliferation potential was observed when compared to low passage OK cells. Increased time in culture was accompanied by an increased total, membrane and cytosol protein content. The Na(+)-insensitive [(14)C]-L-leucine uptake was promoted almost exclusively thought LAT1 (~ 90 vs 80%, high versus low passage OK cells). The increased LAT1 protein abundance in high passage OK cells correlated positively with enhanced ability to take up [(14)C]-L-leucine, despite a 4.3-fold decrease in affinity for the substrate. The Na(+)-sensitive [(14)C]-L-alanine transport was decreased by 2.5-fold in high passage OK cells. However, no differences in ASCT2 expression were observed between high and low passage OK cells. It is concluded that OK cells show functional differences in both L-leucine and L-alanine uptake as a function of passage time in culture. The increased expression and activity of LAT1 in high passage OK cells may correspond to a mechanism enabling the cell to develop the hypertrophy response to prolonged cell passaging, when the function of ASCT2 is markedly depressed. PMID:21125315

Pinho, Maria João; Cabral, José Miguel; Silva, Elisabete; Serrão, Maria Paula; Soares-da-Silva, Patrício



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

SciTech Connect

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.

Hada, Masaki; Ibuki, Sachi; Ninomiya, Satoshi; Matsuo, Jiro [Quantum Science and Engineering Center, Kyoto University, Uji 611-0011 (Japan); Hontani, Yusaku; Yamamoto, Yasuyuki; Ichiki, Kazuya; Seki, Toshio [Department of Nuclear Engineering, Kyoto University, Kyoto 606-8501 (Japan); Aoki, Takaaki [Department of Electronic Science and Engineering, Kyoto University, Kyoto 615-8530 (Japan)



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

NASA Technical Reports Server (NTRS)

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.

Bonner, W. A.



Polypeptide Grafted Hyaluronan: Synthesis and Characterization  

PubMed Central

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 ?-sheet conformation for aggregates of poly(L-leucine).

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



[The transport mechanism of antibiotics using microvillous membrane vesicles (placental transport of fosfomycin)].  


Using the rapid filtration technique, the uptake of fosfomycin into microvillous membrane vesicles isolated from human term placental trophoblast was investigated. The microvillous membrane vesicles exhibited the uptake of fosfomycin into an osmotically reactive intravesicular space and it was indicated that the uptake of fosfomycin by microvillous membrane vesicles represented transport into membrane vesicles. The uptake of fosfomycin by microvillous membrane vesicles was not dependent on the Na+ electrochemical gradient or membrane potential. The initial uptake of fosfomycin by microvillous membrane vesicles did not exhibit saturation kinetics with respect to fosfomycin concentration, and increased linearly as the fosfomycin concentration increased. These results indicated that fosfomycin was transported across the microvillous membrane by simple diffusion. L-alanine, L-valine, L-lysine, inorganic phosphate or D-glucose did not inhibit the uptake of fosfomycin into microvillous membrane vesicles. On the other hand, fosfomycin did not inhibit the uptake of L-alanine, L-valine, L-lysine inorganic phosphate or D-glucose into microvillous membrane vesicles. These results revealed that fosfomycin did not affect the placental transport activity of other nutrients. PMID:3782953

Iioka, H; Moriyama, I; Kyuma, M; Tsuji, Y; Ichijo, M



Exploring Solute-Solvent Interactions of ? -Amino Acids in Aqueous [EPyBF4 ] Arrangements by Volumetric, Viscometric, Refractometric, and Acoustic Approach  

NASA Astrophysics Data System (ADS)

Qualitative and quantitative analysis of molecular interaction prevailing in glycine, uc(l)-alanine, uc(l)-valine, and aqueous solution of ionic liquid (IL) [1-ethylpyridinium tetrafluoroborate (EPyBF4) ] have been investigated by thermophysical properties. The apparent molar volume (? V ), viscosity B -coefficient, molal refraction (RM ), and adiabatic compressibility (? _{ K} ) of glycine, uc(l)-alanine, and uc(l)-valine have been studied in 0.001 mol {\\cdot } dm^{-3} , 0.003 mol {\\cdot } dm^{-3} , and 0.005 mol {\\cdot } dm^{-3} aqueous 1-ethylpyridinium tetrafluoroborate [EPyBF4 ] solutions at 298.15 K from the values of densities (? ) , viscosities (? ), refractive index (nD) , and speed of sound (u) , respectively. The extent of interaction, i.e., the solute-solvent interaction is expressed in terms of the limiting apparent molar volume (? V^0 ) , viscosity B -coefficient, and limiting apparent molar adiabatic compressibility (? K^0) . The limiting apparent molar volumes (? V^0 ) , experimental slopes (SV^*) derived from the Masson equation, and viscosity A - and B -coefficients using the Jones-Dole equation have been interpreted in terms of ion-ion and ion-solvent interactions, respectively. Molal refractions (RM) have been calculated with the help of the Lorentz-Lorenz equation. The role of the solvent (aqueous IL solution) and the contribution of solute-solute and solute-solvent interactions to the solution complexes have also been analyzed through the derived properties.

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



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


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

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



The role of methionine and ?-chymotrypsin-catalysed reactions  

PubMed Central

1. The reaction of ?-chymotrypsin with sodium periodate at pH5·0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The Km values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.

Knowles, J. R.



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

PubMed Central

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

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



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

PubMed Central

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

Olli, Sudar; Rangaraj, Nandini; Nagaraj, Ramakrishnan



Whole cell biotransformation for reductive amination reactions.  


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

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



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

PubMed Central

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

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



Metabolic fate of the carboxyl-carbon of valine  

SciTech Connect

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

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



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


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

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



Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding.  

PubMed Central

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k (cat)/ K (m) values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K (s). Values of k (cat)/ K (m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k (cat)/ K (m) for the Cys(B71) mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 A resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S(1) subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity.

Morillas, Manuel; McVey, Colin E; Brannigan, James A; Ladurner, Andreas G; Forney, Larry J; Virden, Richard



[Determination of intracellular pH by the distribution of benzoic acid in S. cerevisiae. Amino acid transport and proton gradient].  


The internal pH (pHi) of Saccharomyces cerevisiae, wild type strain and its mutant rho- has been measured by the intra-extracellular distribution of 14C-benzoic acid. The values of pHi (external pH 4.5) change with the yeast strain and depend on the cellular metabolic conditions. The values of pHi and proton gradient in the wild type yeast are higher in energized than in starved cells: in energized cells pHi, 6.15 to 6.40, delta pH 1.65 to 1.90 or -97 to -112 mV; starved cells pH 5.90, delta pH 1.40 or -82 mV. In the rho- mutant, the values are lower than in the wild type yeast, in the same metabolic conditions. Energized rho- mutant cells, pH 6.05, delta pH 1.55 or -91 mV; starved cells, pHi 5.70, delta pH 1.20 or -71 mV. The proton conductors, DNP and PCP produce a decrease in pHi and delta pH and inhibition of L-leucine entrance by system S1, high affinity and low velocity and system S2, low affinity and high velocity. The obtained values of delta pH decrease and L-leucine transport inhibition, demonstrate that there is no strict relationship between the proton gradient across the cell membrane and the process of transport of L-leucine in yeast. PMID:2845476

de Bongioanni, L C; Ramos, E H



Effects of "Bioactive" amino acids leucine, glutamate, arginine and tryptophan on feed intake and mRNA expression of relative neuropeptides in broiler chicks  

PubMed Central

Feed intake control is vital to ensuring optimal nutrition and achieving full potential for growth and development in poultry. The aim of the present study was to investigate the effects of L-leucine, L-glutamate, L-tryptophan and L-arginine on feed intake and the mRNA expression levels of hypothalamic Neuropeptide involved in feed intake regulation in broiler chicks. Leucine, glutamate, tryptophan or arginine was intra-cerebroventricularly (ICV) administrated to 4d-old broiler chicks respectively and the feed intake were recorded at various time points. Quantitative PCR was performed to determine the hypothalamic mRNA expression levels of Neuropeptide Y (NPY), agouti related protein (AgRP), pro-opiomelanocortin (POMC), melanocortin receptor 4 (MC4R) and corticotrophin releasing factor (CRF). Our results showed that ICV administration of L-leucine (0.15 or 1.5???mol) significantly (P?L-leucine and L-glutamate could act within the hypothalamus to influence food intake, and that both orexigenic and anorexigenic Neuropeptide genes might contribute directly to these effects.



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

NASA Technical Reports Server (NTRS)

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

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



Comparison of the effects of leucines, non-metabolizable leucine analogues and other insulin secretagogues on the activity of glutamate dehydrogenase  

Microsoft Academic Search

Summary  Glutamate dehydrogenase (GLDH) from bovine liver was employed in a model system for testing a possible role of GLDH in insulin\\u000a release. The ability of different insulin secretagogues to stimulate the activity of the diethylstilbestrol-inhibited enzyme\\u000a was tested. The two insulin-releasing amino acids, L-leucine and its non-metabolizable analogue 2-aminobicyclo(2,2,1)heptane-2-carboxylic\\u000a acid [b(?)-BCH], were the best stimulators of GLDH activity. The non-secreting

Erik Gylfe



The screening of the enzyme and isoenzyme patterns in seeds of Allium cepa L  

Microsoft Academic Search

The screening of enzyme patterns in seeds ofAllium cepa cv. Všetatská revealed the presence of the following enzymes: alcohol dehydrogenase, lactate dehyd ogenase, NAD+- and NADP+-glyceraldehyde-3-phosphate\\u000a dehydrogenase, glucose-6-phosphate dehydrogenase NAD+- and NADP+-malate dehydrogenase, NADH2- and NADPH2-tetrazolium reductase catalase, Superoxide dismutase, acid and alkaline phosphatase, L-leucine aminopeptidase, glutamate\\u000a dehydrogenase, non-specific esterase, and cholinesterase. Altogether 17 enzymes were detected in onion seeds,

V?ra Hada?ová; Eva Klozová; Kv?ta Pitterová; V?ra Turková



The first example of linear peptides containing a N-trifluoroethylated backbone amide linkage and the surprising solution dynamics observed by 19F NMR  

Microsoft Academic Search

The ?-amino group of (l)phenylalanine methyl ester was trifluoroethylated using (2,2,2-trifluoroethyl)phenyliodonium N,N-bis(trifluoromethylsulfonyl)imide. A dipeptide Gly(l)Phe containing a trifluoroethylated peptide bond was synthesized by removing the ?-amino proton of N?-trifluoroethyl (l)phenylalanine methyl ester followed by coupling with N?-phthaloyl glycine acid fluoride. The dipeptide was further coupled with (l)leucine methyl ester under conventional carboxyl activation conditions to provide two diastereomers of the

Changqing Lu; Darryl D. DesMarteau



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

PubMed Central

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

Oliver, A E; Deamer, D W



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


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. PMID:24157279

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



Physiology of rickettsiae. VII. Amino acid incorporation by Coxiella burnetii and by infected hosts.  


Protein synthesis, in terms of (14)C-labeled amino acid incorporation into a hot trichloroacetic acid fraction, was studied in cell-free preparations of Coxiella burnetii, and in uninfected and Q fever-infected guinea pig and chick embryo hosts. Purified and disrupted suspensions of C. burnetii incorporated (14)C-labeled l-leucine, l-phenylalanine and algal hydrolysate. Livers of infected guinea pigs and chick embryos had a greater incorporation rate at the height of infection than comparable preparations from uninfected animals. As chick embryonic development continued during infection, the rate of incorporation progressively decreased below that of uninfected embryos. PMID:6025436

Mallavia, L P; Paretsky, D



A novel leucine-rich repeat receptor-like kinase gene in potato, StLRPK1 , is involved in response to diverse stresses  

Microsoft Academic Search

A potato gene, StLRPK1 (Solanum tuberosum L. leucine-rich-repeat receptor-like protein kinases 1), encoding a protein belonging to leucine-rich repeat receptor-like\\u000a kinases (LRR-RLKs) was identified. It encodes 796 amino acids with 88% of identity to SRF3 of Arabidopsis thaliana and contains a signal peptide, five LRR motifs, a transmembrane domain, two proline-rich regions and a serine\\/threonine protein\\u000a kinase domain. The transcripts

Tian Wu; Zhendong Tian; Jun Liu; Conghua Xie



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


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

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



Characterization of humus–protease complexes extracted from soil  

Microsoft Academic Search

Pyrophosphate (140mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: AI with nominal molecular weight (nmw)>100kD, AII with nmw between 10kD and 100kD and R with nmw<10kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific

Manuel Bonmatí; Brunello Ceccanti; Paolo Nannipieri; Jordi Valero



Characterization of three aminopeptidases purified from human placenta.  


Three aminopeptidases purified from the human placenta were characterized and compared with each other. Aminopeptidase II1 preferred L-arginine- and L-lysine-beta-naphthylamides or p-nitroanilides as substrate, with low or negligible hydrolysis of other amino acid derivatives. It was inhibited by L-arginine, L-lysine and L-methionine. This enzyme activity was highly sensitive to heat treatment, N-ethylmaleimide, p-chloromercuribenzoate, puromycin, bestatin, epsilon-amino-n-caproic acid (EACA) and EDTA. After EDTA, this enzyme could be reactivated by Co2+. It is concluded that aminopeptidase II1 is identical with arginine aminopeptidase (EC from other mammalian tissues. Aminopeptidase II2 preferred L-alanine-beta-naphthylamide and p-nitroanilide as substrates. It was also able to hydrolyse L-leucine, L-arginine, L-methionine and L-lysine derivatives but only very weakly L-cystine and Bz-L-cysteine substrates. This enzyme was inhibited by L-arginine, L-alanine, L-lysine and most strongly by L-leucine and L-methionine. It was resistant to bestatin and heat treatment but sensitive to EACA. EDTA caused a marked suppression, which could be prevented by Co2+ and Zn2+. These characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-) found in other tissues. The third enzyme was the only one clearly particle bound and was therefore called PB-aminopeptidase. It preferred L-leucine derivatives as substrate but also readily hydrolysed other amino acid-beta-naphthylamides and p-nitroanilides including L-cystine and Bz-L-cysteine substrates. Among the amino acids L-cysteine, L-leucine and L-methionine were inhibitory. Bestatin and thiol reagents were without effect and EACA was only moderately inhibitory. EDTA caused a strong suppression, which could be prevented by Co2+ and Zn2+. These properties are equal to those previously described for the placental cystine aminopeptidase (oxytocinase) (EC All three enzymes had an optimum close to neutral pH but apparent differences in their Km and Vmax values with various substrates. These findings suggest that the three purified aminopeptidases are distinct enzymes. Two of these (aminopeptidases II1 and II2) have not previously been isolated and characterized in the human placenta. PMID:6424106

Lampelo, S; Lalu, K; Vanha-Perttula, T



mer-Hydridotris(tri-methyl-phosphane-?P)(d-valinato-?(2) N,O)iridium hexa-fluorido-phosphate di-chloro-methane 0.675-solvate.  


The title compound, [Ir(C5H10NO2)H(C3H9P)3]PF6·0.675CH2Cl2, an iridium compound with a meridional arrangement of PMe3 groups, O,N-bidentate coordination of d-valine and with a hydride ligand trans to the N atom is compared with the l-valine complex reported previously. As expected, the complexes from the corresponding l and d isomers of valine crystallize in enanti-omorphic space groups (P43 and P41, respectively). In the crystal, N-H?O and N-H?F hydrogen bonding is observed, the N-H to carbonyl oxygen hydrogen bond producing a helical motif that proceeds along the 41 screw of the c axis. PMID:24764947

Merola, Joseph S; Slebodnick, Carla; Berg, Michael; Ritchie, Melissa K



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

NASA Astrophysics Data System (ADS)

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

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



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

NASA Astrophysics Data System (ADS)

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.

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



Practical synthesis of fluorous oxazolidinone chiral auxiliaries from alpha-amino acids.  


[reaction: see text] A series of new fluorous-supported oxazolidinone chiral auxiliaries has been prepared using a versatile and general five-step pathway, starting from readily available chiral alpha-amino acids. The key feature of this synthesis is the efficient generation of a suitably active perfluoroalkyllithium species. By use of this protocol, the auxiliaries can be obtained in high enantiomeric purity and on multigram scales from L-phenylalanine and L-valine with overall yields as high as 55%. The new methodology also incorporates fluorous solid-phase extraction on the large scale, allowing bulk quantities (up to 25 g) of fluorous compounds to be purified from the crude reaction mixture. PMID:16292825

Hein, Jason E; Geary, Laina M; Jaworski, Ashley A; Hultin, Philip G



Structure of cymbidine A, a monomeric peptidoglycan-related compound with hypotensive and diuretic activities, isolated from a higher plant, Cymbidium goeringii (Orchidaceae).  


The structure of a new monomeric peptidoglycan-related compound with hypotensive and diuretic activities, cymbidine A (1) isolated from the orchid Cymbidium goeringii, was elucidated mainly by spectroscopic analysis. The structure of 1 was shown to involve four amino acids (D-alanin, meso-diaminopimelic acid, D-gultamic acid, and L-valine) and two amino sugars (N-acetylglucosamine and 1,6-anhydro-N-acetylmuramic acid). The sequence of the amino acids and amino sugars was determined by the analysis of 2D NMR data. The absolute stereochemistries of the three amino acids (D-Ala, D-Glu and L-Val) were determined by the modified Marfey's method, and the (6S,10R) configurations of meso-diaminopimelic acid in 1 were indicated on the basis of the CD analysis. The absolute stereochemistry of 1,6-anhydro-N-acetylmuramic acid was also determined by CD data. PMID:17473468

Watanabe, Kinzo; Tanaka, Rina; Sakurai, Hitomi; Iguchi, Kazuo; Yamada, Yasuji; Hsu, Chau-Shin; Sakuma, Chiseko; Kikuchi, Hiroyuki; Shibayama, Humio; Kawai, Tadahide



Engineering Corynebacterium glutamicum for the production of pyruvate.  


A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM L: -valine, 28 mM L: -alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum ?aceE ?pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD(+)-dependent L: -lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (?C-T IlvN). The latter modification abolished overflow metabolism towards L: -valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum ?aceE ?pqo ?ldhA ?C-T ilvN produced about 190 mM pyruvate with a Y (P/S) of 1.36 mol per mol of glucose; however, it still secreted significant amounts of L: -alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced L: -alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0-5% dissolved oxygen), the newly constructed strain C. glutamicum ?aceE ?pqo ?ldhA ?C-T ilvN ?alaT ?avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g ((CDW)) (-1)  h(-1) (i.e., 0.08 g g((CDW)) (-1) h(-1)) in the production phase. PMID:22228312

Wieschalka, Stefan; Blombach, Bastian; Eikmanns, Bernhard J



Valine-spectrophotometric readout dosimeter (1 Gy 50 kGy)  

NASA Astrophysics Data System (ADS)

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

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



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

SciTech Connect

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.

Reizer, J.; Panos, C.



Crystal structure of a novel N-substituted L-amino acid dioxygenase from Burkholderia ambifaria AMMD.  


A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of ?-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).?-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded ?-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The ?-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).?-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids. PMID:23724013

Qin, Hui-Min; Miyakawa, Takuya; Jia, Min Ze; Nakamura, Akira; Ohtsuka, Jun; Xue, You-Lin; Kawashima, Takashi; Kasahara, Takuya; Hibi, Makoto; Ogawa, Jun; Tanokura, Masaru



Mode of Action of the Toxin from Pseudomonas phaseolicola  

PubMed Central

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

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



A sensitive, versatile microfluidic assay for bacterial chemotaxis.  


We have developed a microfluidic assay for bacterial chemotaxis in which a gradient of chemoeffectors is established inside a microchannel via diffusion between parallel streams of liquid in laminar flow. The random motility and chemotactic responses to L-aspartate, L-serine, L-leucine, and Ni(2+) of WT and chemotactic-mutant strains of Escherichia coli were measured. Migration of the cells was quantified by counting the cells accumulating in each of 22 outlet ports. The sensitivity of the assay is attested to by the significant response of WT cells to 3.2 nM L-aspartate, a concentration three orders of magnitude lower than the detection limit in the standard capillary assay. The response to repellents was as robust and easily recorded as the attractant response. A surprising discovery was that L-leucine is sensed by Tar as an attractant at low concentrations and by Tsr as a repellent at higher concentrations. This assay offers superior performance and convenience relative to the existing assays to measure bacterial tactic responses, and it is flexible enough to be used in a wide range of different applications. PMID:12704234

Mao, Hanbin; Cremer, Paul S; Manson, Michael D



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


The aim of this study was to develop a spray dried submicrometer powder formulation suitable for the excipient enhanced growth (EEG) application. Combination particles were prepared using the Buchi Nano spray dryer B-90. A number of spray drying and formulation variables were investigated with the aims of producing dry powder formulations that were readily dispersed upon aerosolization and maximizing the fraction of submicrometer particles. Albuterol sulfate, mannitol, L-leucine, and poloxamer 188 were selected as a model drug, hygroscopic excipient, dispersibility enhancer and surfactant, respectively. Formulations were assessed by scanning electron microscopy and aerosol performance following aerosolization using an Aerolizer dry powder inhaler (DPI). In vitro drug deposition was studied using a realistic mouth-throat (MT) model. Based on the in vitro aerosolization results, the best performing submicrometer powder formulation consisted of albuterol sulfate, mannitol, L-leucine and poloxamer 188 in a ratio of 30:48:20:2, containing 0.5% solids in a water:ethanol (80:20%, v/v) solution which was spray dried at 70 °C. The submicrometer particle fraction (FPF(1 ?m/ED)) of this final formulation was 28.3% with more than 80% of the capsule contents being emitted during aerosolization. This formulation also showed 4.1% MT deposition. The developed combination formulation delivered a powder aerosol developed for the EEG application with high dispersion efficiency and low MT deposition from a convenient DPI device platform. PMID:23313343

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



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

PubMed Central

The aim of this study was to develop a spray dried submicrometer powder formulation suitable for the excipient enhanced growth (EEG) application. Combination particles were prepared using the Buchi Nano spray dryer B-90. A number of spray drying and formulation variables were investigated with the aims of producing dry powder formulations that were readily dispersed upon aerosolization and maximizing the fraction of submicrometer particles. Albuterol sulfate, mannitol, L-leucine, and poloxamer 188 were selected as a model drug, hygroscopic excipient, dispersibility enhancer and surfactant, respectively. Formulations were assessed by scanning electron microscopy and aerosol performance following aerosolization using an Aerolizer® dry powder inhaler (DPI). In vitro drug deposition was studied using a realistic mouth-throat (MT) model. Based on the in vitro aerosolization results, the best performing submicrometer powder formulation consisted of albuterol sulfate, mannitol, L-leucine and poloxamer 188 in a ratio of 30:48:20:2, containing 0.5% solids in a water:ethanol (80:20% v/v) solution which was spray dried at 70 °C. The submicrometer particle fraction (FPF1?m/ED) of this final formulation was 28.3% with more than 80% of the capsule contents being emitted during aerosolization. This formulation also showed 4.1% MT deposition. The developed combination formulation delivered a powder aerosol developed for the EEG application with high dispersion efficiency and low MT deposition from a convenient DPI device platform.

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



JPH203, an L-type amino acid transporter 1-selective compound, induces apoptosis of YD-38 human oral cancer cells.  


Compared to most normal cells that express L-type amino acid transporter 2, L-type amino acid transporter 1 is highly expressed in cancer cells and presumed to support their elevated growth and proliferation. This study examined JPH203, a potent and selective L-type amino acid transporter 1 inhibitor, and its ability to suppress YD-38 human oral cancer cell growth. The YD-38 cells express L-type amino acid transporter 1 with its associating protein 4F2 heavy chain, but not L-type amino acid transporter 2. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, completely inhibited l-leucine uptake in YD-38 cells. As expected, the intrinsic affinity of JPH203 to inhibit l-leucine uptake was far more efficient than BCH. Likewise, JPH203 and BCH inhibited YD-38 cell growth, with JPH203 being superior to BCH. JPH203 up-regulated the population of apoptotic YD-38 cells through the activation of apoptotic factors, including caspases and PARP. These results suggest that the inhibition of L-type amino acid transporter 1 activity via JPH203, which may act as a potential novel anti-oral-cancer agent, leads to apoptosis by inducing the intracellular depletion of the neutral amino acids essential for cancer cell growth in YD-38 human oral cancer cells. PMID:24492461

Yun, Dae-Woong; Lee, Seul Ah; Park, Min-Gyeong; Kim, Jae-Sung; Yu, Sun-Kyoung; Park, Mi-Ra; Kim, Su-Gwan; Oh, Ji-Su; Kim, Chun Sung; Kim, Heung-Joong; Kim, Jin-Soo; Chun, Hong Sung; Kanai, Yoshikatsu; Endou, Hitoshi; Wempe, Michael F; Kim, Do Kyung



Characterization of ?-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis  

PubMed Central

Background Alpha-isopropylmalate synthase (?-IPMS) is the key enzyme that catalyzes the first committed step in the leucine biosynthetic pathway. The gene encoding ?-IPMS in Mycobacterium tuberculosis, leuA, is polymorphic due to the insertion of 57-bp repeat units referred to as Variable Number of Tandem Repeats (VNTR). The role of the VNTR found within the M. tuberculosis genome is unclear. To investigate the role of the VNTR in leuA, we compared two ?-IPMS proteins with different numbers of amino acid repeats, one with two copies and the other with 14 copies. We have cloned leuA with 14 copies of the repeat units into the pET15b expression vector with a His6-tag at the N-terminus, as was previously done for the leuA gene with two copies of the repeat units. Results The recombinant His6-?-IPMS proteins with two and 14 copies (?-IPMS-2CR and ?-IPMS-14CR, respectively) of the repeat units were purified by immobilized metal ion affinity chromatography and gel filtration. Both enzymes were found to be dimers by gel filtration. Both enzymes work well at pH values of 7–8.5 and temperatures of 37–42°C. However, ?-IPMS-14CR tolerates pH values and temperatures outside of this range better than ?-IPMS-2CR does. ?-IPMS-14CR has higher affinity than ?-IPMS-2CR for the two substrates, ?-ketoisovalerate and acetyl CoA. Furthermore, ?-IPMS-2CR was feedback inhibited by the end product l-leucine, whereas ?-IPMS-14CR was not. Conclusion The differences in the kinetic properties and the l-leucine feedback inhibition between the two M. tuberculosis ?-IPMS proteins containing low and high numbers of VNTR indicate that a large VNTR insertion affects protein structure and function. Demonstration of l-leucine binding to ?-IPMS-14CR would confirm whether or not ?-IPMS-14CR responds to end-product feedback inhibition.



Biosynthesis of the defensive alkaloid cicindeloine in Stenus solutus beetles  

NASA Astrophysics Data System (ADS)

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.

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



Low uplift rates and terrace reoccupation inferred from mollusk aminostratigraphy, Coquimbo Bay area, Chile  

NASA Astrophysics Data System (ADS)

Mollusk aminostratigraphy of Quaternary marine terrace sediments at Coquimbo Bay, Chile, combined with recently available electron spin resonance (ESR) ages, necessitates revision of the northern Chilean relative sea-level and terrace chronology. Protothaca and MuliniaD-alloisoleucine/ L-isoleucine values define four aminozones which are consistent with available ESR ages. Terrace reoccupation during successive high sea-level stands is inferred on the basis of litho- and aminostratigraphically defined unconformities in terrace sediments. ESR data and a nonlinear kinetic racemization model give approximate numerical ages for the aminozones and thus yield estimates of net uplift rates. These rates, averaged over intervals of one to several hundred thousand years, have ranged from less than 0.1 m/1000 yr to no more than 0.2 m/1000 yr. Such slow uplift is the cause of terrace reoccupation, as the amount of uplift between successive glacioeustatic high sea-level stands is frequently not sufficient to isolate an earlier-formed abrasion platform from rising sea level during a subsequent high stand.

Leonard, Eric M.; Wehmiller, John F.



A steroidal molecule present in the egg wax of the tick Rhipicephalus (Boophilus) microplus inhibits bacterial biofilms.  


The cattle tick Rhipicephalus (Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization. PMID:23419060

Zimmer, Karine R; Macedo, Alexandre J; Giordani, Raquel B; Conceição, Jordan M; Nicastro, Gianlucca G; Boechat, Ana Laura; Baldini, Regina L; Abraham, Wolf-Rainer; Termignoni, Carlos



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


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

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



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

PubMed Central

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

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



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

PubMed Central

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

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



Injectable delivery system of 2-methoxyestradiol for breast cancer therapy using biodegradable thermosensitive poly(organophosphazene) hydrogel.  


2-Methoxyestradiol (2-ME) has been reported to have antiangiogenic and antitumor activity. Its biomedical application is limited due to its poor water solubility resulting in its low bioavailability. Poly(organophosphazenes) containing l-isoleucine ethyl ester, ethyl-2-(O-glycyl)lactate, and ?-amino-?-methoxy-poly(ethylene glycol) 550 were synthesized having M(W) of 35-38?kDa and polydispersity index of 2.38-2.73. Using a viscometer, the thermosensitivity useful for locally injectable drug delivery was verified. The aqueous polymer solution showed a sol state at a low temperature and transformed to a gel state at body temperature. The polymer solution (10 wt%) enhanced the solubility of 2-ME by about 10(4) times compared to that of phosphate buffered saline. 2-ME was released from the hydrogel mainly by diffusion, hydrophobic interaction, and surface erosion of the matrix. This release profile could be confirmed through an in vitro release test as a function of polymers and the concentration of 2-ME in hydrogels. By monitoring tumor volume and CD31 immunohistochemical staining in mouse orthotopic breast tumor (MDA-MB-231) model, it was found that the hydrogel containing a relatively low concentration (15?mg/kg) of 2-ME showed the improved antitumor and antiangiogenic activity relative to the original formulation. This research suggests that the developed formulation of poly(organophosphazenes) may have injectable carrier potentials for 2-ME and other lipophilic drugs. PMID:20608785

Cho, Jung-Kyo; Hong, Ki-Yun; Park, Jung Won; Yang, Han-Kwang; Song, Soo-Chang



Doxorubicin-polyphosphazene conjugate hydrogels for locally controlled delivery of cancer therapeutics.  


Poly(organophosphazene)-doxorubicin (DOX) conjugate bearing hydrophobic L-isoleucine ethyl ester (IleOEt) and hydrophilic alpha-amino-omega-methoxy-poly(ethylene glycol) with molecular weight of 550 Da (AMPEG 550) along with carboxylic acid as a functional group was synthesized to create a drug delivery system, which is based on locally injectable, biodegradable, and thermosensitive hydrogels. In addition to the evaluation of the in vitro and in vivo antitumor activities, the physicochemical properties, hydrolytic degradation, and DOX release profile of the poly(organophosphazene)-DOX conjugate were determined. The aqueous solution of the polymer-DOX conjugate showed a sol-gel transition behavior depending on temperature changes. Based on the in vivo antitumor activities of the locally injected poly(organophosphazene)-DOX conjugate into the tumor-induced nude mice, the conjugate hydrogel after the local injection at the tumor site was shown to inhibit tumor growth more effectively with less toxicity and much longer than doxorubicin and saline as controls, indicating that tumor active DOX from the conjugate hydrogel is released slowly over a longer period of time and effectively accumulated locally in the tumor sites. These results suggest that the poly(organophosphazene)-doxorubicin conjugates hold great potential for use in preclinical and clinical studies as single and/or combination therapies. PMID:19520429

Chun, Changju; Lee, Sun M; Kim, Chang W; Hong, Ki-Yun; Kim, Sang Y; Yang, Han K; Song, Soo-Chang



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

PubMed Central

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.

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



Enzymatic determination of carbon-14 labeled L-alanine in biological samples  

SciTech Connect

A method for determination of L-alanine-specific radioactivity in biological samples is presented. This method is based on the specific enzymatic transformation of L-alanine to pyruvic acid hydrazone catalyzed by the enzyme L-alanine dehydrogenase, formation of the pyruvic acid 2,4-dinitrophenylhydrazone derivative, and quantitative trapping in Amberlite XAD-7 columns, followed by radioactivity counting of the lipophilic eluate. No interferences from other UC-labeled materials such as D-glucose, glycerol, L-lactate, L-serine, L-glutamate, L-phenylalanine, glycine, L-leucine, and L-arginine were observed. This inexpensive and high-speed method is applicable to the simultaneous determination of L-alanine-specific radioactivity for a large number of samples.

Serra, F.; Palou, A.; Pons, A.



New Ras CAAX mimetics: design, synthesis, antiproliferative activity, and RAS prenylation inhibition.  


Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed replacing cysteine (C) by 2-hydroxymethylbenzodioxane or 2-aminomethylbenzodioxane, respectively etherified and amidified with 2'-methyl or 2'-methoxy substituted 2-carboxy-4-hydroxybiphenyl and 2,4-dicarboxybiphenyl. These pluri-substituted biphenyl systems, used as internal spacer and AA dipeptide bioisoster, were linked to the methyl ester of l-methionine, glycine or l-leucine by an amide bond. The resultant twelve pairs of stereoisomers at the dioxane C-2 were tested for antiproliferative effect finding the maximum activity for derivatives with methyleneoxy linker between benzodioxane and 2'-methylbiphenyl. Of these compounds, the one with terminal methionine and S configuration proved a good Ras prenylation inhibitor in a cell-based assay. PMID:19666221

Bolchi, Cristiano; Pallavicini, Marco; Fumagalli, Laura; Ferri, Nicola; Corsini, Alberto; Rusconi, Chiara; Valoti, Ermanno



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

SciTech Connect

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.

Raghavan, C.M.; Sankar, R.; Mohan Kumar, R. [Crystal Growth Centre, Anna University, Chennai 600 025 (India); Jayavel, R. [Crystal Growth Centre, Anna University, Chennai 600 025 (India)], E-mail:



Modification of Microbial Polymalic Acid With Hydrophobic Amino Acids for Drug-Releasing Nanoparticles  

PubMed Central

Microbial poly(?, l-malic acid) was modified with either l-leucine ethyl ester (L) or l-phenylalanine methyl ester (F) to produce amphiphylic copolymers. The degradation of these copolymers in aqueous buffer took place under physiological conditions in a few weeks by hydrolysis of the side chain ester group followed by cleavage of the main chain. Spherical nanoparticles with diameters ranging between 70 and 230 nm were prepared from these copolymers by the dialysis-precipitation method. No alteration of the cell viability was observed after incubation of these nanoparticles in different cell lines. Anticancer drugs temozolomide and doxorubicin were encapsulated in the nanoparticles. Temozolomide was released within several hours whereas doxorubicin took several weeks to be completely liberated.

Lanz-Landazuri, Alberto; Portilla-Arias, Jose; de Ilarduya, Antxon Martinez; Holler, Eggehard; Ljubimova, Julia; Munoz-Guerra, Sebastian



Comparison of the structure of key variants of microcystin to vasopressin.  


Microcystins (MCs) are cyclic heptapeptide compounds [where X(2) (position 2) and Z(4) (position 4) are variable l-aminoacids] produced by cyanobacteria and responsible for severe liver damage in animals ingesting acute doses of the toxic compounds. Certain variants of microcystins are more toxic than others, the differences being commonly ascribed to the hydrophobic nature of the variant. Microcystin-LR (MCLR) [X = l-leucine (L); Z = l-arginine (R); R1 = R2 = CH(3)] is the most toxic of all the microcystins investigated to date. This study investigates the similarity of the structures of MCLR and selected MC variants to the liver specific hormone vasopressin. Structures were compiled in HyperChem(®) (professional version 5.1). Initial comparisons of the MCLR and vasopressin indicated comparable volumes, surface areas and masses. Further studies using RMS overlays show that the microcystin derivative MCLR(Dha(7)) is comparably similar to vasopressin in terms of tertiary structure. PMID:21783489

Gehringer, Michelle M; Milne, Pieter; Lucietto, Franco; Downing, Tim G



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

NASA Technical Reports Server (NTRS)

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.

Holstege, Gert; Blok, Bertil F.



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


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. PMID:12882170

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



Purification of three aminopeptidases from human maternal serum.  


Three aminopeptidases (I--III) were purified from maternal serum using sequential chromatographic fractionations. Aminopeptidase I was specific for N-terminal alpha-L-dicarboxylic acid residues and activated by alkaline earth metals (Ba2+, Ca2+, Sr2+). It is concluded that aminopeptidase I is aminopeptidase A (L-alpha-aspartyl-(L-alpha-glutamyl)-peptide hydrolase, EC Aminopeptidase II hydrolysed all tested substrates including L-cystine and Bz-L-cysteine derivatives but preferred L-leucine derivatives. The properties of aminopeptidase II are equal to those described for the cystine aminopeptidase (oxytocinase) (EC Aminopeptidase III preferred L-alanine derivatives as substrates. It was activated by Co2+, but strongly inhibited by amastatin, puromycin and L-methionine. The characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-). PMID:4076525

Lalu, K; Lampelo, S; Vanha-Perttula, T



Exploring the interest of 1,2-dithiolane ring system in peptide chemistry. Synthesis of a chemotactic tripeptide and x-ray crystal structure of a 4-amino-1,2-dithiolane-4-carboxylic acid derivative.  


Due to their relevant biological functions and specific chemical reactivity 1,2-dithiolanes (five-membered cyclic disulfides) represent an emerging class of heterocyclic compounds. However, despite the extensive research centered on lipoic acid and its analogues, only very few data are at the present available on peptides containing this ring system. We report here synthesis, conformation and bioactivity of a fMLF-OMe analogue, namely For-Met-Adt-Phe-OMe (7), in which the residue of the 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) (4) replaces the central L-leucine. The crystal conformation of the synthetic intermediate Boc-Adt-OMe (5) is also described and compared to that of lipoic acid (R-1,2-dithiolane-3-pentanoic acid) (3) and asparagusic acid (1,2-dithiolane-4-carboxylic acid) (2). PMID:11738616

Morera, Enrico; Lucente, Gino; Ortar, Giorgio; Nalli, Marianna; Mazza, Fernando; Gavuzzo, Enrico; Spisani, Susanna



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


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

Stressler, Timo; Eisele, Thomas; Schlayer, Michael; Fischer, Lutz



Astrocyte-microglia cooperation in the expression of a pro-inflammatory phenotype.  


Glial cells not only serve supportive and nutritive roles for neurons, but also respond to protracted stress and insults by up-regulating inflammatory processes. The complexity of studying glial activation in vivo has led to the widespread adoption of in vitro approaches, for example the use of the bacterial toxin lipopolysaccharide (LPS, a ligand for toll-like receptor 4 (TLR4)) as an experimental model of glial activation. Astrocyte cultures frequently contain minor numbers of microglia, which can complicate interpretation of responses. In the present study, enriched (?5% microglia) astrocytes cultured from neonatal rat cortex and spinal cord were treated with the lysosomotropic agent L-leucyl-L-leucine methyl ester to eliminate residual microglia, as confirmed by loss of microglia-specific marker genes. L-Leucyl-L-leucine methyl ester treatment led to a loss of LPS responsiveness, in terms of nitric oxide and cytokine gene up-regulation and mediator (pro-inflammatory cytokines, nitric oxide) output into the culture medium. Surprisingly, when astrocyte/microglia co-cultures were then reconstituted by adding defined numbers of purified microglia to microglia-depleted astrocytes, the LPS-induced up-regulation of pro-inflammatory gene and mediator output far exceeded that observed from cultures containing the same numbers of microglia only. Similar behaviors were found when examining interleukin-1? release caused by activation of the purinergic P2X7 receptor. Given that astrocytes greatly outnumber microglia in the central nervous system, these data suggest that a similar interaction between microglia and astrocytes in vivo may be an important element in the evolution of an inflammatory pathology. PMID:23574172

Barbierato, Massimo; Facci, Laura; Argentini, Carla; Marinelli, Carla; Skaper, Stephen D; Giusti, Pietro



Enterohepatic circulation of bacterial chemotactic peptide in rats with experimental colitis  

SciTech Connect

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

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



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


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

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



Measurement of very low density and low density lipoprotein apolipoprotein (Apo) B-100 and high density lipoprotein Apo A-I production in human subjects using deuterated leucine. Effect of fasting and feeding  

SciTech Connect

Six normolipidemic male subjects, after an 8-h overnight fast, were given a bolus injection and then a 15-h constant intravenous infusion of (D3)L-leucine. Subjects were studied in the fasted state and on a second occasion in the fed state (small, physiological meals were given every hour for 15 h). Apolipoproteins were isolated by preparative gradient gel electrophoresis from plasma lipoproteins separated by sequential ultracentrifugation. Incorporation of (D3)L-leucine into apolipoproteins was monitored by negative ionization, gas chromatography-mass spectrometry. Production rates were determined by multiplying plasma apolipoprotein pool sizes by fractional production rates calculated as the rate of isotopic enrichment (IE) of each protein as a fraction of IE achieved by VLDL (d less than 1.006 g/ml) apo B-100 at plateau. VLDL apo B-100 production was greater, and LDL (1.019 less than d less than 1.063 g/ml) apo B-100 production was less in the fed compared with the fasted state (9.9 +/- 1.7 vs. 6.4 +/- 1.7 mg/kg per d, P less than 0.01, and 8.9 +/- 1.2 vs. 13.1 +/- 1.2 mg/kg per d, P less than 0.05, respectively). No mean change was observed in high density lipoprotein apo A-I production. We conclude that: (a) this stable isotope, endogenous-labeling technique, for the first time allows for the in vivo measurement of apolipoprotein production in the fasted and fed state; and (b) since LDL apo B-100 production was greater than VLDL apo B-100 production in the fasted state, this study provides in vivo evidence that LDL apo B-100 can be produced independently of VLDL apo B-100 in normolipidemic subjects.

Cohn, J.S.; Wagner, D.A.; Cohn, S.D.; Millar, J.S.; Schaefer, E.J. (Tufts Univ., Boston, MA (USA))



Molecular interactions of ?-amino acids insight into aqueous ?-cyclodextrin systems.  


Qualitative and quantitative analysis of molecular interaction prevailing in glycine, L-alanine, L-valine and aqueous solution of ?-cyclodextrin (?-CD) have been probed by thermophysical properties. Density (?), viscosity (?), 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 ?-CD) and the contribution of solute-solute and solute-solvent interactions to the solution complexes have also been analyzed through the derived properties. PMID:23760675

Ekka, Deepak; Roy, Mahendra Nath



Investigation of the synergistic effect with amino acid-derived chiral ionic liquids as additives for enantiomeric separation in capillary electrophoresis.  


Recently, chiral ionic liquids (ILs) have drawn more and more attention in chiral separation by capillary electrophoresis (CE). In this paper, two chiral ILs based on amino acid derivatives, L-alanine and L-valine tert butyl ester bis (trifluoromethane) sulfonimide, were applied for the first time in CE to evaluate their potential synergistic effects with classical chiral selectors (?-cyclodextrin derivatives) for enantiomeric separation. As observed, improved separation of tested drug enantiomers was obtained with the presence of chiral ILs compared to the conventional ?-cyclodextrin derivatives separation system. Parameters such as type and proportion of organic modifier, type and concentration of chiral ILs, concentration of chiral selector, buffer pH and applied voltage were systematically investigated with Me-?-CD/chiral ILs as model system to optimize the novel synergistic system, and the best results were obtained when 15 mM chiral ILs were introduced into the 30 mM sodium citrate/citric acid (20% organic modifier included) buffer solution containing 20 mM Me-?-CD at pH 5.0 with a 20 kV applied voltage for naproxen, pranoprofen and warfarin. PMID:24119759

Zhang, Jinjing; Du, Yingxiang; Zhang, Qi; Chen, Jiaquan; Xu, Guangfu; Yu, Tao; Hua, Xiaoyi



Superdormant spores of Bacillus species have elevated wet-heat resistance and temperature requirements for heat activation.  


Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15 degrees C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations. PMID:19592590

Ghosh, Sonali; Zhang, Pengfei; Li, Yong-qing; Setlow, Peter



DNA binding and nuclease activity of an oxovanadium valinato-Schiff base complex.  


A new oxovanadium complex [VO(sal-l-val)(phen)] (sal-l-val=Schiff base derived from salicylaldehyde and l-valine; phen=1,10-phenanthroline) has been designed and synthesized with the aim of developing potential DNA nuclease. The interaction of DNA with this structurally characterized oxovanadium complex has been studied by various physicochemical tools like UV-vis, fluorescence, viscosity and circular dichroism (CD). The intrinsic binding constant of the complex with DNA is determined by electronic absorption studies and calculated to be (4.74±0.02)×10(5)M(-1). The spectroscopic studies and the viscosity measurements indicate that the complex binds calf thymus DNA (CT-DNA) by intercalative mode. The ability of the complex to induce DNA cleavage was studied by gel electrophoresis techniques. The complex has been found to promote cleavage of pUC19 plasmid DNA from the super coiled (SC) form I to nicked coiled (NC) relaxed form II with good efficiency. PMID:24560950

Saha, Urmila; Mukherjea, Kalyan K



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


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

Huang, Mei; Joseph, Jamie W



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

PubMed Central

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.

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



The dependence of the enthalpy of solution of L-methionine on the composition of water-alcohol binary solvents at 298.15 K  

NASA Astrophysics Data System (ADS)

The integral enthalpies of solution of L-methionine in water-methanol, water-ethanol, water- n-propanol, and water- iso-propanol mixtures were measured calorimetrically at alcohol concentrations x 2 = 0-0.4 mole fractions. The standard enthalpies of solution (?sol H o) and transfer of L-methionine (?tr H o) from water to a binary solvent were calculated. The influence of the structure and properties of L-methionine and the composition of aqueous-organic mixtures on its enthalpy characteristics was considered. The enthalpic pair interaction coefficients ( h xy ) between L-methionine and alcohol molecules were calculated; they were positive and increased in the series methanol (MeOH), ethanol (EtOH), n-propanol ( n-PrOH), iso-propanol ( i-PrOH). The enthalpy characteristics of solution and transfer of L-methionine were compared with those of glycine, L-threonine, L-alanine, and L-valine in similar binary solvents.

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



Simultaneous determination of atenolol and amiloride by capillary electrophoresis with capacitively coupled contactless conductivity detection (C4D).  


Capillary electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE-C(4)D) has been employed for the determination of the ?-blocker drugs (atenolol and amiloride) in pharmaceutical formulations. 150 mM acetic acid 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 an internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28 °C, 25 kV, and using hydrodynamic injection (25 s). The separation was effected in a bare fused-silica capillary 75 ?m × 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 ?g mL(-1) for the studied analytes. The relative standard deviations of intra- and inter-day precisions of 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 the ?-blocker drugs in different pharmaceutical tablets. PMID:22976091

AL Azzam, Khaldun M; Aboul-Enein, Hassan Y



The action of trypsin on synthetic chromogenic arginine substrates.  


A new arginine derivative, N-benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide hydrochloride (ZPVAPA.HCl) was synthesized by the condensation of N-benzyloxy-carbonyl-L-phenylalanyl-L-valine and L-arginine-p-nitroanilide dihydrochloride using dicyclohexylcarbodiimide as a coupling reagent and 1-hydroxy-benzotriazole as an additive. L-ZPVAPA.HCl was split by trypsin more readily than Na-benzyloxycarbonyl-L-arginine-p-nitroanilide hydrochloride (L-ZAPA, HCl), Na-benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA.HCl), Na-tosyl-L-arginine-p-nitroanilide hdyrochloride (L-TAPA.HCl) and Na-benzoyl-DL-arginine-p-nitroanilide hydrochloride (DL-BAPA.HCl) by factors of 100, 400, 600, and 1,200, respectively. Low concentrations of dimethyl formamide (DMF) enhanced the trypsin-catalyzed hydrolyses of L-ZAPA.HCl and L-TAPA.HCl, contrary to the findings of other authors that DMF has no effect on the tryptic hydrolysis. PMID:762040

Somorin, O; Tokura, S; Nishi, N; Noguchi, J



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

PubMed Central

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

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



Facile synthesis of multiamino vinyl poly(amino acid)s for promising bioapplications.  


We presented a general and facile strategy to prepare biocompatible multiamino polymers. Series of new monomers were synthesized by esterification of 2-hydroxyethyl methacrylate (HEMA) and Boc-amino acids, such as Boc-l-phenylalanine, Boc-glycine, Boc-l-alanine, Boc-l-valine, and Boc-l-lysine. Subsequent vinyl polymerization of monomers gave rise to vinyl poly(amino acid)s with a side primary amino group at each unit if deprotected. Both atom transfer radical polymerization (ATRP) and conventional free radical polymerization (FRP) were employed to prepare the multiamino polymers. A well controlled effect upon molecular weight with the standard first-order kinetics was achieved in cases of ATRP, and high molecular weight polymers were obtained via FRP. MTT assay showed that cell survival rates for the multiamino polymers were almost maintained above 90% and that their cytotoxicities were much lower than that of linear PEI (PEI 25000). Zeta potential measurements demonstrated that the vinyl poly(amino acid)s are electropositive, and AFM measurements showed that the vinyl poly(amino acid)s could tightly condense DNA into granular structures at a suitable concentration. The combination of facile availability, controlled productivity, low cytotoxicity and strong binding ability with DNA promises the great potential of the novel multiamino polymers in bioapplications. PMID:21114313

Sun, Haiyan; Gao, Chao



Identification and Biosynthesis of Novel Male Specific Esters in the Wings of the Tropical Butterfly, Bicyclus martius sanaos.  


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

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



Growth Response of Nitrosomonas europaea to Amino Acids  

PubMed Central

Growth responses of Nitrosomonas europaea to individual amino acids or vitamins was observed in log-phase cultures, as was the incorporation of carbon-14 labeled amino acids. Nitrite formation and protein synthesis were increased by l-glutamic acid, l-aspartic acid, l-serine, and l-glutamine. l-Lysine, l-histidine, l-threonine, l-valine, l-methionine, and l-arginine were inhibitory. The other amino acids had no effect on growth. All of the uniformly labeled amino acids added at low concentrations were taken up by growing cells and distributed into cell fractions. From 1 to 12% of the added radioactivity was present in cells analyzed in late log phase, depending on the amino acid; glycine and l-serine caused accumulation of the label to the greatest extent, whereas l-aspartic and l-glutamic acids were among those incorporated to the least extent. Aspartic acid increased both cell protein and nitrite values, but did not alter the ratio of protein to nitrite from that found in controls.

Clark, Connie; Schmidt, E. L.



Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production?  

PubMed Central

2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter?1) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter?1) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.

Krause, Felix S.; Blombach, Bastian; Eikmanns, Bernhard J.



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


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

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



NIR spectroscopy as a process analytical technology (PAT) tool for monitoring and understanding of a hydrolysis process.  


The use of near infrared spectroscopy was investigated as a process analytical technology to monitor the amino acids concentration profile during hydrolysis process of Cornu Bubali. A protocol was followed, including outlier selection using relationship plot of residuals versus the leverage level, calibration models using interval partial least squares and synergy interval partial least squares (SiPLS). A strategy of four robust root mean square error of predictions (RMSEP) values have been developed to assess calibration models by means of the desirability index. Furthermore, multivariate quantification limits (MQL) values of the optimum model were determined using two types of error. The SiPLS(3) models for L-proline, L-tyrosine, L-valine, L-phenylalanine and L-lysine provided excellent accuracies with RMSEP values of 0.0915 mg/mL, 0.1605 mg/mL, 0.0515 mg/mL, 0.0586 mg/mL and 0.0613 mg/mL, respectively. The MQL ranged from 90 ppm to 810 ppm, which confirmed that these models can be suitable for most applications. PMID:23611702

Wu, Zhisheng; Peng, Yanfang; Chen, Wei; Xu, Bing; Ma, Qun; Shi, Xinyuan; Qiao, Yanjiang



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

NASA Astrophysics Data System (ADS)

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

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



Biochemical characterization of Santalum album (Chandan) leaf peroxidase.  


The Santalum peroxidase was extracted from the leaves and precipitated with double volume of chilled acetone. The optimum percent relative activity for the Santalum peroxidase was observed at pH 5.0 and 50 °C temperature. The Santalum peroxidase per cent relative activity was stimulated in the presence of phenolic compounds like ferrulic acid and caffeic acids; however, indole-3-acetic acid (IAA) and protocatechuic acid act as inhibitors. All divalent cations Fe(2+), Mn(2+), Mg(2+), Cu(2+) and Zn(2+) stimulate the relative activity of the Santalum peroxidase at concentration of 2.0 ?M. Amino acids like L-alanine and L-valine activate the per cent relative activity, while L-proline and DL-methionine showed moderate inhibition for the Santalum peroxidase. However, a very low a concentration of cysteine acts as a strong inhibitor of Santalum peroxidase at the concentration of 0.4 mM. Native polyacrylamide gel electrophoresis (Native-PAGE) was performed for isoenzyme determination and two bands were observed. Km and Vmax values were calculated from Lineweaver-Burk graph. The apparent Vmax/Km value for O-dianisidine and H2O2 were 400 and 5.0?×?105 Units/min/mL respectively. PMID:23573005

Kumar, Pradeep; Kamle, Madhu; Singh, Jagtar



Metabonomic Profiling of TASTPM Transgenic Alzheimer's Disease Mouse Model  

SciTech Connect

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

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



Inhibitor coordination interactions in the binuclear manganese cluster of arginase.  


Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. The structure and stability of the binuclear manganese cluster are critical for catalytic activity as it activates the catalytic nucleophile, metal-bridging hydroxide ion, and stabilizes the tetrahedral intermediate and its flanking states. Here, we report X-ray structures of a series of inhibitors bound to the active site of arginase, and each inhibitor exploits a different mode of coordination with the Mn(2+)(2) cluster. Specifically, we have studied the binding of fluoride ion (F(-); an uncompetitive inhibitor) and L-arginine, L-valine, dinor-N(omega)-hydroxy-L-arginine, descarboxy-nor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid. Some inhibitors, such as fluoride ion, dinor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid, cause the net addition of one ligand to the Mn(2+)(2) cluster. Other inhibitors, such as descarboxy-nor-N(omega)-hydroxy-L-arginine, simply displace the metal-bridging hydroxide ion of the native enzyme and do not cause any net change in the metal coordination polyhedra. The highest affinity inhibitors displace the metal-bridging hydroxide ion (and sometimes occupy a Mn(2+)(A) site found vacant in the native enzyme) and maintain a conserved array of hydrogen bonds with their alpha-amino and -carboxylate groups. PMID:15248756

Cama, Evis; Pethe, Stéphanie; Boucher, Jean-Luc; Han, Shoufa; Emig, Frances A; Ash, David E; Viola, Ronald E; Mansuy, Daniel; Christianson, David W



Direct and individual analysis of stress-related phytohormone dispersion in the vascular system of Cucurbita maxima after flagellin 22 treatment.  


• The stress-related phytohormones, salicylic acid (SA) and abscisic acid (ABA), and the three jasmonates, jasmonic acid (JA), cis-12-oxo-phytodienoic acid (cis-OPDA), and (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), were investigated in phloem and xylem exudates of Cucurbita maxima. • Phloem and xylem exudates were separately collected and analysed via liquid chromatography-mass spectrometry. • We show direct evidence for all three jasmonates, ABA, and SA in both phloem and xylem exudates of C. maxima. JA and JA-Ile concentrations are higher in xylem (JA: c(xylem) ? 199.5 nM, c(phloem) ? 43.9 nM; JA-Ile: c(xylem) ? 7.9 nM, c(phloem) ? 1.6 nM), whereas ABA and SA concentrations are higher in phloem exudates (ABA: c(xylem) ? 37.1 nM, c(phloem) ? 142.6 nM; SA: c(xylem) ? 61.6 nM, c(phloem) ? 1319 nM). During bacteria-derived flagellin 22 (flg22)-triggered remote root-to-shoot signalling, phytohormone concentration changed rapidly both in phloem and xylem. • The unequal distribution of phytohormones suggests that phloem and xylem have distinct roles in defence responses. Our data shed light on systemic phytohormone signalling and help explain how plants cope with environmental challenges by lateral exchange between phloem and xylem. Our analysis is a starting point for further investigations of how phytohormones contribute to phloem- and xylem-based defence signalling. PMID:24387138

Furch, Alexandra C U; Zimmermann, Matthias R; Kogel, Karl-Heinz; Reichelt, Michael; Mithöfer, Axel



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

NASA Astrophysics Data System (ADS)

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.

Meijer, T.; Cleveringa, P.



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

PubMed Central

Previously established radiometric techniques were used to assess the effectiveness of combined antimicrobial drug-inhibitory drug (drug-inhibitor) treatment on two clinical isolates of the Mycobacterium avium complex representing three colony variants: smooth opaque (dome) (SmO), smooth transparent (SmT), and rough (Rg). All variants were identified as members of the M. avium complex; however, only the SmT colony type of strain 373 possessed characteristic serovar-specific glycopeptidolipid (GPL) antigens. MICs, determined radiometrically, of drugs with the potential to inhibit the biosynthesis of GPL antigens or other cell envelope constituents were similar for all strains. These drugs included cerulenin, N-carbamyl-DL-phenylalanine, N-carbamyl-L-isoleucine, trans-cinnamic acid, ethambutol, 1-fluoro-1-deoxy-beta-D-glucose, 2-deoxy-D-glucose, and m-fluoro-phenylalanine. The MICs of the antimicrobial drugs amikacin, sparfloxacin, and clarithromycin varied, but overall the MICs for the SmO variant were the lowest. Radiometric assessment of drug-inhibitor combinations by using established x/y determinations revealed enhanced activity when either ethambutol or cerulenin were used in combination with all antimicrobial agents for all variants except the Rg variant of strain 424, for which ethambutol was not effective. Enhanced activity with amino acid analogs was observed with the Rg colony variants of strains 373 and 424. Two potential sites for drug targeting were identified: fatty acid synthesis, for all strains assayed, and peptide biosynthesis, particularly for Rg colony variants that possess previously identified phenylalanine-containing lipopeptides as potential targets for future drug development. Images

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



Evolved Cobalamin-Independent Methionine Synthase (MetE) Improves the Acetate and Thermal Tolerance of Escherichia coli  

PubMed Central

Acetate-mediated growth inhibition of Escherichia coli has been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance of E. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in the E. coli K-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treated E. coli cells among three mutant MetE proteins. E. coli WE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine in l-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45°C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (Tm) but an enhanced in vivo stability. Thus, we demonstrate improved E. coli growth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when an E. coli WE strain carrying the MetE mutant was combined with a previously found MetA (homoserine o-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45°C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by l-methionine.

Mordukhova, Elena A.



Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling  

PubMed Central

SUMMARY Jasmonates (JAs) are fatty acid-derived signaling compounds that control diverse aspects of plant growth, development, and immunity. The F-box protein COI1 functions both as a receptor for jasmonoyl-L-isoleucine (JA-Ile) and as the component of an E3-ubiquitin ligase complex (SCFCOI1) that targets JAZ transcriptional regulators for degradation. A key feature of JAZ proteins is the C-terminal Jas motif that mediates JA-Ile-dependent interaction with COI1. Here, we show that most JAZ genes from evolutionarily diverse plants contain a conserved intron that splits the Jas motif into 20 N-terminal and 7 C-terminal (X5PY) amino acid submotifs. In most members of the Arabidopsis JAZ family, alternative splicing events involving retention of this intron generate proteins that are truncated before the X5PY sequence. In vitro pull-down and yeast two-hybrid assays indicate that these splice variants have reduced capacity to form stable complexes with COI1 in the presence of the bioactive stereoisomer of the hormone, (3R,7S)-JA-Ile. cDNA overexpression studies showed that some, but not all, truncated splice variants are dominant repressors of JA signaling. We also show that strong constitutive expression of an intron-containing JAZ10 genomic clone is sufficient to repress JA responses. These findings provide evidence for functional differences between JAZ isoforms, and establish a direct link between alternative splicing of JAZ pre-mRNA and dominant repression of JA signal output. We propose that production of dominant JAZ repressors by alternative splicing reduces the negative consequences associated with inappropriate or hyperactivation of the JA response pathway.

Chung, Hoo Sun; Cooke, Thomas F.; DePew, Cody L.; Patel, Lalita C.; Ogawa, Narihito; Kobayashi, Yuichi; Howe, Gregg A.



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


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-?-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 ?-ketobutyrate, a product of L-methionine-?-lyase activity. ?-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-?-lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing L-methionine-?-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. PMID:23402686

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



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

NASA Astrophysics Data System (ADS)

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

Euliss, Larken E.


Modification of fetal plasma amino acid composition by placental amino acid exchangers in vitro  

PubMed Central

Fetal growth is dependent on both the quantity and relative composition of amino acids delivered to the fetal circulation, and impaired placental amino acid supply is associated with restricted fetal growth. Amino acid exchangers can alter the composition, but not the quantity, of amino acids in the intra- and extracellular amino acid pools. In the placenta, exchangers may be important determinants of the amino acid composition in the fetal circulation. This study investigates the substrate specificity of exchange between the placenta and the feto-placental circulation. Maternal–fetal transfer of radiolabelled amino acids and creatinine were measured in the isolated perfused human placental cotyledon. Transfer of l-[14C]serine or l-[14C]leucine, and [3H]glycine, were measured in the absence of amino acids in the fetal circulation (transfer by non-exchange mechanisms) and following 10–20 ?mol boluses of unlabelled amino acids into the fetal circulation to provide substrates for exchange (transfer by exchange and non-exchange mechanisms). The ability of fetal arterial boluses of l-alanine and l-leucine to stimulate release of amino acids from the placenta was also determined using HPLC in order to demonstrate the overall pattern of amino acid release. Experiments with radiolabelled amino acids demonstrated increased maternal–fetal transfer of l-serine and l-leucine, but not glycine, following boluses of specific amino acids into the fetal circulation. l-[14C]Leucine, but not l-[14C]serine or [3H]glycine, was transferred from the maternal to the fetal circulation by non-exchange mechanisms also (P < 0.01). HPLC analysis demonstrated that fetal amino acid boluses stimulated increased transport of a range of different amino acids by 4–7 ?mol l?1 (P < 0.05). Amino acid exchange provides a mechanism to supply the fetus with amino acids that it requires for fetal growth. This study demonstrates that these transporters have the capacity to exchange micromolar amounts of specific amino acids, and suggests that they play an important role in regulating fetal plasma amino acid composition.

Cleal, Jane K; Brownbill, Paul; Godfrey, Keith M; Jackson, John M; Jackson, Alan A; Sibley, Colin P; Hanson, Mark A; Lewis, Rohan M



Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.  


Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined. PMID:19101583

Ciscotto, P; Machado de Avila, R A; Coelho, E A F; Oliveira, J; Diniz, C G; Farías, L M; de Carvalho, M A R; Maria, W S; Sanchez, E F; Borges, A; Chávez-Olórtegui, C



Investigation of Isovaline Enantiomeric Excesses and Other C5 Amino Acids in Carbonaceous Meteorites  

NASA Technical Reports Server (NTRS)

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.

Dworkin, Jason P.; Glavin, Daniel P.



Significance of Peptide Transporter 1 in the Intestinal Permeability of Valacyclovir in Wild-Type and PepT1 Knockout Mice  

PubMed Central

The purpose of this study was to quantitatively determine the contribution of PepT1 [peptide transporter 1 (SLC15A1)] to the intestinal permeability of valacyclovir, an ester prodrug of the antiviral drug acyclovir. In situ single-pass intestinal perfusions were employed (pH 6.5 × 90 minutes) to assess the effective permeability (Peff) of 100 ?M [3H]valacyclovir in wild-type and PepT1 knockout mice. Acyclovir pharmacokinetics was also evaluated after oral administration of 25 nmol/g valacyclovir. In wild-type mice, jejunal uptake of valacyclovir was best described by both saturable (Km = 10.2 mM) and nonsaturable components where the saturable pathway accounted for 82% of total transport. Valacyclovir Peff was 2.4 × 10?4 cm/s in duodenum, 1.7 × 10?4 cm/s in jejunum, 2.1 × 10?4 cm/s in ileum, and 0.27 × 10?4 cm/s in colon. In Pept1 knockout mice, Peff values were about 10% of that in wild-type animals for these small intestinal segments. Valacyclovir Peff was similar in the colon of both genotypes. There were no differences in valacyclovir Peff between any of the intestinal segments of PepT1 knockout mice. Valacyclovir Peff was significantly reduced by the dipeptide glycylsarcosine and the aminocephalosporin cefadroxil, but not by the amino acids l-valine or l-histidine, the organic acid p-aminohippurate, or the organic base tetraethylammonium (all at 25 mM). PepT1 ablation resulted in 3- to 5-fold reductions in the in vivo rate and extent of valacyclovir absorption. Our findings conclusively demonstrate, using in situ and in vivo validations in genetically modified mice, that PepT1 has a major influence in improving the oral absorption of valacyclovir.

Yang, Bei



Chiral changes of simple amino acids in early Earth's ocean by meteorite impacts: Experimental simulations  

NASA Astrophysics Data System (ADS)

It has been recognized that meteorite impacts on early Earth ocean may have contributed significantly for molecules related to the origin of life to originate and evolve. We have already established the formation of simple biomolecules from inorganic materials through oceanic impacts that may have occurred at late heavy bombardment. These simple molecules including amino acids need to be subjected to further developments to initiate life on the Earth. The chirality of terrestrial amino acids constructing proteins is only L-type. In order to make clear the the point that biomolecules are formed by oceanic impacts of meteorites, it wll be crucial to determine how they select the chirality. In order to investigate the basic chemistry on chirality of simple amino acids, we tried to simulate experimentally the chiral change of some amino acids present in ocean at that time under shock loading. Each aqueous solution (0.1 M) of L- and D-valine was prepared and used as mixtures of olivine powders and solutions in sealed steel containers. We performed shock recovery experiments at an impact condition where samples were compressed at ~5 GPa. The analytical results of shock recovered solutions indicate that valine survives significantly (~10%) and that L- and D-valines transform partially to D- and L-valine, respectively. The transformation rate varied with the chemical species present in solutions. These results imply that meteorite impacts as well as the surrounding conditions play important roles to control the chirality of simple amino acids that may have been formed at that time.

Takase, A.; Sekine, T.; Furukawa, Y.; Kakegawa, T.



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

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.

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



Regulation and compartmentalization of ?-lactam biosynthesis  

PubMed Central

Summary Penicillins and cephalosporins are ??lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l???aminoadipic acid, l?cysteine and l?valine to form the tripeptide ??l???aminoadipyl?l?cysteinyl?d?valine catalysed by the non?ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl?CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two?component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains.

Martin, Juan F.; Ullan, Ricardo V.; Garcia-Estrada, Carlos



Blockade of maitotoxin-induced endothelial cell lysis by glycine and L-alanine.  


The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs) is a model for oncotic/necrotic cell death. The cascade is initiated by an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which is followed by the biphasic uptake of vital dyes. The initial phase of dye entry reflects activation of large pores and correlates with surface membrane bleb formation; the second phase reflects cell lysis. In the present study, the effect of the cytoprotective amino acid glycine was examined. Glycine had no effect on MTX-induced change in [Ca(2+)](i) or on the first phase of vital dye uptake but produced a concentration-dependent (EC(50) approximately 1 mM) inhibition of the second phase of dye uptake. No cytoprotective effect was observed with l-valine, l-proline, or d-alanine, whereas l-alanine was equieffective to glycine. Furthermore, glycine had no effect on MTX-induced bleb formation. To test the hypothesis that glycine specifically blocks formation of a lytic "pore," the loss of fluorescence from BAECs transiently expressing GFP and concatemers of GFP ranging in size from 27 to 162 kDa was examined using time-lapse videomicroscopy. MTX-induced loss of GFP was rapid, correlated with the second phase of dye uptake, and was relatively independent of molecular size. The MTX-induced loss of GFP from BAECs was completely blocked by glycine. The data suggest that the second "lytic" phase of MTX-induced endothelial cell death reflects formation of a novel permeability pathway that allows macromolecules such as GFP or LDH to escape, yet can be prevented by the cytoprotective agents glycine and l-alanine. PMID:12477666

Estacion, Mark; Weinberg, Justin S; Sinkins, William G; Schilling, William P



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

NASA Astrophysics Data System (ADS)

We present the theoretical description and experimental demonstration of a zero-quantum stochastic dipolar recoupling (ZQ-SDR) technique for solid state nuclear magnetic resonance (NMR) studies of 13C-labeled molecules, including proteins, under magic-angle spinning (MAS). The ZQ-SDR technique combines zero-quantum recoupling pulse sequence blocks with randomly varying chemical shift precession periods to create randomly amplitude- and phase-modulated effective homonuclear magnetic dipole-dipole couplings. To a good approximation, couplings between different 13C spin pairs become uncorrelated under ZQ-SDR, leading to spin dynamics (averaged over many repetitions of the ZQ-SDR sequence) that are fully described by an orientation-dependent N × N polarization transfer rate matrix for an N-spin system, with rates that are inversely proportional to the sixth power of internuclear distances. Suppression of polarization transfers due to non-commutivity of pairwise couplings (i.e., dipolar truncation) does not occur under ZQ-SDR, as we show both analytically and numerically. Experimental demonstrations are reported for uniformly 13C-labeled L-valine powder (at 14.1 T and 28.00 kHz MAS), uniformly 13C-labeled protein GB1 in microcrystalline form (at 17.6 T and 40.00 kHz MAS), and partially labeled 13C-labeled protein GB1 (at 14.1 T and 40.00 kHz MAS). The experimental results verify that spin dynamics under ZQ-SDR are described accurately by rate matrices and suggest the utility of ZQ-SDR in structural studies of 13C-labeled solids.

Qiang, Wei; Tycko, Robert



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

PubMed Central

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.

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



Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus.  


Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine; Gln, l-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid; and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ± 30 nmol min(-1) mg(-1)·protein and 136 ± 6 nmol min(-1) mg(-1)·protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa. PMID:23218487

Arai, Toshinobu; Noguchi, Atsushi; Takano, Eriko; Kino, Kuniki



1D cadmium(II) thiocyanate systems: Synthesis and characterization of three new polymeric 1D cadmium(II) thiocyanato complexes  

NASA Astrophysics Data System (ADS)

Three new cadmium(II) thiocyanato complexes, [{Cd(NCS) 2(val)}·H 2O] n1, [Cd(NCS) 2(3-ampy) 2] n2, and [Cd(NCS) 2(pyrazolinone)] n3, (val = D, L-valine, 3-ampy = 3-aminopyridine and pyrazolinone = 3-methyl-1-phenyl-2-pyrazolin-5-one) have been synthesized and structurally characterized. The X-ray structure analysis revealed di-?-N,S thiocyanato bridges connecting cadmium centers in a 1D chain with the co-ligand blocking the remaining coordination sites. The structure of complex 1 features six coordinate Cd(II) centers, each cadmium is surrounded by two N atoms and two S atoms from two bridging N,S-thiocyanato groups giving rise to a zigzag 1D chain and two oxygen atoms of the alternating chelating ?-O,O'-valine that coordinates as zwitterionic terminal amino acid. The structure of complex 2 consists of octahedral Cd(II) centers, connected by di-?-N,S-bridging NCS groups, thus forming a 1D chain system along the [1 0 1] direction. The amino-groups are forming one intra-chain N sbnd H⋯N hydrogen bond and one interchain N sbnd H⋯N hydrogen bond to N-atoms of adjacent chains. The structure of 3 reveals di-?-N,S-NCS doubly bridged unusual penta-coordinated cadmium centers with the alternating monodentate pyrazolinone ligand blocking the fifth coordination site. IR spectra and thermal properties of complexes are reported.

Saber, Mohamed R.; Abu-Youssef, Morsy A. M.; Goher, Mohamed A. S.; Sabra, Berry A.; Hafez, Afaf K.; Badr, Ahmed M.-A.; Mautner, Franz A.



Baceridin, a cyclic hexapeptide from an epiphytic bacillus strain, inhibits the proteasome.  


A new cyclic hexapeptide, baceridin (1), was isolated from the culture medium of a plant-associated Bacillus strain. The structure of 1 was elucidated by HR-HPLC-MS and 1D and 2D NMR experiments and confirmed by ESI MS/MS sequence analysis of the corresponding linear hexapeptide 2. The absolute configurations of the amino acid residues were determined after derivatization by GC-MS and Marfey's method. The cyclopeptide 1 consists partially of nonribosomal-derived D- and allo-D-configured amino acids. The order of the D- and L-leucine residues within the sequence cyclo(-L-Trp-D-Ala-D-allo-Ile-L-Val-D-Leu-L-Leu-) was assigned by total synthesis of the two possible stereoisomers. Baceridin (1) was tested for antimicrobial and cytotoxic activity and displayed moderate cytotoxicity (1-2 ?g?mL(-1)) as well as weak activity against Staphylococcus aureus. However, it was identified to be a proteasome inhibitor that inhibits cell cycle progression and induces apoptosis in tumor cells by a p53-independent pathway. PMID:24692199

Niggemann, Jutta; Bozko, Przemyslaw; Bruns, Nicole; Wodtke, Anne; Gieseler, Marc Timo; Thomas, Kevin; Jahns, Christine; Nimtz, Manfred; Reupke, Inge; Brüser, Thomas; Auling, Georg; Malek, Nisar; Kalesse, Markus



Regulation of amino acid/carnitine transporter B 0,+ (ATB 0,+) in astrocytes by protein kinase C: independent effects on raft and non-raft transporter subpopulations.  


Neutral and basic amino acid transporter B(0,+) belongs to a Na,Cl-dependent superfamily of proteins transporting neurotransmitters, amino acids and osmolytes, known to be regulated by protein kinase C (PKC). The present study demonstrates an increased phosphorylation of B(0,+) on serine moiety after treatment of rat astrocytes with phorbol 12-myristate 13-acetate, a process correlated with an augmented activity of l-leucine transport and an enhanced presence of the transporter at the cell surface. After solubilization with Triton X-100 and sucrose gradient centrifugation, B(0,+) was detected in non-raft as well as in detergent-resistant raft fractions under control conditions, while phorbol 12-myristate 13-acetate treatment resulted in a complete disappearance of the transporter from the raft fraction. B(0,+) was observed to interact with caveolin-1 and flotillin-1 (reggie-2) proteins, the markers of detergent-resistant microdomains of plasma membrane. As verified in immunocytochemistry and immunoprecipitation experiments, modification of PKC activity did not affect these interactions. It is proposed that PKC reveals different effects on raft and non-raft subpopulations of B(0,+). Phorbol ester treatment results in trafficking of the transporter from the intracellular pool to non-raft microdomains and increased activity, while B(0,+) present in raft microdomains undergoes either internalization or is transferred laterally to non-raft domains. PMID:20977479

Samluk, ?ukasz; Czeredys, Magdalena; Na??cz, Katarzyna A



Hybrid polypeptide micelles loading indocyanine green for tumor imaging and photothermal effect study.  


Indocyanine green (ICG) is a near-infrared (NIR) fluorescence dye for extensive applications; however, it is limited for further biological application due to its poor aqueous stability in vitro, concentration-dependent aggregation, rapid elimination from the body, and lack of target specificity. To overcome its limitations, ICG was encapsulated in the core of a polymeric micelle, which self-assembled from amphiphilic PEG-polypeptide hybrid triblock copolymers of poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu), with PLLeu as the hydrophobic core and PEG as the hydrophilic shell. The ICG was associated with the hydrophobic core via hydrophobic interaction and also the hydrophilic heads through electrostatic attractive interaction. Compared with free ICG, PEG-PLL-PLLeu-ICG micelles significantly improved quantum yield and fluorescent stability. The cellular uptake experiments showed that PEG-PLL-PLLeu-ICG micelles have a high cellular uptake rate. And the in vivo experiments revealed the excellent passive tumor targeting ability and long circulation time of PEG-PLL-PLLeu-ICG. The above results indicated the broad prospects of PEG-PLL-PLLeu-ICG application in the fields of tumor diagnosis and imaging. In addition, temperature measurements under NIR laser irradiation and in vitro photothermal ablation studies proved the potential application of PEG-PLL-PLLeu-ICG in tumor photothermal therapy. PMID:23941524

Wu, Lei; Fang, Shengtao; Shi, Shuai; Deng, Jizhe; Liu, Bin; Cai, Lintao



Characterization of proteins produced in vitro by periattachment bovine conceptuses  

SciTech Connect

Bovine conceptuses from Days 16 (n = 4), 19 (n = 6), 22 (n = 3), and 24 (n = 4), and chorion from Day 69 (estrus/mating = Day 0) were cultured for 24 h in modified minimum essential medium (MEM) in the presence of radioactive L-leucine ((/sup 3/H) leucine) to characterize de novo synthesis and release of proteins. Proteins released into MEM were identified by two-dimensional polyacrylamide gel electrophoresis, fluorography, and gel and ion exchange chromatography. Major polypeptides identified in MEM were different from those identified in conceptus and chorionic tissues. Both uptake of (/sup 3/H) leucine and quality of polypeptides produced de novo and released into MEM were related to stage of conceptus development. Percent retention of (/sup 3/H) leucine in MEM was lowest in Day 16 cultures, increased in Days 19 and 22 cultures, and decreased in Day 24 cultures. Complexity of polypeptides increased after Day 16. Days 16, 19, 22 and 24 conceptus culture MEM was enriched in low-Mr, acidic polypeptides (Mr/isoelectric point ranges: 22K-26K/6.5-5.6, 20K-26K/5.5-5.4, and 16K-20K/5.0-4.5), which were not prominent products of Day 29 and 69 tissues. A high-Mr (Mr +/- SEM; 735K +/- 22K) glycoprotein was produced by all conceptus and chorionic tissues. The transient nature of production of low-Mr polypeptides suggests that they may be required during the periattachment period.

Bartol, F.F.; Roberts, R.M.; Bazer, F.W.; Lewis, G.S.; Godkin, J.D.; Thatcher, W.W.



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

PubMed Central

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.

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



Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases  

SciTech Connect

Amino acid racemases inherently catalyze the exchange of alpha-hydrogen of amino acids with deuterium during racemization in /sup 2/H/sub 2/O. When the reactions catalyzed by alanine racemase and L-alanine dehydrogenase (EC, which is pro-R specific for the C-4 hydrogen transfer of NADH, are coupled in /sup 2/H/sub 2/O, (4R-2H)NADH is exclusively produced. Similarly, (4S-2H)NADH is made in /sup 2/H/sub 2/O with amino-acid racemase with low substrate specificity and L-leucine dehydrogenase, which is pro-S specific. We have established a simple procedure for the in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with either of the above two couples of enzymes in the same reaction mixture. When the C-4 hydrogen of NAD+ is fully retained after sufficient incubation, the stereospecificity of hydrogen transfer by a dehydrogenase is the same as that of alanine dehydrogenase or leucine dehydrogenase. However, when the C-4 hydrogen of NAD+ is exchanged with deuterium, the enzyme to be examined shows the different stereospecificity from alanine dehydrogenase or leucine dehydrogenase. Thus, we can readily determine the stereospecificity by /sup 1/H NMR measurement without isolation of the coenzymes and products.

Esaki, N.; Shimoi, H.; Nakajima, N.; Ohshima, T.; Tanaka, H.; Soda, K.



A high-throughput assay for screening L- or D-amino acid specific aminotransferase mutant libraries.  


Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening ?-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the L-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either L- or D-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 D-amino acid aminotransferase displaying a decrease in k(cat)/K(M) of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate L-leucine than L-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering. PMID:23871995

Walton, Curtis J W; Chica, Roberto A



Long polypeptide 310-helices at atomic resolution  

PubMed Central

The crystal-state preferred conformation of the terminally blocked homooctapeptide from the C?,?-dimethylated ?-aminoisobutyric acid (Aib) residue, pBrBz-(Aib)8-OBut, in which pBrBz is para-bromobenzoyl and OBut is tert-butoxy, determined by x-ray diffraction analysis using direct methods, was found to be a 310-helix stabilized by six consecutive intramolecular N—H....O=C hydrogen bonds of the C10-III (or III?) type. This is the first observation at atomic resolution of a regular 310-helix longer than two complete turns. The solid-state structural analysis was extended to the terminally blocked, ?-aminoisobutyric acid-rich octapeptide corresponding to the 2-9 sequence of the peptaibol antibiotics emerimicins III and IV, pBrBz-Aib3-L-Val-Gly-L-Leu-Aib2-OMe. Again, this peptide adopts a (right-handed) 310-helical structure, although slightly distorted at the level of the L-leucine residue. The role of specific amino acid sequence and peptide main-chain length in stabilizing either the 310- or the ?-helical conformation and their possible implications on the nature of the channel formed by peptaibol antibiotics in the membrane are also briefly discussed.

Bavoso, Alfonso; Benedetti, Ettore; Di Blasio, Benedetto; Pavone, Vincenzo; Pedone, Carlo; Toniolo, Claudio; Bonora, Gian Maria



Effects of conversion of the zinc-binding motif sequence of thermolysin, HEXXH, to that of dipeptidyl peptidase III, HEXXXH, on the activity and stability of thermolysin.  


Most zinc metalloproteinases have the consensus zinc-binding motif sequence HEXXH, in which two histidine residues chelate a catalytic zinc ion. The zinc-binding motif sequence of thermolysin, H(142)ELTH(146), belongs to this motif sequence, while that of dipeptidyl peptidase III (DPP III), H(450)ELLGH(455), belongs to the motif sequence HEXXXH. In this study, we examined effects of conversion of HEXXH to HEXXXH in thermolysin on its activity and stability. Thermolysin variants bearing H(142)ELLGH(146) or H(142)ELTGH(146) (designated T145LG and T145TG respectively) were constructed by site-directed mutagenesis and were produced in Escherichia coli cells by co-expressing the mature and pro domains separately. They did not exhibit hydrolyzing activity for casein or N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but exhibited binding ability to a substrate analog glycyl-D-phenylalanine (Gly-D-Phe). The apparent denaturing temperatures based on the ellipticity at 222 nm of T145LG and T145TG were 85 ± 1 °C and 86 ± 1 °C respectively, almost the same as that of wild-type thermolysin (85 ± 1 °C). These results indicate that conversion of HEXXH to HEXXXH abolishes thermolysin activity, but does not affect its binding ability to Gly-D-Phe or its stability. Our results are in contrast to ones reported previously, that DPP III variants bearing H(450)ELGH(455) exhibit activity. PMID:24018667

Menach, Evans; Hashida, Yasuhiko; Yasukawa, Kiyoshi; Inouye, Kuniyo



Crystal structures and magnetic properties of a set of dihalo-bridged oxalamidato copper(ii) dimers.  


A set of four copper(ii) complexes, and (X = Cl, Br; = N-(l-leucine methyl ester)-N'-((2-pyridin-2-yl)methyl)oxalamide and = N-benzyl-N'-((2-pyridin-2-yl)methyl)oxalamide), have been synthesized and characterized by X-ray structural analysis, electron paramagnetic resonance (EPR) spectroscopy on single crystals and by SQUID magnetization measurements. X-ray diffraction studies show one-dimensional hydrogen bonded networks of dimeric copper(ii)-complexes bridged by two halide ions and with the two metal centers 3.44-3.69 Å apart. The geometry at each copper(ii) atom is ideal or near ideal square pyramidal. EPR and SQUID studies indicate that all complexes exhibit weak antiferromagnetic interactions between the Cu(ii) paramagnetic centers, with exchange parameter |J| ? 1 cm(-1). Magneto-structural comparisons among similar dihalo-bridged Cu(ii) dinuclear complexes are also provided, and a possible correlation has been established. PMID:24965121

Zili?, Dijana; Rakvin, Boris; Mili?, Dalibor; Paji?, Damir; Dilovi?, Ivica; Cametti, Massimo; Džoli?, Zoran



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

PubMed Central

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

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



Comparison of Immunogenicity between Codon Optimized HIV-1 Thailand Subtype B gp140 and gp145 Vaccines  

PubMed Central

HIV-1 pandemic posed an unprecedent challenge to the global health and it is believed that an effective vaccine will be the final solution against HIV-1. HIV-1 envelope is the primary immunogen in developing neutralization antibody based HIV vaccine. To define the suitable Env derived immunogen, we systemically compared the immunogenicity of gp140 and gp145 in a DNA vaccination alone and a prime-boost modalities. 2 DNA vaccines and 2 recombinant Tiantan vaccinia vaccines (rTTV) were constructed for vaccination of female Balb/c mice. Elispot assay was used to read out the T cell immunity and ELISA assay was used to quantify antibody immunity. PLL (poly-L-Leucine)-ELISA assay was used in linear antibody epitope mapping. Mice primed with gp145 tended to elicit more Env-specific T cells responses than those primed with gp140, significant difference was observed in DNA immunization alone. The ultimate T cell responses in prime-boost regimen tends to be determined mainly by the priming efficacy. Linear antibody epitope mapping displayed that sera raised by gp145 priming were vigorously reactive to more peptides than that by gp140. Our data demonstrated HIV-1 Thailand B-derived gp145 may raise higher T-cell responses and broader linear peptide-specific antibody responses than gp140 does. However, it remains to be determined that how these observations are relevant to neutralization antibody activities.

Wan, Yanmin; Wu, Lan; Liu, Lianxing; Xu, Jianqing; Liu, Ying; Liu, Yong; Shao, Yiming



Poly(ester amide) blend microspheres for oral insulin delivery.  


This study developed a novel oral insulin formulation centered on microspheres consisting of a blend of biodegradable poly(ester amide) (PEA). In the formulation, L-lysine-/L-leucine-based PEA with pendant COOH groups (PEA-COOH) was used as a pH-responsive material for the protection of insulin from the harsh environmental conditions of the stomach. Arginine-based PEA (Arg-PEA) was introduced to improve the intestinal absorption of the drug. The influence of both the hydrophobicity of PEA-COOH and the content of Arg-PEA was investigated in detail on microsphere surface morphology, drug loading, and the in vitro release profile of insulin. The PEA-COOH/Arg-PEA blend microspheres protected the loaded insulin in simulated gastric fluid and released insulin in a fast and sustained manner in simulated intestinal fluid. The in vivo test demonstrated that the oral administration of insulin-loaded PEA blend microspheres could effectively suppress the blood glucose level in diabetic rats for 10h, and the oral bioavailability was improved to 5.89+1.84% in healthy rats. These results indicate that the PEA blend microspheres are promising vehicles for the oral delivery of insulin. PMID:23876502

He, Pan; Liu, Huaiyu; Tang, Zhaohui; Deng, Mingxiao; Yang, Yan; Pang, Xuan; Chen, Xuesi



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

SciTech Connect

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.

Hu,G.; Lin, G.; Wang, M.; Dick, L.; Xu, R.; Nathan, C.; Li, H.



Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin.  


We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions. PMID:11853866

Christoffers, Keith H; Khokhar, Arshia; Chaturvedi, Kirti; Howells, Richard D



Synthesis of new poly(ether-urethane-urea)s based on amino acid cyclopeptide and PEG: study of their environmental degradation.  


Conventional polyurethanes (PUs) are among biomaterials not intended to degrade but are susceptible to hydrolytic, oxidative and enzymatic degradation in vivo. Biodegradable PUs are typically prepared from polyester polyols, aliphatic diisocyanates and chain extenders. In this work we have developed a degradable monomer based on ?-amino acid to accelerate hard segment degradation. Thus a new class of degradable poly(ether-urethane-urea)s (PEUUs) was synthesized via direct reaction of 4,4'-methylene-bis(4-phenylisocyanate) (MDI), L-leucine anhydride (LA) and polyethylene glycol with molecular weight of 1,000 (PEG-1000) as polyether soft segment. The resulting polymers are environmentally biodegradable and thermally stable. Decomposition temperatures for 5 % weight loss occurred above 300 °C by TGA in nitrogen atmospheres. Some structural characterization and physical properties of these polymers before and after degradation in soil, river water and sludge are reported. The environmental degradation of the polymer films was investigated by SEM, FTIR, TGA, DSC, GPC and XRD techniques. A significant rate of degradation occurred in PEUU samples under river water and sludge condition. The polymeric films were not toxic to E. coli (Gram negative), Staphylococcus aureus and Micrococcus (Gram positive) bacteria and showed good biofilm formation on polymer surface. Our results show that hard segment degraded selectively as much as soft segment and these polymers are susceptible to degradation in soil and water. Thus our study shows that new environment-friendly polyurethane, which can degrade in soil, river water and sludge, is synthesized. PMID:22833157

Rafiemanzelat, Fatemeh; Fathollahi Zonouz, Abolfazl; Emtiazi, Giti



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


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

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



Endotoxin contamination in wound dressings made of natural biomaterials.  


Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately. PMID:12808594

Nakagawa, Y; Murai, T; Hasegawa, Chie; Hirata, M; Tsuchiya, T; Yagami, T; Haishima, Y



Impact of epiphytic and endogenous enzyme activities of senescent maize leaves and roots on the soil biodegradation process.  


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 ?-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, ? 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. PMID:22078739

Zafar Amin, Bilal Ahmad; Beaugrand, Johnny; Debeire, Philippe; Chabbert, Brigitte; Bertrand, Isabelle



Seasonal and Spatial Distribution of Extracellular Enzymatic Activities and Microbial Incorporation of Dissolved Organic Substrates in Marine Sediments  

PubMed Central

Seasonal and spatial distributions of extracellular enzymatic activities and microbial incorporations of dissolved organic substrates were followed in sediments of the brackish water Kiel Bight (Baltic Sea, Federal Republic of Germany). Enzymatic hydrolysis of polymeric organic compounds was determined by means of fluorogenic substrates (4-methylumbelliferyl-?-d-glucoside, l-leucine-4-methylcoumarinyl-7-amide hydrochloride); incorporation of dissolved organic substrates into microbial biomass was measured by using tritiated substances (acetate, leucine, and thymidine). Based on a recently developed core injection technique, substrates were injected in microliter portions into undisturbed sediment cores. Enzymatic and incorporation activities underwent strong seasonal variations related to the enrichment of organic material in the sediment surface following sedimentation events. The input of the phytoplankton bloom during autumn caused stimulation of both enzymatic hydrolysis of polymeric organic compounds and microbial incorporation of dissolved organic substrates. Following input by spring phytoplankton bloom, mainly incorporation activities were stimulated. In late spring the development of the benthic fauna obviously greatly influenced microbial activities. During summer individual periods of high microbial activities were observed which might be traced back to short-term sedimentation events. The high microbial incorporation of leucine and thymidine during winter demonstrated that the nutrient supply rather than temperature is the dominating factor determining microbial production. Stimulation of microbial activities arose from the sediment surface and spread out relatively quickly into deeper horizons. Generally, the sediments were characterized by distinct patterns of interrelationships between the individual parameters of microbial activities measured.

Meyer-Reil, Lutz-Arend



Synthesis and spectroscopic analysis of modified bile salts.  


For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation. PMID:6658870

Ballatore, A M; Beckner, C F; Caprioli, R M; Hoffman, N E; Liehr, J G



Carbon Catabolism and Synthesis of Macromolecules During Spore Germination of Microsporum gypseum1  

PubMed Central

Either d- or l-leucine (10?3m) and unsaturated long-chain fatty acids such as oleic, linoleic, and arachidonic (10?4m) significantly stimulated macroconidia germination of Microsporum gypseum. Saturated long-chain fatty acids did not affect germination, whereas saturated short-chain fatty acids such as caprylic, hexanoic, and butyric were completely inhibitory. Germination was followed by an increase in endogenous respiration and a decrease in dry weight of approximately 5% at 4 hr. Endogenous fatty acids and soluble carbohydrates were utilized significantly during germination. Tritiated leucine, uridine, and thymidine were incorporated respectively into protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) fractions within the first 5 min of germination. Incorporation of oleic-1-C14 into RNA and protein was significantly increased after germ tube development. Net synthesis of RNA and protein started prior to germ tube protrusion. Increase in DNA could be detected only later. A significant increase in RNA and protein during the 4th hr of germination was correlated with vegetative development. Inhibition of respiration and incorporation of leucine-H3 and uridine-H3 into corresponding macromolecules by dl-fluorophenylalanine and phenethyl alcohol started before germ tube appearance. Griseofulvin significantly inhibited incorporation of uridine-H3 and thymidine-H3, but not of leucine-H3. This inhibition occurred only after initial vegetative development. In contrast to the two other inhibitors, which substantially inhibited germination, griseofulvin only slightly retarded the period of germination and did not affect respiration.

Barash, I.; Conway, M. Loretto; Howard, D. H.



Absence of glucose-stimulated transport in yeast protoplasts.  


Protoplasts of Saccharomyces cerevisiae prepared by snail-gut juice treatment were compared in their transport properties with intact cells. 1. Constitutive monosaccharide transport (D-xylose, 6-deoxy-D-glucose), as well as inducible transport of D-galactose, were unaltered. 2. Phosphorylation-associated transport of 2-deoxy-D-glucose was enhanced in protoplasts, possibly as a consequence of removal of the unstirred layer of the cell wall. 3. Proton-driven transports of trehalose, L-leucine, L-proline and monophosphate could not be activated by preincubation with D-glucose, apparently owing to lack of proton-solute coupling in transport. Utilization of glucose was not depressed but respiration was reduced by about 50% while acidification of the external medium after glucose addition was inhibited by more than 90%. This may be related to the inability of protoplast plasma membrane H-ATPase to be activated by glucose and hence to impaired proton-translocating capacity. Uranyl ions inhibited generally much less in protoplasts than in intact cells although their binding to protoplasts was greater (maximum 0.68 fmol per cell but 3.2 fmol per protoplast). PMID:2860054

Kotyk, A; Michaljanicová, D; Struzinský, R; Baryshnikova, L M; Sychrová, H



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

SciTech Connect

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

Aschner, M.; Clarkson, T.W.



A novel leucine-rich repeat receptor-like kinase gene in potato, StLRPK1, is involved in response to diverse stresses.  


A potato gene, StLRPK1 (Solanum tuberosum L. leucine-rich-repeat receptor-like protein kinases 1), encoding a protein belonging to leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. It encodes 796 amino acids with 88% of identity to SRF3 of Arabidopsis thaliana and contains a signal peptide, five LRR motifs, a transmembrane domain, two proline-rich regions and a serine/threonine protein kinase domain. The transcripts were present at high levels in flowers and young leaves, while low in other tested organs. The mRNA of StLRPK1 was inducible in potato leaves by Phytophthora infestans, a pathogen causing late blight disease, and showed different profiles after treatment with salicylic acid, methyl jasmonate, ethylene, abscissic acid, wounding, 40 degrees C, 4 degrees C and a salinity stress. The results suggest that StLRPK1 may participate in the responses against environmental stresses and disease resistance in potato. PMID:19214776

Wu, Tian; Tian, Zhendong; Liu, Jun; Xie, Conghua



Bidirectional Transport of Amino Acids Regulates mTOR and Autophagy  

PubMed Central

SUMMARY Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.

Nicklin, Paul; Bergman, Philip; Zhang, Bailin; Triantafellow, Ellen; Wang, Henry; Nyfeler, Beat; Yang, Haidi; Hild, Marc; Kung, Charles; Wilson, Christopher; Myer, Vic E.; MacKeigan, Jeffrey P.; Porter, Jeffrey A.; Wang, Y. Karen; Cantley, Lewis C.; Finan, Peter M.; Murphy, Leon O.



Endocytosis and intracellular trafficking properties of transferrin-conjugated block copolypeptide vesicles.  


Block polypeptides are an emerging class of materials that have the potential to be used in many biomedical applications, including the field of drug delivery. We have previously developed a negatively charged block copolypeptide, poly(L-glutamate)60-b-poly(L-leucine)20 (E60L20), which forms spherical vesicles in aqueous solution. Since these vesicles are negatively charged, they are minimally toxic toward cells. However, the negative charge also inhibits these vesicles from effectively being internalized by cells, which can be problematic as many therapeutics have intracellular targets. To overcome this limitation of the E60L20 vesicles, transferrin (Tf) was conjugated onto the vesicle surface, since the receptor for Tf is overexpressed on cancer cells. The enhanced uptake of the Tf-conjugated vesicle was verified through confocal microscopy. Furthermore, endocytosis and immunostaining experiments confirmed that the Tf conjugated on the vesicle surface plays a critical role in the internalization and subsequent intracellular trafficking behavior of the vesicles. PMID:23581747

Choe, Uh-Joo; Rodriguez, April R; Lee, Brian S; Knowles, Scott M; Wu, Anna M; Deming, Timothy J; Kamei, Daniel T



Effects of amino acids on membrane potential and 86Rb+ fluxes in pancreatic beta-cells  

SciTech Connect

The membrane potential of beta-cells was studied with microelectrodes in mouse islets and their potassium permeability was evaluated by measuring 86Rb+ fluxes in rat islets. In the absence of glucose, L-leucine, its metabolite ketoisocaproate, and its nonmetabolized analogue 2-aminonorbornane-2-carboxylic acid (BCH) depolarized beta-cells and triggered bursts of electrical activity like glucose. The effect of leucine was weak, but was potentiated by a low concentration of glucose or by theophylline; the effect of ketoisocaproate was stronger and faster than that of an equimolar concentration of glucose. Arginine alone produced only a fast depolarization of beta-cells, insufficient to trigger electrical activity. Leucine and arginine potentiated the activity induced by glucose. In a glucose-free medium, alanine only slightly depolarized beta cells, whereas isoleucine and phenylalanie had no effect. Leucine, ketoisocaproate, and BCH reversibly decreased 86Rb+ efflux from islets perifused in the absence of glucose and increased 86Rb+ uptake. By contrast, both in the absence or presence of glucose, arginine increased 86Rb+ efflux and decreased 86Rb+ uptake. It is proposed that leucine, ketoisocaproate, and BCH, as glucose, deplolarize beta-cells by decreasing their potassium permeability, whereas arginine acts differently. The appearance of bursts of electrical activity with secretagogues unrelated to glucose suggests that they reflect an intrinsic property of the beta-cell membrane.

Henquin, J.C.; Meissner, H.P.



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

SciTech Connect

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

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



Studies on mass attenuation coefficient, effective atomic number and electron density of some amino acids in the energy range 0.122-1.330 MeV  

NASA Astrophysics Data System (ADS)

The total mass attenuation coefficients of some amino acids, such as Glycine (C2H5NO2), DL-Alanine (C3H7NO2), Proline (C5H9NO2), L-Leucine (C6H13NO2 ), L-Arginine (C6H14N4O2) and L-Arginine Monohydrochloride (C6H15ClN4O2), were measured at 122, 356, 511, 662, 1170, 1275 and 1330 keV photon energies using a well-collimated narrow beam good geometry set-up. The gamma rays were detected using NaI (Tl) scintillation detection system with a resolution of 10.2% at 662 keV. The attenuation coefficient data were then used to obtain the effective atomic numbers (Zeff) and effective electron densities (Neff) of amino acids. It was observed that the effective atomic number (Zeff) and effective electron densities (Neff) tend to be almost constant as a function of gamma-ray energy. The results show that, the experimental values of mass attenuation coefficients, effective atomic numbers and effective electron densities are in good agreement with the theoretical values with less than 1% error.

Pawar, Pravina P.; Bichile, Govind K.



Endocytosis and Intracellular Trafficking Properties of Transferrin-Conjugated Block Copolypeptide Vesicles  

PubMed Central

Block polypeptides are an emerging class of materials that have the potential to be used in many biomedical applications, including the field of drug delivery. We have previously developed a negatively charged block copolypeptide, poly(L-glutamate)60-b-poly(L-leucine)20 (E60L20), which forms spherical vesicles in aqueous solution. Since these vesicles are negatively charged, they are minimally toxic toward cells. However, the negative charge also inhibits these vesicles from effectively being internalized by cells, which can be problematic as many therapeutics have intracellular targets. To overcome this limitation of the E60L20 vesicles, transferrin (Tf) was conjugated onto the vesicle surface, since the receptor for Tf is overexpressed on cancer cells. The enhanced uptake of the Tf-conjugated vesicle was verified through confocal microscopy. Furthermore, endocytosis and immunostaining experiments confirmed that the Tf conjugated on the vesicle surface plays a critical role in the internalization and subsequent intracellular trafficking behavior of the vesicles.

Choe, Uh-Joo; Rodriguez, April R.; Lee, Brian S.; Knowles, Scott; Wu, Anna M.; Deming, Timothy J.; Kamei, Daniel T.



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

PubMed Central

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.

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



Leucyl-tRNA Synthetase Regulates Lactation and Cell Proliferation via mTOR Signaling in Dairy Cow Mammary Epithelial Cells  

PubMed Central

The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, ?-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration.

Wang, Lina; Lin, Ye; Bian, Yanjie; Liu, Lili; Shao, Li; Lin, Lin; Qu, Bo; Zhao, Feng; Gao, Xuejun; Li, Qingzhang



Leucyl-tRNA synthetase regulates lactation and cell proliferation via mTOR signaling in dairy cow mammary epithelial cells.  


The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, ?-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration. PMID:24722568

Wang, Lina; Lin, Ye; Bian, Yanjie; Liu, Lili; Shao, Li; Lin, Lin; Qu, Bo; Zhao, Feng; Gao, Xuejun; Li, Qingzhang



Purification and Characterization of an l-Amino Amidase from Mycobacterium neoaurum ATCC 25795  

PubMed Central

An l-amino amidase from Mycobacterium neoaurum ATCC 25795 responsible for the enantioselective resolution of dl-?-methyl valine amide was purified and characterized. The purification procedure included ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography, which resulted in a homogeneous preparation of the enzyme with a native molecular mass of 136 kDa and a subunit molecular mass of 40 kDa. The purified enzyme displayed the highest activity at 50°C and at pH 8.0 and 9.5. The enzyme was strongly inhibited by the metal-chelating agent 1,10-phenanthroline, the disulfide-reducing agent dithiothreitol, and the cysteine proteinase inhibitor iodoacetamide. The purified amino amidase showed a unique l-enantioselective activity towards a broad range of both ?-H- and ?-alkyl-substituted amino acid amides, with the highest activity towards the cyclic amino acid amide dl-proline amide. No activity was measured with dl-mandelic acid amide nor with the dipeptide l-phenylalanine-l-leucine. The highest catalytic efficiency (kcat/Km ratio) was measured with dl-?-allyl alanine amide, dl-?-methyl phenylalanine amide, and dl-?-methyl leucine amide.

Hermes, H. F. M.; Tandler, R. F.; Sonke, T.; Dijkhuizen, L.; Meijer, E. M.



Identification of Immunopotentiating Lactic Acid Bacteria that Induce Antibody Production by in vitro Stimulated Human Peripheral Blood Mononuclear Cells  

PubMed Central

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.

YAMASHITA, Makiko; HITAKA, Akira; FUJINO, Himiko; MATSUMOTO, Takashi; HASEGAWA, Takanori; MORIMATSU, Fumiki; FUJIKI, Tsukasa; KATAKURA, Yoshinori



Initial adhesion of Listeria monocytogenes to fine polished stainless steel under flow conditions is determined by prior growth conditions.  


Listeria monocytogenes is a food-borne pathogen known to persist in food production environments, where it is able to attach and form biofilms, potentially contaminating food products ready for consumption. In this study the first step in the establishment of L. monocytogenes in a food-processing environment was examined, namely the initial adhesion to stainless steel under specific dynamic flow conditions. It was found that the intrinsic ability of L. monocytogenes to adhere to solid surfaces under flow conditions is dependent on nutrient availability. The addition of L-leucine to the growth medium altered the fatty acid composition of the L. monocytogenes cells and increased adhesion. The growth conditions resulting in the highest adhesion (growth medium with added glucose) had cells with the highest electron donating and lowest electron accepting properties, whereas growth conditions resulting in lowest adhesion (growth medium with added mannose) had cells with the lowest electron donating properties and highest electron accepting properties. The highest and lowest adhesion conditions correlated with differences in expression of cell surface protein of L. monocytogenes and among these the autolysin amidase (Ami). This study implies that food composition influences the adhesion of L. monocytogenes to solid surfaces during dynamic flow conditions. PMID:23685728

Skovager, Anne; Larsen, Marianne Halberg; Castro-Mejia, Josue Leonardo; Hecker, Michael; Albrecht, Dirk; Gerth, Ulf; Arneborg, Nils; Ingmer, Hanne



Duodenal juice total protein and pancreatic enzyme synthesis, turnover, and secretion in patients after acute pancreatitis.  

PubMed Central

It is controversial whether acute pancreatitis has longterm effects on pancreatic function. Pancreatic enzyme synthesis, turnover, and secretion were measured in 10 patients in clinical remission who had had one or more (one to six) attacks of acute alcoholic pancreatitis. The studies were done between two and 29 months after the most recent attack. A control group included five patients with no evidence of pancreatic disease. A four hour primed/continuous intravenous infusion of [14C]L-leucine tracer was given with secretin (2 U/kg/h) and cholecystokinin (0.5 U/kg/h) and secreted duodenal juice aspirated. Amylase and trypsin were extracted from duodenal juice by affinity chromatography, permitting measurement of the rate of isotope incorporation into total protein, amylase, and trypsin. The results showed non-parallel changes in enzyme synthesis and turnover with decreases in total enzyme protein and amylase synthesis and turnover but preservation of trypsin synthesis and turnover. The low turnover rates may be ascribed to continuing pancreatic cell malfunction after recovery from acute alcoholic pancreatitis and suggest that the decreased amylase secretion rates are partly a consequence of impaired amylase synthesis and not simply because of loss of pancreatic tissue.

Ogden, J M; O'Keefe, S J; Louw, J A; Adams, G; Marks, I N



In vivo exposure of Mytilus edulis to living enteric bacteria: a threat for immune competency?  


Mussels are widespread in coastal environments and experience various physical, chemical, and bacteriological conditions. Owing to the increase of coastal urbanization, mussels are now commonly exposed not only to indigenous bacteria, but also to enteric bacteria originating from pulsed and chronic sewage discharges into coastal environments. Due to its broad resilience to environmental variations, the blue mussel Mytilus edulis is commonly used as an indicator of environmental quality in bio-monitoring programs. However, since mussel immune system capabilities may be affected by the presence of exogenous fecal bacteria in coastal seawater subjected to sewage discharges, we aimed to determine the effect of in vivo bacterial challenges on mussels' immune competency by using two exogenous enteric bacterial strains, Escherichia coli and Enterococcus faecalis, and an indigenous bacterial strain Vibrio splendidus (as control). Bacterial strains were tested individually, by injection into the posterior adductor muscle at three different cell densities (10(2), 10(3), and 10(4) cells). Unlike classic in vitro experiments using higher bacterial concentrations, neither the enteric bacteria nor the indigenous strain induced significant increase or decrease of either cell-mediated (phagocytosis, reactive oxygen species, and NO(x) production) or humoral components (prophenoloxidase-like, acid phosphatase, and L-leucine-aminopeptidase production) of the immune system. This study demonstrates that, at low concentrations, E. coli and E. faecalis do not represent an additional threat that could impair M. edulis immune competency and, as a consequence, its potential of survival in coastal areas subjected to sewage discharges. PMID:23014953

Gauthier-Clerc, Sophie; Boily, Isabelle; Fournier, Michel; Lemarchand, Karine



MAGUKs, scaffolding proteins at cell junctions, are substrates of different proteases during apoptosis.  


A major feature of apoptotic cell death is gross structural changes, one of which is the loss of cell-cell contacts. The caspases, executioners of apoptosis, were shown to cleave several proteins involved in the formation of cell junctions. The membrane-associated guanylate kinases (MAGUKs), which are typically associated with cell junctions, have a major role in the organization of protein-protein complexes at plasma membranes and are therefore potentially important caspase targets during apoptosis. We report here that MAGUKs are cleaved and/or degraded by executioner caspases, granzyme B and several cysteine cathepsins in vitro. When apoptosis was induced by UV-irradiation and staurosporine in different epithelial cell lines, caspases were found to efficiently cleave MAGUKs in these cell models, as the cleavages could be prevented by a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone. Using a selective lysosomal disrupting agent L-leucyl-L-leucine methyl ester, which induces apoptosis through the lysosomal pathway, it was further shown that MAGUKs are also cleaved by the cathepsins in HaCaT and CaCo-2 cells. Immunohistological data showed rapid loss of MAGUKs at the sites of cell-cell contacts, preceding actual cell detachment, suggesting that cleavage of MAGUKs is an important step in fast and efficient cell detachment. PMID:21368887

Ivanova, S; Gregorc, U; Vidergar, N; Javier, R; Bredt, D S; Vandenabeele, P; Pardo, J; Simon, M M; Turk, V; Banks, L; Turk, B



Assessment of recovery of the intestine after acute radiation injury  

SciTech Connect

Several aspects of intestinal function and morphology are affected by acute radiation damage, including changes in the activity of proliferative cells in the crypts, immune cell populations, and the transport of various substrates. This study was designed to compare the time course of the recovery of intestinal proliferation, transport, and leukocyte population following radiation injury. Rats received a single dose of 6 Gy to the abdomen from a /sup 137/Cs source and were studied 3, 7, and 14 days later. No changes in the passive uptake of L-glucose or D-leucine were observed in the jejunum. Active transport of D-glucose and maximal water uptake were reduced at 3 days but had returned to normal by 7 days, whereas L-leucine uptake required more than 7 days to return to control levels. Mucosal permeability, assessed by an in vivo potential difference technique, remained increased 7 days after irradiation. Ornithine decarboxylase, an indicator of DNA synthetic activity, was elevated following radiation treatment and remained so even after 14 days. By comparison, myeloperoxidase activity, used as a quantitative monitor of granulocyte numbers, was still reduced after 7 days. These data indicate that while certain parameters of gut function may return to normal soon after radiation injury, the recovery of other factors is more prolonged. Thus the return of transport function to normal values post irradiation may be viewed as an adaptive change rather than simply the recovery of the tissue.

Baer, A.R.; Cheeseman, C.I.; Thomson, A.B.



Inhibition of proteasome activity sensitizes dopamine neurons to protein alterations and oxidative stress.  


Impairment in the capacity of the ubiquitin-proteasome pathway to clear unwanted proteins has been implicated in the cell death that occurs in Parkinson's disease (PD). In support of this concept, defects in proteasomal structure and function, as well as protein aggregates and increased levels of oxidized proteins are found in the substantia nigra of PD patients. We have previously demonstrated that inhibition of proteasome activity in mesencephalic cultures induces degeneration of dopaminergic neurons coupled with the formation of proteinaceous intracellular inclusions. In this study we examined the effect of proteasome inhibition on cultured dopamine neurons when combined with oxidative stress and protein misfolding, in order to better simulate the condition in PD. We demonstrate that two structurally unrelated inhibitors of proteasome activity, lactacystin and carbobenzoxy-L-leucul-L-leucyl-L-leucinal (MG132), cause dose-dependent cell loss that preferentially affects dopaminergic neurons. Conditions that promote protein damage and misfolding such as oxidative stress, heat shock, and canavanine also induce neuronal degeneration with preferential loss of dopamine neurons and cell death is markedly increased when any of these is combined with a proteasome inhibitor. These studies demonstrate a synergistic effect between conditions that promote the formation of damaged proteins and those in which proteasomal function is impaired, and provide further support for the notion that cell loss in PD could be related to a defect in protein handling. PMID:15480836

Mytilineou, C; McNaught, K St P; Shashidharan, P; Yabut, J; Baptiste, R J; Parnandi, A; Olanow, C W



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)

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.

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



Expression, purification, crystallization and preliminary X-ray analysis of a novel N-substituted branched-chain L-amino-acid dioxygenase from Burkholderia ambifaria AMMD.  


Ferrous ion- and ?-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria AMMD (SadA) catalyzes the C3-hydroxylation of N-substituted branched-chain L-amino acids, especially N-succinyl-L-leucine, coupled to the conversion of ?-ketoglutarate to succinate and CO(2). SadA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of selenomethionine-substituted SadA were obtained using a reservoir solution containing PEG 3000 as the precipitant at pH 9.5 and diffracted X-rays to 2.4 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient (V(M) = 2.1 Å(3) Da(-1), 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit. PMID:22949196

Qin, Hui-Min; Miyakawa, Takuya; Nakamura, Akira; Xue, You-Lin; Kawashima, Takashi; Kasahara, Takuya; Hibi, Makoto; Ogawa, Jun; Tanokura, Masaru



Kinetics and conformational stability studies of recombinant leucine aminopeptidase.  


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) ?M(-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. PMID:24368112

Hernández-Moreno, Ana V; Villaseñor, Francisco; Medina-Rivero, Emilio; Pérez, Néstor O; Flores-Ortiz, Luis F; Saab-Rincón, Gloria; Luna-Bárcenas, Gabriel



dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.  

PubMed Central

Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties. Images

Henrich, B; Becker, S; Schroeder, U; Plapp, R



Chiral micellar electrokinetic chromatography (CMEKC)-atmospheric pressure photoionization of benzoin derivatives using mixed molecular micelles  

PubMed Central

In the present work we report, for the first time, the successful on-line coupling of chiral micellar electrokinetic chromatography (CMEKC) to atmospheric pressure photo-ionization mass spectrometry (APPI-MS). Four structurally similar neutral test solutes (e.g., benzoin derivatives) were successfully ionized by APPI-MS. The mass spectra in the positive ion mode showed that the protonated molecular ions of benzoins are not the most abundant fragment ions. Simultaneous enantioseparation by CMEKC and on-line APPI-MS detection of four photoinitiators: hydrobenzoin (HBNZ), benzoin (BNZ), benzoin methyl ether (BME), benzoin ethyl ether (BEE), were achieved using an optimized molar ratio of mixed molecular micelle of two polymeric chiral surfactants (polysodium N-undecenoxy carbonyl-L-leucinate and polysodium N-undecenoyl-L,L-leucylvalinate). The CMEKC conditions, such as voltage, chiral polymeric surfactant concentration, buffer pH, and BGE concentration, were optimized using a multivariate central composite design (CCD). The sheath liquid composition (involving % v/v methanol, dopant concentration, electrolyte additive concentration, and flow rate) and spray chamber parameters (drying gas flow rate, drying gas temperature, and vaporizer temperature) were also optimized with CCD. Models built based on the CCD results and response surface method was used to analyze the interactions between factors and their effects on the responses. The final overall optimum conditions for CMEKC-APPI-MS were also predicted and found in agreement with the experimentally optimized parameters.

He, Jun; Shamsi, Shahab A.



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


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

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



Leucine transport by the larval midgut of the parasitoid Aphidius ervi (Hymenoptera).  


The larval midgut of the hymenopteran parasitoid Aphidius ervi accomplishes a large transport of nutrients from the lumen to the haemocoel, providing most of the organic molecules necessary for rapid insect development. l-amino acids in general, and leucine in particular, are efficiently accumulated in the larval body. We show here that the intact midgut of early 3rd instar larvae incubated in vitro can take up [(3)H]l-leucine from the basolateral side of the epithelium by transporters insensitive to the presence of monovalent cations. When the midgut is opened and the apical membrane of the absorbing epithelial cells is exposed to the medium containing radiolabelled leucine, a sodium-dependent uptake of the amino acid becomes apparent, disclosing the presence of a symport mechanism. Inhibition experiments of leucine uptake by a 100-fold excess of different amino acids, selected according to the properties of their side chain, revealed that this apical sodium-dependent mechanism is a broad spectrum transport system with a specialization for the absorption of aliphatic amino acids, that can also transfer glutamine and proline, but not phenylalanine, lysine and arginine. Altogether the experimental results obtained with intact- and open-gut preparations suggest that leucine transport across the basolateral membrane is mediated by both an uniporter and an obligatory amino acid exchange mechanism. PMID:19799906

Fiandra, L; Caccia, S; Giordana, B; Casartelli, M



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


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

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



Molecular Dynamics of Peptide Folding at Aqueous Interfaces  

NASA Technical Reports Server (NTRS)

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.

Pohorille, Andrew; Chipot, Christophe; Chang, Sherwood (Technical Monitor)



Site density of mouse intestinal glucose transporters declines with age.  


To evaluate the effect of age on nutrient transport, the absorption rates of D-glucose, D-fructose, L-alanine, L-aspartate, L-leucine, L-lysine, L-proline, folic acid, and nicotinamide were determined in isolated jejunal tissues of young (6.7 mo old) and aged (23.7 and 27.0 mo old) mice (COBS:SFW). D-Glucose and D-fructose uptakes per milligram tissue were approximately 20-120% higher in the proximal jejunum and 15-50% higher in the distal jejunum of young mice. Amino acid and vitamin uptakes per milligram were also higher in young mice, but differences were not statistically significant. The number of Na(+)-D-glucose transporters per milligram tissue as estimated by specific phlorizin binding decreased with age. There was no age-related change in passive L-glucose permeability, in Kd of specific phlorizin binding, in transporter turnover rate, and in the molecular weight of the Na(+)-D-glucose transporter. Thus a reduction in D-glucose transporter site density fully accounts for the age-related decline in D-glucose transport rate per milligram small intestine. PMID:8447410

Ferraris, R P; Hsiao, J; Hernandez, R; Hirayama, B



Effects of excess dietary leucine and leucine catabolites on growth and immune responses in weanling pigs.  


Two experiments with weanling pigs were conducted to study the effects on growth and immune responses of excess dietary L-leucine (LEU) and dietary supplementation with the LEU catabolites, alpha-ketoisocaproic acid (KIC) and beta-hydroxymethyl butyrate (HMB). In Exp. 1, 80 pigs were randomly allocated according to initial BW and ancestry to five replications of four dietary treatments (four pigs/pen). The control diet contained wheat, oat groats, menhaden fish meal, and dried whey and provided 1.12% LEU. Treatment diets were the control plus 1.12% LEU, 1.12% KIC, or .4% HMB. The experiment lasted 6 wk. In Exp. 2, 36 pigs were randomly allocated to nine replications of four dietary treatments in a 2 x 2 factorial arrangement. Treatments consisted of two concentrations of dietary LEU and a daily i.m. injection of dexamethasone (DEX) or saline. Pigs were fed a control corn-soybean meal and dried whey diet (1.56% LEU) or the control diet plus 1.56% of crystalline LEU. Pigs were individually penned and the experiment lasted 4 wk. Growth performance, plasma free amino acids, plasma urea nitrogen, and humoral and cellular immune responses were measured. Results indicated that LEU concentrations in practical diets and supplementation with KIC and HMB (Exp. 1) did not detrimentally affect growth and immune response. The high LEU concentration and DEX injection used in Exp. 2, however, were detrimental to both growth and immune response. PMID:7601729

Gatnau, R; Zimmerman, D R; Nissen, S L; Wannemuehler, M; Ewan, R C



Functional Characterization of Two M42 Aminopeptidases Erroneously Annotated as Cellulases  

PubMed Central

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.

Dutoit, Raphael; Brandt, Nathalie; Legrain, Christianne; Bauvois, Cedric



Standard-Free Quantitation of Mixtures using Clusters Formed by Electrospray Mass Spectrometry  

PubMed Central

Ion abundances in electrospray ionization mass spectra depend on many factors, including molecular hydrophobicity, basicity, solution composition, and instrumental parameters. A recently introduced method that uses nonspecific cluster ion abundances to obtain solution-phase molar fractions of analytes directly from ESI mass spectra without using standards was evaluated using solutions containing 0.03% to 24% L-threonine, D-threonine, L-leucine, L-lysine, L-glutamic acid or diglycine with L-serine as a major component. Because of the propensity of serine clusters to exhibit “magic” numbers, which can be chirally selective, these experiments provide a rigorous test of this standard-free cluster quantitation method, which requires that clusters form statistically from analytes in solution. For each of these solutions, the compositions of clusters containing ? 32 molecules reflect the solution molar fractions of each component. From the abundances of these larger clusters, the solution molar fraction can be determined to better than 10% accuracy over nearly three orders of magnitude in concentration. In contrast, the ionization/detection efficiency of the individual amino acids differs by as much as a factor of 460 in these experiments. The protonated octamer incorporates some molecules statistically but efficiently excludes other molecules that have significantly different properties or chirality. This standard-free quantitation method may be most advantageous for rapidly characterizing mixtures, such as products of chemical synthesis, which contain unknown products or molecules for which suitable standards are not readily available.

Flick, Tawnya G.; Leib, Ryan D.; Williams, Evan R.



Nutrient metabolism in pancreatic islets from protein malnourished rats.  


The metabolism of D-[5-3H]glucose, D-[3,4-14C]glucose, [2-3H]glycerol, L-[U-14C]glutamine, L-[1-14C]-leucine, and L-[U-14C]leucine was investigated in pancreatic islets isolated from either control rats or animals fed a low-protein isocaloric diet, containing 8% instead of 20% protein, during both fetal and postnatal life. In the latter animals, decreases in body weight, plasma insulin concentration, and insulinogenic index were associated with two major anomalies of islet nutrient metabolism. First, an imbalance between oxidative and anaerobic glycolysis was found in the islets of rats fed the low-protein isocaloric diet. It coincided with a decreased circulation in the glycerol phosphate shuttle, as judged by the generation of 3HOH from [2-3H]glycerol, and was probably attributable to the deficiency of mitochondrial FAD-linked glycerophosphate dehydrogenase previously documented in islet homogenates of the rats fed low protein. Second, the transamination of L-leucine to 2-ketoisocaproate was decreased in the low-protein-fed rats, while the oxidative decarboxylation of the 2-keto acid and the further catabolism of isovaleryl CoA occurred at normal rates when expressed relative to the initial transamination rate. These metabolic anomalies may account, in part at least, for the impairment of insulin release in protein malnutrition. PMID:8902196

Sener, A; Reusens, B; Remacle, C; Hoet, J J; Malaisse, W J



Microplasma discharge ionization source for ambient mass spectrometry.  


In this paper, we demonstrate the first use of a microplasma ionization source for ambient mass spectrometry. This device is a robust, easy-to-operate microhollow discharge that enables ambient direct analysis of gaseous, liquid, and solid-phase samples with minimum requirements in terms of operating power and high purity gas consumption. The initial performance of the microplasma device has been evaluated by ionizing samples containing dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methyl salicylate, caffeine, l-leucine, l-histidine, loratadine, ibuprofen, acetaminophen, acetylsalicylic acid, and cocaine in various forms. These molecules are diverse in nature, but almost all have relatively high proton affinities. Thus, the major species observed in all obtained mass spectra corresponded to protonated molecules. Though these microplasmas are known to produce significant densities of metastable species and electrons with mean energies greater than several electronvolt, minimal fragmentation was observed. Background spectra showed prominent signals corresponding to H(+)(H(2)O)(2) ions and a distinct lack of H(3)O(+). Small water cluster ions are likely the dominant proton transfer agents, giving rise to mass spectral data very similar to that obtained using other plasma-based ambient ionization techniques. The simplicity, low cost, low power, low rate of gas consumption, and possibility of being batch-fabricated, makes these microplasma devices attractive candidates as ion sources for miniaturized mass spectrometry and other field detection applications. PMID:20020764

Symonds, Joshua M; Galhena, Asiri S; Fernández, Facundo M; Orlando, Thomas M



Geochemical gradients within modern and fossil shells of Concholepas concholepas from northern Chile: an insight into U-Th systematics and diagenetic/authigenic isotopic imprints in mollusk shells  

NASA Astrophysics Data System (ADS)

Seriate geochemical measurements through shells of one modern, one Holocene, and two Sangamonian Concholepas concholepas, from marine terraces of Northern Chile, were performed to document diagenetic vs. authigenic geochemical signatures, and to better interpret U-series ages on such material. Subsamples were recovered by drilling from the outer calcitic layer to the inner aragonitic layer of each of the studied shells. Unfortunately, this sampling procedure induces artifacts, notably the convertion of up to ˜20% of calcite into aragonite, and of up to ˜6% of aragonite into calcite, as well as in the epimerization of a few percent of isoleucine into D-alloisoleucine/ L-isoleucine. Negligible sampling artifacts were noticed for stable isotope and total amino acid contents. Diagenetic effects on the geochemical properties of the shells are particularly pronounced in the inner aragonitic layer and more discrete in the outer calcitic layer. The time-dependent decay of the organic matrix of the shell is illustrated by a one order of magnitude lower total amino acid content in the Sangamonian specimens by comparison with the modern shell. Conversely, the Sangamonian shells U contents increase by a similar factor and 13C- 18O enrichments as high as 2 to 3‰ seem also to occur through the same time interval possibly due to partial replacement of aragonite by gypsum. The decay of the organic matrix of the aragonitic layer of the shell is thought to play a major role with respect to U-uptake processes and stable isotope shifts. Nevertheless, asymptotic 230Th-ages (˜100 ka) in the inner U-rich layers of the Sangamonian shells, and 234U/ 238U ratios compatible with a marine origin for U, suggest U-uptake within a short diagenetic interval, when marine waters were still bathing the embedding sediment. Thus, U-series ages on fossil mollusks from such a hyper-arid environment should not differ much from the age of the corresponding marine unit deposition. However, the diagenetic enrichments in stable isotopes raise concerns about their use for paleoenvironmental reconstructions under such climate conditions.

Labonne, Maylis; Hillaire-Marcel, Claude



Sequential Induction of Phenylalanine Ammonia-lyase and a Lyase-inactivating System in Potato Tuber Disks  

PubMed Central

The light induced synthesis of phenylalanine ammonia-lyase in disks cut from potato tubers is very sensitive to cycloheximide. Synthesis is inhibited 50% in disks cultured on 5 ?m cycloheximide instead of water and almost completely in disks aged in the presence of 10 ?m inhibitor. Inhibition is irreversible. Fresh disks exposed only 1 hour to 10 ?m cycloheximide do not synthesize enzyme during the subsequent 24 hours. Normally a maximal enzyme activity develops in disks about 24 hours after being cut from the tuber. Thereafter enzyme activity declines. The disappearance of enzyme is not affected by concentrations of cycloheximide sufficient to inhibit the synthesis of enzyme initially. No disappearance of enzyme is noted during the initial phase of induction if enzyme synthesis is inhibited by cycloheximide. However, enzyme does disappear from the tissue if more than half the maximal enzyme content is allowed to form before synthesis is inhibited. If cycloheximide at a concentration 10-fold that needed to inhibit synthesis completely is added to disks after they have attained a maximal enzyme level, then subsequent loss of enzyme activity from the tissue is prevented. The initial stability of the enzyme in the absence of further synthesis and the inhibition of enzyme disappearance by high concentrations of cycloheximide suggest A) that early phases of induction involve synthesis of enzyme protein in the absence of turnover, B) that a system capable of degrading or inactivating the lyase subsequently forms in the tissue, and C) that the formation of the degrading or inactivating system requires protein synthesis. The effect of cycloheximide on uptake and incorporation of l-isoleucine-U-14C into soluble and insoluble proteins of tuber disks was also examined. During induction the rate of uptake increased 3 to 4-fold, and the rate of incorporation into protein, corrected for change in uptake, increased 25-fold. Cycloheximide inhibited incorporation of isoleucine-14C into proteins of fresh disks more than 80%. It did not prevent activation of general protein synthesis during induction and inhibited incorporation in induced disks only 20%. At all times incorporation of amino acid into the soluble, lyase-rich, protein fraction was more sensitive to cycloheximide than the insoluble fraction.

Zucker, Milton



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


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. PMID:19060158

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



Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus?  

PubMed Central

Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar Vmax values (between 47 and 58 ?mol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

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



Combining cosmogenic radionuclides and amino acid racemization to date late Pliocene glacial deposits exposed on Baffin Island, Nunavut, Canada  

NASA Astrophysics Data System (ADS)

Sequences of glacial deposits spanning the Quaternary are valuable archives recording the effects of glaciation on landscapes through time, but determining the age of such deposits has long challenged geologists. The recent advances in cosmogenic radionuclide (CRN) measurement has made it possible to date some of these deposits, but dating buried glacial sediments in most settings remains problematic. Here we explore a new approach to date the oldest glacial deposits in the Plio-Pleistocene Clyde Foreland Formation of Baffin Island. This formation, approximately 40 m thick, includes interlayered shell-bearing marine, glaciomarine, and glacial sediments deposited along the northern margin of the Laurentide Ice Sheet and earlier continental ice sheets. Previous work on foraminifera assemblages suggests that the deposits span the last ?2 Ma. By combining CRN measurements (10Be and 26Al) from the glacial units and measurements of the D-alloisoleucine:L-isoleucine ratios (A/I) in valves of the mollusk Hiatella arctica in the marine units overlying a particular glacial deposit, we can calculate the age of the glacial deposit. Because the post-burial temperature history for the mollusks preserved in the Clyde Foreland Formation is poorly constrained, A/I ratios alone cannot be used to determine absolute ages. Instead, we use A/I ratios to identify sediment packages of discrete ages and define a step-wise burial history function for glacial units. A/I ratios of all packages (<0.3 for the total hydrolysate fraction) fall within the A/I interval characterized by linear racemization kinetics, so the age of each package in the burial history function can simply be defined as a fractional age with respect to the total burial age for the glacial deposit of interest. The long duration of burial (26Al/10Be as low as 1.6±0.6 at 2?) and low initial CRN inventories require that post-burial muogenic production is accounted for using the burial history function. We apply a numerical model to calculate the duration of burial from the measured CRN concentrations for a given inherited CRN inventory. But because this initial inventory is unknown, a single CRN sample/burial history combination will not provide a unique age solution. Instead, measurements from multiple localities where a particular glacial deposit has differing burial histories (i.e., the thickness of overlying units or ages of overlying units differ) are required to statistically determine the total burial age that most closely matches the observed CRN inventories and burial histories.

Refsnider, K. A.; Miller, G. H.



The enigma of 3400 years BP coastal oolites in tropical northwest Western Australia… why then, why there?  

NASA Astrophysics Data System (ADS)

Oolites crop out along the northwestern coast of Western Australia at Port Smith, about 80 km SW of Broome. An oolitic coastal ridge truncated by marine erosion exposes subtidal, intertidal, and supratidal (aeolian) facies. The deposits are firmly indurated and composed of about 75% tangentially and moderately thickly layered, aragonitic ooid grains with over 90% quartz nuclei. Subtidal sedimentary structures are exposed about a metre above the present high tide mark, hinting that sea level may have been somewhat higher when the shoreline was formed. However, the macrotidal range of up to 7 m, and the possibility of cyclonic surges along the coast, precludes unequivocal determinations on this point. Whole-rock amino acid racemisation (AAR) geochronology (epimerisation of isoleucine: D-alloisoleucine/ L-isoleucine or A/I) on each facies of the oolite outcrop averaged 0.106 ± 0.013 ( N = 10). The modern beach contains fewer ooids (˜ 30%), and nearly half of these are stained brown, grey, or black, perhaps as a result of burial, reduction and/or mineralization. A higher (older) mean and large standard deviation in whole-rock amino acid ratio of 0.145 ± 0.067 ( N = 2) supports our inference that ooids on the modern beach were reworked from fossil deposits. Reverse phase chronostratigraphy (RPC) on individual ooid grains holds tremendous promise in this preliminary study. RPC results show a narrow variation of D/ L values (CV = 6-11%), and yield nearly identical D/ L Asp means from light coloured fossil ooids (0.307 ± 0.018 ( N = 17)); light (0.323 ± 0.026 ( N = 12)) and dark coloured ooids (0.298 ± 0.027 ( N = 10)) from the active beach face. When compared to A/I ratios from 14C dated mid-Holocene ooids in the Bahamas, the mean A/I from Port Smith reflects an age of ca. 3500-4500 years that is in agreement with a calibrated AMS 14C age of 3370 ± 50 calendar years BP on the same material. Thus, the ooids were formed, transported, emplaced, strongly cemented, and largely eroded from the beach ridge in only 3400 years. The environmental conditions that underlie a pulse of ooid deposition during a brief period of the mid-Holocene almost certainly involve extensive tidal inlets in the area, a possible mid-Holocene oscillation 1-2 m above present, and a subsequent fall to present level that terminated the process. Apparently, a unique combination of factors produced ooids here, and in only a handful of other sites in Australia and the Indian Ocean.

Hearty, Paul; O'Leary, Michael; Donald, Andrew; Lachlan, Terry



Characterization of rapid and high-affinity uptake of L-serine in neurons and astrocytes in primary culture.  


The non-essential amino acid L-serine was shown to be required to support the survival of rat cerebellar Purkinje neurons because of lack of the expression of the L-serine biosynthesis enzyme 3-phosphoglycerate dehydrogenase in them. In the present study, we investigated L-[(3)H]serine uptake in primary cultures of neurons and astrocytes from the rat telencephalon. In both neurons and astrocytes, L-[(3)H]serine uptake was dependent on temperature and Na(+) ions, and exhibited a single component of high-affinity uptake sites (K(m)=15.0 and 17.2 micro M for neurons and astrocytes, respectively). Kinetic analysis of L-[(3)H]serine uptake also revealed that the uptake into neurons was faster than that into astrocytes. The selectivity of inhibition by amino acids of the L-[(3)H]serine uptake resembled that of the system ASC transporters ASCT1 and ASCT2. Neutral amino acids L-alanine, L-serine, L-cysteine, and L-threonine strongly inhibited the uptake by both cell types. Furthermore, in astrocytes, but not in neurons, L-valine and L-proline also inhibited L-[(3)H]serine uptake. Neither alpha-methyl aminoisobutyric acid (a system A-specific substrate) nor 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (a system L-specific substrate) inhibited the uptake of L-[(3)H]serine in both neurons and astrocytes. Expression of ASCT transporters in both neurons and astrocytes was examined by use of reverse transcriptase polymerase chain reaction and immunoblot analysis. Whereas transcripts (mRNAs) of both ASCT1 and ASCT2 transporters were detected in astrocytes, only the mRNA of the former subtype was detected in neurons. Immunoblot analysis confirmed the presence of ASCT1 in both neurons and astrocytes. These findings indicate that neurons accumulate a high level of L-serine by using a Na(+)-dependent, high-affinity transport system, operating predominantly through the ASCT1 transporter subtype. PMID:12885409

Yamamoto, Toshifumi; Nishizaki, Itone; Furuya, Shigeki; Hirabayashi, Yoshio; Takahashi, Kenzo; Okuyama, Shigeru; Yamamoto, Hideko



Global Regulation of Food Supply by Pseudomonas putida DOT-T1E? †  

PubMed Central

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 ?crc, ?crp, ?cyoB, and ?ptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d-fructose is influenced by CyoB, and l-arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for N-acetyl-l-cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival.

Daniels, Craig; Godoy, Patricia; Duque, Estrella; Molina-Henares, M. Antonia; de la Torre, Jesus; del Arco, Jose Maria; Herrera, Carmen; Segura, Ana; Guazzaroni, M. Eugenia; Ferrer, Manuel; Ramos, Juan Luis



Mixed ligand complex formation of 2-aminobenzamide with Cu(II) in the presence of some amino acids: synthesis, structural, biological, pH-metric, spectrophotometric and thermodynamic studies.  


Mixed ligand Cu(II) complexes of 2-aminobenzamide (2AB) and amino acids viz., glycine (gly), L-alanine (ala), L-valine (val) and L-phenylalanine (phe) have been synthesised and characterized by various physico-chemical and spectral techniques. The calculated g-tensor values for Cu(II) complexes at 77 K and 300 K, show the distorted octahedral geometry which has been confirmed from the absorption studies. Consequently, the thermal studies illustrate that the loss of water and acetate molecules in the initial stage which are followed by the decomposition of organic residues. The powder X-ray diffraction and SEM analysis reflect that all the complexes have well-defined crystallinity nature with homogeneous morphology. The binding activities of CT DNA with CuAB complexes have been examined by absorption studies. Further, the oxidative cleavage interactions of 2-aminobenzamide and CuAB complexes with DNA were studied by gel electrophoresis method in H2O2 medium. Also, the complex formation of Cu(II) involving 2-aminobenzamide and amino acids were carried out by a combined pH-metric and spectrophotometric techniques in 50% (v/v) water-ethanol mixture at 300, 310, 320 and 330±0.1 K with I=0.15 mol dm(-3) (NaClO4). In solution, CuAB and CuAB2 species has been detected and the binding modes of 2-aminobenzamide and amino acids in both binary and mixed ligand complexes are same. The calculated stabilization value of ?logK, log X and log X' indicates higher stabilities for the mixed ligand complexes rather than their binary species. The thermodynamic parameters like ?G, ?H and ?S have been determined from temperature dependence of the stability constant. In vitro biological activities of 2-aminobenzamide, CuA and CuAB complexes show remarkable activities against some bacterial and fungal strains. The percentage distribution of various binary and mixed ligand species in solution at dissimilar pH intervals were also evaluated. PMID:23811147

Dharmaraja, Jeyaprakash; Esakkidurai, Thirugnanasamy; Subbaraj, Paramasivam; Shobana, Sutha



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

PubMed Central

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

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



Genotype to phenotype: identification of diagnostic vibrio phenotypes using whole genome sequences.  


Vibrios are ubiquitous in the aquatic environment and can be found in association with animal or plant hosts. The range of ecological relationships includes pathogenic and mutualistic associations. To gain a better understanding of the ecology of these microbes, it is important to determine their phenotypic features. However, the traditional phenotypic characterization of vibrios has been expensive, time-consuming and restricted in scope to a limited number of features. In addition, most of the commercial systems applied for phenotypic characterization cannot characterize the broad spectrum of environmental strains. A reliable and possible alternative is to obtain phenotypic information directly from whole genome sequences. The aim of the present study was to evaluate the usefulness of whole genome sequences as a source of phenotypic information. We performed a comparison of the vibrio phenotypes obtained from the literature with the phenotypes obtained from whole genome sequences. We observed a significant correlation between the previously published phenotypic data and the phenotypic data retrieved from whole genome sequences of vibrios. Analysis of 26 vibrio genomes revealed that all genes coding for the specific proteins involved in the metabolic pathways responsible for positive phenotypes of the 14 diagnostic features (Voges-Proskauer reaction, indole production, arginine dihydrolase, ornithine decarboxylase, utilization of myo-inositol, sucrose and L-leucine, and fermentation of D-mannitol, D-sorbitol, L-arabinose, trehalose, cellobiose, D-mannose and D-galactose) were found in the majority of the vibrios genomes. Vibrio species that were negative for a given phenotype revealed the absence of all or several genes involved in the respective biochemical pathways, indicating the utility of this approach to characterize the phenotypes of vibrios. The absence of the global regulation and regulatory proteins in the Vibrio parahaemolyticus genome indicated a non-vibrio phenotype. Whole genome sequences represent an important source for the phenotypic identification of vibrios. PMID:24505074

Amaral, Gilda Rose S; Dias, Graciela M; Wellington-Oguri, Michiyo; Chimetto, Luciane; Campeão, Mariana E; Thompson, Fabiano L; Thompson, Cristiane C



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

PubMed Central

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

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



Light-Activated Amino Acid Transport Systems in Halobacterium halobium Envelope Vesicles: Role of Chemical and Electrical Gradients  

NASA Technical Reports Server (NTRS)

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.

MacDonald, Russell E.; Greene, Richard V.; Lanyi, Janos K.



Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices.  


Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca(2+)-dependent transglutaminase (TG, EC activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-?-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally. PMID:23076251

Beninati, Simone; Iorio, Rosa Anna; Tasco, Gianluca; Serafini-Fracassini, Donatella; Casadio, Rita; Del Duca, Stefano



Global Analyses of Transcriptomes and Proteomes of a Parent Strain and an l-Threonine-Overproducing Mutant Strain  

PubMed Central

We compared the transcriptome, proteome, and nucleotide sequences between the parent strain Escherichia coli W3110 and the l-threonine-overproducing mutant E. coli TF5015. DNA macroarrays were used to measure mRNA levels for all of the genes of E. coli, and two-dimensional gel electrophoresis was used to compare protein levels. It was observed that only 54 of 4,290 genes (1.3%) exhibited differential expression profiles. Typically, genes such as aceA, aceB, icdA, gltA, glnA, leu operon, proA, thrA, thrC, and yigJ, which are involved in the glyoxylate shunt, the tricarboxylic acid cycle, and amino acid biosynthesis (l-glutamine, l-leucine, proline, and l-threonine), were significantly upregulated, whereas the genes dadAX, hdeA, hdeB, ompF, oppA, oppB, oppF, yfiD, and many ribosomal protein genes were downregulated in TF5015 compared to W3110. The differential expression such as upregulation of thr operon and expression of yigJ would result in an accumulation of l-threonine in TF5015. Furthermore, two significant mutations, thrA345 and ilvA97, which are essential for overproduction of l-threonine, were identified in TF5015 by the sequence analysis. In particular, expression of the mutated thrABC (pATF92) in W3110 resulted in a significant incremental effect on l-threonine production. Upregulation of aceBA and downregulation of b1795, hdeAB, oppA, and yfiD seem to be linked to a low accumulation of acetate in TF5015. Such comprehensive analyses provide information regarding the regulatory mechanism of l-threonine production and the physiological consequences in the mutant stain.

Lee, Jin-Ho; Lee, Dong-Eun; Lee, Bheong-Uk; Kim, Hak-Sung



Effects of site-directed mutagenesis of the loop residue of the N-terminal domain Gly117 of thermolysin on its catalytic activity.  


In the N-terminal domain of thermolysin, two polypeptide strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122, are connected by a short loop, Asn116-Gly117, to form an anti-parallel ?-sheet. The Asn112-Trp115 strand is located in the active site, while the Ser118-Tyr122 strand and the Asn116-Gly117 loop are located outside the active site. In this study, we explored the catalytic role of Gly117 by site-directed mutagenesis. Five variants, G117A (Gly117 is replaced by Ala), G117D, G117E, G117K, and G117R, were produced by co-expressing in Escherichia coli the mature and pro domains as independent polypeptides. The production levels were in the order G117E > wild type > G117K, G117R > G117D. G117A was hardly produced. This result is in contrast to our previous one that all 72 active-site thermolysin variants were produced at the similar levels whether they retained activity or not (M. Kusano et al. J. Biochem., 145, 103-113 (2009)). G117E exhibited lower activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and higher activity in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester than the wild-type thermolysin. G117K and G117R exhibited considerably reduced activities. This suggests that Gly117 plays an important role in the activity and stability of thermolysin, presumably by affecting the geometries of the Asn112-Trp115 and Ser118-Tyr122 strands. PMID:21150094

Menach, Evans; Yasukawa, Kiyoshi; Inouye, Kuniyo



Asymmetric induction in hydrogen-mediated reductive aldol additions to alpha-amino aldehydes catalyzed by rhodium: selective formation of syn-stereotriads directed by intramolecular hydrogen-bonding.  


Rhodium-catalyzed hydrogenation of methyl vinyl ketone and ethyl vinyl ketone in the presence of N-Boc-alpha-aminoaldehydes 3a-8a at ambient temperature and pressure results in reductive C-C coupling to furnish aldol adducts 3b-8b and 3c-8c, respectively, which incorporate stereotriads that embody high levels of syn-aldol selectivity accompanied by high levels of anti-Felkin-Anh control. The collective data are consistent with a catalytic mechanism involving addition of the Z(O)-rhodium enolate to the sterically less-encumbered aldehyde pi-face of an intramolecularly hydrogen-bonded chelate through a Zimmerman-Traxler type transition structure. Stereochemical assignments are supported by single-crystal X-ray diffraction analysis of 5b-O-3,5-dinitrobenzoate, iso-5b, N-Me-iso-5b-O-3,5-dinitrobenzoate, and 7b. As revealed by HPLC analysis, optical purity of the stereochemically labile alpha-aminoaldehydes is completely preserved under the conditions of hydrogen-mediated aldol coupling. Deletion of the intramolecular hydrogen bond, as in the case of N-methyl-N-Boc-l-leucinal N-Me-5a, inverts stereoselectivity to furnish the Felkin-Anh product N-Me-iso-5b in 17% yield. Additionally, reactions performed in the presence of tert-amyl alcohol (10 equiv) exhibit markedly lower levels of anti-Felkin-Anh control (7:1 versus > or = 20:1). The collective studies suggest that intramolecular hydrogen bonding plays a key role in both activating the alpha-aminoaldehyde toward addition and directing facial selectivity. PMID:17177457

Jung, Cheol-Kyu; Krische, Michael J



Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells  

PubMed Central

The loss of tyrosinase, the key enzyme in melanin synthesis, has been implicated in the dedifferentiation of malignant melanocytes. The presence of tyrosinase transcripts and antigenic peptides in melanoma tumors prompted us to investigate whether the basis for the loss of the enzyme was proteolytic degradation. Toward this aim, we followed the kinetics of synthesis, degradation, processing, chaperone binding, inhibitor sensitivity, and subcellular localization of tyrosinase in normal and malignant melanocytes. We found that, in amelanotic melanoma cell lines, tyrosinase failed to reach the melanosome, the organelle for melanin synthesis, because it was retained in the endoplasmic reticulum (ER) and then degraded. Tyrosinase appeared mostly as a 70-kDa core-glycosylated, endoglycosidase H-sensitive, immature form bound to the ER chaperone calnexin and had a life-span of only 25% of normal. Maturation and transit from the ER to the Golgi compartment was facilitated by lowering the temperature of incubation to 31°C. Several proteasome inhibitors caused the accumulation of an ?60-kDa tyrosinase doublet that was more prominent in malignant than in normal melanocytes and promoted, to various degrees, the maturation of tyrosinase in melanoma cells and the translocation of the enzyme to melanosomes. The appearance of ubiquitinated tyrosinase after treatment of normal melanocytes with N-acetyl-l-leucinyl-l-leucinal-l-norleucinal reinforced our notion that some tyrosinase is normally degraded by proteasomes. Proteolysis of tyrosinase by proteasomes is consistent with the production of antigenic tyrosinase peptides that are presented to the immune system by major histocompatibility complex class I molecules.

Halaban, Ruth; Cheng, Elaine; Zhang, Yuhua; Moellmann, Gisela; Hanlon, Douglas; Michalak, Marek; Setaluri, Vijayasaradhi; Hebert, Daniel N.



Kynurenate production by cultured human astrocytes.  


In the rodent brain, astrocytes are known to be the primary source of kynurenate (KYNA), an endogenous antagonist of both the glycine(B) and the alpha7 nicotinic acetylcholine receptor. In the present study, primary human astrocytes were used to examine the characteristics and regulation of de novo KYNA synthesis in vitro. To this end, cells were exposed to KYNA's bioprecursor L-kynurenine, and newly formed KYNA was recovered from the extracellular milieu. The production of KYNA was stereospecific and rose with increasing L-kynurenine concentrations, reaching a plateau in the high microM range. In an analogous experiment, astrocytes also readily produced and liberated the potent, specific glycine(B) receptor antagonist 7-chlorokynurenate from L-4-chlorokynurenine. KYNA synthesis was dose-dependently reduced by L-leucine or L-phenylalanine, two amino acids that compete with L-kynurenine for cellular uptake, and by aminooxyacetate, a non-specific aminotransferase inhibitor. In contrast, KYNA formation was stimulated by 5 mM pyruvate or oxaloacetate, which act as co-substrates of the transamination reaction. Aglycemic or depolarizing (50 mM KCl or 100 microM veratridine) conditions had no effect on KYNA synthesis. Subsequent studies using tissue homogenate showed that both known cerebral kynurenine aminotransferases (KAT I and KAT II) are present in astrocytes, but that KAT II appears to be singularly responsible for KYNA formation under physiological conditions. Taken together with previous results, these data suggest that very similar mechanisms control KYNA synthesis in the rodent and in the human brain. These regulatory events are likely to influence the neuromodulatory effects of astrocyte-derived KYNA in the normal and diseased human brain. PMID:12541009

Kiss, C; Ceresoli-Borroni, G; Guidetti, P; Zielke, C L; Zielke, H R; Schwarcz, R



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

PubMed Central

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

Jabeen, Qaiser; Aslam, Naveed



A new protocol for the treatment of the chronic venous ulcers of the lower limb.  


Venous leg ulcer is a pathological condition afflicting prevalently elderly patients, which has been found to have a major impact on individuals' health and social aspects of quality of life. Actually, the best practice treatment is recommended to include wound dressing and multilayer compression therapy. In this study, we have tested the effectiveness and safety of Vulnamin(®), a novel dressing in the form of a metal cellulose gel containing the amino acids glycine, L: -lysine, L: -proline, L: -leucine, and hyaluronic acid, and elastic compressive bandages in the treatment of chronic venous ulcers of the lower limbs. The study has been conducted in two groups of patients, one treated with Vulnamin(®) plus Ca-alginate (ulcer duration = 25.4 ± 6.2 weeks; mean baseline ulcer area = 13.9 ± 4.5 cm(2)) and a control group treated with Ca-alginate alone (ulcer duration = 23.4 ± 4.2 weeks; mean baseline ulcer area = 15.1 ± 4.7 cm(2)). Results have shown that after 70 days of treatment patients significantly ameliorate their pathological condition if they are treated with Vulnamin(®), as compared with patients treated with Ca-alginate alone. In fact, at the end of the treatment, complete healing closure was 61% in the group treated with Vulnamin(®) and, respectively, 27% in the control group. Moreover, ulcer areas showed a significant reduction in patients treated with Vulnamin(®) (mean ulcer area = 3.04 ± 0.8 cm(2)), as compared with controls (mean ulcer area = 10.96 ± 3.8 cm(2)). Overall, the results of this study indicate that Vulnamin(®) together with elastocompression is safe and more effective than standard dressing together with elastocompression in inducing a faster healing in chronic venous ulcers of the lower limb. PMID:21559987

Maggio, Giulio; Armenio, Andrea; Ruccia, Francesca; Giglietto, Domenico; Pascone, Michele; Ribatti, Domenico



Hormonal regulation of leucine catabolism in mammary epithelial cells.  


Branched-chain amino acids (BCAA) are actively taken up and catabolized by the mammary gland during lactation for syntheses of glutamate, glutamine and aspartate. Available evidence shows that the onset of lactation is associated with increases in circulating levels of cortisol, prolactin and glucagon, but decreases in insulin and growth hormone. This study determined the effects of physiological concentrations of these hormones on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 3 mM D-glucose, 0.5 mM L-leucine, L-[1-14C]leucine or L-[U-14C]leucine, and 0-50 ?U/mL insulin, 0-20 ng/mL growth hormone 0-200 ng/mL prolactin, 0-150 nM cortisol or 0-300 pg/mL glucagon. Increasing extracellular concentrations of insulin did not affect leucine transamination or oxidative decarboxylation, but decreased the rate of oxidation of leucine carbons 2-6. Elevated levels of growth hormone dose dependently inhibited leucine catabolism, ?-ketoisocaproate (KIC) production and the syntheses of glutamate plus glutamine. In contrast, cortisol and glucagon increased leucine transamination, leucine oxidative decarboxylation, KIC production, the oxidation of leucine 2-6 carbons and the syntheses of glutamate plus glutamine. Prolactin did not affect leucine catabolism in the cells. The changes in leucine degradation were consistent with alterations in abundances of BCAA transaminase and phosphorylated levels of branched-chain ?-ketoacid dehydrogenase. Reductions in insulin and growth hormone but increases in cortisol and glucagon with lactation act in concert to stimulate BCAA catabolism for glutamate and glutamine syntheses. These coordinated changes in hormones may facilitate milk production in lactating mammals. PMID:22707151

Lei, Jian; Feng, Dingyuan; Zhang, Yongliang; Dahanayaka, Sudath; Li, Xilong; Yao, Kang; Wang, Junjun; Wu, Zhenlong; Dai, Zhaolai; Wu, Guoyao



Endocannabinoid-mediated long-term depression of afferent excitatory synapses in hippocampal pyramidal cells and GABAergic interneurons.  


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-? (DGL-?), 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-? 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 endocannabinoid-LTD contributes to cortical information processing during distinct network activity patterns in a cell type-specific manner. PMID:23055515

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



The TonB3 System in the Human Pathogen Vibrio vulnificus Is under the Control of the Global Regulators Lrp and Cyclic AMP Receptor Protein  

PubMed Central

TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position ?59 to ?509, cAMP-CRP binds exclusively in a region centered at position ?122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between “feast” and “famine”). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms.

Crosa, Jorge H.



Benzo[a]pyrene Uptake by Bacteria and Yeast  

PubMed Central

Moore, B. G. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Arthur P. Harrison, Jr. Benzo[a]pyrene uptake by bacteria and yeast. J. Bacteriol. 90:989–1000. 1965.—Various Enterobacteriaceae and yeast incubated in a medium containing 25 ?g/ml of H3-benzo[a]pyrene (30% serum in the medium dissolves the hydrocarbon) retain radioactivity after washings with fresh 30% serum-medium. This radioactivity is defined as bound and represents intact benzo[a]pyrene. Factors relating to the binding of benzo[a]pyrene (benzo[a]pyrene uptake) have been studied in detail with Escherichia coli Ma, a triple auxotroph requiring l-leucine, uracil, and thymine. In defined medium, benzo[a]pyrene uptake by normally growing cells is 10?10 to 2 × 10?10 ?g per cell. Uptake is the same in suspensions lacking leucine and containing chloramphenicol where there is neither measurable protein synthesis nor cell division. Uptake is diminished, but not eliminated, by autoclaving the cells; thus, some uptake occurs in the absence of enzymatic activity. Uptake is enhanced by heat shock, thymine deprivation, uracil deprivation, and exposure to penicillin. Thus, uptake is affected by the physiological state of the cells. Either the cells play a direct (enzymatic) role in uptake, or they affect uptake indirectly by increasing or altering the benzo[a]pyrene-binding structure. Physical fractionation of cells demonstrates that this structure is associated with the cell wall-membrane complex. All but 1% of the bound radioactivity is extracted with ethyl alcohol-ether. This residual radioactivity is defined as fixed, and may be associated with cell protein. The extracted radioactivity is identified as benzo[a]pyrene. Very little hydrocarbon is metabolized. Adverse photodynamic effects, increase in mutation, and dimunition in bacteriophage replication (in whole cells) have not been observed in the benzo[a]pyrene cultures.

Moore, B. G.; Harrison, Arthur P.



Downregulation of LAT1 expression suppresses cholangiocarcinoma cell invasion and migration.  


Currently, there is no effective treatment for cholangiocarcinoma (CCA), which is the most prevalent in the northeastern part of Thailand. A new molecular target for the treatment of CCA is, therefore, urgently needed. Although L-type amino acid transporter 1 (LAT1) is highly expressed in CCA cells, its role in malignant phenotypes of CCA cells remains unclear. This study aimed to investigate the impact of LAT1 on proliferation, migration, and invasion of KKU-M213 cells, the CCA cells derived from Thai patients with intrahepatic cholangiocarcinoma. Results showed that KKU-M213 cells expressed all LAT isoforms (LAT1, LAT2, LAT3 and LAT4). The expressions of LAT1 and its associated protein 4F2hc were highest whereas those of LAT2 and LAT4 were extremely low. Treatment with 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced L-leucine uptake concomitant with an inhibition of cell motility and, to a lesser extent, on cell proliferation. It also induced a time dependent up-regulation of LAT1 and 4F2hc expressions. Similarly, cell migration and invasion, but not proliferation, were reduced in LAT1 knockdown KKU-M213 cells. In addition, silencing of LAT1 inhibited the expressions of 4F2hc mRNA and protein whereas the expression of microRNA-7, the 4F2hc down-regulator, was increased. Furthermore, the phosphorylation levels of ERK1/2 and p70S6K were reduced after LAT1 knockdown. Collectively, these results suggest that suppression of cell invasion and migration in LAT1 knockdown KKU-M213 cells may be partly mediated through the inhibition of the 4F2hc-signaling pathway by the up-regulation of microRNA-7. Based on this finding, LAT1 may be a potential therapeutic target for treating CCA. PMID:24726839

Janpipatkul, Keatdamrong; Suksen, Kanoknetr; Borwornpinyo, Suparerk; Jearawiriyapaisarn, Natee; Hongeng, Suradej; Piyachaturawat, Pawinee; Chairoungdua, Arthit



Molecular imaging of urogenital diseases.  


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 therapeutic options for the treatment of metastatic and advanced prostate cancer, improved functional imaging of prostate cancer beyond the limitations of conventional CT and bone scan is becoming increasingly important for both clinical management and drug development. PET radiotracers, apart from ¹?F-FDG, for prostate cancer are ¹?F-sodium fluoride, ¹¹C-choline, and ¹?F-fluorocholine, and (¹¹C-acetate. Other emerging and promising PET radiotracers include a synthetic l-leucine amino acid analogue (anti-¹?F-fluorocyclobutane-1-carboxylic acid), dihydrotestosterone analogue (¹?F-fluoro-5?-dihydrotestosterone), and prostate-specific membrane antigen-based PET radiotracers (eg, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-¹?F-fluorobenzyl-l-cysteine, ??Zr-DFO-J591, and ??Ga [HBED-CC]). Larger prospective and comparison trials of these PET radiotracers are needed to establish the role of PET/CT in prostate cancer. Although renal cell cancer imaging with FDG-PET/CT is available, it can be limited, especially for detection of the primary tumor. Improved renal cell cancer detection with carbonic anhydrase IX (CAIX)-based antibody (¹²?I-girentuximab) and radioimmunotherapy targeting with ¹??Lu-cG250 appear promising. Evaluation of renal injury by imaging renal perfusion and function with novel PET radiotracers include p-¹?F-fluorohippurate, hippurate m-cyano-p-¹?F-fluorohippurate, 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 is also becoming available for clinical translation. PMID:24484747

Cho, Steve Y; Szabo, Zsolt



Dry powder inhalation of macromolecules using novel PEG-co-polyester microparticle carriers.  


This study investigated optimizing the formulation parameters for encapsulation of a model mucinolytic enzyme, ?-chymotrypsin (?-CH), within a novel polymer; poly(ethylene glycol)-co-poly(glycerol adipate-co-?-pentadecalactone), PEG-co-(PGA-co-PDL) which were then applied to the formulation of DNase I. ?-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). ?-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 ?-CH-CHF/PLF68, 1% produced the highest bioactivity, enzyme encapsulation (20.08±3.91%), loading (22.31±4.34 ?g/mg), FPF (fine particle fraction) (37.63±0.97%); FPD (fine particle dose) (179.88±9.43 ?g), MMAD (mass median aerodynamic diameter) (2.95±1.61 ?m), 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 ?g/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. PMID:23124106

Tawfeek, Hesham M; Evans, Andrew R; Iftikhar, Abid; Mohammed, Afzal R; Shabir, Anjum; Somavarapu, Satyanarayana; Hutcheon, Gillian A; Saleem, Imran Y



Evaluation and Modification of Commercial Dry Powder Inhalers for the Aerosolization of a Submicrometer Excipient Enhanced Growth (EEG) Formulation  

PubMed Central

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

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



Spray-dried porcine plasma reduces the effects of staphylococcal enterotoxin B on glucose transport in rat intestine.  


We investigated the intestinal transport of D-glucose (D-Glc) and 3 essential amino acids in a model of intestinal inflammation, and the effects of dietary supplementation with animal plasma proteins on this function. Wistar Lewis rats were fed a diet containing an isonitrogenous amount of milk protein (control group) or a diet supplemented with either spray-dried animal plasma (SDAP) or immunoglobulin concentrate (IC) from porcine plasma, from d 21 of life (weaning) until d 35. On d 30 and 33, rats were challenged intraperitoneally with Staphylococcus aureus enterotoxin B (SEB; groups SEB, SEB-SDAP, and SEB-IC) and on d 35, brush border membrane vesicles (BBMVs) were prepared and used for transport and binding studies. Administration of SEB reduced D-Glc transport across sodium glucose transporter 1 [SGLT1; 20% reduction in maximal transport rate (Vmax); P < 0.05], without affecting the Michaelis constant (Km). The results from specific phlorizin binding, Western blot, and immunohistochemistry supported the view that the effects of SEB are due to reduced expression of D-Glc transporters in the apical membrane. SEB increased the passive diffusion constant (Kd) for D-Glc 3-fold (P < 0.05). SEB did not affect mediated or passive amino acid fluxes of L-leucine, L-methionine, or L-lysine. Dietary SDAP increased the D-Glc Vmax in the SEB group without affecting the passive component. Changes in d-Glc Vmax due to SEB and to the dietary treatments were correlated with changes in the number of SGLT1 transporters present in the BBMVs (r = 0.9468; P < 0.05). Dietary IC had no observed effect. We estimate that, in rats challenged with SEB, SDAP supplementation can increase glucose absorption by 8-9% during the interdigestive periods. PMID:15987845

Garriga, Carles; Pérez-Bosque, Anna; Amat, Concepció; Campbell, Joy M; Russell, Louis; Polo, Javier; Planas, Joana M; Moretó, Miquel



?-alanine and l-histidine transport across the inner blood-retinal barrier: potential involvement in L-carnosine supply.  


The supply of L-carnosine, a bioactive dipeptide of ?-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]?-alanine and [(3)H]l-histidine compared with that of a paracellular marker, respectively. The RUI of [(3)H]?-alanine was taurine- and ?-aminobutyric acid-sensitive, and the in vitro uptake by TR-iBRB2 cells showed time- concentration- and temperature-dependent [(3)H]?-alanine uptake, suggesting that a carrier-mediated process was involved in ?-alanine transport across the inner BRB. [(3)H]?-Alanine uptake was inhibited by taurine and ?-guanidinopropionic acid, suggesting that taurine transporter (TAUT/SLC6A6) is responsible for the influx transport of ?-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 ?-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. PMID:23773890

Usui, Takuya; Kubo, Yoshiyuki; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi



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

PubMed Central

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

Chougule, Mahavir; Padhi, Bijay; Misra, Ambikanandan



Hypoxia stabilizes type 2 deiodinase activity in rat astrocytes.  


T(4) activation into T(3) is catalyzed by type 2 deiodinase (D2) in the brain. The rapid induction of D2 in astrocytes by transient brain ischemia has prompted us to explore the effects of hypoxia on D2 in cultures of astrocytes. Hypoxia (2.5% O(2)) of cultured astrocytes increased D2 activity, alone or in association with agents stimulating the cAMP pathway. Hypoxia had no effect on D2 mRNA accumulation. Cycloheximide did not block the effect of hypoxia on D2 activity and D2 half-life was enhanced under hypoxia demonstrating a posttranslational action of hypoxia. Furthermore, the D2 activity increase by hypoxia was not additive with the increase promoted by the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132). This strongly suggests that hypoxia leads to stabilization of D2 by slowing its degradation by the proteasome pathway. Hypoxia, in contrast to MG132, did not block the T(4)-induced D2 inactivation. A contribution of prolyl hydroxylase to the hypoxia effects on D2 was also suggested on the basis of increased D2 activity after addition of different prolyl hydroxylase inhibitors (cobalt chloride, desferrioxamine, dimethyloxalylglycine, dimethylsuccinate). Specific inhibitors of ERK, p38 MAPK, or phosphatidylinositol 3-kinase pathways were without any effect on hypoxia-increased D2 activity, eliminating their role in the effects of hypoxia. Interestingly, diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase inhibited the hypoxia-increased D2 indicating a role for some reactive oxygen species in the mechanism of D2 increase. Further studies are required to clarify the precise molecular mechanisms involved in the D2 stabilization by hypoxia. PMID:17615150

Lamirand, Audrey; Mercier, Gilles; Ramaugé, Martine; Pierre, Michel; Courtin, Françoise



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


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

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



Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.  


The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled ?-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies. PMID:24078041

Mas, Guillaume; Crublet, Elodie; Hamelin, Olivier; Gans, Pierre; Boisbouvier, Jérôme



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

PubMed Central

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

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



Proteasome inhibition induces hsp30 and hsp70 gene expression as well as the acquisition of thermotolerance in Xenopus laevis A6 cells  

PubMed Central

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.

Young, Jordan T. F.



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


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

Chougule, Mahavir; Padhi, Bijay; Misra, Ambikanandan



Effects of pH, temperature, and alcohols on the remarkable activation of thermolysin by salts.  


The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5 M) of neutral salts. The activation is due to an increase in the molecular activity, k(cat), while the Michaelis constant, K(m), is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by k(cat)/K(m), is pH-dependent, being controlled by two functional residues with pK(a) values of 5.4 and 7.8 in the absence of NaCl. The acidic pK(a) is shifted from 5.4 to 6.7 by the addition of 4 M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35 degrees C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts. PMID:9378714

Inouye, K; Lee, S B; Nambu, K; Tonomura, B



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


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

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



Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation.  

PubMed Central

We investigated the regulation of system-L amino acid transporter (LAT1) during T-cell activation. In quiescent T-cells, L-leucine transport is mediated mainly by the system-L amino acid transport system and is increased significantly during T-cell activation by PMA and ionomycin. In quiescent T-cells, the LAT1 protein was heterocomplexed with 4F2 heavy chain (4F2hc) in the plasma membrane. During T-cell activation, the amounts of 4F2hc and LAT1 heterocomplex were significantly elevated compared with those in quiescent T-cells. In addition, by Northern-blot analysis, these increments were found to be due to elevated levels of LAT1 and 4F2hc mRNA. Transient expression of constructs comprising various LAT1 gene promoter fragments, which contained all three of the GC boxes, was sufficient for promoting luciferase expression in Jurkat T-cells, but the promoter of the LAT1 gene did not respond to PMA and ionomycin. Similar observations were observed in the human 4F2hc gene promoter. In nuclear run-on assay, the LAT1 and 4F2hc genes were actively transcribed even in quiescent T-cells, but the low levels of both transcripts were shown to be the result of a block to transcription elongation within the exon 1 intron 1 regions. These findings indicated that a removal of the block to mRNA elongation stimulates the induction of system-L amino acid transporter gene transcripts (LAT1 and 4F2hc) in activated T-cells.

Nii, T; Segawa, H; Taketani, Y; Tani, Y; Ohkido, M; Kishida, S; Ito, M; Endou, H; Kanai, Y; Takeda, E; Miyamoto Ki



Abrin Immunotoxin: Targeted Cytotoxicity and Intracellular Trafficking Pathway  

PubMed Central

Background Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy. Methods Protein synthesis assay using 3[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins. Results We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells. Conclusions This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.

Gadadhar, Sudarshan; Karande, Anjali A.



Myocardial oxidative metabolism and protein synthesis during mechanical circulatory support by extracorporeal membrane oxygenation  

PubMed Central

Extracorporeal membrane oxygenation (ECMO) provides essential mechanical circulatory support necessary 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 metabolism and protein synthesis. 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 1) the fractional contribution of leucine (FcLeucine) and pyruvate to mitochondrial acetyl-CoA formation by nuclear magnetic resonance and 2) 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 h 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. The heart under ECMO shows reduced oxidative metabolism of substrates, including amino acids, while maintaining 1) metabolic flexibility indicated by ability to respond to pyruvate and 2) a normal or increased capacity for global protein synthesis.

Priddy, Colleen M. O?Kelly; Kajimoto, Masaki; Ledee, Dolena R.; Bouchard, Bertrand; Isern, Nancy; Olson, Aaron K.; Rosiers, Christine Des



Transpulmonary flux of S-nitrosothiols and pulmonary vasodilation during nitric oxide inhalation: role of transport.  


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. PMID:22323364