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1

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

SciTech Connect

Short-lived radicals in ..gamma..-irradiated aqueous solutions of either L-isoleucine or L-leucine were converted into relatively stable spin adducts by means of spin trapping with 2-methyl-2-nitrosopropane. The spin adducts were separated and identified individually by means of high-performance liquid chromatography combined with ESR spectroscopy. The structures of the spin adducts assigned in this study are as follows: for L-isoleucine, t-BuN(O.)CH(COO-)CH(CH/sub 3/)C/sub 2/H/sub 5/, t-BuN(O.)C(NH/sub 3//sup +/)(COO-)CH(CH/sub 3/)C/sub 2/H/sub 5/, t-BuN(O.)CH(CH/sub 3/)CH(CH/sub 3/)CH(NH/sub 3//sup +/)COO-, t-BuN(O.)CH/sub 2/CH/sub 2/CH(CH/sub 3/)CH(NH/sub 3//sup +/)COO-, t-BuN(O.)CH/sub 2/CH(C/sub 2/H/sub 5/)CH(NH/sub 3//sup +/)COO- and t-BuN(O.)C(CH/sub 3/)(C/sub 2/H/sub 5/)CH(NH/sub 3//sup +/)COO- (a pair of diastereomers); for L-leucine, t-BuN(O.)CH(COO-)CH/sub 2/CH(CH/sub 3/)/sub 2/, t-BuN(O.)CH/sub 2/CH(NH/sub 3//sup +/)COO- (I), t-BuN(O.)C(NH/sub 3//sup +/)(COO-)CH/sub 2/CH(CH/sub 3/)/sub 2/, t-BuN(O.)CH(CH(NH/sub 3//sup +/)COO/sup -/)CH(CH/sub 3/)/sub 2/, t-BuN(O.)CH/sub 2/CH(CH/sub 3/)CH/sub 2/CH(NH/sub 3//sup +/)COO-, and t-BuN(O.)C(CH/sub 3/)/sub 2/CH/sub 2/CH(NH/sub 3//sup +/)COO-. The spin adduct I is produced by C-C cleavage in L-leucine caused by direct effect of ..gamma..-rays which exhibits quite the same spectrum as that obtained from L-alanine. The stability of the spin adducts such as those formed by the H abstraction from the backbone carbons of L-isoleucine and L-leucine (chiral carbons of the amino acids) could be discussed by comparing the spectra obtained here with those of other studies. The mechanism which causes line-width alternation in the spectra could also be studied with regard to a combination of two ..beta.. hydrogens and the positions (..beta.. or ..gamma..) of chiral carbons of the spin adducts. In addition, differences in the spectra of various kinds of diastereomeric spin adducts obtained here could be studied in detail.

Makino, K.

1980-07-24

2

l-Leucine Methyl Ester: The Female-Produced Sex Pheromone of the Scarab Beetle, Phyllophaga lanceolata  

Microsoft Academic Search

The female-produced sex pheromone of the scarab beetle Phyllophagalanceolata was identified as the methyl ester of an essential amino acid, l-leucine. During field testing, 239 male P. lanceolata were caught in traps baited with l-leucine methyl ester. l-Isoleucine and l-valine methyl esters, similar in structure to l-leucine methyl ester and previously identified as female-produced sex pheromone compounds employed by other

Satoshi Nojima; Paul S. Robbins; Glenn A. Salsbury; Bruce D. Morris; Wendell L. Roelofs; Michael G. Villani

2003-01-01

3

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

PubMed

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

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

2013-06-05

4

Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine  

PubMed Central

Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ?ilvA ?panBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l-isoleucine could relieve the valine effect on VAL1 whereas l-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

Lange, C.; Rittmann, D.; Wendisch, V. F.; Bott, M.; Sahm, H.

2003-01-01

5

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.

2002-08-20

6

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

PubMed

L-isoleucine is synthesized from 2-ketobutyrate and pyruvate in Corynebacterium glutamicum, and the supplies of these two precursors are important for L-isoleucine synthesis. C. glutamicum YILW?alaT with alaT gene deletion (encoding alanine aminotransferase, a principal enzyme for L-alanine synthesis) was constructed to increase intracellular pyruvate availability, and the thrABC genes from Escherichia coli (encoding bifunctional aspartate kinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase) were overexpressed in C. glutamicum YILW and YILW?alaT to increase the supply of intracellular 2-ketobutyrate. In the fed-batch fermentation, YILWpXMJ19thrABC, YILW?alaT, and YILW?alaTpXMJ19thrABC exhibited 5.3, 17.6, and 8.4 % higher L-isoleucine production than the original strain, respectively. Both YILWpXMJ19thrABC and YILW?alaT excreted lower concentrations of L-lysine, L-alanine, and L-valine. YILW?alaTpXMJ19thrABC exhibited a cumulative reduction of these by-products excretion, which indicated that thrABC overexpression combined with alaT deletion resulted in the metabolic flux redistribution from 2-ketobutyrate and pyruvate to L-isoleucine synthesis, and decreased the fluxes to by-products synthesis accordingly. PMID:23813403

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

2013-06-30

7

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

PubMed

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

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

2013-04-02

8

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

2013-01-01

9

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.

10

Optical Properties of TGS Crystal with L-Valine Admixture  

SciTech Connect

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

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

2010-11-15

11

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

PubMed

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

1999-04-01

12

Thirteen-Week Oral Toxicity Study of BranchedChain Amino Acids in Rats  

Microsoft Academic Search

Branched-chain amino acids (L-isoleucine, L-valine, and L-leucine) are being increasingly used in sport supplements. This study evaluated toxicological and behavioral effects of L-isoleucine (Ile), L-valine (Val), and L-leucine (Leu) during a dosing study with male and female Sprague-Dawley rats. The amino acids were incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w\\/w). A control group

Shoji Tsubuku; Kazuhisa Hatayama; Toyohisa Katsumata; Nobuo Nishimura; Kazunori Mawatari; Miro Smriga; Takeshi Kimura

2004-01-01

13

The fruits of molecular physiology: engineering the l-isoleucine biosynthesis pathway in Corynebacterium glutamicum  

Microsoft Academic Search

The known processes for microbial l-isoleucine production include: (i)the conversion of precursors; (ii)the use of classically derived strains to overcome the regulation within biosynthesis; and (iii)the recent use of recombinant strains. The consistent application of genetic engineering in close conjunction with physiological studies has resulted in a fully defined set of recombinant isoleucine producers of Corynebacterium glutamicum. The best strain

Lothar Eggeling; Susanne Morbach; Hermann Sahm

1997-01-01

14

Time Series Analysis of Fed-batch Fermentation Process for L-valine Production  

Microsoft Academic Search

Fed-batch fermentation processes are some of the most efficient and wildly applied types of cultivation for industrial production of most amino acids including L-valine. Time series analysis is an important tool for description of the experimental data. This article deals with statistical inference from the time series analysis of generalised stoichiometric equations as a hypothesis for modelling and optimisation. The

Tzanko Georgiev; Velitchka Ivanova; Julia Kristeva; Ignat Dimov; Alexander Ratkov

2006-01-01

15

Swine liver L-leucine aminopeptidase: improved purification procedure.  

PubMed

An L-leucine aminopeptidase, having a specificity toward the substrate L-leucine amide, was purified 1084-fold from swine liver with a yield of 50.7 per cent. Purification procedure was carried out using successively centrifugation at 105 000 X g, fractionation by ammonium sulfate, DEAE Sephacel chromatography and zonal ultracentrifugation. Enzyme homogeneity and purity studies were carried out by analytical ultracentrifugation and polyacrylamide gel electrophoresis. In SDS-gel polyacrylamide, a single band was observed. It corresponded to a 55 000 molecular weight protein. PMID:6733155

Vincent-Fiquet, O; Rogez, J C; Plaquet, R

1984-02-01

16

L-Valine and L-Proline--solid-state IR-LD spectroscopic study.  

PubMed

Spectral investigation including IR-characteristic bands assignment of the amino acids zwitterions (L)-Valine ((L)-Val) and (L)-Proline ((L)-Pro) was carried out by linear-dichroic infrared (IR-LD) spectroscopy of oriented solid sample as a nematic liquid crystal suspension. The obtained experimental IR-LD results (transition moment directions) were compared with known crystal X-ray data for molecules orientation in the unit cells of the studied compounds, confirming the applicability of the used spectral method for structural determination. The influence of the protonation on the IR-spectroscopic patterns of the both amino acids is discussed. PMID:20236087

Chapkanov, A G; Zareva, S Y

2010-03-01

17

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

2013-06-01

18

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

PubMed Central

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

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

2011-01-01

19

Separation of L-valine from fermentation broths using a supported liquid membrane.  

PubMed

A carrier-mediated counter transport process is proposed to separate and to purify an amino acid produced by microbial fermentation. The case of L-valine permeation through a liquid membrane, constituted by a solution of Aliquat 336 in decanol and supported by a hydrophobic microporous membrane, is reported. A mathematical model was developed to estimate distribution coefficients and permeabilities and to predict the influence of hydrodynamic and pH conditions on supported liquid membrane (SLM) performances. Optimum conditions for the transport and the concentration of valine were achieved with synthetic aqueous valine solutions. Series of experiments on fermentation broths, where molasses and biomass contents were varied, permitted pointing out the role of the broth composition on the kinetics and yields of separation. The selectivity of transport of valine by an Aliquat 336/decanol liquid membrane was about 10 toward molasses dyes, 100 toward glucose, and beyond 1000 toward sucrose. This allowed us to achieve the recovery and one step of purification of the product in a single operation. The stability of the Aliquat 336/decanol liquid membrane was sufficient to ensure a selective transport of valine during a continuous run lasting 18 days. PMID:18592501

Deblay, P; Minier, M; Renon, H

1990-01-20

20

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

PubMed

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

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

2012-05-05

21

Human liver L-leucine aminopeptidase: evidence for two forms compared to pig liver enzyme.  

PubMed

Two forms of L-leucine aminopeptidase (E.C. 3.4.11.1) having a specific activity toward L-leucine amide and L-leucylpeptides substrates but not toward chromogenic substrates: L-leucyl paranitroanilide or L-leucyl beta naphthylamide have been evidenced from human liver. Human liver enzymes have been distinguished from pig liver enzyme by DEAE Sephacel chromatography and analytical electrophoresis on cellulose acetate strips. We compared enzymic properties of L-leucine aminopeptidases from human liver with pig liver enzyme: they were activated by Mg2+ and Mn2+ and inhibited by Zn2+ and Co2+, EDTA and citric acid. The optimum pH's were 10. Both human liver L-leucine aminopeptidases were less sensitive to heat elevation than pig liver enzyme. PMID:6414529

Ledeme, N; Vincent-Fiquet, O; Hennon, G; Plaquet, R

1983-07-01

22

Characterization of L-leucine Induced Germination of Trichophyton mentagrophytes microspores.  

National Technical Information Service (NTIS)

L-Leucine and several amino acids proved to be effective germination inducers of microspores of Trichophyton mentagrophytes. During germination phase darkening and swelling occurred concomitantly with the alterations of several spore properties inherent t...

T. Hashimoto C. D. R. Wu H. J. Blumenthal

1970-01-01

23

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

PubMed

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. PMID:23583879

Nirmala, L Ruby; Thomas Joseph Prakash, J

2013-03-25

24

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

PubMed

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

2010-09-17

25

3H-L-leucine transport by the promiscuous crustacean dipeptide-like cotransporter.  

PubMed

The crustacean intestine and hepatopancreas display a variety of solute transport mechanisms for transmembrane transfer of dietary contents from lumen to epithelial cytosol. An in vitro intestinal perfusion apparatus was used to characterize mucosal to serosoal (MS) and serosal to mucosal (SM) Zn(2+) -dependent (3)H-L-leucine transport by the intestine of the American lobster, Homarus americanus. Transmural 20?µM MS (3)H-L-leucine fluxes across lobster intestine were a hyperbolic function of luminal zinc concentration (1-50?µM) following Michaelis-Menten kinetics (K(m) = 2.67 ± 0.74?µM; J(max) = 19.56 ± 2.22?pmol/cm(2) ×min). Transmural 20?µM SM (3)H-L-leucine fluxes were not affected by serosal zinc, resulting in a highly significant stimulation of net amino acid transfer to the blood by luminal metal. MS fluxes of 20?µM (3)H-L-leucine were also hyperbolic functions of luminal [Cu(2+)], [Mn(2+)], [Na(+)], and [H(+)]. MS flux of (3)H-L-leucine was a sigmoidal function of luminal [L-leucine] and was stimulated by the addition of 20?µM luminal zinc at both pH 7.0 and 5.5. A greater enhanced amino acid transport occurred at the lower pH 5.5. MS flux of 20?µM (3)H-L-leucine in the presence of 20?µM zinc was significantly inhibited by addition of 100?µM luminal glycylsarcosine, and MS flux of 20?µM (3)H-glycylsarcosine was inhibited by 100?µM L-leucine in the presence of 20?µM zinc. Results suggest that (3)H-L-leucine and metals form a complex (e.g., Leu-Zn-Leu] that may functionally mimic dipeptides and use a dipeptide-like transporter during MS fluxes as suggested for fish and mammals. PMID:21732547

Obi, I; Wells, A L; Ortega, P; Patel, D; Farah, L; Zanotto, F P; Ahearn, G A

2011-07-05

26

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

2003-01-01

27

Stimulation of Lipid Peroxidation in Vitro in Rat Brain by the Metabolites Accumulating in Maple Syrup Urine Disease  

Microsoft Academic Search

In this study we investigated the in vitro effects of the metabolites accumulating in maple syrup urine disease on lipid peroxidation in brain of young rats. Chemiluminescence and thiobarbituric acid-reactive substances were measured in brain homogenates from 7- and 30-day-old rats in the presence of 10 mM of the branched-chain amino acids L-leucine, L-isoleucine, or L-valine; their keto acids L-2-ketoisocaproic

Fernanda U. Fontella; Edson Gassen; Vânia Pulrolnik; Clóvis M. D. Wannmacher; Adriane B. Klein; Moacir Wajner; Carlos S. Dutra-Filho

2002-01-01

28

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

Microsoft Academic Search

Far infrared spectra of zwitterionic, cationic, and anionic forms of aliphatic amino acids in solid state have been studied experimentally. Measurements were done on glycine, L-alanine, L-valine, L-leucine, and L-isoleucine powder samples and film samples obtained from dried solutions prepared at pH ranging from 1 to 13. Solid state density functional theory calculations were also performed, and detailed potential energy

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

2010-01-01

29

Purification and enzymatic properties of an L-leucine aminopeptidase from swine liver.  

PubMed

An L-leucine aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1), having a specificity toward the substrate L-leucine amide, but not toward L-leucyl beta-naphthylamide or L-leucyl p-nitroanilide, has been purified 332-fold from swine liver, with a yield of 8.6%. This is the first purification of this enzyme from hepatic tissue. The purified enzyme submitted to analytical electrophoresis on cellulose acetate strips or in polyacrylamide gel showed a single band after straining with Ponceau S Red dye or Amido black, respectively. Purified swine liver L-leucine aminopeptidase, a cytosol enzyme, exhibited a molecular weight of 268 000 +/- 50 000 by gel filtration. It hydrolyzed L-leucine amide substrate and L-leucyl peptides. It was activated by Mg2+ and Mn2+ and inhibited by Co2+ and Zn2+. The optimum pH was 10. It was rather sensitive to heat elevation. Swine liver L-leucine aminopeptidase was inhibited by EDTA, citric acid, isocaproic acid, dodecylamine, aliphatic alcohols and p-chloromercuribenzoate but unaffected by monoiodoacetic acid and diisopropyl fluorophosphate. PMID:7284403

Ledeme, N; Hennon, G; Vincent-Fiquet, O; Plaquet, R

1981-08-13

30

Adenosine 5?-Triphosphate-Yielding Pathways of Branched-Chain Amino Acid Fermentation by a Marine Spirochete  

PubMed Central

The metabolic pathways utilized by an obligately anaerobic marine spirochete (strain MA-2) to ferment branched-chain amino acids were studied. The spirochete catabolized l-leucine to isovaleric acid, l-isoleucine to 2-methylbutyric acid, and l-valine to isobutyric acid, with accompanying CO2 production in each fermentation. Cell extracts of spirochete MA-2 converted l-leucine, l-isoleucine, and l-valine to 2-ketoisocaproic, 2-keto-3-methylvaleric, and 2-ketoisovaleric acids, respectively, through mediation of 2-ketoglutarate-dependent aminotransferase activities. The branched-chain keto acids were decarboxylated and oxidized to form isovaleryl coenzyme A, 2-methylbutyryl coenzyme A, and isobutyryl coenzyme A, respectively, in the presence of sulfhydryl coenzyme A and benzyl viologen. The acyl coenzyme A's were converted to acyl phosphates by phosphate branched-chain acyltransferase enzymatic activities. Branched-chain fatty acid kinase activities catalyzed formation of isovaleric, 2-methylbutyric, and isobutyric acids from isovaleryl phosphate, 2-methylbutyryl phosphate, and isobutyryl phosphate, respectively. Adenosine 5?-triphosphate was formed during conversion of branched-chain acyl phosphates to branched-chain fatty acids. The results indicate that conversion of l-leucine, l-isoleucine, and l-valine to branched-chain fatty acids by spirochete MA-2 results in adenosine 5?-triphosphate generation. The metabolic pathways utilized for this conversion involve amino acid amino-transferase, 2-keto acid oxidoreductase, phosphate acyltransferase, and fatty acid kinase activities.

Harwood, C. S.; Canale-Parola, E.

1981-01-01

31

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

2013-01-01

32

Stereoselective recognition of dipeptide derivatives in molecularly imprinted polymers which incorporate an l-valine derivative as a novel functional monomer  

Microsoft Academic Search

A polymerizable l-valine derivative was synthesized for use as a novel functional monomer in molecular imprinting applications. This monomer is expected to form multiple hydrogen bonds with peptide derivatives, thus allowing a specific molecular memory in molecularly imprinted polymers. Several dipeptide derivatives were imprinted and the resulting polymers were used as stationary phases for liquid chromatography. It was shown that

Kazuyoshi Yano; Takeshi Nakagiri; Toshifumi Takeuchi; Jun Matsui; Kazunori Ikebukuro; Isao Karube

1997-01-01

33

Asymmetric epoxidation of chalcone catalyzed by reusable poly- l-leucine immobilized on hydrotalcite  

Microsoft Academic Search

Nanohybrid materials based on polyamino acids immobilized onto inorganic materials are of interest for their potential applications in protein engineering, biomedicine and catalysis. We developed an efficient and eco-friendly new protocol for the immobilization of synthesized poly-l-leucine (PLL) onto rehydrated hydrotalcite (HTr). To do this, we synthesized different PLLs containing both C-terminal and N-terminal groups and compared them with a

Ronald-Alexander Miranda; Jordi Llorca; Francisco Medina; Jesús E. Sueiras; Anna M. Segarra

2011-01-01

34

Immobilization of bromelain onto porous copoly(?-methyl- l-glutamate\\/ l-leucine) beads  

Microsoft Academic Search

Water-insoluble bromelain was prepared by immobilizing bromelain onto the surface of porous copoly(?-methyl-l-glutamate\\/l-leucine) (ML) beads with and without spacer. The mode of the immobilization between bromelain and porous copolypeptide ML beads was covalent fixation. The relative activity and the stability of the immobilized bromelain was investigated. The retained activity of the bromelain covalently immobilized by the azide method was found

Shinya Yodoya; Tetsuya Takagi; Michiyoshi Kurotani; Takanori Hayashi; Masakazu Furuta; Masahito Oka; Toshio Hayashi

2003-01-01

35

Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester  

Microsoft Academic Search

Lysosomes are fundamental for cell growth, and thus inhibition of the lysosomal function often leads to cell death. L-Leucyl-L-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. LeuLeuOMe is taken up through receptor-mediated endocytosis, and then converted into (LeuLeu)n-OMe (n>3) by dipeptidyl peptidase I (DPPI) in lysosomes, which reportedly causes rupture of the lysosomes

Takayuki Uchimoto; Hiroyuki Nohara; Rieko Kamehara; Michiko Iwamura; Naoko Watanabe; Yoshiro Kobayashi

1999-01-01

36

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

Microsoft Academic Search

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

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

2007-01-01

37

Precision neutron diffraction structure determination of protein and nucleic acid components. XV. Crystal and molecular structure of the amino acid L-valine hydrochloride  

Microsoft Academic Search

A neutron diffraction study of L-valine · HCl has been carried out: space group P21, a = 10.382(2), b = 7.066(1), c = 5.4407(9) Å, ? = 91.40(2)°, Z = 2. The structure has been refined by full-matrix least-squares techniques with anisotropic temperature factors for all atoms and with a Type II anisotropic extinction correction, leading to a conventional R

Thomas F. Koetzle; Ljubo Golic; Mogens S. Lehmann; Jacques J. Verbist; Walter C. Hamilton

1974-01-01

38

Precision neutron diffraction structure determination of protein and nucleic acid components. XV. Crystal and molecular structure of the amino acid L-valine hydrochloride  

Microsoft Academic Search

A neutron diffraction study of L-valine . HCl has been carried out: space group P21, a = 10.382(2), b = 7.066(1), c = 5.4407(9) Å, beta = 91.40(2)°, Z = 2. The structure has been refined by full-matrix least-squares techniques with anisotropic temperature factors for all atoms and with a Type II anisotropic extinction correction, leading to a conventional R

Thomas F. Koetzle; Ljubo Golic; Mogens S. Lehmann; Jacques J. Verbist; Walter C. Hamilton

1974-01-01

39

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

PubMed

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

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

2009-10-29

40

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

PubMed

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

Makino, K; Riesz, P

1982-06-01

41

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

PubMed

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

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

2013-07-08

42

An improved bioprocess for extracellular L-leucine amino peptidase production using Streptomyces gedanensis.  

PubMed

A bioprocess was developed for the production of L-leucine aminopeptidase under solid-state fermentation (SSF) by cultivating Streptomyces gedanensis in an inert support impregnated with a minimal medium. Response surface methodology of Box Behnken design was used to derive the optimum level of significant factors (3 ml inoculum (1.2 × 10(9) CFU/ml); 0.275% w/v (NH(4))(2)SO(4); 0.275% w/v MgSO(4)·7H(2)O and 0.55% w/v Tryptone) for maximum LAP production (489 IU/g PUF) as compared to the initial level of 176.3 ± 0.02 IU/g PUF. The high level of extracellular aminopeptidase yield achieved in this work showed the technical feasibility of LAP production under SSF using inert support and is the first report of this kind. The ability of Streptomyces amino peptidase to release particular N-terminal amino acids made them interesting for controlling the degree of hydrolysis and flavor development for a wide range of substrates in food like industries. PMID:21104085

Rahulan, Raji; Pandey, Ashok; Nampoothiri, K Madhavan

2010-11-21

43

Utilization of amino acids by bacteria from the pig small intestine  

Microsoft Academic Search

This study determined the utilization of amino acids (AA) by bacteria from the lumen of the pig small intestine. Digesta samples\\u000a from different segments of the small intestine were inoculated into media containing 10 mmol\\/L each of select AA (l-lysine, l-threonine, l-arginine, l-glutamate, l-histidine, l-leucine, l-isoleucine, l-valine, l-proline, l-methionine, l-phenylalanine or l-tryptophan) and incubated for 24 h. The previous 24-h culture served

Zhao-Lai Dai; Jing Zhang; Guoyao Wu; Wei-Yun Zhu

2010-01-01

44

Spectroscopic studies of amino acid ionic liquid-supported Schiff bases.  

PubMed

Amino acid ionic liquid-supported Schiff bases, derivatives of salicylaldehyde and various amino acids (L-threonine, L-valine, L-leucine, L-isoleucine and L-histidine) have been investigated by means of various spectroscopic techniques (NMR, UV-Vis, IR, MS) and deuterium isotope effects on ¹³C-NMR chemical shifts. The results have shown that in all studied amino acid ionic liquid-supported Schiff bases (except the L-histidine derivative) a proton transfer equilibrium exists and the presence of the COO? group stabilizes the proton transferred NH-form. PMID:23629755

Ossowicz, Paula; Janus, Ewa; Schroeder, Grzegorz; Rozwadowski, Zbigniew

2013-04-29

45

Expression of NAD(H) kinase and glucose-6-phosphate dehydrogenase improve NADPH supply and L-isoleucine biosynthesis in Corynebacterium glutamicum ssp. lactofermentum.  

PubMed

Corynebacterium glutamicum is the workhorse for the production of amino acids, including L-isoleucine (Ile). During Ile biosynthesis, NADPH is required as a crucial cofactor. In this study, four NADPH-supplying strategies based on NAD kinase, NADH kinase, glucose-6-phosphate dehydrogenase, and NAD kinase coupling with glucose-6-phosphate dehydrogenase were compared, and their influences on Ile biosynthesis were examined. PpnK is a NAD kinase of C. glutamicum ssp. lactofermentum JHI3-156 that predominantly phosphorylates NAD(+) to produce NADP(+). Pos5 is a NADH kinase of Saccharomyces cerevisiae that predominantly phosphorylates NADH to produce NADPH. Zwf is a glucose-6-phosphate dehydrogenase of JHI3-156. The ppnK, POS5, zwf, and zwf-ppnK genes were overexpressed in the Ile-producing strain JHI3-156. The expression of all four genes increased intracellular NADPH concentration and Ile production. The increase of NADPH concentration and Ile production in a POS5-expressing strain (229 and 75.6 %, respectively) was higher than that in a ppnK-expression strain. The expression of zwf also increased NADPH supply and Ile biosynthesis, but the constitutive expression of zwf was not as effective as the inducible expression of zwf. Coexpression of zwf and ppnK genes greatly enhanced NADPH supply and thus improved Ile production by up to 85.9 %, indicating that this strategy was the most effective one. These results are helpful for improving Ile biosynthesis and other biosynthetic processes. PMID:23868449

Shi, Feng; Li, Kun; Huan, Xiaojing; Wang, Xiaoyuan

2013-07-19

46

A route to anionic hydrophilic films of copolymers of l-leucine, l-aspartic acid and l-aspartic acid esters  

Microsoft Academic Search

A series of copolymers of l-leucine and ?-benzyl-l-aspartate [Leu\\/Asp(OBz)] covering the range 30–70 mol % of l-leucine, was synthesized by the N-carboxyanhydride (NCA) method. The copolymers were characterized by elemental analysis, infra-red spectroscopy and viscometry. For all compositions high molecular weight copolymers were prepared with excellent film-forming properties. Tercopolymers of l-leucine, ?-benzyl-l-aspartate and ?-methyl-l-aspartate [Leu\\/Asp(OBz)\\/Asp(OMe)] were obtained after an ester

W. L. Sederel; A. Bantjes; J. Feijen

1975-01-01

47

Measurement of activity coefficients of amino acids in aqueous electrolyte solutions: experimental data for the systems (H 2O + NaBr + glycine) and (H 2O + NaBr + l-valine) at T=298.15 K  

Microsoft Academic Search

Electrochemical cells with two ion-selective electrodes, a cation ion-selective electrode against an anion ion-selective electrode, were used to measure the activity coefficient of amino acids in aqueous electrolyte solutions. Activity coefficient data were measured for (H2O+NaBr+glycine) and (H2O+NaBr+l-valine) at T=298.15 K. The maximum concentrations of sodium bromide, glycine, and l-valine were (1.0, 2.4, and 0.4) mol·kg?1, respectively. The results show

A. Khavaninzadeh; H. Modarress; V. Taghikhani; M. K. Khoshkbarchi

2003-01-01

48

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.

49

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

PubMed

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

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

2013-10-03

50

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

PubMed Central

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

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

2013-01-01

51

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

PubMed

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20 mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p < 0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J max in the same medium. These results suggest that shrimp BBMV transport (3)H-L-leucine by a single L-methionine- and L-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na(+) or K(+) acting as co-transport drivers binding to shared activator sites. PMID:23615795

Duka, Ada; Ahearn, Gregory A

2013-04-25

52

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

PubMed

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

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

2013-01-10

53

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

NASA Astrophysics Data System (ADS)

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

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

2013-04-01

54

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 H2CCHC(O)N(H)C system, CCCN, CCCO and (C)CC(O)N(H)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 OCC(H)N(H) torsion angle is?sp, cys (?28(1)°). The CO bond distance at amide is 1.240(3) Å, whereas the CO 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

2012-12-01

55

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

2012-10-01

56

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

NASA Astrophysics Data System (ADS)

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

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

2011-11-01

57

Immobilazation of Aerobic Microorganisms on Glassy Sintered Material, Illustrated by the Example of the Production of L Leucine Using Corynebacterium Glutamicum.  

National Technical Information Service (NTIS)

The aim of this study was to develop the carrier fixation of aerobic microorganisms on open-pore sintered glass material. The fermentative production of L-leucine from (alpha) cetonic isocaproic acid with Corynebacterium glutamicum was chosen as an exampl...

J. Buechs

1988-01-01

58

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

PubMed Central

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

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

2012-01-01

59

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

PubMed

We have studied the amino acid L-leucine (LEU) using inelastic neutron scattering, X-rays and neutron diffraction, calorimetry and Raman scattering as a function of temperature, focusing on the relationship between the local dynamics of the NH(3), CH(3), CH(2) and CO(2) moieties and the molecular structure of LEU. Calorimetric and diffraction data evidenced two novel phase transitions at about 150 K (T(1)) and 275 K (T(2)). The dynamical susceptibility function, obtained from the inelastic neutron scattering results, shows a re-distribution of the intensity of the vibrational bands that can be directly correlated with the phase transitions observed at T(1) and T(2), as well as with the already reported phase transition at T(3) = 353 K. Through the analysis of the Raman modes, the new structural arrangement observed below T(1) was related to conformational modifications of the CH and CH(3) groups, while the behavior of the N-H stretching vibration, ?(NH(3)), gave insight into the intermolecular N-H…O interactions. The observation of changes in the translational symmetry in the crystalline lattice, as well as anharmonic dynamics, allows for localized motions in LEU. PMID:21384001

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

2011-03-07

60

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

NASA Astrophysics Data System (ADS)

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

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

2012-12-01

61

Production of ?-ketoisocaproate via free-whole-cell biotransformation by Rhodococcus opacus DSM 43250 with L-leucine as the substrate.  

PubMed

This work aims to produce ?-ketoisocaproate (KIC) from L-leucine via the free-whole-cell biotransformation of Rhodococcus opacus DSM 43250. The effects of temperature, pH, substrate concentration, cell concentration, and rotating speed on KIC production were examined. Furthermore, the biotransformation conditions were optimized with response surface methodology (RSM). The optimal biotransformation conditions were as follows: temperature 43.7 °C, pH 8.4, L-leucine concentration 5.1g/L, cell concentration 30.4 g/L, and rotating speed 170 rpm. The maximal KIC production predicted by RSM model reached 1275 mg/L at the optimal conditions, and in the validated experiments, the maximal KIC production reached 1264 mg/L, which was very close to the predicted KIC production. The current work provides an alternative for the production of KIC and the results obtained here are useful for the biotransformatic KIC production on a large scale. PMID:22112557

Zhu, Yuhong; Li, Jianghua; Liu, Long; Du, Guocheng; Chen, Jian

2011-06-15

62

Biodegradability and tissue reaction of random copolymers of L-leucine, L-aspartic acid, and L-aspartic acid esters  

Microsoft Academic Search

A series of copoly(?-amino acids) with varying percentages of hydrophilic (l-aspartic acid) and hydrophobic monomers (l-leucine, ß-methyl-l-aspartate, and ß-benzyl-l-aspartate) were implanted subcutaneously in rats and the macroscopic degradation behavior was studied. Three groups of materials (A, B, C) with different ranges of hydrophilicity were distinguished: A) hydrophobic materials showed no degradation after 12 weeks; B) more hydrophilic materials revealed a

K. W. Marck; Ch. R. H. Wildevuur; W. L. Sederel; A. Bantjes; J. Feijen

1977-01-01

63

Intestinal absorption of D-galactose and L-leucine and intestinal disaccharidase activities in growing chickens fed different raw legume diets.  

PubMed

A significant (P less than .01) impairment in the rate of growth, along with a significant (P less than .01) inhibition in the rate of in vivo intestinal absorption of D-galactose and L-leucine, and in the in vitro intestinal absorption of D-galactose, was found in growing chickens fed ad libitum over a 60-day period, diets containing the raw legumes Vicia faba, Glycine soja, Vicia ervilia, and Phaseolus vulgaris as the main source of protein. Furthermore, a significant (P less than .01) reduction in the intestinal disaccharidase activity was found in the legume-fed chickens. The possible nature of these effects was discussed. PMID:7301750

Santidrian, S; Lasheras, B; Cenarruzabeitia, M N; Bolufer, J; Larralde, J

1981-04-01

64

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

2002-01-01

65

Incorporation of N-Acetyl-[14C]D-Glucosamine and [3H]L-Leucine by Isolated Pig Gastric Mucosal Cells  

Microsoft Academic Search

Glycoprotein and protein production of isolated pig gastric mucosal cells were determined by the incorporation of N-acetyl-[14C]D-glucosamine ([14C]G1cNAc) and [3H]L-leucine ([3H]Leu) into acid-insoluble macromolecules (AIM). In four cell fractions (F1-F4), obtained by counterflow centrifugation, specific [14C]G1cNAc incorporation was greatest in the mucous cell-enriched F2. Tracer incorporation by F2 cells, proceeded lineraly up to 20 h, was inhibited by cycloheximide or

Hans-Karl Heim; Anja Oestmann; Helga Thiele; Karl-Friedrich Sewing

1989-01-01

66

New optically active poly(amide-imide)s derived from N, N?-(4,4-diphthaloyl)-bis- l-leucine and hydantoin derivatives: Synthesis and properties  

Microsoft Academic Search

Six new optically active poly(amide-imide)s (5a–f) were synthesized through the direct polycondensation reaction of N,N?-(4,4?-diphthaloyl)-bis-l-leucine (3) with six hydantoin derivatives (4a–f). Triphenyl phosphite (TPP)\\/pyridine in the presence of calcium chloride (CaCl2) and N-methyl-2-pyrrolidone (NMP) were successfully applied for direct polycondensation. The polycondensation reactions produce a series of new poly(amide-imide)s (5a–f) in high yields, and inherent viscosity between 0.42 and 0.55dL\\/g.

Khalil Faghihi

2009-01-01

67

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

PubMed

?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

2013-03-01

68

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

PubMed Central

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.

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

1990-01-01

69

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

2009-01-01

70

Synthesis and characterization of new optically active poly(amide-imide)s containing 1,3,4-oxadiazole moiety in the main chain  

Microsoft Academic Search

Six new optically active poly(amide-imide)s were synthesized by poly condensation reaction of 2,5-bis(4-aminophenyl)-1,3,4-oxadiazole\\u000a (8) with six chiral N,N?-(bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic)-bis-l-amino acids (3a–f) in a medium consisting of N-methyl-2-pyrrolidone (NMP), triphenylphosphite (TPP), calcium chloride (CaCl2), and pyridine. Chiral N,N?-(bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic)-bis-l-amino acids (3a–f) were obtained by the reaction of bicyclo[2.2.2]-oct-7-ene-2,3,5,6-tetracarboxylic dianhydride (1) with two equimolar of l-alanine (2a), l-valine (2b), l-leucine (2c), l-isoleucine (2d), l-phenyl

Khalil Faghihi; Hassan Moghanian

2010-01-01

71

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

2010-01-01

72

Methyl 2-(Methylthio)Benzoate: A Sex Attractant for the June Beetles, Phyllophaga tristis and P. apicata  

PubMed Central

Male antennae of Phyllophaga tristis (Fabricius) (Coleoptera: Scarabaeidae: Melolonthinae) were tested using a coupled gas chromatograph-electroantennogram detector (GC-EAD) system for electrophysiological responses to five sex pheromones identified from other Phyllophaga species including L-valine methyl ester, L-isoleucine methyl ester, L-leucine methyl ester, methyl 2(methylthio)benzoate and methyl 2-amino benzoate. Male antennae responded only to methyl 2(methylthio)benzoate. In a 2003 field test near Greensburg, Kansas, cross-vane traps baited with rubber septa containing 1 mg of methyl 2-(methylthio)benzoate captured 466 male P. tristis. Control traps baited with rubber septa loaded with only hexane captured none. Similarly, in a field test in 2010 in Gainesville, Florida, 265 male P. apicata Reinhard were captured in traps baited with 1 mg of methyl 2-(methylthio)benzoate whereas control traps captured only a single male.

Robbins, Paul S.; Salsbury, Glenn A.; Woodruff, Robert E.; Lapointe, Stephen L.; Linn, Charles E.

2011-01-01

73

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

PubMed

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

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

2008-12-01

74

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

NASA Astrophysics Data System (ADS)

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

Guidara, Sameh; Feki, Habib; Abid, Younes

2013-11-01

75

Vibrational spectral studies and non-linear optical properties of l-leucine l-leucinium picrate: A Density Functional Theory approach.  

PubMed

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

Guidara, Sameh; Feki, Habib; Abid, Younes

2013-07-01

76

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

PubMed

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

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

2012-07-31

77

Interaction of L-leucyl-L-leucyl-L-leucine thin film with water and organic vapors: receptor properties and related morphology.  

PubMed

The ability of highly ordered tripeptide structures to keep or change their morphology in contact with organic vapors was studied. A thin film of tripeptide L-leucyl-L-leucyl-L-leucine (LLL) was prepared having microcrystals and nanocrystals on its surface, which are stable upon vacuum drying but become objects of selective morphology change after a contact with vapors of organic solvents. Fine separate LLL crystals and their agglomerates of submicron and larger dimensions were observed by atomic force microscopy and scanning electron microscopy. After saturation with guest vapors, these crystals can remain intact or change their morphology with the increase in size or complete destruction depending on the guest molecular structure. The crystals completely lose their shape after the binding of pyridine vapors. The other studied guests produce much smaller transformations or have no effect on crystal morphology despite being sorbed by solid LLL, which was shown using quartz crystal microbalance sensor. The observed size-exclusion effect for guest sorption by LLL was found to be broken by the same guests that can change the initial crystal shape. This helps to explain the morphology changes of LLL crystals after the guest sorption and release. PMID:22389009

Ziganshin, Marat A; Efimova, Irina G; Gorbatchuk, Valery V; Ziganshina, Sufia A; Chuklanov, Anton P; Bukharaev, Anastas A; Soldatov, Dmitry V

2012-03-02

78

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.

1986-11-01

79

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.

1988-11-01

80

Lipid-lowering efficacy of 3,4-di(OH)-phenylpropionic L-leucine in high-cholesterol fed rats.  

PubMed

A preliminary study revealed that 3,4-di(OH)-hydrocinnamate (HC), a polyphenolic compound, lowered the plasma lipids in high-cholesterol fed rats. Accordingly, this study was designed to test the lipid-lowering efficacy of a synthetic derivative, 3,4-di(OH)-phenylpropionic (L-leucine) amide (PPLA), in rats fed a high-cholesterol (1%, wt/wt) diet. As such, HC or PPLA was given as supplement to a high-cholesterol diet for 6 weeks at a dose of 0.137 mmol/100 g diet. The supplementation of HC and PPLA significantly lowered the plasma and hepatic cholesterol and triglyceride levels compared to the control group. The activities of hepatic HMG-CoA reductase (164 +/- 9.12 and 124.74 +/- 17.09 pmol/min/mg protein vs. 245.41 +/- 13.01 pmol/min/mg protein, p < 0.05) and ACAT (411.49 +/- 11.48 and 334.35 +/- 17.68 pmol/min/mg protein vs. 490.41 +/- 16.69 pmol/min/mg protein, p < 0.05) were significantly lower in the HC- and PPLA-supplemented groups than in the control group. However, PPLA was more effective in inhibiting the enzyme activities than HC. The excretion of neutral sterol was significantly higher in HC- and PPLA-supplemented groups than in the control group. Therefore, these results indicate that PPLA, a leucine-attached version of HC, exhibited a similar significant hypocholesterolemic effect to HC in rats fed a high-cholesterol diet. PMID:15736153

Kim, Soon-Ja; Bok, Song-Hae; Lee, Sangku; Lee, Mi-Kyung; Park, Yong Bok; Kim, Hye-Jin; Choi, Myung-Sook

2005-01-01

81

Synthesis of the amino acid conjugates of epi-jasmonic acid.  

PubMed

The TES ether of the C6-hydroxy derivative of naturally occurring epi-jasmonic acid (epi-JA) was designed as epimerization-free equivalent of epi-JA. The TES ether was synthesized from (1R,4S)-4-hydroxycyclopent-2-enyl acetate in 13 steps. The acid part of the ether was activated with ClCO2Bui and subjected to condensation with L-amino acid at room temperature for 48 h. The TES group in the condensation product was removed in HCO2H (0°C, 30 min) and the resulting hydroxyl group was oxidized with Jones reagent (acetone, 0°C, 30 min) to furnish the amino acid conjugate of epi-JA. The amino acids examined are L-isoleucine, L-leucine, L-alanine, L-valine, and D-allo-isoleucine, which afforded the conjugates in 48-68% yields with 89-96% diastereomeric purity over the trans isomers. Similarly, the possible three stereoisomers of epi-JA were condensed with L-isoleucine successfully, producing the corresponding stereoisomers in good yields. PMID:21562820

Ogawa, N; Kobayashi, Y

2011-05-12

82

Interactions in L-phenylalanine/L-leucine/L-glutamic Acid/L-proline + 2 M aqueous NaCl/2 M NaNO3 systems at different temperatures  

NASA Astrophysics Data System (ADS)

Density (?) and speed of sound ( u) in 2 M aqueous NaCl and 2 M NaNO3 solutions of amino acids: L-phenylalanine, L-leucine, L-glutamic acid, and L-proline have been measured for several molal concentrations of amino acids at different temperatures. The ? and u data have been used to calculate the values of isothermal compressibility and internal pressure at different temperatures. The trends of variations of ? T and P i with an increase in molal concentration of amino acid and temperature have been discussed in terms of solute-solvent and solute-solute interactions in the systems.

Riyazuddeen, Imran Khan; Afrin, Sadaf

2012-12-01

83

Thirteen-week oral toxicity study of branched-chain amino acids in rats.  

PubMed

Branched-chain amino acids (L-isoleucine, L-valine, and L-leucine) are being increasingly used in sport supplements. This study evaluated toxicological and behavioral effects of L-isoleucine (Ile), L-valine (Val), and L-leucine (Leu) during a dosing study with male and female Sprague-Dawley rats. The amino acids were incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine stability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals. No significant, dose-related effects on body weight were found in rats fed a Leu- and Ile-supplemented diet. Val mixed into a diet at 5.0% (w/w) decreased slightly, but significantly body weight gain in females, but not males. Ile (5.0% w/w) affected the urine electrolytes, protein, ketone bodies, urine glucose, and urobilinogen in both genders, yet the observed changes remained mostly within the range observed in controls. The random findings in hepatology and ophthalmology at the 13-week sacrifice were not considered toxicologically relevant to effects of the tested amino acids. No significant changes in organ weights were recorded. We estimate the no-observed-adverse-effect level (NOAEL) for Ile at 2.5% for both genders (male, 1.565 +/- 0.060 g/kg/day; females, 1.646 +/- 0.095 g/kg/day), Val at 5.0% for males (3.225 +/- 0.135 g/kg/day) and 2.5% for females (1.853 +/- 0.060 g/kg/day), and Leu at 5.0% for both genders (males, 3.333 +/- 0.101 g/kg/day: females, 3.835 +/- 0.257 g/kg/day). PMID:15204732

Tsubuku, Shoji; Hatayama, Kazuhisa; Katsumata, Toyohisa; Nishimura, Nobuo; Mawatari, Kazunori; Smriga, Miro; Kimura, Takeshi

84

Determination of isotopic ratios of L-leucine and L-phenylalanine and their stable isotope labeled analogues in biological samples by gas chromatography/triple-stage quadrupole mass spectrometry.  

PubMed

A gas chromatographic/triple-stage quadrupole mass spectrometric (GC/MS/MS) method for measuring very low levels of enrichment of [5,5,5-2H3]-L-leucine and [ring-13C6]-L-phenylalanine in plasma and lipoprotein hydrolysates is described. The amino acids were derivatized to their N-heptafluorobutyryl isobutyl ester derivatives and the isotope ratio was determined by GC/MS/MS in the negative-ion chemical ionization mode. Parent ions were the [M-HF]- ions and fragment ions used for quantification were [P-2HF-C3H7]- (leucine) and [P-HF-OC4H9]- (phenylalanine), respectively. The limit of quantification was about 10 pg of the labeled compound co-eluting with 20 ng of the endogenous compound. The calibration curves were linear in the investigated range from 0.1% to 100% of the labeled compound. In biological samples, the higher selectivity of GC/MS/MS compared with GC/MS was demonstrated. PMID:8799305

Schweer, H; Watzer, B; Seyberth, H W; Steinmetz, A; Schaefer, J R

1996-07-01

85

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

PubMed

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

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

1999-05-19

86

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

PubMed Central

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.

Eikmanns, Bernhard J

2011-01-01

87

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

SciTech Connect

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

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

1996-05-29

88

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

NASA Astrophysics Data System (ADS)

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

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

2010-03-01

89

Characterisation of the ArmA adenylation domain implies a more diverse secondary metabolism in the genus Armillaria.  

PubMed

The armA-gene, encoding a tridomain enzyme reminiscent of nonribosomal peptide synthetases, was identified in the genome of the basidiomycete Armillaria mellea. Heterologously expressed enzyme and the ATP-pyrophosphate exchange assay were used for the in vitro biochemical characterisation of the ArmA adenylation domain. l-leucine was the preferred substrate, while l-threonine, l-valine, l-alanine, and l-isoleucine were turned over at lower rates (83 %, 62 %, 56 %, and 44 %, respectively). Other proteinogenic amino acids, 2-oxo acids, and benzoic acid derivatives were not accepted. As the substrate range of ArmA is incompatible with the secondary metabolites known from the genus Armillaria, our results imply greater natural product diversity in this genus. This is the first biochemical characterisation of a basidiomycete amino acid-adenylating domain, and our results may help refine computer algorithms to predict substrate specificities for basidiomycete nonribosomal peptide synthetases whose genes are discovered through genome sequencing. PMID:21802058

Misiek, Mathias; Braesel, Jana; Hoffmeister, Dirk

2011-06-30

90

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

PubMed

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

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

2006-01-01

91

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

PubMed Central

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

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

2006-01-01

92

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.

2011-01-01

93

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

PubMed

This study offers experimental evidence for the existence of three pheromone races of the northern genitalic form of Phyllophaga anxia: one race in which females produce and males respond mainly to L-valine methyl ester, a second producing and responding to L-isoleucine methyl ester, and a third producing and responding to an intermediate range of blends of the two compounds. At Franklinville, NY, pheromone gland contents of females were analyzed using coupled gas chromatography-electroantennogram detection. Two types of females were found, one that produced greater than 99% L-valine methyl ester and another that produced greater than 99% L-isoleucine methyl ester. Capture-mark-release-recapture field tests with males at Franklinville established that most males were recaptured in traps baited with the same blends with which they were originally captured. The populations characterized at Franklinville, NY, have also been found at numerous locations from eastern Canada and the northeast and north central USA, sometimes in allopatry and sometimes in sympatry. At a site in Carver, MA, P. anxia males responded to blends of the methyl esters of L-valine and L-isoleucine, and Carver females produced blends similar to those to which the males responded. Populations responding to blends have been identified only from southeastern Massachusetts and Rhode Island. At a field site near Waterloo, NY, the addition of small proportions of L-isoleucine methyl ester to lures containing L-valine methyl ester did not affect trap captures, but higher proportions of L-isoleucine methyl ester were inhibitory, decreasing trap captures. PMID:18213495

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

2008-01-24

94

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

PubMed

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

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

2013-01-23

95

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

PubMed

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

Mourad, G; Williams, D; King, J

1995-01-01

96

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

PubMed Central

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

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

1977-01-01

97

Characterization of neutral amino acid transport in a marine pseudomonad.  

PubMed Central

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

Fein, J E; MacLeod, R A

1975-01-01

98

Production of essential L-branched-chain amino acids in bioreactors containing artificial cells immobilized multienzyme systems and dextran-NAD+.  

PubMed

We prepared artificial cells each containing leucine dehydrogenase (EC 1.4.1.9), urease (EC 3.5.1.5), soluble dextran-NAD(+), and one of the following coenzyme regenerating dehydrogenases: glucose dehydrogenase (EC 1.1.1.47); yeast alcohol dehydrogenase (EC 1.1.1.1); malate dehydrogenase (EC 1.1.1.37); or lactate dehydrogenase (EC 1.1.1.27). Artificial cells were packed in small columns. L-Leucine, L-valine, and L-isoleucine were continuously produced with simultaneous dextran-NADH regeneration. The maximum production ratios depended on the coenzyme regenerating systems used: 83-93% for D-glucose and glucose dehydrogenase system; 90% for ethanol and yeast alcohol dehydrogenase system; 45-55% for L-malate and malate dehydrogenase system; and 64-78% for L-lactate and lactate dehydrogenase system. Kinetic experiments were also carried out. The apparent K(m) values are as follows: 0.33 mM for alpha-ketoisocaproate (KIC); 0.51 mM for alpha-ketoisovalerate (KIV); 0.58 mM for DL-alpha-keto-beta-methyl-n-valerate (KMV); 3.52 mM for urea; 27.82 mM for D-glucose; 3.89 mM for ethanol; 3.02 mM for L-malate; and 16.67 mM for L-lactate. Kinetic analysis showed that KIC, KIV, and KMV were all competitive inhibitors in the reactions catalyzed by leucine dehydrogenase. Their inhibitor constants were the corresponding K(m) values. PMID:18595077

Gu, K F; Chang, T M

1990-07-01

99

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

PubMed

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

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

2007-11-01

100

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

PubMed Central

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

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

2008-01-01

101

The effect of ethanol on intestinal L-leucine absorption in rats.  

PubMed

The chronic effect of ethanol on leucine absorption by the whole rat intestine (between duodenum and rectum) was studied using an in vivo multiple-pass perfusion technique. Leucine concentrations in the perfusion medium were 5, 10 and 25 mM respectively in successive passes. Ethanol was administered in drinking water during a one month induction period and then for a four week period of ad libitum ingestion of 30% ethanol solution. The results were compared with ad libitum-fed control rats. The total calorie consumption due to the chow diet plus ethanol increased in the rats which had ingested ethanol when compared with that of the controls. The daily protein intake in ethanol-fed rats was less than that of the controls. No significant differences in morphometric tissue parameters were found between the two experimental groups. Chronic ethanol ingestion provoked a slight (but not significant) decrease in net leucine absorption at 5 mM leucine concentration. In contrast, minor increases in the absorption values were found at 10 and 25 mM leucine concentrations. These findings suggest that the diminished active mechanisms of leucine absorption provoked by ethanol ingestion are compensated for by the enhanced diffusive processes, the passage of the nutrients through the whole intestine, and that the low protein consumption of ethanol-fed rats in ad libitum conditions isn't enough to provoke significant decreases in leucine absorption by the whole intestine. PMID:7684271

Carreras, O; Vazquez, A L; Rubio, J M; Delgado, M J; Murillo, M L

102

The upstream activating sequence for L-leucine gene regulation in Saccharomyces cerevisiae.  

PubMed Central

The upstream activating sequence (UAS) conferring leucine-specific regulation of transcription in Saccharomyces cerevisiae was identified by analysis of the LEU2 promoter and by comparison to other genes regulated by leucine. The UAS was localized with deletions and cloned synthetic DNA. Point mutations and sequence rearrangements were used to identify important basepairs and to construct an improved UAS with increased regulation and expression. The improved UAS contains a core ten basepair, GC-rich, palindromic sequence, which is sufficient to confer minimal levels of activation and regulation, within a 36 basepair palindromic sequence which confers maximal activation and regulation. Deletions downstream of the UAS indicated that the UAS must act in conjunction with at least one other site, perhaps a TATAA region, in order to confer high levels of activation. Tandem copies of the UAS in front of LEU2 increased expression and regulation. Tandem UAS elements in trans on a multi-copy 2 mu-based plasmid decreased expression and regulation. These results are consistent with a model that the UAS serves as the DNA-binding site for diffusible activation factor(s), possibly the LEU3 gene product.

Tu, H; Casadaban, M J

1990-01-01

103

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

2011-01-01

104

Complexation, liquid–liquid extraction, and transport through a liquid membrane of protonated peptides using crown ethers  

Microsoft Academic Search

By means of an ion-pairing mechanism, some protonated peptides (glycyl-glycine, glycyl-l-?-alanine, glycyl-l-valine, glycyl-l-leucine, glycyl-l-phenylalanine, glycyl-glycyl-glycine, glycyl-l-aspartic acid, and l-leucyl-glycyl-glycine) were extracted by crown ethers from an aqueous phase into a chloroform phase and transported though a chloroform liquid membrane in the presence of counterions. The peptides under study exhibited good extractability by crown ethers from the aqueous phase into the

Hans-Jürgen Buschmann; Lucia Mutihac

2002-01-01

105

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

Microsoft Academic Search

A series of new ternary zinc(II) complexes [Zn(L1–10)(phen)], where phen is 1,10-phenanthroline and H2L1–10=tridentate Schiff base ligands derived from the condensation of amino acids (glycine, l-phenylalanine, l-valine, l-alanine, and l-leucine) and salicylaldehyde-5-sulfonates (sodium salicylaldehyde-5-sulfonate and sodium 3-methoxy-salicylaldehyde-5-sulfonate), have been synthesized. The complexes were characterized by elemental analysis, IR, UV–vis, 1H NMR, and 13C NMR spectra. The IR spectra of the

Davar M. Boghaei; Mehrnaz Gharagozlou

2007-01-01

106

Thermodynamic study of solvation of some amino acids, diglycine and lysozyme in aqueous and mixed aqueous solutions  

Microsoft Academic Search

Apparent molar adiabatic compressibilities and viscosities of glycine, dl-?-alanine, dl-?-amino-n-butyric acid, l-valine, l-leucine and diglycine have been determined in aqueous and mixed aqueous solutions of mB=1.0, 2.0, 3.0, 4.0 and 5.0 aqueous n-propanol solutions at 298.15K. From these data the partial molar adiabatic compressibilities and viscosity B-coefficients have been evaluated to calculate the corresponding transfer functions. The partial molar adiabatic

Tarlok S. Banipal; Gagandeep Singh

2004-01-01

107

Antitumor active monofunctional platinum(II) complexes: Synthesis, structural characterization and reactivity towards biomolecules  

Microsoft Academic Search

Monofunctional platinum(II) complexes represent a class of antitumor agents that do not obey the classical structure–activity relationships. We report herein two novel platinum(II) complexes [PtL1Cl] (L1=l-valine-N-8-quinolylamide) and [PtL2Cl] (L2=l-leucine-N-8-quinolylamide) in which Cl? acts as the only potential leaving group. These complexes showed comparable cytotoxicity to cisplatin against the murine leukemia cell line (P-388) and the human non-small-cell lung cancer cell

Xinliu Gao; Xiaoyong Wang; Jian Ding; Liping Lin; Yizhi Li; Zijian Guo

2006-01-01

108

Higher-order complexity through R-group effects in self-assembled tripeptide monolayers.  

PubMed

Self-assembled monolayers of tri-L-leucine and tri-L-valine formed on highly ordered pyrolytic graphite (HOPG) substrates have been examined using scanning tunneling microscopy. These monolayers exhibit markedly different structures, even though the tripeptides differ by only a minor change in the amino acid R-group. This minor change in R-group apparently affects the balance between hydrogen bonding and van der Waals interactions that control the monolayer structures. Implications of this effect for evolution of molecular complexity in prebiotic synthesis on environmental surfaces are discussed. PMID:20602532

Rampulla, David M; Oncel, Nuri; Malcolm, Stuart A; Bernasek, Steven L

2010-11-01

109

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

2011-01-01

110

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

PubMed Central

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

Tak, Aijaz Ahmad; Arjmand, Farukh

2002-01-01

111

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

SciTech Connect

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

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

1988-08-01

112

Spectroscopy, cytotoxicity and DNA-binding of the lanthanum(III) complex of an l-valine derivative of 1,10-phenanthroline  

Microsoft Academic Search

The interaction of the lanthanum(III) La(III)–L (L=N,N?-bis-(1-carboxy-2-methylpropyl)-1,10-phenanthroline-2,9-dimethanamine) complex with calf thymus DNA was studied by electronic spectra, fluorescence spectra and circular dichroic spectra. The La(III)–L complex was assayed for antitumor activity in vitro against the HL-60 (the human leucocytoma) cells, HCT-8 (the human coloadenocarcinoma) cells, BGC-823 (the human carcinoma of stomach) cells, Bel-7402 (the human liver carcinoma) cells and KB

Zhong-Ming Wang; Hua-Kuan Lin; Shou-Rong Zhu; Tian-Fu Liu; Yun-Ti Chen

2002-01-01

113

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

PubMed

A sensitive method for the determination of S- and R-3-methyl-2-oxopentanoate enantiomers (KMV, alpha-keto-beta-methylvalerate) 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 are formed from S- and R-3-methyl-2-oxopentanoate, respectively, are then quantified by amino acid analysis. The method was used for determination of the R-IS-3-methyl-2-oxopentanoate ratio in plasma of healthy subjects and patients with diabetes mellitus and maple syrup urine disease. Applicability for gas chromatographic-mass spectrometric analysis of 13C-label enrichment in plasma S-3-methyl-2-oxopentanoate is demonstrated. PMID:8844412

Schadewaldt, P; Wendel, U; Hammen, H W

1996-07-12

114

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

PubMed Central

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

Shibata, Y; Fujimura, S; Nakamura, T

1993-01-01

115

Sodium-stimulated amino acid uptake into isolated membrane vesicles from Balb/c 3T3 cells transformed by simian virus 40.  

PubMed Central

Mediated uptake of amino acids by membrane vesicles isolated from Balb/c 3T3 cells transformed by simian virus 40 has been demonstrated. Initial rates of transport of radioactively labeled L-leucine and alpha-aminoisobutyric acid were enhanced by the addition of NaCl (100 mM) to the reaction mixture at the start of the uptake process. This enhancement included a prominent "overshoot" during initial uptake. Slight stimulation of alpha-aminoisobutyric acid uptake was seen with K+, but none with Li+. The mediated nature of the uptake event for L-leucine was shown by saturation kinetics and by inhibition with L-valine. The transport assay measured predominantly intravesicular amino acid uptake rather than binding, as shown by the variation of uptake in response to changes in extravesicular osmolarity. Electron microscopy confirmed the presence of closed vesicles. Thus, amino acid transport has been characterized in an in vitro membrane vesicles system which should prove useful for studies of growth control. Images

Quinlan, D C; Parnes, J R; Shalom, R; Garvey, T Q; Isselbacher, K J; Hochstadt, J

1976-01-01

116

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

PubMed Central

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 activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (Km = 68 ?M, k cat = 31 s?1) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate.

Kim, Jihoe; Darley, Daniel; Selmer, Thorsten; Buckel, Wolfgang

2006-01-01

117

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

2010-03-01

118

Purification and structure analysis of mycolic acids in Corynebacterium glutamicum.  

PubMed

Corynebacterium glutamicum is widely used for producing amino acids. Mycolic acids, the major components in the cell wall of C. glutamicum might be closely related to the secretion of amino acids. In this study, mycolic acids were extracted from 5 strains of C. glutamicum, including ATCC 13032, ATCC 13869, ATCC 14067, L-isoleucine producing strain IWJ-1, and L-valine producing strain VWJ-1. Structures of these mycolic acids were analyzed using thin layer chromatography and electrospray ionization mass spectrometry. More than twenty molecular species of mycolic acid were observed in all 5 strains. They differ in the length (20-40 carbons) and saturation (0-3 double bonds) of their constituent fatty acids. The dominant species of mycolic acid in every strain was different, but their two hydrocarbon chains were similar in length (14-18 carbons), and the meromycolate chain usually contained double bonds. As the growth temperature of cells increased from 30°C to 34°C, the proportion of mycolic acid species containing unsaturated and shorter hydrocarbon chains increased. These results provide new information on mycolic acids in C. glutamicum, and could be useful for modifying the cell wall to increase the production of amino acids. PMID:22538651

Yang, Yang; Shi, Feng; Tao, Guanjun; Wang, Xiaoyuan

2012-04-27

119

Optimization and validation of a capillary electrophoresis laser-induced fluorescence method for amino acids determination in human plasma: application to bipolar disorder study.  

PubMed

Quantitative and qualitative analysis of amino acids in biofluids offers relevant information in diagnosis of diseases, evaluation of nutritional state, and in elucidating metabolic influences on physiology. A simple, rapid, and robust procedure in terms of sample treatment, separation, and quantitation based on CE-LIF has been optimized for use in human plasma samples. Time required for derivatization was 15 min and analysis time was 35 min. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was the labeling agent used for obtaining fluorescent derivatives. Electrophoretic conditions were: 175 mM borate buffer at pH 10.25 prepared with 12.5 mM ?-cyclodextrin. The voltage applied was +21 kV. Fourteen amino acids could be quantified: L-proline, L-phenylalanine, L-leucine, L-isoleucine, L-ornithine, D-ornithine, L-glutamine, L-alanine, L-threonine, glycine, L-serine, D-serine, taurine and L-glutamate. With this chiral CE-LIF method, L- and D-amino acids are adequately separated. The method was validated for a representative group of amino acids in human plasma: L-proline, L-isoleucine, L-ornithine, L-glutamine, L-alanine L-threonine, glycine, L-serine, D-serine, and glutamate. The method has been successfully applied to human plasma from patients with bipolar disorder, all of whom were taking lithium as a mood stabilizer. Eleven amino acids were quantified in plasma from nine patients, aged 24-55 years. The results were in accordance to published values for the bipolar patients. The method is useful particularly in studies where plasma amino acid levels can be used as biomarkers for diagnosis of diseases, evaluating the disease progression, and monitoring response to drug therapy. PMID:23512402

Lorenzo, Ma Paz; Villaseñor, Alma; Ramamoorthy, Anuradha; Garcia, Antonia

2013-05-08

120

Transport of L-phenylalanine and related amino acids at the ovine blood-brain barrier.  

PubMed Central

1. Unidirectional influx of amino acids at the blood-brain barrier was studied in the lamb and sheep under barbiturate anaesthesia using the single-pass indicator-dilution technique. 2. In the lamb, influx of both L-phenylalanine (14 +/- 1 nmol g-1 min-1) and L-alanine (12 +/- 2 nmol g-1 min-1) was greater than in the sheep: L-phenylalanine influx, 9 +/- 1 nmol g-1 min-1; L-alanine influx, 5 +/- 1 nmol g-1 min-1 (P less than 0.01). This difference reflected higher blood concentrations of these amino acids in the younger animal. 3. The kinetic parameters of transport for L-phenylalanine were determined in the lamb and sheep from measurements of influx over a range of blood concentrations. The concentration dependence of L-phenylalanine influx was best described by a model with a saturable and non-saturable component. Maximum influx (Jmax) was higher and apparent transport constant (km, app) lower in the lamb. Values obtained (mean +/- S.E.M.) were: lamb, Jmax, 138 +/- 6 nmol g-1 min; km, app, 0.85 +/- 0.10 mmol l-1; sheep, Jmax, 107 +/- 7 nmol g-1 min-1; km, app, 2.25 +/- 0.25 mmol l-1. 4. L-Phenylalanine inhibited influx of L-leucine, L-tyrosine, L-valine and L-glutamine but not L-arginine and L-lysine. Its influx was inhibited by L-histidine, L-valine and L-leucine, but not by L-glutamine or L-alanine. In the lamb, L-phenylalanine inhibited L-histidine influx with an apparent inhibitor constant (kh) of 139 mumol l-1, and a maximum inhibition of 92%. In the sheep, L-phenylalanine inhibited L-methionine influx with an apparent kh of 33 mumol l-1 and a maximum inhibition of 82%. 5. Fractional extraction of phenylalanine and alanine was stereospecific with preference for the L-enantiomer. In the lamb, fractional extraction values (mean +/- S.E.M.) were: L-phenylalanine, 0.58 +/- 0.03; D-phenylalanine, 0.20 +/- 0.02; L-alanine, 0.16 +/- 0.03; D-alanine, 0.05 +/- 0.02. Self-inhibition of extraction was evident for L-phenylalanine and L-alanine in both lamb and sheep.

Brenton, D P; Gardiner, R M

1988-01-01

121

A Hydrolase from Lactobacillus sakei Moonlights as a Transaminase  

PubMed Central

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

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

2013-01-01

122

A hydrolase from Lactobacillus sakei moonlights as a transaminase.  

PubMed

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

Sinz, Quirin; Freiding, Simone; Vogel, Rudi F; Schwab, Wilfried

2013-01-25

123

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

PubMed Central

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

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

2013-01-01

124

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

2011-03-01

125

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

PubMed Central

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

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

1997-01-01

126

Effects of alpha-ketomonocarboxylic acids upon insulin secretion and metabolism of isolated pancreatic islets.  

PubMed

In perifused isolated pancreatic islets alpha-ketoisocaproic acid (KIC) or alpha-ketocaproic acid (KC) induced a high insulin secretion rate and a steep increase of the fluorescence of reduced pyridine pyridine nucleotides [NAD(P)H] which fell again to almost prestimulatory levels 6 min after medium change. Insulin release in response to alpha-ketooctanoic (KO) acid started slowly and was accompanied by a decrease of the NAD(P)H-fluorescence trace. Beta-phenylpyruvate which is known to initiate insulin release also caused a fluorescence decrease. Alpha-keto-isovaleric (KIV) acid or pyruvate had no significant effects upon insulin secretion or NAD(P)H-fluorescence. In contrast to l-leucine, l-norleucine or l-valine did not enhance insulin release or fluorescence of NAD(P)H. KIV, alpha-keto-beta-methylvaleric acid (KMV), KIC and KC raised the production their corresponding amino acids by islet cells. From these results it is concluded that alpha-ketomonocarboxylic acids as such trigger insulin release by acting upon receptor sites which differ from those occupied by amino acids. PMID:1687

Panten, U

1975-01-01

127

A Molecular Dynamics Simulation Study of Two Dipeptide Based Molecular Micelles: Effect of Amino Acid Order  

PubMed Central

Molecular dynamics (MD) simulations were used to compare the structures of the chiral molecular micelles (MM) poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)) and poly-(sodium undecyl-(L,L)-valine-leucine) (poly (SUVL)). Both MM contained polymerized surfactant monomers tenninated by chiral dipeptide headgroups. The study was undertaken to investigate why poly(SULV) is generally a better chiral selector compared to poly(SUVL) in electrokinetic chromatography separations. When comparing poly(SULV) to poly(SUVL), poly(SULV) had the more conformational flexible dipeptide headgroup and hydrogen bond analyses revealed that the poly(SULV) headgroup conformation allowed a larger number of intramolecular hydrogen bonds to form between monomer chains. In addition, a larger number of water molecules surrounded the chiral centers of the poly(SULV) molecular micelle. Poly(SULV) was also found to have a larger solvent accessible surface area (SASA) than poly(SUVL) and fluctuations in the poly(SULV) SASA during the MD simulation allowed dynamic monomer chain motions expected to be important in chiral recognition to be identified. Finally, approximately 50% of the Na+ counterions were found in the first three solvation shells surrounding both MM, with the remainder located in the bulk. Overall the MD simulations point to both greater headgroup flexibility and solvent and analyte access to the chiral centers of the dipeptide headgroup as factors contributing to the enhanced chiral selectivity observed with poly(SULV).

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

2013-01-01

128

Simultaneous separation of jasmonic acid conjugates with amino acids by MEKC.  

PubMed

Jasmonic acid (JA) conjugates with amino acids (AAs) are a group of plant hormone in the family of jasmonates. The separation of stereoisomers of JA-AA conjugates is a very challenging work since these stereoisomers have similar chromatographic and electrophoretic behavior. Simultaneous separation of ten (±)-JA conjugates with five AAs including L-Tyr (tyrosine), L-leucine, L-Ile (isoleucine), L-valine, and L-phenylalanine and their stereoisomers has been achieved by MEKC with diode array detector in this work. Optimum separation of the analytes was obtained on a 61.5 cm × 75 ?m id capillary using a running buffer containing 80 mM SDS and 50 mM phosphates (pH 7.0) at +18 kV applied voltage and capillary temperature of 35°C. Ten stereoisomers of JA conjugates with five AAs are completely separated in 13 min. The RSDs of the migration times and peak areas of the ten stereoisomers were in the range of 0.48-1.03% and 1.03-2.07%, respectively. In the tested concentration range, good linear relationships (correlation coefficients above 99%) between peak areas and concentrations of the analytes were observed. The proposed method has been successfully applied to the analysis of spiked rice floret sample and original reaction solution of (±)-JA-Ile conjugate and (±)-JA-Tyr conjugate. The recoveries ranged from 91.7 to 107.6% for the rice floret sample and 92.9 to 107.2% for the original reaction solution. PMID:23483735

Chen, Ying; Chen, Zilin

2013-03-01

129

Atmospheric oxygen and other conditions affecting the production of cereulide by Bacillus cereus in food.  

PubMed

Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography-ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation. Oxygen was essential for production of the emetic toxin by B. cereus. When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 microg ml(-1) or g(-1) wet wt) of cereulide accumulated during 4-day storage at room temperature. In parallel cultures and foods, stored under nitrogen atmosphere (> 99.5% N2), less than 0.05 microg of cereulide ml(-1) or g(-1) wet wt accumulated. The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans. Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans. Adding l-leucine and l-valine (0.3 g l(-1)) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar. When the B. cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days. In broth culture, the production of cereulide started later than 16-24 h. Anoxic storage prevented cereulide production also when the amino acids had been supplied. Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food. PMID:15358508

Jääskeläinen, E L; Häggblom, M M; Andersson, M A; Salkinoja-Salonen, M S

2004-10-01

130

Sex Pheromone of the Scarab Beetle Phyllophaga elenans and Some Intriguing Minor Components  

Microsoft Academic Search

Three amino acid-derived compounds were identified in extracts from the pheromone glands of the scarab beetle Phyllophaga elenans, i.e., L-isoleucine methyl ester (LIME), N-formyl L-isoleucine methyl ester (For-LIME), and N-acetyl L-isoleucine methyl ester (Ac-LIME). The compounds were characterized from their spectral data (MS and IR), confirmed by synthesis, and their absolute configurations were assigned by gas chromatography with a chiral

Walter S. Leal; Allan C. Oehlschlager; Paulo H. G. Zarbin; Eduardo Hidalgo; Philip J. Shannon; Yasuhiro Murata; Lilliana Gonzalez; Romano Andrade; Mikio Ono

2003-01-01

131

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

2011-01-01

132

Chloride and ethyl ester morpholine thiourea derivatives and their Ni(II) complexes. Crystal and molecular structures of the thiourea derivative L-leucine methyl ester and its complexes with Cu(II) and Pt(II). Growth of the pathogenic fungus Botrytis cinerea  

Microsoft Academic Search

We have synthesized a series of ligands (1, 3, 4, 6 and 7) and some of their complexes with Ni (II), Cu (II) and Pt (II) (2, 5, 8 and 9). These compounds were studied and characterized by elemental analysis, IR and UV–Vis spectra, conductivity measurements in solution, FAB+\\/MS, 1H and 13C NMR, ESR, etc. Compound 7 crystallized in the

E. Rodr??guez-Fernández; Eva Garc??a; M. R. Hermosa; A. Jiménez-Sánchez; M. Mar Sánchez; Enrique Monte; Julio J. Criado

1999-01-01

133

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.

2012-01-01

134

On the mechanism of L-alloisoleucine formation: studies on a healthy subject and in fibroblasts from normals and patients with maple syrup urine disease.  

PubMed

L-Alloisoleucine formation from L-isoleucine was studied in vitro and in vivo. When a healthy subject was loaded with L-isoleucine, plasma levels of L-isoleucine and 3-methyl-2-oxopentanoate (KMV), as well as L-alloisoleucine, increased. Peak values were reached successively and were in the order L-isoleucine much greater than KMV much greater than L-alloisoleucine. Metabolic clearance of L-isoleucine and KMV was rapid; clearance of L-alloisoleucine was considerably delayed. When human skin fibroblast cultures were challenged with L-isoleucine, KMV accumulated at a gradually decreased rate, whereas L-alloisoleucine accumulated at a gradually accelerated rate. KMV and L-alloisoleucine formation were related and depended on the L-isoleucine concentration applied. In cell lines derived from MSUD patients (classical form), metabolite formation was only about 2-fold higher than in control strains. The relatively small difference between normal and MSUD fibroblasts in vitro as opposed to the striking differences between healthy subjects and MSUD patients in vivo are discussed with respect to the significance of physiological mechanisms participating in the formation and degradation of L-alloisoleucine in man. PMID:2116545

Schadewaldt, P; Hammen, H W; Dalle-Feste, C; Wendel, U

1990-01-01

135

Cypridina Bioluminescence I: Structure of Cypridina Luciferin.  

National Technical Information Service (NTIS)

The complete structure of Cypridina luciferin is examined. Luciferin is composed of tryptamine, arginine and L-isoleucine moieties, and the extended dihydropyrazine structure is responsible for the oxidation during the luminescence. (Author)

Y. Kishi T. Goto Y. Harata O. Shimomura F. H. Johnson

1966-01-01

136

Synthesis of coronafacic acid via TBAF-assisted elimination of the mesylate and its conversion to the isoleucine conjugate.  

PubMed

An aldol reaction followed by elimination of the derived mesylate was used to construct the side chain that was designed to afford the cyclohexene ring of coronafacic acid via intramolecular alkylation. Elimination of the mesylate proceeded with TBAF. The alkylation was achieved with t-BuOK in THF, and then hydrolysis afforded coronafacic acid, which upon condensation with unprotected L-isoleucine using ClCO(2)Bu(i) furnished coronafacoyl-L-isoleucine, the L-Ile conjugate. PMID:21766797

Kosaki, Yusuke; Ogawa, Narihito; Wang, Qian; Kobayashi, Yuichi

2011-07-18

137

21 CFR 172.320 - Amino acids.  

Code of Federal Regulations, 2013 CFR

...Monohydrochloride Monopotassium L -glutamate L-Tyrosine L-Valine (2) As found in âSpecifications and Criteria for Biochemical Compounds,â NAS/NRC Publication, 3rd Ed. (1972), which is incorporated by reference (Copies are...

2013-04-01

138

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

139

Low temperature specific heat anomaly of D-Valine  

Microsoft Academic Search

The low temperature specific heat ofD-Valine andL-Valine has been measured by differential scanning calorimetry in the temperature region between 77–300K. It was found that an obvious lambda transition at 272±1K. X-ray diffraction crystallographic data showed that no crystal lattice changed C,H,N element analysis proved the high purity of the sample ofD andL-Valine. We propose that the shape of the jump

Wenqing Wang; Xiangrong Sheng; Hongshun Yang; Zhizhong Zhuang; Fenming Lou; Zhaojia Chen

1995-01-01

140

The avermectin-producing Streptomyces avermitilis possesses an inducible valine dehydrogenase  

Microsoft Academic Search

Valine dehydrogenase (VDH) is believed to be absent in Streptomyces avermitilis. In the present study, a VDH (Mr, 72 000) was detected by activity measurement and activity staining on a native-PAGE gel. The enzyme activity was induced by L-valine and repressed by ammonia. VDH activity was found to be significantly lower than L-valine transaminase activity. The results suggest that one

Kien Trung Nguyen; Lieu Thi Nguyen; Vladislav B?hal

1995-01-01

141

The Relationship between Dipeptidase Activity Variation and Larval Viability in Drosophila melanogaster  

PubMed Central

The enzyme dipeptidase-A (DIP-A) in Drosophila melanogaster is coded by a second chromosome locus that is polymorphic for three allozymes in natural populations. DIP-A appears to be the only enzyme in D. melanogaster capable of hydrolyzing the dipeptide glycyl-l-isoleucine, since flies homozygous for null alleles at this locus have no detectable glycyl- l-isoleucine-ase activity. DIP-A activity occurs in many tissues and throughout development, but is particularly high in the larval midgut, suggesting an important role in protein digestion. These observations suggested an experimental design for investigating the adaptive significance of genetic variation in DIP-A activity. Fitness components of DIP-A variants could be estimated and compared under two environmental conditions (defined diets under axenic conditions). In the restrictive environment, the essential amino acid l-isoleucine is provided only in the form of glycyl-l-isoleucine, whereas in the permissive environment, l-isoleucine is provided in free form. We predicted that DIP-A activity would be essential in the restrictive, but not in the permissive environment. The results reported here clearly contradict this prediction. Two stocks homozygous for DIP-A null alleles from different geographic locations are each viable on the restrictive diet. Furthermore, relative viability experiments in which null allele larvae compete with larvae having DIP-A activity provide no evidence for even a partial reduction in egg to adult survival on the restrictive diet. Apparently, the null allele larvae have some alternative mechanism for obtaining l-isoleucine from the dipeptide, even though no glycyl-l-isoleucine-ase activity can be detected in vitro. These results, along with the viability of null alleles for many other enzymes, support the idea that eukaryotes have an intricate network of alternative biochemical pathways through which the same necessary function may be achieved. Such "buffering capacity" makes it very difficult to analyze the effects of enzyme variants on fitness components.

Hiraizumi, Kazuo; Laurie, Cathy C.

1987-01-01

142

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug  

Microsoft Academic Search

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N?-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O?-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M,

Ronald L Hanson; Zhongping Shi; David B Brzozowski; Amit Banerjee; Thomas P Kissick; Janak Singh; Annie J Pullockaran; Jeffrey T North; Junying Fan; Jeffrey Howell; Susan C Durand; Michael A Montana; David R Kronenthal; Richard H Mueller; Ramesh N Patel

2000-01-01

143

Small molecule functional discrimination of the kinases required for the microbial synthesis of threonine and isoleucine  

Microsoft Academic Search

The biosynthesis of l-threonine and l-isoleucine in bacteria and in fungi requires the action of 2 amino acid kinases: aspartate kinase and homoserine kinase. Although these kinases bind similar substrates and catalyze analogous phosphotransfer chemistry, they do not show high amino acid sequence homology. We show that despite this difference, both kinases form a ternary complex consisting of enzyme- adenosine

David Bareich; Kalinka Koteva; Ishac Nazi; Gerard D Wright

2004-01-01

144

Agency Response Letter GRAS Notice No. GRN 000308  

Center for Food Safety and Applied Nutrition (CFSAN)

... that L-leucine is GRAS, through scientific procedures, for use as an ingredient in non-milk and milk based meal replacements, sports and isotonic ... More results from www.fda.gov/food/ingredientspackaginglabeling/gras

145

BACTERIOPLANKTON DYNAMICS IN A SUBTROPICAL ESTUARY: EVIDENCE FOR SUBSTRATE LIMITATION  

EPA Science Inventory

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

146

Relationship between Protein Deficiency in the Ration of Rats during Early Ontogeny and Function of Enzyme Systems of Digestive and Non-Digestive Organs in Adult Life  

Microsoft Academic Search

Low protein content in the ration of rat pups during transfer from mixed to definitive nutrition (days 21-30 of life) has a negative impact on digestive function of the small intestine and trophic and barrier functions of the large intestine, liver, and kidneys and increases (sucrase, glycyl-L-leucin dipeptidase) or decreases (alkaline phosphatase, aminopeptidase M, glycyl-L-leucine dipeptidase) enzyme activities in these

N. M. Timofeeva; A. A. Nikitina; V. V. Egorova; L. A. Gordova

2004-01-01

147

Relationship between protein deficiency in the ration of rats during early ontogeny and function of enzyme systems of digestive and non-digestive organs in adult life  

Microsoft Academic Search

Low protein content in the ration of rat pups during transfer from mixed to definitive nutrition (days 21–30 of life) has\\u000a a negative impact on digestive function of the small intestine and trophic and barrier functions of the large intestine, liver,\\u000a and kidneys and increases (sucrase, glycyl-L-leucin dipeptidase) or decreases (alkaline phosphatase, aminopeptidase M, glycyl-L-leucine\\u000a dipeptidase) enzyme activities in these

N. M. Timofeeva; A. A. Nikitina; V. V. Egorova; L. A. Gordova

2004-01-01

148

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

NASA Astrophysics Data System (ADS)

The surface of L-leucine films irradiated with an Ar5000 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; Ibuki, Sachi; Ichiki, Kazuya; Ninomiya, Satoshi; Seki, Toshio; Aoki, Takaaki; Matsuo, Jiro

2011-01-01

149

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

PubMed

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

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

2006-09-27

150

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

PubMed

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

2011-09-27

151

Kinetic evidence for interdomain communication in the allosteric regulation of alpha-isopropylmalate synthase from Mycobacterium tuberculosis.  

PubMed

The enzyme alpha-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been identified as a possible target for the design of new antitubercular 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 canonical allosteric mechanisms, including changes to the quaternary 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 more than 50 A. 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 interdomain communication of the inhibitory signal from the regulatory to catalytic domain and implicate the interactions between the linker and catalytic domains as critical determinants of inhibitory signal transmission. PMID:19166329

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

2009-03-10

152

Saccharomyces cerevisiae aspartate kinase mechanism and inhibition  

Microsoft Academic Search

Aspartate kinase (AK) from Saccharomyces cerevisiae (AKsc) catalyzes the first step in the aspartate pathway responsible for biosynthesis of L-threonine, L-isoleucine, and L-methionine in fungi. Little was known about amino acids important for AKsc substrate binding and catalysis. Hypotheses about important amino acids were tested using site directed mutagenesis to substitute these amino acids with others having different properties. Steady

David Christopher Bareich

2003-01-01

153

Fungal aspartate kinase mechanism and inhibition  

Microsoft Academic Search

Aspartate kinase (AK) from Saccharomyces cerevisiae (AKSc) catalyzes the first step in the aspartate pathway responsible for biosynthesis of L-threonine, L-isoleucine, and L-methionine in fungi. Little was known about amino acids important for AKSc substrate binding and catalysis. Hypotheses about important amino acids were tested using site directed mutagenesis to substitute these amino acids with others having different properties. Steady

David C Bareich

2003-01-01

154

Stratigraphy and timing of eolianite deposition on Rottnest Island, Western Australia  

Microsoft Academic Search

Over 100 whole-rock amino acid racemization (AAR) ratios from outcrops around Rottnest Island (32.0° S Latitude near Perth) indicate distinct pulses of eolian deposition during the late Quaternary. Whole-rock d-alloisoleucine\\/l-isoleucine (A\\/I) ratios from bioclastic carbonate deposits fall into three distinct modal classes or “aminozones.” The oldest, Aminozone E, averages 0.33 ± 0.04 (n = 21). Red palaeosol and thick calcrete

Paul J. Hearty

2003-01-01

155

Boulder Deposits from Large Waves during the Last Interglaciation on North Eleuthera Island, Bahamas  

Microsoft Academic Search

Seven boulders measuring 100 to 1000 m3are scattered along the coastal ridge of north Eleuthera. Some are situated on ridge crests up to 20 m above present sea level. The boulders were probably deposited during oxygen-isotope substage 5e or 5d, as shown by their stratigraphic setting and by amino acid racemization ratios.d-alloisoleucine\\/l-isoleucine ratios were determined for land snails, and oolite

Paul J. Hearty

1997-01-01

156

Was natural ? radioactivity of carbon-14 the origin of optical one-handedness in life?  

Microsoft Academic Search

14C labelled solid D- and L-leucine decomposes with significantly different rates by auto-radiolysis. The -decarboxylation ratio (103xCO2%)D\\/(103xCO2%)L was found to be (2.3±0.2)\\/(1.2±0.2)= 1.9±0.5 for samples kept in evacuated tubes at room temperature for 1 year \\/sp. activity: 0.9 MBq g–1; -dose: 224 Gy\\/. EPR indicates a 10% higher radical concentration in the stored solid D-leucine samples than in L-leucine. The

R. K. Tokay; B. Nordén; J.-O. Liljenzin; S. Andersson

1986-01-01

157

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)

2011-01-07

158

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

PubMed

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

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

159

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

Microsoft Academic Search

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

WILLIAM B. KINTER; T. HASTINGS WILSON

1965-01-01

160

Quantitative HPLC Determination of Ciphelin  

Microsoft Academic Search

N-[N-Acetyl-p-di(2-chloroethyl)amino-D,L-phenylalanine]-D,L-valine ethyl ester (ciphelin) is an original domestic antitumor preparation possessing alkylating activity, which was synthesized and characterized at the Blokhin Oncological Research Center (Moscow) [1 – 3]. With respect to the antitumor activity spectrum, ciphelin is fully analogous to sarcolysin and cyclophosphane. A distinguishing feature of these drugs is that, while producing the same action upon tumor, ciphelin exhibits

A. P. Arzamastsev; N. V. Valova; N. A. Oborotova

2001-01-01

161

Synthesis of Substituted Phenyl Esters of Amino Acids and Polycondensation in Langmuirblodgett Films  

Microsoft Academic Search

Long-alkyl-chain phenyl esters of ?-alanine, glycine, and L-valine were prepared, and their monolayer properties were correlated with their molecular structures. These compounds formed stable monolayers on acidic subphases. In particular, the p-hexadecylphenyl esters of ?-alanine and glycine were remarkably stable, and their monolayers could be deposited on calcium fluoride plates as Y-type film by Blodgett's technique. The polycondensation of multilayers

Kenji Hanabusa; Takekazu Oumi; Toshiki Koyama; Hirofusa Shirai; Tadao Hayakawa; Akio Kurose

1989-01-01

162

21 CFR 172.829 - Neotame.  

Code of Federal Regulations, 2013 CFR

...that reasonably required to accomplish the intended technical effect, in foods for which standards of identity established under...do not preclude such use. (d) When neotame is used as a sugar substitute tablet, L-leucine may be used as a...

2013-04-01

163

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

PubMed

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

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

1999-12-01

164

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

Microsoft Academic Search

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmo- spheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibri- onaceae. 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

MICHELE NEMECEK-MARSHALL; CHERYL WOJCIECHOWSKI; WILLIAM P. WAGNER; RAY FALL

1999-01-01

165

21 CFR 172.829 - Neotame.  

Code of Federal Regulations, 2010 CFR

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

2009-04-01

166

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

1981-01-01

167

Identification, purification, and characterization of a novel amino Acid racemase, isoleucine 2-epimerase, from lactobacillus species.  

PubMed

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. PMID:24039265

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

2013-09-13

168

Sex pheromone of the scarab beetle Phyllophaga (Phytalus) georgiana (horn).  

PubMed

The sex pheromone of Phyllophaga (Phytalus) georgiana was characterized as valine methyl ester, tentatively the L-enantiomer. This is the first sex pheromone identified from the Phyllophaga subgenus Phytalus. The pheromone was extracted from female glands, the active component isolated by coupled gas chromatography-electroantennogram detection analysis, characterized by mass spectrometry, and shown to be active in field tests. The seasonal flight pattern was determined for P. georgiana as well as for three other species, P. anxia (both northern and southern genitalic forms), P. gracilis, and P. postrema. The latter three species were captured in traps baited with L-isoleucine methyl ester. PMID:19247715

Robbins, Paul S; Nojima, Satoshi; Polavarapu, Sridhar; Koppenhöfer, Albrecht M; Rodriguez-Saona, Cesar; Holdcraft, Robert J; Consolie, Nancy H; Peck, Daniel C; Roelofs, Wendell L

2009-02-27

169

(Methanol-?O)(methano-lato-?O)oxido[N-(2-oxidobenzyl-idene)isoleucinato-?3 O,N,O?]vanadium(V)  

PubMed Central

In the title complex, [V(C13H15NO3)O(CH3O)(CH3OH)], the VV atom is six-coordinated by a tridentate O,N,O?-donor ligand, derived from the condensation of salicyl­aldehyde and l-isoleucine, a vanadyl O atom, a methano­late O atom and a methanol O atom in a distorted octa­hedral geometry. The asymmetric unit contains two complex mol­ecules. In the crystal, inter­molecular O—H?O and C—H?O hydrogen bonds connect the mol­ecules into a one-dimensional chain along [100].

Wang, Chengyuan; Guo, Zhenghua; Dong, Jianfang; Li, Lianzhi

2012-01-01

170

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

2010-01-01

171

Biodegradable functional poly(ester amide)s with pendant hydroxyl functional groups: Synthesis, characterization, fabrication and in vitro cellular response  

Microsoft Academic Search

The synthesis of a new family of biodegradable ?-amino acid poly(ester amide)s (AA-PEAs) with pendant benzyl ether groups and hydroxyl functional groups is reported. The synthetic strategy employs the ring opening reaction of O-benzyl-l-serine-N-carboxyanhydride with di-p-toluenesulfonic acid salts of bis-l-valine butane-1,4-diester, followed by solution polycondesation reactions with di-p-nitrophenyl sebacate in N,N-dimethylacetamide. Catalytic hydrogenation of the resulting benzyl ether protected AA-PEAs

Mingxiao Deng; Jun Wu; Cynthia A. Reinhart-King; Chih-Chang Chu

2011-01-01

172

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

NASA Astrophysics Data System (ADS)

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

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

2003-12-01

173

Total synthesis and conformational studies of hapalosin, N-desmethylhapalosin and 8-Deoxyhapalosin 1 Dedicated with affection and respect to the memory of Professor Sir Derek H. R. Barton in recognition of his distinguished contributions to science and in sincere gratitude for the immense degree of inspiration and encouragement he provided to one of us (J. Zhu). 1  

Microsoft Academic Search

Hapalosin (2), a 12-membered cyclic depsipeptide possessing MDR-reversing activity, and analogues (3) and (4) have been synthesized using macrolactamization as an important ring-forming step. Three building blocks: (2S, 3R)-3-(tert-butyldimethylsilyloxy)-2-methyl-decanoic acid (13), benzyl (S)-2-hydroxy-3-methylbutanate (14), and (4S,3R)-4-(benzyloxycarbonyl-methylamino)-3-methoxymethoxy-5-phenyl-pentanoic acid (28) were prepared from Evans’s chiral imide (9), l-valine, and l-N-Boc phenylalanine (17), respectively, and were assembled together by applying twice Yamaguchi’s coupling

Björn Wagner; Gabriel Islas Gonzalez; Marie Elise Tran Hun Dau; Jieping Zhu

1999-01-01

174

Regulation of Hemoglobin ?-Chain Synthesis in Bone Marrow Erythroid Cells by ? Chains  

PubMed Central

Synthesis of ? and ? chains of hemoglobin was studied in vitro in intact reticulocytes and bone marrow cells. The cells were from rabbits having a variant form of hemoglobin in which L-isoleucine is in the ? but not in the ? chains. This characteristic permitted a selective inhibition of ?-chain synthesis to be produced by addition to the incubation medium of L-O-methylthreonine, an inhibitor of protein synthesis that is a specific antagonist of L-isoleucine. In studies with reticulocytes, 25 mM L-O-methylthreonine produced a 60-70% inhibition of ?-chain synthesis, but ?-chain synthesis was unaffected even after incubation times for 4 hr. Because reticulocytes contain a pool of uncombined ? chains which might have obscured the demonstration of an ? chain-dependent mechanism for ?-chain synthesis, subsequent studies were done with bone marrow cells. The latter had little or no detectable ?-chain pool. A substantial inhibition of ?-chain synthesis by the bone marrow cells was produced by the isoleucine antagonist but was also accompanied by a significantly decreased rate of ?-chain synthesis. These findings suggest that the coordinated synthesis of the complementary ?- and ?-globin chains of hemoglobin may reflect in part a modifying effect of ?-chain synthesis on the synthesis of ? chains.

Wolf, Jeffrey L.; Mason, R. George; Honig, George R.

1973-01-01

175

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

PubMed

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

Bhushan, Ravi; Agarwal, Charu

2010-06-18

176

Thermal Studies of the Dibutyltin(IV) Complexes of Schiff Bases Derived from Amino Acids  

Microsoft Academic Search

The thermal decomposition using TG, DTG and DTA, of seven complexes of the types Bu2SnL(I) and Bu2SnL(II) (where H2L(I)=Schiff base derived from acetylacetone and glycine [H2L-1(I)] or L-leucine [H2L-4(I)] or methionine [H2L-5(I)] or phenylglycine [H2L-6(I)]; H2L(II)=Schiff base derived from o-hydroxynaphthaldehyde and ?-alanine [H2L-2(II)] or DL-valine [H2L-3(II)] or L-leucine [H2L-4(II)] is shown to fall into one of two categories, viz, (1)

M. Nath; R. Yadav

1999-01-01

177

Influence of metabolic and physical factors on production of diacetoxyscirpenol by Fusarium sambucinum Fuckel.  

PubMed Central

Fusarium sambucinum Fuckel 8099-1 was grown on Czapek-Dox peptone-supplemented medium at 15 degrees C for 14 days, and the cultures were investigated for diacetoxyscirpenol (DAS) production by liquid-liquid extraction and gas chromatography. The addition of 150 mg of sorbic acid, a tricarboxylic acid cycle inhibitor, per liter stimulated both fungal growth and DAS production. Among the beta-hydroxy-beta-methylglutaryl coenzyme A precursors tested, isovaleric acid completely inhibited fungal growth and DAS production, ethyl isovalerate did not support a significant increase in DAS production, and L-leucine partially inhibited DAS production, showing that L-leucine and isovaleric acid catabolisms do not induce trichothecene biosynthesis. Solid particles (cork powder) were necessary for DAS production in stationary cultures but did not influence DAS production in shaken cultures. Shaking strongly stimulated DAS production and fungal growth.

Monnet, D; Vidal, D; Creach, O

1988-01-01

178

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

1990-01-01

179

Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12.  

PubMed

Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied. PMID:26411

Turková, J; Bláha, K; Malaníková, M; Vancurová, D; Svec, F; Kálal, J

1978-05-11

180

Measurement of very low density and low density lipoprotein apolipoprotein (Apo) B100 and high density lipoprotein Apo AI production in human subjects using deuterated leucine. Effect of fasting and feeding  

Microsoft Academic Search

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

J. S. Cohn; D. A. Wagner; S. D. Cohn; J. S. Millar; E. J. Schaefer

1990-01-01

181

Effects of leucine and phenylalanine supplementation during intermittent periods of food restriction and refeeding in adult rats  

Microsoft Academic Search

Although many studies have shown that amino acid ingestion acutely stimulates protein anabolism, only few studies have investigated whether long-term supplementation promotes changes in body composition. We therefore tested the hypothesis that l-leucine (LEU) and l-phenylalanine (PHE) supplementation might have a positive impact on the body composition of rats submitted to intermittent periods of food restriction and refeeding (weight cycling

Jose Donato; Rogerio Graça Pedrosa; Jonas Alves de Araújo; Ivanir Santana de Oliveira Pires; Julio Tirapegui

2007-01-01

182

A sensitive, versatile microfluidic assay for bacterial chemotaxis  

Microsoft Academic Search

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 Ni2+ of WT and chemotactic-mutant strains of Escherichia coli were measured. Migration of the cells was quantified by counting

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

2003-01-01

183

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

PubMed

Production of nitric oxide through the action of nitric oxide synthase (NOS) has been detected in the islets of Langerhans. The inducible isoform of NOS (iNOS) is induced by cytokines and might contribute to the development of type-1 diabetes, while the constitutive isoform (cNOS) is thought to be implicated in the physiological regulation of insulin secretion. In the present study we have detected and quantified islet cNOS- and iNOS-derived NO production concomitant with measuring its influence on insulin secretion in the presence of different secretagogues: glucose, L-arginine, L-leucine and ?-ketoisocaproic acid (KIC) both during fasting and freely fed conditions. In intact islets from freely fed mice both cNOS- and iNOS-activity was greatly increased by glucose (20 mmol/l). Fasting induced islet iNOS activity at both physiological (7 mmol/l) and high (20 mmol/l) glucose concentrations. NOS blockade increased insulin secretion both during freely fed conditions and after fasting. L-arginine stimulated islet cNOS activity and did not affect islet iNOS activity. l-leucine or KIC, known to enter the TCA cycle without affecting glycolysis, did not affect either islet cNOS- or iNOS activity. Accordingly, insulin secretion stimulated by L-leucine or KIC was unaffected by addition of L-NAME both during feeding and fasting. We conclude that both high glucose concentrations and fasting increase islet total NO production (mostly iNOS derived) which inhibit insulin secretion. The insulin secretagogues L-leucine and KIC, which do not affect glycolysis, do not interfere with the islet NO-NOS system. PMID:22120830

Eckersten, Dag; Henningsson, Ragnar

2011-11-24

184

Inhibition of Rat Mammary Gland Carcinogenesis by Simultaneous Targeting of Cyclooxygenase2 and Peroxisome Proliferator-activated Receptor  

Microsoft Academic Search

We examined the effect of celecoxib, a cyclooxygenase-2 (COX-2) in- hibitor, and N-(9-fluorenyl-methyloxycarbonyl)-L-leucine (F-L-Leu), a peroxisome proliferator-activated receptor (PPAR) agonist, separately and combined, on the development of methylnitrosourea (MNU)-induced rat mammary gland carcinogenesis. Celecoxib and F-L-Leu significantly reduced tumor incidence and multiplicity (P < 0.05). Combining both agents exerted higher (synergistic) cancer inhibition than separate treat- ments (P < 0.05).

Alaa F. Badawi; Mazen B. Eldeen; Yingying Liu; Eric A. Ross; Mostafa Z. Badr

2005-01-01

185

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

Microsoft Academic Search

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

H K Heim; A Oestmann; K F Sewing

1990-01-01

186

Proteasome Inhibition Enhances AAV-Mediated Transgene Expression in Human Synoviocytes in Vitro and in Vivo  

Microsoft Academic Search

To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in

Kristi Jennings; Takako Miyamae; Russell Traister; Anthony Marinov; Shigeki Katakura; Dawn Sowders; Bruce Trapnell; James M. Wilson; Guangping Gao; Raphael Hirsch

2005-01-01

187

3Methyl1-butanol production in Escherichia coli : random mutagenesis and two-phase fermentation  

Microsoft Academic Search

Interest in producing biofuels from renewable sources has escalated due to energy and environmental concerns. Recently, the\\u000a production of higher chain alcohols from 2-keto acid pathways has shown significant progress. In this paper, we demonstrate\\u000a a mutagenesis approach in developing a strain of Escherichia coli for the production of 3-methyl-1-butanol by leveraging selective pressure toward l-leucine biosynthesis and screening for

Michael R. Connor; Anthony F. Cann; James C. Liao

2010-01-01

188

Protease activities in normal and schizophrenic human prefrontal cortex and white matter  

Microsoft Academic Search

Endo- and exopeptidase activities have been measured in normal post-mortem human prefrontal cortex and subjacent white matter to estimate their relative capabilities for protein and peptide degradation. Cathepsin D and three dipeptidases versus leucyl-glycine, glycyl-l-leucine and glycyl-glycine) were assayed in serial, microtome prepared frozen sections (± 125 µg fresh weight) and related to histological composition (Nissl stain), dry weight, total

Alfred Pope; Judith A. Amelotte; Howard Belfer; Ralph A. Nixon

1981-01-01

189

Effect of magnesium acetate on the volumetric and transport behavior of some amino acids in aqueous solutions at 298.15 K  

Microsoft Academic Search

Densities, ?, and viscosities, ?, of glycine, dl-?-alanine, dl-?-amino-n-butyric acid, l-leucine and l-phenylalanine in 0.5, 1.0, 1.5 and 2.0 mB of aqueous magnesium acetate solutions at 298.15K have been measured as a function of concentration of amino acids using vibrating tube-digital densimeter and Ubbelohde capillary type viscometer, respectively. The apparent molar volumes, V?, and relative viscosities, ?r, of amino acids

Tarlok S. Banipal; Damanjit Kaur; Parampaul K. Banipal

2006-01-01

190

Cycling of organic nitrogen in marine plankton communities studied in enclosed water columns  

Microsoft Academic Search

Concentrations of fluorescamine-positive substances (primary amines) and turnover rates of L-leucine pools were measured concurrently in seawater samples taken from 1300 m3 plastic enclosures moored in Saanich Inlet, British Columbia, Canada. Concentration and turnover rates of dissolved free amino acids were calculated and then used to determine the instantaneous flux of dissolved free amino acids, which ranged from 0.09 to

J. T. Hollibaugh; A. B. Carruthers; J. A. Fuhrman; F. Azam

1980-01-01

191

Synthesis, Biological Activity and Decomposition Studies of Amino Acid Phosphomonoester Amidates of Acyclovir  

Microsoft Academic Search

Highly stable and water soluble amino acid phosphomonoester amidates of acyclovir (ACV) were synthesized and shown to function predominantly as prodrugs of AC V and not acyclovir monophosphate (AC V-MP) with activities within two fold of the amino acid prodrug of ACV, valaciclovir (VACV). Metabolism studies revealed that incubation of cell-free extracts of Vero cells with the L-leucine phosphomonoester amidate

Timothy W. Abraham; Edward J. Mclntee; Vidhya V. Iyer; Raymond F. Schinazi; Carston R. Wagne

1997-01-01

192

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

2006-01-01

193

Behavior of tetrahydrolipstatin in biological model membranes and emulsions  

Microsoft Academic Search

Tetrahydrolipstatin (orlistat) (S)-I-{ (ZS,3S)-3-hexyl- 4-oxooxetan-Z-yl)methyl)dodecyl N-formyl-L-leucinate, a po- tent inhibitor of pancreatic lipase, is hydrophobic, amphi- pathic, and water-insoluble. It binds irreversibly to pancreatic lipases and inhibits fat absorption. The focus of this investiga- tion is on the distribution of orlistat in emulsified fat andvesicu- lar membranes such as might be present in the intestine during fat absorption. The models

John KO; Donald M. Small

194

Yeast genomics on food flavours  

Microsoft Academic Search

The appearance and concentration of the fusel alcohol 3-methyl-1-butanol is important for the flavour of fermented foods. 3-Methyl-1-butanol is formed by yeast during the conversion of L-leucine. Identification of the enzymes and genes involved in the formation of 3-methyl-1-butanol is a major prerequisite to optimize and control the final food flavour. To identify genes involved in this metabolic route, cDNA

Sung Ah Schoondermark-Stolk

2005-01-01

195

CHEMOSENSORY RESPONSES TO AMINO ACIDS AND CERTAIN AMINES BY THE CILIATE TETRAHYMENA: A FLAT CAPILLARY ASSAY  

Microsoft Academic Search

An assay for chemosensory responsesby the ciliate Tetrahymenathermophilais described that uses glass capillaries with a rectangular cross-section (inner dimensions, 20 X 2 X 0.2 mm). Thesehaveopticalandgeometricalpmpertiespermittingconvenient observation of cell behavior within the capillaries. Washedcells, starvedfor 12 h, accumulatedpreferentiallyin capifiariescontaining L-methionine, L-leucine, L-cysteine, L-histidine, L-histamine, cimetidine, agmatine, and berenil at concentrations of i03 M or less. They avoided capillaries containing tripelennamine,

M. LEVANDOWSKY; T. CHENG; A. KEHR; J. KIM; L GARDNER; L SILVERN; G. LAI L TSANG; C. CHUNG

196

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

Microsoft Academic Search

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

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

2003-01-01

197

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

1997-01-01

198

The Perseus-Exobiology experiment onboard MIR  

NASA Astrophysics Data System (ADS)

Two amino acids, L-leucine and "?-methyl-L-leucine; a cyclic dipeptide, L-leucine-diketopiperazine, and an activated tripeptide L-trileucine thioethylester, were exposed for three months to space conditions onboard the MIR station during the Perseus-Exobiology mission in 1999. These samples were exposed in order to study the exogeneous hypothesis for the origin of some of the important biological building blocks of life. The four compounds were exposed both free and associated with basalt, clay and meteorite powder to simulate the effects of potential meteorite protection. Post-flight analyses did not reveal any racemization or polymerisation of the exposed compounds. Approximately half of the amino acids were photolyzed with decarboxylation apparently the primary cause. Peptides were less sensitive to photolysis which mainly occurred by decarbonylation, but were partly lost by natural degradation or sublimation. The best mineral protection for the samples was ensured by the meteorite powder, which exhibits the highest absorption in VUV, whereas clay, almost transparent in VUV was the least efficient. By varying the thickness of the meteorite layer, it was determined that a 5 ?m film was necessary to ensure efficient protection against UV radiation.

Barbier, B.; Boillot, F.; Chabin, A.; Buré, C.; Venet, M.; Belsky, A.; Jacquet, R.; Bertrand-Urbaniak, M.; Delmas, A.; Brack, A.

2002-11-01

199

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

2010-01-01

200

Polypeptide Grafted Hyaluronan: Synthesis and Characterization  

SciTech Connect

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

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

2010-01-01

201

The radiolysis and radioracemization of amino acids on clays  

NASA Astrophysics Data System (ADS)

L-Leucine and its hydrochloride salt have been deposited on the clay minerals kaolin and bentonite, and the amino acid/clay preparations have been irradiated in a 3000 Ci60Co ?-ray source for radiation dosages that achieved 2 89% radiolysis of the leucine. The undecomposed leucine was thereupon recovered and both percent radiolysis and percent radioracemization were determined. Similar studies were made using solid L-leucine and its hydrochloride, and L-leucine in 0.1 M aqueous solution. It has been found that radiolysis and radio-racemization in these and the previously studied leucine systems follow pseudo-first-order rate laws, and the corresponding specific rate constants are evaluated and compared. Leucine and its hydrochloride salt proved to be the most stable to both radiolysis and radioracemization, followed by leucine and its HCl salt on kaolin, followed by leucine and its HCl salt on bentonite, with leucine (and its HCl and Na salts) in aqueous solution being least stable to both radiolysis and (except for the HCl salt) radioracemization. Implications of these observations as regards the Vester-Ulbricht mechanism for the origin of optical activity are discussed.

Bonner, William A.; Hall, Hillary; Chow, George; Liang, Yi; Lemmon, Richard M.

1985-06-01

202

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)

2011-11-01

203

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

2012-01-01

204

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

PubMed Central

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

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

1972-01-01

205

Asymmetric transfer hydrogenation of ketones catalyzed by amino acid derived rhodium complexes: on the origin of enantioselectivity and enantioswitchability.  

PubMed

Amino acid based thioamides, hydroxamic acids, and hydrazides have been evaluated as ligands in the rhodium-catalyzed asymmetric transfer hydrogenation of ketones in 2-propanol. Catalysts containing thioamide ligands derived from L-valine were found to selectively generate the product with an R configuration (95 % ee), whereas the corresponding L-valine-based hydroxamic acids or hydrazides facilitated the formation of the (S)-alcohols (97 and 91 % ee, respectively). The catalytic reduction was examined by performing a structure-activity correlation investigation with differently functionalized or substituted ligands and the results obtained indicate that the major difference between the thioamide and hydroxamic acid based catalysts is the coordination mode of the ligands. Kinetic experiments were performed and the rate constants for the reduction reactions were determined by using rhodium-arene catalysts derived from amino acid thioamide and hydroxamic acid ligands. The data obtained show that the thioamide-based catalyst systems demonstrate a pseudo-first-order dependence on the substrate, whereas pseudo-zero-order dependence was observed for the hydroxamic acid containing catalysts. Furthermore, the kinetic experiments revealed that the rate-limiting steps of the two catalytic systems differ. From the data obtained in the structure-activity correlation investigation and along with the kinetic investigation it was concluded that the enantioswitchable nature of the catalysts studied originates from different ligand coordination, which affects the rate-limiting step of the catalytic reduction reaction. PMID:19750526

Ahlford, Katrin; Ekström, Jesper; Zaitsev, Alexey B; Ryberg, Per; Eriksson, Lars; Adolfsson, Hans

2009-10-26

206

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

PubMed

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

2011-06-10

207

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

NASA Astrophysics Data System (ADS)

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

Lefevre, Joseph W.

1998-10-01

208

Small molecule functional discrimination of the kinases required for the microbial synthesis of threonine and isoleucine.  

PubMed

The biosynthesis of l-threonine and l-isoleucine in bacteria and in fungi requires the action of 2 amino acid kinases: aspartate kinase and homoserine kinase. Although these kinases bind similar substrates and catalyze analogous phosphotransfer chemistry, they do not show high amino acid sequence homology. We show that despite this difference, both kinases form a ternary complex consisting of enzyme- adenosine triphosphate- amino acid to accomplish phosphoryl transfer. With this similarity in mind, we set out to identify molecules that could lead to inhibitors with activity against both kinases in the pathway. We synthesized a series of aspartic acid-adenosine bisubstrate compounds separated by a variable length alkyl linker that we hypothesized could bind to these kinases. These bisubstrate compounds only inhibited the bacterial aspartate kinase. These results reveal unexpected differences in small molecule interactions among these functionally similar enzymes. PMID:14759741

Bareich, David; Koteva, Kalinka; Nazi, Ishac; Wright, Gerard D

2004-02-15

209

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

2013-01-01

210

Experimental evidence for a metallohydrolase mechanism in which the nucleophile is not delivered by a metal ion: EPR spectrokinetic and structural studies of aminopeptidase from Vibrio proteolyticus.  

PubMed

Metallohydrolases catalyse some of the most important reactions in biology and are targets for numerous chemotherapeutic agents designed to combat bacterial infectivity, antibiotic resistance, HIV infectivity, tumour growth, angiogenesis and immune disorders. Rational design of inhibitors of these enzymes with chemotherapeutic potential relies on detailed knowledge of the catalytic mechanism. The roles of the catalytic transition ions in these enzymes have long been assumed to include the activation and delivery of a nucleophilic hydroxy moiety. In the present study, catalytic intermediates in the hydrolysis of L-leucyl-L-leucyl-L-leucine by Vibrio proteolyticus aminopeptidase were characterized in spectrokinetic and structural studies. Rapid-freeze-quench EPR studies of reaction products of L-leucyl-L-leucyl-L-leucine and Co(II)-substituted aminopeptidase, and comparison of the EPR data with those from structurally characterized complexes of aminopeptidase with inhibitors, indicated the formation of a catalytically competent post-Michaelis pre-transition state intermediate with a structure analogous to that of the inhibited complex with bestatin. The X-ray crystal structure of an aminopeptidase-L-leucyl-L-leucyl-L-leucine complex was also analogous to that of the bestatin complex. In these structures, no water/hydroxy group was observed bound to the essential metal ion. However, a water/hydroxy group was clearly identified that was bound to the metal-ligating oxygen atom of Glu152. This water/hydroxy group is proposed as a candidate for the active nucleophile in a novel metallohydrolase mechanism that shares features of the catalytic mechanisms of aspartic proteases and of B2 metallo-beta-lactamases. Preliminary studies on site-directed variants are consistent with the proposal. Other features of the structure suggest roles for the dinuclear centre in geometrically and electrophilically activating the substrate. PMID:17238863

Kumar, Amit; Periyannan, Gopal Raj; Narayanan, Beena; Kittell, Aaron W; Kim, Jung-Ja; Bennett, Brian

2007-05-01

211

Cation-dependent nutrient transport in shrimp digestive tract.  

PubMed

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

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

2011-10-09

212

Intracellular proteases of Bacillus stearothermophilus.  

PubMed

Cell-free extracts of Bacillus stearothermophilus have been shown to exhibit proteolytic activity toward casein as well as specific activity to catalyze the hydrolysis of furylacryloylglycyl-l-leucine amide, furylacryloylglycine, and carbobenzoxyl-glycine-p-nitrophenyl ester, indicating the presence of a neutral proteinase, a carboxypeptidase-like enzyme, and an alkaline proteinase. The neutral proteinase and carboxypeptidase-like activities were separated by gel filtration over Bio-Gel P-60, and both were reversibly inhibited by 1, 10-phenanthroline. The esterase activity was inhibited by diisopropylfluorophosphate, which did not affect other enzymatic activities and was insensitive to 1, 10-phenanthroline and ethylenediaminetetra-acetic acid. PMID:5002894

Feder, J; Ladenburg, K; Delente, J; Wildi, B S

1971-12-01

213

Intracellular Proteases of Bacillus stearothermophilus  

PubMed Central

Cell-free extracts of Bacillus stearothermophilus have been shown to exhibit proteolytic activity toward casein as well as specific activity to catalyze the hydrolysis of furylacryloylglycyl-l-leucine amide, furylacryloylglycine, and carbobenzoxyl-glycine-p-nitrophenyl ester, indicating the presence of a neutral proteinase, a carboxypeptidase-like enzyme, and an alkaline proteinase. The neutral proteinase and carboxypeptidase-like activities were separated by gel filtration over Bio-Gel P-60, and both were reversibly inhibited by 1, 10-phenanthroline. The esterase activity was inhibited by diisopropylfluorophosphate, which did not affect other enzymatic activities and was insensitive to 1, 10-phenanthroline and ethylenediaminetetra-acetic acid.

Feder, Joseph; Ladenburg, Kurt; Delente, Jacques; Wildi, B. S.

1971-01-01

214

Initial Experience with the Radiotracer Anti1-amino-3-[ 18 F]Fluorocyclobutane-1Carboxylic Acid (Anti[ 18 F]FACBC) with PET in Renal Carcinoma  

Microsoft Academic Search

Purpose  Assessment of renal masses with conventional imaging may be challenging. Anti-1-amino-3-[18F]fluorocyclobutane-1-carboxylic acid (anti-[18F]FACBC) is a synthetic l-leucine analog with relatively little renal excretion. The present study examines anti-[18F]FACBC positron emission tomography uptake in patients with renal masses.\\u000a \\u000a \\u000a \\u000a Procedures  Six patients with seven renal lesions were imaged dynamically for 2 h after injection of 10–10.9 mCi (370–403 MBq) anti-[18F]FACBC. Lesions were evaluated qualitatively and quantitatively

David M. Schuster; Jonathon A. Nye; Peter T. Nieh; John R. Votaw; Raghuveer K. Halkar; Muta M. Issa; Weiping Yu; Julio Sepulveda; Wanzhen Zeng; Andrew Young; Mark M. Goodman

2009-01-01

215

Initial Experience with the Radiotracer Anti1- Amino3-18F-Fluorocyclobutane-1Carboxylic Acid with PET\\/CT in Prostate Carcinoma  

Microsoft Academic Search

Conventional imaging techniques have serious limitations in the detection, staging, and restaging of prostate carcinoma. Anti- 1-amino-3-18F-fluorocyclobutane-1-carboxylicacid(anti-18F-FACBC) is a synthetic L-leucine analog that has excellent in vitro uptake within the DU-145 prostate carcinoma cell line and orthotopically implanted prostate tumor in nude rats. There is little renal excretion compared with 18F-FDG. The present study examines anti-18F-FACBC uptake in patients with

David M. Schuster; R. Votaw; Peter T. Nieh; Weiping Yu; A. Nye; Viraj Master; F. DuBois Bowman; Muta M. Issa; Mark M. Goodman

216

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

Microsoft Academic Search

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

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

1988-01-01

217

Synthesis and asymmetric polymerization of chiral maleimides bearing an aza crown ether  

Microsoft Academic Search

Chiral N-substituted maleimide derivatives bearing L-phenylalanine- or L-leucine-introduced aza crown ether ((S)-A15C5PAMI, (S)-A15C5LMI) were synthesized from maleic anhydride, corresponding amino acids and aza crown ether, and polymerized with an organometal\\/chiral ligand. The number-average-molecular weights and the specific optical rotations of the polymers were 700–5600 and ?105.8° to ?38.3°, respectively. The specific optical rotations of all polymers tended to a positive

Motohisa Azechi; Jun Iwai; Kazuhiro Yamabuki; Kenjiro Onimura; Tsutomu Oishi

2011-01-01

218

Effects of Histamine on Protein and Glycoprotein Production of Isolated Pig Gastric Mucosal Cells  

Microsoft Academic Search

The production of glycoprotein and protein by isolated pig gastric non-parietal cells was measured by incorporation of N-acetyl-[14C]-D-glucosamine ([14C]GlcNAc) and [3H]-L-leucine ([3H]Leu), respectively, into acid insoluble material (AIM). Histamine enhanced incorporation of the tracers into cellular and released AIM in a concentration-dependent manner. The H2 receptor antagonist ranitidine completely blocked the effects of histamine (100 ?mol\\/l) on [3H]Leu incorporation into

H. K. Heim; A. Oestmann; K.-F. Sewing

1990-01-01

219

Amplification of Molecular Information through Self-Assembly: Nanofibers Formed from Amino Acids and Cyanine Dyes by Extended Molecular Pairing  

Microsoft Academic Search

Experimental Section Materials. Cationic cyanine dye, 5-chloro-2-(3-(5-chloro-3-ethyl-2(3H)-benzothiazolylidene)- 2-methyl-1-propenyl)-3-ethyl-, iodine (1) was purchased from Hayashibara Biochemical Laboratories, Inc.. D-glutamic acid, L-alanine, L-lysine hydrochloride, L-arginine, L-leucine, L-serine and ortho-phthalaldehyde (OPA) were purchased from Wako Pure Chemical, Ltd.. D-alanine, L-phenylalanine and 2-mercaptoethanesulfonic acid, sodium salt (MES) were obtained from Tokyo Chemical Industry Co., Ltd., Nacalai Tesque, Inc. and Aldrich Chem. Co., respectively. Glycine

Tomohiro Shiraki; Masa-aki Morikawa; Nobuo Kimizuka

2008-01-01

220

The physiology of Clostridium sporogenes NCIB 8053 growing in defined media.  

PubMed

The physiology of Clostridium sporogenes was investigated in defined, minimal media. In batch culture, the major end products of glucose dissimilation were acetate, ethanol and formate. When L-proline was present as an electron acceptor, acetate production was strongly enhanced at the expense of ethanol. As judged by assay of the relevant enzymes, glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The growth energetics of Cl. sporogenes were investigated in glucose- or L-valine-limited chemostat cultures. In the former case, the addition of L-proline to the medium caused a significant increase in the molar growth yield (as calculated by extrapolation to infinite dilution rate). This finding adds weight to the view that the reduction of L-proline by Cl. sporogenes is coupled to the conservation of free energy. PMID:3571035

Lovitt, R W; Kell, D B; Morris, J G

1987-01-01

221

The specificity of proteinases from Streptomyces griseus (pronase)  

PubMed Central

Purification of pronase by ion-exchange chromatography gave four proteolytically active fractions. Fraction A2 contained an endopeptidase that attacks poly l-valine. Fraction B contained an endopeptidase, an aminopeptidase and carboxypeptidases. The activities against hippuryl-l-arginine and hippuryl-l-phenylalanine could be inhibited to a considerable extent by di-isopropyl phosphorofluoridate and by EDTA. Fraction C contained an endopeptidase resembling bovine trypsin. The pure enzyme was completely inactivated by di-isopropyl phosphorofluoridate and pancreatic trypsin inhibitor and to about 90% by other naturally occurring trypsin inhibitors. Fraction D contained an apparently homogeneous endopeptidase, inhibited by diisopropyl phosphorofluoridate, that adsorbed to and hydrolysed elastin. The activity of all these fractions was tested qualitatively against a wide range of small peptides and synthetic substrates.

Trop, Moshe; Birk, Yehudith

1970-01-01

222

Separation of the enantiomers of substituted putrescine and cadaverine analogues by gas chromatography on chiral and achiral stationary phases.  

PubMed

Capillary gas chromatography (GC) on chiral stationary phases, i.e., Chirasil-Val [L-valine-tert.-(R)-alpha-butylamide] and XE-60-S-valine-(R)-alpha-phenylethylamide, has been applied to the resolution of various substituted analogues of putrescine as their N,N'-perfluoroacyl derivatives. The influence of the nature of the substituent on the retention behaviour and on the resolution of the enantiomers was studied. The results are discussed in terms of volatility and interaction with the chiral stationary phase. The 1,4-disubstituted putrescine analogues with two chiral centres were also clearly resolved into their corresponding stereoisomers. When the chain length between the two amino groups was increased, no clear resolution was obtained of the monosubstituted cadaverine analogues as their N,N'-perfluoroacyl derivatives. However, resolution was obtained after derivatization of the cadaverine analogues with (-)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride, followed by GC analysis on an achiral phase. PMID:3624364

Gaget, C; Wolf, E; Heintzelmann, B; Wagner, J

1987-06-12

223

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

SciTech Connect

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

Romanova, V.S.; Tsyryapkin, V.A.; Vol`pin, M.E. [A.N. Nesmeyanov Institute of Organoelement Compounds, Moscow (Russian Federation)] [and others

1994-12-01

224

The NRPS Enzyme DdaD Tethers N?-fumaramoyl-DAP for Fe(II)/?-ketoglutarate-Dependent Epoxidation by DdaC During Dapdiamide Antibiotic Biosynthesis  

PubMed Central

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

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

2010-01-01

225

Nocardiamides A and B, two cyclohexapeptides from the marine-derived actinomycete Nocardiopsis sp. CNX037.  

PubMed

Two new cyclic hexapeptides, nocardiamides A (1) and B (2), were isolated from the culture broth of marine-derived actinomycete CNX037 strain that was identified as a Nocardiopsis species. The planar structures of nocardiamides A (1) and B (2) were assigned on the basis of 1D and 2D NMR and HRESIMS spectroscopic analyses. Their absolute configurations were deduced by the advanced Marfey's method and chiral-phase HPLC analysis. The challenge of locating two d- and one l-valine residue in 1 and 2 was accomplished by total synthesis using solid-phase peptide synthetic methods. Both 1 and 2 showed negligible antimicrobial activities against seven indicator strains and exhibited no cytotoxicity against HCT-116. PMID:23586970

Wu, Zheng-Chao; Li, Sumei; Nam, Sang-Jip; Liu, Zhong; Zhang, Changsheng

2013-04-15

226

Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis.  

PubMed

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

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

2012-08-17

227

Valine-spectrophotometric readout dosimeter (1 Gy 50 kGy)  

NASA Astrophysics Data System (ADS)

In this method 20 mg unirradiated/irradiated L-valine powder [(CH3)2CH·NH2CH·COOH] is dissolved in 10 ml of a solution containing 4×10-4 mol dm-3 Fe2+ 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 Fe2+ 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 Fe2+ 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.

2005-07-01

228

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

PubMed Central

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

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

2012-01-01

229

Immunoreactivity and biologic activity of semisynthetic [LeuB-30]-insulin: potential value in the treatment of insulin antibody-mediated insulin resistance.  

PubMed

Insulin analogues with different amino acids, including threonine, alanine, L-leucine, D-leucine, L-leucine amide, phenylalanine, tri-alanine, or desalanine, at the B-30 position were semisynthesized from pork insulin by the new enzymatic method. The order of ability of the insulin analogues to bind to anti-insulin sera was [AlaB-30] greater than desalanine greater than [ThrB-30] greater than [Ala-Ala-AlaB-30] greater than [D-LeuB-30], [Leu-NH2B-30],[PheB-30] greater than desoctapeptide greater than or equal to [LeuB-30]. The ability of insulin analogues with different amino acids at B-30 to bind to receptors, as well as their biologic potency tested with glucose uptake in isolated rat adipocytes, was comparable among the analogues. These results suggest that [LeuB-30]-insulin demonstrated the least immunoreactivity and has full activity in receptor binding and biologic effect, and that it may be useful for treatment of anti-insulin antibody-mediated insulin resistance. PMID:7014315

Kobayashi, M; Ohgaku, S; Iwasaki, M; Shigeta, Y; Oka, T; Morihara, K

1981-06-01

230

Microbial degradation of dissolved proteins in seawater  

SciTech Connect

An experimental protocol using radiolabeled proteins was developed to investigate the rates and mechanisms whereby dissolved proteins are degraded in natural marine plankton communities. The results of field observations and laboratory experiments indicate that proteins are degraded by a particle-bound, thermolabile system, presumably bacteria-associated enzymes, with an apparent half-saturation constant of ca. 25 ..mu..g bovine serum albumin (BSA) per liter. Gel permeation chromatography indicated that peptides of chain length intermediate between BSA and the final products of degradation (MW<700) do not accumulate in the medium. Competition experiments indicate that the system is relatively nonspecific. Turnover rates for the protein pool in samples collected in the Southern California Bight were of the same order of magnitude as the turnover rate of the L-leucine pool and were correlated with primary productivity, chlorophyll a concentrations, bacterial abundance and biomass, and L-leucine turnover rate. These data suggest that amino acids derived from proteins are utilized preferentially and do not completely mix with the amino acids in the bulk phase.

Hollibaugh, J.T.; Azam, F.

1983-11-01

231

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

NASA Astrophysics Data System (ADS)

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

Hitz, Thomas H.; Luisi, Pier L.

2004-02-01

232

/sup 3/H-cyclosporine internalization and secretion by human fetal pancreatic islets  

SciTech Connect

Human fetal pancreatic islets were isolated from 16- to 20-week-old fetuses by a collagenase technique and cultured 48 hr in RPMI 1640 containing 10% human adult serum and unlabeled 0 to 5 micrograms cyclosporine A (CsA)/ml. Insulin secretory capacity of human fetal islets was expressed as a fractional stimulatory ratio FSR = F2/F1 of the fractional secretion rates during two successive 1 hr static incubations first with 2 mM glucose (F1) to stabilize secretion followed by maximal stimulus, i.e., 25 mM glucose plus 10 mM L-leucine and 10 mM L-arginine (F2). Unlabeled CsA at the above concentrations had no significant effects on the insulin secretory capacity expressed by FSR-values. Studies of net uptake of 3H-CsA by islets cultured for varying periods up to 40 hr and expressed as picomole 3H-CsA per picomole islet insulin content demonstrated that uptake rate was slow and did not reach isotopic equilibrium over the 40 hr of culture. When isolated fetal islets were cultured for 48 hr in the presence of 3H-CsA and varying concentrations of unlabeled CsA it was found during two successive 1 hr static incubations that fetal islets secrete insulin concomitantly with 3H-CsA following maximal stimulus for secretion. An optimal secretory molar ratio of 3H-CsA to insulin of 4.0 +/- 1.3 (n = 7) was found after islets were cultured 48 hr in the presence of a saturating 2.128 micrograms 3H-CsA per milliliter culture medium. In three successive 30-min static incubations of 3H-CsA loaded islets, first with low glucose, followed by high glucose plus L-arginine and L-leucine, and finally with high glucose plus L-arginine and L-leucine and 10 mM theophylline, the proportional fractional secretion rates of insulin and 3H-CsA were of the same magnitude.

Formby, B.; Walker, L.; Peterson, C.M.

1988-10-01

233

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

PubMed Central

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

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

2011-01-01

234

Response of rice to insect elicitors and the role of OsJAR1 in wound and herbivory-induced JA-Ile accumulation.  

PubMed

Plants produce jasmonic acid (JA) and its amino acid conjugate, jasmonoyl-L-isoleucine (JA-Ile) as major defense signals in response to wounding and herbivory. In rice (Oryza sativa), JA and JA-Ile rapidly increased after mechanical damage, and this increase was further amplified when the wounds were treated with oral secretions from generalist herbivore larvae, lawn armyworms (Spodoptera mauritia), revealing for the first time active perception mechanisms of herbivore-associated elicitor(s) in rice. In the rice genome, two OsJAR genes can conjugate JA and Ile and form JA-Ile in vitro; however, their function in herbivory-induced accumulation of JA-Ile has not been investigated. By functional characterization of TOS17 retrotransposon-tagged Osjar1 plants and their response to simulated herbivory, we show that OsJAR1 is essential for JA-Ile production in herbivore-attacked, field-grown plants. In addition, OsJAR1 was required for normal seed development in rice under field conditions. Our results suggest that OsJAR1 possesses at least two major functions in rice defense and development that cannot be complemented by the additional OsJAR2 gene function, although this gene previously showed overlapping enzyme activity in vitro. PMID:23621526

Fukumoto, Kaori; Alamgir, Kabir; Yamashita, Yuko; Mori, Izumi C; Matsuura, Hideyuki; Galis, Ivan

2013-08-01

235

Synthesis, Characterization, and Antibacterial Studies of Mixed Ligand Dioxouranium Complexes with 8-Hydroxyquinoline and Some Amino Acids  

PubMed Central

Mixed ligand complexes of dioxouranium (VI) of the type [UO2(Q)(L)·2H2O] have been synthesized using 8-hydroxyquinoline (HQ) as a primary ligand and amino acids (HL) such as L-threonine, L-tryptophan, and L-isoleucine as secondary ligands. The metal complexes have been characterized by elemental analysis, electrical conductance, magnetic susceptibility measurements, and spectral and thermal studies. The electrical conductance studies of the complexes indicate their nonelectrolytic nature. Magnetic susceptibility measurements revealed diamagnetic nature of the complexes. Electronic absorption spectra of the complexes show intraligand and charge transfer transitions, respectively. Bonding of the metal ion through N- and O-donor atoms of the ligands is revealed by IR studies, and the chemical environment of the protons is confirmed by NMR studies. The thermal analysis data of the complexes indicate the presence of coordinated water molecules. The agar cup and tube dilution methods have been used to study the antibacterial activity of the complexes against the pathogenic bacteria S. aureus, C. diphtheriae, S. typhi, and E. coli.

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

2011-01-01

236

Catabolism and deactivation of the lipid-derived hormone jasmonoyl-isoleucine.  

PubMed

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

Koo, Abraham J K; Howe, Gregg A

2012-02-07

237

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

2010-01-01

238

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

PubMed

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

2010-07-07

239

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

PubMed

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

Dave, Anuja; Graham, Ian A

2012-03-08

240

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

2012-08-01

241

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

PubMed Central

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

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

1992-01-01

242

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

SciTech Connect

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

Fletcher, R.R.; Wehmiller, J.F.; Martin, R.E.; Johnson, B.J. (Univ. of Delaware, Newark (United States))

1991-03-01

243

A thermo-sensitive poly(organophosphazene) hydrogel used as an extracellular matrix for artificial pancreas.  

PubMed

A poly(organophosphazene) bearing alpha-amino-omega-methyl-poly(ethylene glycol) (AMPEG) and hydrophobic L-isoleucine ethyl ester (IleOEt) side groups has been synthesized. This material exhibited 4 phase transitions in an aqueous solution on gradually increasing the temperature, i.e., a transparent sol, a transparent gel, an opaque gel and a turbid sol. A 10 wt% buffered solution of the polymer was employed to entrap islets of Langerhans in an artificial pancreas. Rat islets entrapped in the gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free rat islets and islets entrapped in other types of polymer gels. Over a 28-day culture period, the rat islets in the poly(organophosphazene) hydrogel maintained higher cell viability and insulin production versus rat islets in different hydrogels and free islets. This thermo-sensitive injectable, biodegradable matrix can be used with several cell types, including nerve cells, to promote nerve regeneration. PMID:16370242

Park, Keun-Hong; Song, Soo-Chang

2005-01-01

244

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

SciTech Connect

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

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

1989-03-01

245

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

PubMed Central

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

He, Jun; Shamsi, Shahab A.

2009-01-01

246

Separation of rat muscle aminopeptidases.  

PubMed

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour. PMID:938487

Parsons, M E; Pennington, R J

1976-05-01

247

Catalytic activity of thermolysin under extremes of pressure and temperature: modulation by metal ions.  

PubMed

The catalytic activity of thermolysin (TL), a Zn-dependent neutral protease from Bacillus thermoproteolyticus, has been studied over a wide interval of pressures (1 bar to 4 kbar) and temperatures (20 degreesC to 80 degreesC) by monitoring hydrolysis of a low-molecular-mass substrate, 3-(2-furylacryloyl)-glycyl-L-leucine amide. This reaction shows a very large negative value for the activation volume and, because of that, simultaneous increase in temperature and pressure leads to a significant (up to 40-fold) acceleration of the reaction. At pressures higher than 2-2.5 kbar, the reaction rate starts to decrease due to disactivation of TL. This disactivation is explained in part by pressure-promoted dissociation of zinc ion from the active site and can be inhibited by adding exogenous zinc. Thus, this thermostable protease does not specifically show a higher stability at high pressure in comparison with small mesophilic proteases. PMID:9675281

Kudryashova, E V; Mozhaev, V V; Balny, C

1998-07-28

248

The specificity of the S1 subsite of papain  

PubMed Central

The specificity of the S1? subsite of the proteolytic enzyme papain was investigated by studying the effect of l-?-amino acid amides on the enzyme-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester and by determining the kinetic parameters for the enzyme-catalysed hydrolysis of some N-benzyloxycarbonylglycyl-l-amino acid amides. These studies showed that the S1? subsite has a predilection for hydrophobic residues, in particular l-leucine and l-tryptophan. The specificity for these residues is manifest in both the binding and acylation steps. N-Benzyloxycarbonylglycine amide is not hydrolysed under comparable conditions, indicating that the amide group adjacent to and on the C-terminal side of the peptide bond about to be cleaved makes an important contribution to the rate of the papain-catalysed hydrolysis of peptides.

Alecio, M. Robert; Dann, Malcolm L.; Lowe, Gordon

1974-01-01

249

Improvement of peptoid chiral stationary phases by modifying the terminal group of selector.  

PubMed

The role of a terminal group of a peptoid selector in a chiral separation was investigated. Six chiral stationary phases (CSPs) with the terminal groups replaced by achiral alkyl groups (n-butyl, t-butyl and diisopropyl) and chiral groups ((1R, 2R)-2-aminocyclohexyl phenylcarbamate, N'-phenyl-L-proline amide and N'-phenyl-L-leucine amide) were synthesized. Achiral terminal groups with different steric hindrances did not broaden the enantioselectivity, but improved the separation factors for some analytes, probably due to the improvement of interactions between the analytes and peptoid chain. Introducing new chiral terminal groups proved an effective way to broaden the applicable spectrum of peptoid CSPs. PMID:23068766

Wu, Haibo; Su, Xiaobing; Li, Kuiyong; Yu, Hui; Ke, Yanxiong; Liang, Xinmiao

2012-09-29

250

Separation of rat muscle aminopeptidases.  

PubMed Central

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.

Parsons, M E; Pennington, R J

1976-01-01

251

Identification of genes involved in the suppression of antibody production from human peripheral blood lymphocytes.  

PubMed

Pretreatment with L-leucyl-L-leucine methyl ester (LLME) is a prerequisite for peripheral blood mononuclear cells (PBMCs) to produce antigen-specific antibodies when sensitized with an antigen. Little information, however, is available regarding the mechanisms involved in LLME-induced augmentation of antibody production from PBMCs that are antigen sensitized. In the present study, we attempted to identify the genes involved in the suppression of antibody production from PBMCs that was not treated with LLME, but sensitized with an antigen. Using subtractive screening, we obtained 63 independent genes, including 17 EST genes, that are specific for LLME-nontreated PBMC. Among these genes, the expression of heavy chain ferritin (H-ferritin), CC chemokine ligand 18 (CCL18), and matrix metalloproteinase 12 (MMP12) were augmented in LLME-nontreated PBMCs, suggesting that inflammatory factors might be involved in the suppression of antibody production in LLME-nontreated PBMCs. PMID:16636465

Aiba, Yoshihiro; Yamashita, Makiko; Katakura, Yoshinori; Furukawa, Yuki; Matsumoto, Shin-ei; Tomimatsu, Kousuke; Teruya, Kiichiro; Shirahata, Sanetaka

2006-04-01

252

Propionibacterium acnes acts as an adjuvant in in vitro immunization of human peripheral blood mononuclear cells.  

PubMed

We have established an in vitro immunization protocol whereby human peripheral blood mononuclear cells (PBMCs) are initially treated with L-leucyl-L-leucine methyl ester (LLME) and subsequently sensitized with antigen in the presence of interleukin (IL)-2, IL-4, and adjuvant. This protocol resulted in the production of antigen-specific antibodies. PBMCs are potentiated to react with exogenous antigens upon treatment with LLME. We are using this system to investigate the immunomodulatory activity of additives. In the present study, we aimed to evaluate the immunomodulatory activity of Propionibacterium acnes (P. acnes), which is known to exhibit various immunomodulatory effects in murine models, using this in vitro immunization protocol. P. acnes was found to augment the production of antigen-specific antibodies by PBMC, possibly through increased production of inflammatory cytokines and/or increased T-B cell interaction. P. acnes hence appears to act as an adjuvant in the antibody response in in vitro immunization. PMID:17690460

Jung, Yeon Suk; Matsumoto, Shin-ei; Yamashita, Makiko; Tomimatsu, Kosuke; Teruya, Kiichiro; Katakura, Yoshinori; Shirahata, Sanetaka

2007-08-07

253

Poly(Glycerol Adipate-co-?-Pentadecalactone) Spray-Dried Microparticles as Sustained Release Carriers for Pulmonary Delivery  

Microsoft Academic Search

Purpose  The aim of this work was to optimize biodegradable polyester poly(glycerol adipate-co-?-pentadecalactone), PGA-co-PDL, microparticles\\u000a as sustained release (SR) carriers for pulmonary drug delivery.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Microparticles were produced by spray drying directly from double emulsion with and without dispersibility enhancers (L-arginine and L-leucine) (0.5–1.5%w\\/w) using sodium fluorescein (SF) as a model hydrophilic drug.\\u000a \\u000a \\u000a \\u000a \\u000a Results  Spray-dried microparticles without dispersibility enhancers exhibited aggregated powders leading

Hesham Tawfeek; Sayed Khidr; Eman Samy; Sayed Ahmed; Mark Murphy; Afzaal Mohammed; Anjum Shabir; Gillian Hutcheon; Imran Saleem

254

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

PubMed

A pair of L-leucine (L-Leu) and D-leucine (D-Leu) was incorporated into a-aminoisobutyric acid (Aib) peptide segments. Thedominant conformations of four hexapeptides, Boc-L-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe (1a), Boc-D-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe(1b), Boc-Aib-Aib-L-Leu-L-Leu-Aib-Aib-OMe (2a), and Boc-Aib-Aib-D-Leu-L-Leu-Aib-Aib-OMe (2b), were investigated by IR,¹H NMR, CD spectra, and X-ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3??-helical structures in solution. X-ray crystallographic analysis revealed that right-handed (P) 3??-helices were present in 1a and 1b and a mixture of right-handed(P) and left-handed (M) 3??-helices was present in 2b in their crystalline states. PMID:22619002

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

2012-05-22

255

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

Microsoft Academic Search

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

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

2007-01-01

256

Identification and interaction of amino acids with leucine-anthracene reagent by TLC and spectrophotometry: experimental and theoretical studies.  

PubMed

A new reagent has been synthesized by coupling anthracene moiety to L-leucine. The reagent is characterized by different analytical techniques. It is capable for easy identification of various amino acids on thin-layer chromatography plates by developing distinguishable colors (detection limit between 0.1-0.5 ?g at cold condition and 0.1-0.4 ?g after heating). This reagent also binds with different amino acids very strongly in solution (methanol). Estimation of equilibrium binding constants of this new reagent with different amino acids has also been carried out. The values of the binding constants are lowest for L-Tyrosine (6.86 × 10³ dm³ mol(-1)) and highest for L-Arginine monohydrochloride (8.86 × 10? dm³ mol(-1)) at 25°C. A theoretical study (Hartree-Fock) has been performed to investigate the interaction of the reagent with a representative amino acid, glycine. PMID:21859542

Sahana, Animesh; Das, Sudipta; Saha, Raja; Gupta, Moumita; Laskar, Subrata; Das, Debasis

2011-09-01

257

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: rjvel@annauniv.edu

2008-02-05

258

Protein properties of mackerel viscera extracted by supercritical carbon dioxide.  

PubMed

The extraction of mackerel viscera using supercritical carbon dioxide (SCO2) was performed under the conditions of temperature range from 35 to 45 degrees C, and constant pressure 25 MPa. The digestive enzyme activities were determined in comparison of untreated and treated SCO2 and solvent treatment. Activities were maintained with high level compared to that of solvent extraction. Also from result of SDS-PAGE, the protein denaturation was minimized when using SCO2 extraction. The major amino acids in the mackerel viscera were determined as glutamic acid, aspartic acid, glycine, leucine, lysine and the free amino acids were taurine, L-alanine, L-leucine, 1-methyl-L-histamine, 3-methyl-L-histidine. PMID:19195377

Park, Ji Yeon; Back, Sung Sin; Chun, Byung Soo

2008-07-01

259

Biphasic Extracellular Proteolytic Enzyme Activity in Benthic Water and Sediment in the Northwestern Mediterranean Sea  

PubMed Central

In this study, we used the fact that bacteria are able to cleave a fluorogenic substrate analog (l-leucine-7-amido-4-methylcoumarin) to determine the maximal ectoproteolytic activities (Vm) and affinities (Km) of natural benthic microbial communities by the multiconcentration kinetic method. This investigation was performed during the winter and summer of 1997 with a set of 36 samples of near-bottom water and sediment collected from a coastal area and an offshore area in the western part of the Gulf of Lions. The existence of biphasic microbial ectoproteolysis was statistically confirmed for both the near-bottom water and the sediment, regardless of the spatial and seasonal conditions. Globally, 72.2% of the entire set of bacterial consortia collected at the water-sediment boundary layer showed biphasic microbial kinetics. A specific estimator of the biphasicity indicated that deep benthic bacterial consortia responded better with episodic nutrient supplies than shallower benthic bacterial consortia responded.

Tholosan, Olivier; Lamy, Francois; Garcin, Jean; Polychronaki, Thalia; Bianchi, Armand

1999-01-01

260

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

PubMed

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

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

2013-08-06

261

Characterization of Urease-Positive Thermophilic Campylobacter Subspecies by Multilocus Enzyme Electrophoresis Typing  

PubMed Central

Thirty-one urease-positive thermophilic Campylobacter (UPTC) isolates, including three reference strains (NCTC12892, NCTC12895 and NCTC12896), and three Campylobacter lari isolates, which were isolated from several countries and sources, were compared genotypically by using multilocus enzyme electrophoresis (MLEE). We examined allelic variation around seven enzyme loci, including the adenylate kinase, alkaline phosphatase, catalase, fumarase, malic enzyme, malate dehydrogenase, and l-phenylalanyl-l-leucine peptidase loci. MLEE typing revealed the presence of 23 different electrophoretic types (ETs) among the 31 UPTC isolates, and 14 isolates shared six electrophoretic profiles. Three different ETs were identified for the three C. lari isolates examined, and no ETs were shared by UPTC and C. lari isolates. Quantitative analyses were subsequently performed by using allelic variation data, and the results demonstrated that the mean genetic diversity was 0.655. In conclusion, MLEE demonstrated that the UPTC isolates examined are genetically hypervariable and form a cluster separate from the C. lari cluster.

Matsuda, Motoo; Kaneko, Aki; Stanley, Timothy; Millar, B. Cherie; Miyajima, M.; Murphy, Philip G.; Moore, John E.

2003-01-01

262

Studies on the soluble and membrane-bound amino acid 2-naphthylamidases in pig and human epidermis.  

PubMed Central

1. Membrane-bound (particulate) and soluble amino acid 2-naphthylamidases (EC 3.5.1.-) were present in subcellular fractions of epidermis from pig and human. 2. The particulate enzymes exhibited Michaelis-Menten kinetics, with Km 5.1x10(-5) (pig) and Km 7.3x10(-5)M (human) for the substrate L-leucine 2-naphthylamide. They were inhibited by puromycin and partially inhibited by EDTA. They did not require heavy metals and were not inhibited by thiol-group-blocking agents. Their pH optima were 7.0 (human) and 6.6 (pig). The particulate enzyme from pig epidermis retained 50% activity after 30 min at 70 degrees C. 3. The soluble amino acid 2-naphthylamidases gave sigmoidal curves for reaction velocity versus substrate concentration, and the kinetic data suggested that there was positive co-operativity between binding sites. This co-operativity was lost after treatment with 0.1mM-p-hydroxymercuribenzoate and the enzymes showed first-order kinetics at low substrate concentrations. The soluble enzymes were inhibited by puromycin and by thiol-group-blocking agents and activated by dithiothreitol. They were inactivated above 60 degrees C and lost activity on storage, but this could be restored with dithiothreitol. 4. The amino acid 2-naphthylamidases of human epidermis were much more active (2.5 times) towards L-alanine 2-naphthylamide than towards the commonly used substrate L-leucine 2-naphthylamide. 5. The kinetics of both the solube and particulate enzymes from epidermis of some elderly patients with either diabetes or ischaemia showed some differences from the kinetics of enzymes from healthy epidermis from younger individuals.

Gray, G M; Tabiowo, A; Trotter, M D

1977-01-01

263

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

SciTech Connect

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

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

1986-03-05

264

Topographic organization of the olivocerebellar projection in normal and mutant mice  

SciTech Connect

An intriguing question in development neurobiology is whether target cell position and number are important in determining the normal organization and distribution of an afferent projection to that target. The olivocerebellar projection provides an ideal system for such study since it possesses a high degree of specificity. Groups of cells in the inferior olivary complex (IO) provide climbing fiber (CF) afferents to Purkinje cells (PCs) in particular parasagittal zones. A further advantage in studying this projection system is the availability of a variety of neurological mutant mice with genetic defects in this primary target cell (i.e., the PC). Three mutants, reeler (rl/rl), weaver (wv/wv; wv/+), and staggerer (sg/sg), were chosen for this study because they have varying degrees of PC abnormalities including reduced numbers and ectopic PCs. To investigate this problem, three types of studies were carried out in normal and mutant mice; (1) a qualitative and quantitative light microscopic study of the IO to determine whether the IO is also abnormal in the mutants, (2) an autoradiographic study in normal mice involving single or multiple injections of /sup 3/H-L-leucine into the IO in an effort to resolve the controversial issue of whether the olivocerebellar projection terminates in a series of parasagittal bands with empty zones between labeled bands or if CFs supply PCs in all cerebellar regions, and (3) using a similar approach in the mutants (i.e., single injections of /sup 3/H-L-leucine into the IO) to determine what effects abnormal target cell position and number have on olivocerebellar organization and topography.

Blatt, G.J.

1986-01-01

265

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.

1988-04-01

266

Amino acid absorption by mouse ascites-tumour cells depleted of both endogenous amino acids and adenosine triphosphate  

PubMed Central

1. Despite the depletion of both their content of exchangeable endogenous amino acids and reserves of ATP, starved hypo-osmotically shocked preparations of the tumour cells accumulated relatively large amounts of 14C-labelled 2-aminoisobutyrate, l-alanine, glycine, l-leucine, l-methionine, l-phenylalanine and l-serine, against their respective concentration gradients, by a process apparently driven by the spontaneous flow of Na+ ions into the cellular phase. Dependent on (a) which compound was used, (b) its concentration and (c) the direction of the Na+ ion gradient, the peak value of the ratio of the cellular to extracellular amino acid concentration varied from about 0.4 to 7. 2. The extent to which ATP increased the ratio was defined for l-methionine. 3. Chemical analysis of the cellular amino acid content showed that this increased in parallel with the absorption of 14C. 4. The accumulation of l-methionine and of glycine, against their own concentration gradients, continued in the presence of either 0.3mm-ouabain or 10?g of oligomycin/ml. Thus the sodium pump was probably not involved in the process when ATP was lacking. 5. l-Leucine caused 0.72±0.12 (s.e.m.; 6) extra equivalents of Na+ to enter the shocked starved tumour cells in parallel with the uptake of leucine itself. Only a small loss of K+ was induced. 6. The influx and efflux of l-methionine in preparations depleted of ATP were both markedly accelerated by the presence of Na+ ions. 7. The observations provide further examples of the application of the ion-gradient hypothesis, according to which Na+ ions act as co-substrates of the amino acid pump. The quantitative importance of parallel Na+-independent systems was studied with a new mathematical model.

Morville, M.; Reid, M.; Eddy, A. A.

1973-01-01

267

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))

1990-03-01

268

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

PubMed

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

2013-08-01

269

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

PubMed Central

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

Rizvi, Syed Asad Ali; Shamsi, Shahab A.

2008-01-01

270

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

NASA Astrophysics Data System (ADS)

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

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

2007-10-01

271

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

PubMed

Identification of molecular mechanisms underlying early stage Alzheimer's disease (AD) is important for the development of new therapies against and diagnosis of AD. In this study, nontargeted metabonomics of TASTPM transgenic AD mice was performed. The metabolic profiles of both brain and plasma of TASTPM mice were characterized using gas chromatography-mass spectrometry and compared to those of wild-type C57BL/6J mice. TASTPM mice were metabolically distinct compared to wild-type mice (Q2Y=0.587 and 0.766 for PLS-DA models derived from brain and plasma, respectively). A number of metabolites were found to be perturbed in TASTPM mice in both brain (D-fructose, L-valine, L-serine, L-threonine, zymosterol) and plasma (D-glucose, D-galactose, linoleic acid, arachidonic acid, palmitic acid and D-gluconic acid). In addition, enzyme immunoassay confirmed that selected endogenous steroids were significantly perturbed in brain (androstenedione and 17-OH-progesterone) and plasma (cortisol and testosterone) of TASTPM mice. Ingenuity pathway analysis revealed that perturbations related to amino acid metabolism (brain), steroid biosynthesis (brain), linoleic acid metabolism (plasma) and energy metabolism (plasma) accounted for the differentiation of TASTPM and wild-type mice. Our results provided insights on the pathogenesis of APP-induced AD and reinforced the role of TASTPM in drug and biomarker development. PMID:23078235

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

2012-10-29

272

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

PubMed

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

2013-06-13

273

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

NASA Astrophysics Data System (ADS)

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

Chan, Jerry C. C.; Tycko, Robert

2003-05-01

274

Genetic basis of destruxin production in the entomopathogen Metarhizium robertsii.  

PubMed

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

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

2012-02-25

275

Asymmetric aerobic oxidation of alpha-hydroxy acid derivatives by C4-symmetric, vanadate-centered, tetrakisvanadyl(V) clusters derived from N-salicylidene-alpha-aminocarboxylates.  

PubMed

A series of chiral vanadyl(V) methoxides bearing 3-t-butyl-5-substituted N-salicylene-L-valinate and L-t-leucinate as chiral auxiliaries has been prepared. In all cases except the 3,5-di-t-butyl analogue, they exist as monomers both in solution and in the single crystal state. In the case of the 3,5-di-t-butyl analogue, the architectural nature of the vanadyl(V) complex highly depends on the base used during the complex formation event. A pentanuclear C4-symmetric complex was formed when potassium salts were employed instead of the corresponding sodium salts. A central vanadate(V) unit serves to grip four identical chiral monomeric vanadyl(V) units together, by which a potassium ion sits on top of the four flanking units through carbonyl coordinations and serves to hold the whole cluster by cooperation with the central vanadate(V) unit. In comparison with the corresponding monomeric vanadyl(V) methoxide complex, the cluster complex was utilized to facilitate the asymmetric aerobic oxidations of various racemic alpha-hydroxyesters, -amides, and -thioesters with excellent selectivity factors (krel 40 to >500). PMID:17915919

Chen, Chien-Tien; Bettigeri, Sampada; Weng, Shiue-Shien; Pawar, Vijay D; Lin, Ya-Hui; Liu, Cheng-Yuan; Lee, Way-Zen

2007-10-04

276

Copper(ii) complexes of a polydentate imidazole-based ligand. pH effect on magnetic coupling and catecholase activity.  

PubMed

A potentially dodecadentate N8O4-donor ligand obtained from 2,2'-biimidazole and l-valine and its tetranuclear Cu(ii) complexes in different degrees of protonation were characterized by chemical and spectroscopic methods. The extensive solution studies performed reveal that the rise in pH media leads successively to the formation of imidazolato (pKa(1) and pKa(2) and hydroxido (pKa(3) and pKa(4)) bridges. A frozen solution EPR study shows a decrease in the signal intensity until an EPR silent spectrum is observed, upon increasing the basicity of the solution. The catalytic performance of the oxidation of 3,5-di-tert-butylcatechol to its corresponding quinone was studied using UV-Vis-NIR absorption spectroscopic methods in CH3CN-H2O and in CH3OH-H2O at pH = 7.5, 8.0 and 8.5. A marked increase in activity, consistent with the formation of the hydroxide bridged species, is observed at pH = 8.5 in both solvent mixtures, but the activity is significantly higher in CH3OH-H2O. PMID:18369492

Mijangos, Edgar; Reedijk, Jan; Gasque, Laura

2008-01-18

277

A design of experiments to optimize a new manufacturing process for high activity protein-containing submicron particles.  

PubMed

A novel method for the manufacture of protein/peptide-containing submicron particles was developed in an attempt to provide particles with increased activity while using high energy input technologies. The method consists of antisolvent co-precipitation from an aqueous solution containing both an amino acid core material (e.g. D,L-valine), and either bovine serum albumin (BSA) or lysozyme (Lys) as model proteins. The aqueous solution was added to the organic phase by means of a nebulizer to increase the total surface area of interaction for the precipitation process. Sonication proved to be an effective method to produce small particle sizes while maintaining high activity of Lys. The use of a polysorbate or sorbitan ester derivatives as stabilizers proved to be necessary to yield submicron particles. Particles with very high yields (approximately 100%) and very high activity after manufacture (approximately 100%) could be obtained. A particle size of 439.0?nm, with a yield of 48.8% and with final remaining activity of98.7% was obtained. By studying various factors using a design of experiments strategy (DoE) we were able to establish the critical controlling factors for this new method of manufacture. PMID:23298292

Morales, Javier O; Joks, Gero M; Lamprecht, Alf; Ross, Alistair C; McConville, Jason T

2013-01-08

278

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

PubMed

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

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

2013-01-16

279

Changes in the metabolic profile of rat liver after ?-tocopherol deficiency as revealed by metabolomics analysis.  

PubMed

Metabolomics is an approach in which the profiles of metabolites in different tissues and/or biofluids are investigated to understand the changes induced following a modulation. We used this approach to investigate the biochemical effects of ?-tocopherol in the liver using a rat model. Rats (21-day-old) were fed either an ?-tocopherol-sufficient control (n?=?10) or an ?-tocopherol-deficient (n?=?10) diet for 2 months before sacrifice. Livers were homogenized in methanol-chloroform-water (3 : 1 : 1, v/v/v), and the polar phase extracts of the liver samples were analyzed using (1) H NMR. Multivariate statistical analysis of the data was performed using principal component analysis and orthogonal partial least squares-discriminant analysis. Identification of (1) H NMR signals was performed primarily using the Human Metabolome Database, Biological Magnetic Resonance Data Bank and previous literature, and confirmed by spiking with metabolites and applying two-dimensional NMR. The statistical analysis revealed that ?-tocopherol deficiency caused an increase in carnitine, choline, L-valine, L-lysine, tyrosine and inosine content and a reduction in glucose and uridine 5'-monophosphate content. Changes in carnitine and glucose suggest a possible shift in energy metabolism. PMID:21674651

Moazzami, Ali A; Andersson, Roger; Kamal-Eldin, Afaf

2010-11-30

280

Spectroscopic studies on the influence of 10 mol% of glycine on nonlinear optical crystal L-valinium picrate.  

PubMed

Nonlinear optics is a fascinating field, which plays a vital role in the emerging field of photonics and optoelectronics. A new nonlinear optical crystal of glycine mixed L-valine picrate (GVP) have been grown from saturated aqueous solution by slow evaporation method at a temperature of 36°C using a constant temperature bath of accuracy of ±0.01°C. The synthesized organic optical material has been purified by repeated recrystallization. Single crystal X-ray diffraction analysis has been made to determine the cell parameters and it confirms the crystal lattice to be orthorhombic. UV-vis-NIR spectrum have recorded for GVP crystals in the range from 190 nm to 1100 nm and it is found that the crystal has cut-off at 450 nm. Fourier transform infrared transmission has confirmed the presence of the functional group present in the title compound. The spectrum has been recorded by KBr pellet technique. The (1)H and (13)C NMR spectra have been recorded to elucidate the molecular structure of GVP crystals. The second harmonic generation (SHG) of the grown crystal have been confirmed by Kurtz-Perry method using Nd:YAG laser as source. PMID:21821468

Antony Joseph, A; Ebenezar, I John David; Ramachandra Raja, C

2011-07-27

281

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

2012-12-07

282

Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production.  

PubMed

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. PMID:20935122

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

2010-10-08

283

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.

2010-07-01

284

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.

2010-01-01

285

Escherichia coli transport mutants lacking binding protein and other components of the branched-chain amino acid transport systems.  

PubMed Central

Further evidence on the role of binding proteins in branched-chain amino acid transport in Escherichia coli was obtained by selecting mutants with altered expression of the binding proteins. The mutator phage Mu was used to induce E. coli L-valine-resistant mutants defective in branched-chain amino acid transport. By making use of mild selective conditions and strain backgrounds with derepressed high-affinity, binding protein-mediated transport systems, we were able to isolate a new class of transport mutants defective in these systems. Mutant strains AE84084 (livK::Mucts) and AE840102 (livJ) were found to be defective in leucine-specific and LIV binding proteins, respectively, by transport assay, in vitro binding activity, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mutant strain AE4107 (livH::Mu), although lacking high-affinity, branched-chain amino acid transport, retained functional binding proteins and therefore evidently codes for an additional component of high-affinity transport. The livH, livJ, and livK mutations were mapped by transduction and shown to be closely linked to each other in the malT region (min 74) of the E. coli genetic map. Images

Anderson, J J; Oxender, D L

1977-01-01

286

Sequence, transcriptional, and functional analyses of the valine (branched-chain amino acid) dehydrogenase gene of Streptomyces coelicolor.  

PubMed Central

The gene encoding the valine (branched-chain amino acid) dehydrogenase (Vdh) from Streptomyces coelicolor has been characterized as follows. The vdh gene was identified by hybridization to a specific oligodeoxynucleotide that was synthesized on the basis of the N-terminal amino acid sequence of purified Vdh. Nucleotide sequence analysis predicts that the vdh gene contains a 364-amino-acid open reading frame that should produce a 38,305-M(r) protein. The deduced amino acid sequence of the Vdh protein is significantly similar to those of several other amino acid dehydrogenases, especially the leucine and phenylalanine dehydrogenases from Bacillus spp. The vdh gene is apparently transcribed from a single major transcriptional start point, separated by only 8 bp from the 5' end of a divergent transcript and located 63 bp upstream from the vdh translational start point. Mutants with a disrupted vdh gene have no detectable Vdh activity and have lost the ability to grow on valine, leucine, or isoleucine as the sole nitrogen source. This vdh mutation does not significantly affect growth or actinorhodin production in a minimal medium, yet the addition of 0.2% L-valine to the medium provokes approximately 32 and 80% increases in actinorhodin production in vdh+ and vdh strains, respectively. Images

Tang, L; Hutchinson, C R

1993-01-01

287

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

PubMed

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 15mM chiral ILs were introduced into the 30mM sodium citrate/citric acid (20% organic modifier included) buffer solution containing 20mM Me-?-CD at pH 5.0 with a 20kV applied voltage for naproxen, pranoprofen and warfarin. PMID:24119759

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

2013-09-25

288

Chiral separation with ligand-exchange micellar electrokinetic chromatography using a D-penicillamine-copper(II) ternary complex as chiral selector.  

PubMed

D-Penicillamine is demonstrated for the first time as a chiral ligand for the enantioseparation of dansyl amino acids based on ligand-exchange micellar electrokinetic chromatography (LE-MEKC). Copper(II) was used as the central ion in the ternary complex. The effect of surfactant on the resolution was significant. A concentration of 20 mM sodium dodecyl sulfate (SDS) was shown to be necessary for the separation. Other important parameters, such as the concentration ratio of D-penicillamine (D-PEN) to Cu2+, the kind of metal central ion, the type and pH value of buffer, were also investigated. N-Acetyl-D-penicillamine and L-valine (Val), with similar structure to D-penicillamine, were applied as their copper(II) complexes as chiral selector and the chiral recognition mechanism is briefly discussed. Under optimum experimental conditions, i.e., 20 mM NH4OAc, pH 6.5, a 2:1 concentration ratio of D-penicillamine to Cu(II), 4 mM CuSO4 and 8 mM D-penicillamine, the chiral separation of eight pairs of different dansyl amino acid enantiomers was accomplished with resolution ranging from 1.1 to 5.9. When L-PEN was used instead of D-PEN, reversal of the migration order was observed. PMID:14679569

Zheng, Zhi-Xia; Lin, Jin-Ming; Qu, Feng; Hobo, Toshiyuki

2003-12-01

289

L-tyrosine fails to potentiate several peripheral actions of the sympathomimetics.  

PubMed

We have recently reported the ability of L-tyrosine (L-TYR) to potentiate the anorectic activity of various mixed-acting sympathomimetics including phenylpropanolamine (PPA), l-ephedrine (EPH) and d-amphetamine (AMP). Included in those studies was the attenuation of L-TYR's effect when coadministered with L-valine, a large neutral amino acid which competes with L-TYR for uptake into the brain, suggesting a central locus for the action of L-TYR. To determine to what extent L-TYR can potentiate peripheral actions, we investigated the effects of L-TYR with either PPA (20 mg/kg), EPH (20 mg/kg) or AMP (1.75 mg/kg) on gastric transit, gastric retention and intrascapular brown adipose tissue thermogenesis. In each of the paradigms studied, PPA, EPH and AMP significantly increased the expected sympathomimetic-mediated response, but no potentiation of L-TYR was observed. These results are consistent with the hypothesis that L-TYR potentiates the anorectic activity of the mixed-acting sympathomimetics largely via an action at a central locus. PMID:1784604

Hull, K M; Maher, T J

1991-07-01

290

Effects of L-tyrosine on mixed-acting sympathomimetic-induced pressor actions.  

PubMed

We previously reported the ability of L-tyrosine (L-TYR) to potentiate the anorectic activity of various mixed-acting sympathomimetics including [R*S*]-(+/-)-norephedrine [phenylpropanolamine (PPA)], [1R,2S]-(-)-ephedrine (EPH), and [S]-(+)-amphetamine (AMPH) in hyperphagic rats. Included in those studies was the attenuation of L-TYR's effect when coadministered with L-valine, a large neutral amino acid that competes with L-TYR for uptake into the brain, suggesting a central locus for the action of L-TYR. Additional studies demonstrated the inability of L-TYR to alter the peripherally mediated PPA-, EPH-, and AMPH-induced increases in gastrointestinal transit time and retention and intrascapular brown adipose tissue thermogenesis. Because the mixed-acting sympathomimetics are known to increase blood pressure, these studies examined the ability of L-TYR to influence the pressor responses to PPA, EPH, and AMPH (0.03-1 mg/kg) in urethane-anesthetized rats. Each of the mixed-acting sympathomimetics significantly increased mean arterial, systolic, and diastolic blood pressures when administered alone, but no potentiation by L-TYR was observed. These results demonstrate the inability of L-TYR to potentiate the peripheral vasopressor effects of PPA, EPH, and AMPH. These data, in conjunction with our previous findings, suggest that the potentiation by L-TYR of the mixed-acting sympathomimetics is largely restricted to centrally mediated responses. PMID:1475286

Hull, K M; Maher, T J

1992-12-01

291

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

PubMed

The effects of L-tyrosine (L-TYR) on the anorectic activity of several mixed-acting sympathomimetics were determined during the dark cycle in rats made hyperphagic by food deprivation. L-TYR (200 mg/kg) significantly potentiated the anorectic activity of phenylpropanolamine, (-)-ephedrine and (+)-amphetamine by 48, 50 and 37%, respectively. When the dose of L-TYR was varied (25-400 mg/kg), a significant dose-dependent relationship was noted. The observed potentiation was positively correlated with increases in brain TYR concentrations; blockade of L-TYR uptake into the brain by the coadministration of L-valine prevented this potentiation. Various other L-amino acids, as well as D-TYR, failed to mimic the potentiating action of L-TYR. As determined by alpha-methyl-p-TYR pretreatment, the L-TYR-induced potentiation was dependent upon increased catecholamine synthesis. Although various other mixed-acting sympathomimetic anorexiants were similarly potentiated by L-TYR, the direct-acting beta-2 adrenoceptor anorexiants, salbutamol and methoxyphenamine, were not. These results indicate that L-TYR specifically potentiates the anorectic activity of the studied mixed-acting sympathomimetics and are consistent with the requirement of the central conversion of L-TYR to catecholamines via TYR hydroxylase for this response. The possibility that the effect of mixed-acting sympathomimetics is normally limited by the availability of L-TYR is suggested. PMID:2243331

Hull, K M; Maher, T J

1990-11-01

292

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

SciTech Connect

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

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

1982-01-01

293

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

SciTech Connect

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

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

1982-01-01

294

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

PubMed Central

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

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

2009-01-01

295

Emerged quaternary marine terraces in southern Peru: Sea level changes and continental margin tectonics over the subducting Nazca Ridge  

SciTech Connect

Because of the arid climate, mollusk shells in the marine cover strata are exceptionally well preserved and provide datable samples for a terrace and sea level chronology. Amino acid racemization was the most extensively used dating method, with three other numerical dating methods, electron spin resonance (ESR), Uranium-series, and radiocarbon ages used to calibrate the amino acid ages. D-alloisoleucine/L-isoleucine (A/I) ratios of >200 mollusks shells belong to six statistically and geomorphically defined aminozones. Aminozone IIa correlates with the last Interglacial (deep-sea oxygen isotope Stage 5e) based on 12 ESR ages and the similarity of the IIa ratios to those determined from calibrated last Interglacial sites on the Pacific Coast, U.S. Calibrated amino acid ages for the other aminozones are; I = >45,000 yr B.P. and {le} 87,000; IIb = 211,000 yr B.P.; III = 297,000 yr B.P.; IV = 496,000 yr B.P.; and V {ge} 945,000 yr B.P. The calibrated ages agree with ESR, Uranium series, and radioactive ages. The pattern of terrace deformation can be approximated by a simple geometric model in which the subconducting Nazca Ridge migrates obliquely southeastward as a rigid trapezoid beneath the forearc. The model correctly predicts a pattern of marine terrace deformation with the highest elevations located above the southern flank of the Nazca Ridge and subsidence above the northern flank. The topography, pattern of marine terrace deformation, and Quaternary rates of uplift of the Paracas Block are determined by oblique subduction of the aseismic Nazca Ridge.

Hsu, J.T.J.

1988-01-01

296

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

PubMed

The study describes a new "one-pot" route to the synthesis of N-9-fluorenylmethyloxycarbonyl (Fmoc) ?-amino diazoketones. The procedure was tested on a series of commercially available free or side-chain protected ?-amino acids employed as precursors. The conversion into the title compounds was achieved by masking and activating the ?-amino acids with a single reagent, namely, 9-fluorenylmethyl chloroformate (Fmoc-Cl). The resulting N-protected mixed anhydrides were reacted with diazomethane to lead to the ?-amino diazoketones, which were isolated by flash column chromatography in very good to excellent overall yields. The versatility of the procedure was verified on lipophilic ?-amino acids and further demonstrated by the preparation of N-Fmoc-?-amino diazoketones also from ?-amino acids containing side-chain masking groups, which are orthogonal to the Fmoc one. The results confirmed that tert-butyloxycarbonyl (Boc), tert-butyl ((t)Bu), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), three acid-labile protecting groups mostly adopted in the solution and solid-phase peptide synthesis, are compatible to the adopted reaction conditions. In all cases, the formation of the corresponding C-methyl ester of the starting amino acid was not observed. Moreover, the proposed method respects the chirality of the starting ?-amino acids. No racemization occurred when the procedure was applied to the synthesis of the respective N-Fmoc-protected ?-amino diazoketones from L-isoleucine and L-threonine and to the preparation of a diastereomeric pair of N-Fmoc-protected dipeptidyl diazoketones. PMID:23146162

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

2012-11-16

297

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

PubMed

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

2013-03-22

298

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

PubMed

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

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

2010-12-01

299

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

PubMed Central

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

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

2010-01-01

300

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

PubMed

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

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

2011-11-15

301

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

USGS Publications Warehouse

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

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

1989-01-01

302

Human Lung Angiotensin Converting Enzyme  

PubMed Central

To enable its immunohistologic localization, angiotensin converting enzyme (EC 3.4.15.1) from human lung was solubilized by trypsinization and purified ?2,660-fold to apparent homogeneity from a washed lung particulate fraction. The specific activity of pure enzyme was estimated to be 117 ?mol/min per mg protein with the substrate hippuryl-l-histidyl-l-leucine. Consistent with previously described lung enzyme studies, catalytic activity was strongly inhibited by EDTA, O-phenanthroline, SQ 20,881, and SQ 14,225 and increased by CoCl2. SQ 20,881 was a somewhat more potent inhibitor than SQ 14,225, unlike rabbit lung enzyme. The Michaelis constant (Km) with hippuryl-l-histidyl-l-leucine was 1.6 mM. The molecular weight was estimated at 150,000 from sucrose density gradient centrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single polypeptide chain estimated at 130,000 daltons. Rabbit antibody to human lung enzyme was prepared by parenteral administration of pure angiotensin-converting enzyme in Freund's adjuvant. Rabbit antibody to human lung angiotensin-converting enzyme appeared to crossreact weakly with the rabbit enzyme and strongly inhibited the catalytic activity of the enzymes from human serum, lung, and lymph node. The specificity of the rabbit antibody and purity of the final human lung enzyme preparation was suggested by the single precipitin lines obtained by radial double immunodiffusion, and by the coincidence of enzyme catalytic activity and immunoreactivity on polyacrylamide gel electrophoresis, with both relatively pure and highly impure enzymes. Generally applicable sensitive analysis of acrylamide gels for immunoreactivity (and subsequently for any other activity) by use of intact gel slices in radial double immunodiffusion was devised. Human lung enzyme was very tightly bound to and catalytically active on anti-human enzyme antibody covalently bound to Sepharose 4B, and could not be readily dissociated without inactivation. Antibody to human lung angiotensin converting enzyme has permitted tissue localization of the enzyme, which appears to be clinically useful in diseases associated with abnormal abundance of angiotensin-converting enzyme in tissues, such as sarcoidosis. Images

Friedland, Joan; Silverstein, Emanuel; Drooker, Martin; Setton, Charlotte

1981-01-01

303

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

PubMed

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

2008-12-11

304

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

SciTech Connect

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

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

1983-10-01

305

Synthesis, chemical and enzymatic hydrolysis, and aqueous solubility of amino acid ester prodrugs of 3-carboranyl thymidine analogs for boron neutron capture therapy of brain tumors.  

PubMed

Various water-soluble L-valine-, L-glutamate-, and glycine ester prodrugs of two 3-Carboranyl Thymidine Analogs (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 48-6600 times compared with parental N5 and N5-2OH. The stability of the amino acid ester prodrugs was evaluated in PBS at pH 7.4, Bovine serum, and Bovine cerebrospinal fluid (CSF). The rate of the hydrolysis in all three incubation media depended primarily on the amino acid promoiety and, to a lesser extend, on the site of esterification at the deoxyribose portion of the 3-CTAs. In general, 3'-amino acid ester prodrugs were less sensitive to chemical and enzymatic hydrolysis than 5'-amino acid ester prodrugs and the stabilities of the latter decreased in the following order: 5'-valine > 5'-glutamate > 5'-glycine. The rate of the hydrolysis of the 5'-amino acid ester prodrugs in Bovine CSF was overall higher than in PBS and somewhat lower than in Bovine serum. Overall, 5'-glutamate ester prodrug of N5 and the 5'-glycine ester prodrugs of N5 and N5-2OH appeared to be the most promising candidates for preclinical BNCT studies. PMID:22889558

Hasabelnaby, Sherifa; Goudah, Ayman; Agarwal, Hitesh K; abd Alla, Mosaad S M; Tjarks, Werner

2012-07-27

306

The bacterial biogenesis of isobutyraldoxime O-methyl ether, a novel volatile secondary metabolite.  

PubMed

Production of the volatile metabolite, isobutyraldoxime O-methyl ether (IBME) by a Moraxella-like organism NCIB 11650 was investigated under a variety of environmental conditions using gas chromatography. Under aerobic conditions up to 10 micrograms IBME ml-1 was produced on mineral salts media containing 0.5% (w/v) glucose or succinate as sole C source with 0.1% (w/v) NH4Cl as sole N source. Exogenous L-valine further stimulated IBME formation up to 25 micrograms ml-1 but supplementation of the medium with D-isomer or other amino acids had little effect on IBME production and did not lead to the appearance of analogues of IBME. Trapping experiments using [14C]valine confirmed that IBME was derived from this amino acid. Several other bacterial species examined, e.g. Alcaligenes sp. NCIB 11652, another Moraxella-like organism NCIB 11651 and Pseudomonas sp. NCIB 11653 also produced IBME under similar conditions. The Alcaligenes strain synthesized up to 20 micrograms ml-1 in the absence of valine and up to 90 micrograms ml-1 in its presence. The product of IBME exhibited many features characteristic of the formation of a secondary metabolite. Thus biosynthesis was confined to a narrower range of temperature than cell division, was almost completely suppressed by 300 mM-phosphate and was inhibited by high concentrations of readily utilizable C sources. Although IBME synthesis in the Moraxella-like organism NCIB 11650 appeared to be growth-related, its formation by both the Alcaligenes sp. and the Moraxella-like organism NCIB 11651 was delayed until the late-exponential and early-stationary phases of growth. The biological significance of this novel class of secondary metabolite is discussed and a possible biosynthetic route proposed. PMID:7142955

Harper, D B; Nelson, J

1982-08-01

307

Constant time tensor correlation experiments by non-gamma-encoded recoupling pulse sequences  

NASA Astrophysics Data System (ADS)

Constant-time tensor correlation under magic-angle spinning conditions is an important technique in solid-state nuclear magnetic resonance spectroscopy for the measurements of backbone or side-chain torsion angles of polypeptides and proteins. We introduce a general method for the design of constant-time tensor correlation experiments under magic-angle spinning. Our method requires that the amplitude of the average Hamiltonian must depend on all the three Euler angles bringing the principal axis system to the rotor-fixed frame, which is commonly referred to as non-gamma encoding. We abbreviate this novel approach as COrrelation of Non-Gamma-Encoded Experiment (CONGEE), which exploits the orientation-dependence of non-gamma-encoded sequences with respect to the magic-angle rotation axis. By manipulating the relative orientation of the average Hamiltonians created by two non-gamma-encoded sequences, one can obtain a modulation of the detected signal, from which the structural information can be extracted when the tensor orientations relative to the molecular frame are known. CONGEE has a prominent feature that the number of rf pulses and the total pulse sequence duration can be maintained to be constant so that for torsion angle determination the effects of systematic errors owing to the experimental imperfections and/or T2 effects could be minimized. As a proof of concept, we illustrate the utility of CONGEE in the correlation between the C' chemical shift tensor and the C?-H? dipolar tensor for the backbone psi angle determination. In addition to a detailed theoretical analysis, numerical simulations and experiments measured for [U-13C, 15N]-L-alanine and N-acetyl-[U-13C, 15N]-D,L-valine are used to validate our approach at a spinning frequency of 20 kHz.

Mou, Yun; Tsai, Tim W. T.; Chan, Jerry C. C.

2012-10-01

308

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Degradation by Alkaline Phosphatase*  

PubMed Central

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous second messenger providing a Ca2+ trigger in a wide range of cell types. However, its metabolism is not well understood. Here, we demonstrate the presence of endogenous NAADP in HeLa cells. CD38, a promiscuous enzyme described to be involved in NAADP metabolism, was not detectable in HeLa cells. In cell-free extracts of HeLa cells, NAADP was degraded to nicotinic acid adenine dinucleotide (NAAD). The enzyme was enriched in membranes (10,000 × g pellet) and displayed characteristics typical of alkaline phosphatase (AP), e.g. pH optimum at 8–9 and sensitivity to the inhibitors l-homoarginine and l-leucine. Importantly, NAADP at physiological concentrations (50–100 nm) was degraded to NAAD. Expression of AP isoenzymes was analyzed in HeLa cells. Based on the results together with inhibitor studies, the placental AP isoform emerged as the best candidate for NAADP degradation in HeLa cells. In contrast to HeLa cells, Jurkat T cells or HEK293 cells did not express any AP isoenzymes and did not display any NAADP 2?-phosphatase activity. Finally, the placental AP isoform was expressed heterologously in HEK293 cells, resulting in reconstitution of NAADP 2?-phosphatase activity in cell-free extracts. On the basis of the results, we provide evidence for AP as the metabolizing enzyme of NAADP in cells that do not express CD38.

Schmid, Frederike; Fliegert, Ralf; Westphal, Tim; Bauche, Andreas; Guse, Andreas H.

2012-01-01

309

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.

2012-01-01

310

Multivariate approach for the enantioselective analysis in MEKC-MS: II. Optimization of 1,1?-binaphthyl-2,2?-diamine in positive ion mode  

PubMed Central

Enantiomeric separation and detection of 1,1?-binaphthyl-2,2?-diamine (BNA) has been successfully optimized by micellar electrokinetic chromatography coupled to electrospray ionization mass spectrometry (MEKC-ESI-MS) using a polymeric surfactant polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) as a pseudostationary phase. In the first step, MEKC conditions were optimized by a five-factor three-level central composite design (CCD) of experiment. All five MEKC factors (buffer pH, percentage of acetonitrile in the running buffer, concentration of surfactant, concentration of NH4OAc, and voltage) were found significant to the responses (measured as the chiral resolution and analysis time). The interactions between MEKC factors were further evaluated using a quadratic model equation which allowed the generation of 3-D response surface image to reach the optimum conditions. To obtain the best signal to noise (S/N) ratio, sheath liquid composition and spray chamber parameters were successfully optimized using the same strategy. Baseline enantiomeric resolution in less than 20 min and optimum mass spectrometry signal of BNA enantiomers (S/N = 45 at 0.4 mg/mL) were ultimately achieved at the optimized conditions. The adequacy of the model was validated by experimental runs at the optimal predicted conditions. The predicted results were found to be in good agreement with the experimental data.

He, Jun; Shamsi, Shahab A.

2009-01-01

311

BETAview: a digital /?-imaging system for dynamic studies of biological phenomena  

NASA Astrophysics Data System (ADS)

We present a digital autoradiography (DAR) system, named BETAview, based on semiconductor pixel detectors and a single particle counting chip, for quantitative analysis of /?-emitting radioactive tracers in biological samples. The system is able to perform a real time monitoring of time-dependent biological phenomena. BETAview could be equipped either with GaAs or with Si semiconductor pixellated detectors. In this paper, we describe the results obtained with an assembly based on a Si detector, 300?m thick, segmented into 64×64 170?m size square pixels. The detector is bump-bonded to the low threshold, single particle counting chip named Medipix1, developed by a CERN-based European collaboration. The sensitive area is about 1cm2. Studies of background noise and detection efficiency have been performed. Moreover, time-resolved cellular uptake studies with radiolabelled molecules have been monitored. Specifically, we have followed in vivo and in real time, the [14C]l-leucine amino acid uptake by eggs of Octopus vulgaris confirming the preliminary results of a previous paper. This opens the field of biomolecular kynetic studies with this new class of semiconductor DAR systems, whose evolution (using the Medipix2 chip, 256×256pixels, 55?m pixel size) is soon to come.

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

2002-02-01

312

Differentiation of microcystin, nodularin, and their component amino acids by drop-coating deposition Raman spectroscopy.  

PubMed

Raman spectra of microcystin-LR (MC-LR), MC-RR, MC-LA, MC-LF, MC-LY, MC-LW, MC-YR, and nodularin collected by drop-coating deposition Raman (DCDR) spectroscopy are sufficiently unique for variant identification. Amino acid spectra of L-phenylalanine, L-leucine, L-alanine, D-alanine, L-glutamic acid, L-arginine, L-tryptophan, L-tyrosine, and N-methyl-D-aspartic acid were collected in crystalline, DCDR, and aqueous forms to aid in cyanotoxin Raman peak assignments. Both peak ratio analysis and principal component analysis (PCA) properly classified 72 DCDR spectra belonging to the eight toxins. Loading plots for the first three principal components (PCs) most heavily weighted the peaks highlighted in the peak ratio analysis, specifically the 760 cm(-1) tryptophan peak, 853 cm(-1) tyrosine peak, and 1006 cm(-1) phenylalanine peak. Peak ratio analyses may be preferred under some circumstances because of the ease and speed with which the ratios can be computed, even by untrained lab technicians. A set of rules was created to mathematically classify toxins using the peak ratios. DCDR methods hold great potential for future application in routine monitoring because portable and hand-held Raman spectrometers are commercially available, DCDR spectra can be collected in seconds for biomolecule mixtures as well as samples containing impurities, and the method requires far fewer consumables than conventional cyanotoxin detection methods. PMID:22035262

Halvorson, Rebecca A; Leng, Weinan; Vikesland, Peter J

2011-11-23

313

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization.  

PubMed

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin. PMID:16169746

Inouye, Kuniyo; Minoda, Masashi; Takita, Teisuke; Sakurama, Haruko; Hashida, Yasuhiko; Kusano, Masayuki; Yasukawa, Kiyoshi

2005-08-24

314

Enzymatic synthesis in biphasic aqueous-organic systems. II. Shift of ionic equilibria.  

PubMed

Ionic equilibria in "water-water-immiscible organic solvent" systems have been studied. It has been shown that in such systems shift of the apparent pK value of acids and bases takes place (compared to aqueous solutions), the value of the shift being rather high, up to 5 and more pH units (with 2,4-dinitrophenyltryptophan as an acid and neutral red as a base). The pK shift of ionogenic reagents observed in biphasic systems can be used in preparative organic synthesis for increasing the yield of end products in enzyme-catalyzed reactions. In connection with this, the physico-chemical reasons for the equilibrium shift in a chemical reaction that involves one or two ionogenic reagents are theoretically analyzed. The above approach has been tested with two alpha-chymotrypsin-catalyzed reactions, i.e., synthesis of N-benzoyl-L-phenylalanine ethyl ester (from NBz-LPhe-OH and ethanol) and synthesis of N-acetyl-L-tryptophanyl-L-leucine amide (from NAc-LTrp-OH and LLeu-NH2). In water the equilibria in these reactions are shifted almost entirely towards the starting reagents with the yield of end product being negligibly low. In biphasic systems consisting of chloroform +5% (v/v) water or ethyl acetate +2% (v/v) water, the yield of both the ester and the dipeptide reaches 100%. PMID:7213763

Martinek, K; Semenov, A N

1981-03-13

315

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.

1994-01-01

316

Effects of mutations of thermolysin, asn116 to asp and asp150 to glu, on salt-induced activation and stabilization.  

PubMed

Neutral salts activate and stabilize thermolysin. We previously found that two single mutations, Asn116?Asp and Asp150?Glu, increase the activity of thermolysin. In the present study, we examined their effects on NaCl-induced activation and stabilization. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, the relative activities (the ratios of the specificity constant, kcat/Km, at x M NaCl to that at 0 M NaCl) at 0.5-4.0 M NaCl of D150E and N116D/D150E were lower than those of wild-type thermolysin (WT) and N116D, respectively. In thermal inactivation at 70 °C, the relative stabilities (the ratios of the first-order rate constant, kobs, at 0 M NaCl to that at x M NaCl) at 0.5-4.0 M NaCl of D150E and N116D/D150E were lower than those of WT and N116D, respectively. These results indicate that unlike Asn116?Asp, Asp150?Glu reduced NaCl-induced activation and stabilization, suggesting that the binding of ions with certain residues of thermolysin is involved in the activation and stabilization. PMID:23563542

Menach, Evans; Yasukawa, Kiyoshi; Inouye, Kuniyo

2013-04-07

317

Microscopical examination of the localisation patterns of two novel rhodamine derivatives in normal and neoplastic colonic mucosa.  

PubMed

Tissue characterisation by fluorescence imaging, using exogenous fluorophores, is a promising method for cancer detection. Histochemical alterations in the composition of mucins, when neoplastic transformations occur, could be exploited to derive more selective fluoroprobes indicative of early malignant transformation. The aim of this work was to develop and examine tumour selective fluoroprobes for colon cancer diagnosis, as well as to determine the morphological components where selective dye accumulation has occurred. Two novel fluoroprobes: rhodamine B-L-leucine amide and rhodamine B-phenylboronic acid were synthesised and examined together with Mayer's mucicarmine, alexa 350-wheat germ agglutinin (WGA) and tetramethyl rhodamine-concanavalin A (ConA). Fluorescence microscopy studies were performed with deparaffinised human colon sections, using an epifluorescence microscope equipped with a colour CCD camera. The intense accumulation of the novel fluoroprobes was localised in the amorphous material in the lumen of neoplastic crypts. To gain insight into the localisation patterns, mucicarmine, alexa 350-WGA and tetramethyl rhodamine-ConA were used. Alexa 350-WGA reacted primarily with mucin secreted in the malignant crypt lumen suggesting that this material is rich in sialic acid and N-acetylglucosaminyl residues. These derivatives clearly and consistently distinguished non-neoplastic from neoplastic human colon tissue sections. The intense accumulation at the altered mucins indicates that they could be used as fluoroprobes of biochemical alterations for carcinoma detection. PMID:11702630

Atlamazoglou, V; Yova, D; Kavantzas, N; Loukas, S

2001-01-01

318

Effect of amino acid residues at the cleavable site of substrates on the remarkable activation of thermolysin by salts.  

PubMed

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced in the presence of high concentrations (1-5 M) of neutral salts [Inouye (1992) J. Biochem. (Tokyo) 112, 335-340]. In this study, the effect of salts on such activity has been examined using a series of substrates, furylacryloyl dipeptide amides, which have various hydrophobic amino acids at the cleavable bond. Although the enzyme activity varies widely depending on the substrate employed, the degree of activation at a given concentration of NaCl is considerably similar. This indicates that the degree of activation is not dependent on the hydrophobicity of the amino acid side chains at the scissile bond of the substrates. The molecular activity, kcat, and Michaelis constant, Km, were evaluated separately for substrates N[3-(2-furyl)acryloyl]-L-leucyl-L-alanine amide and N-[3-(2-furyl)acryloyl]L-phenyl-alanyl-L-alanine amide, and the activation was found to be brought about only by an increase in k(cat'). The effectiveness of monovalent cations on the increase of k(cat) was determined to follow the order of Na(+)>K(+)>Li(+). PMID:8670097

Inouye, K; Lee, S B; Tonomura, B

1996-04-01

319

Peptidase-deficient mutants of Escherichia coli.  

PubMed

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides. PMID:355237

Miller, C G; Schwartz, G

1978-08-01

320

Kinetic analysis of the activation-and-inhibition dual effects of cobalt ion on thermolysin activity.  

PubMed

Thermolysin activity as well as its stability is remarkably enhanced by high concentration of neutral salts consisting of Na(+), K(+), Cl(-) and Br(-) in the synthesis and hydrolysis of N-carbobenzoxy-L-aspertyl-L-phenylalanine methyl ester and hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) [Inouye, K. (1992) J. Biochem. 112, 335-340]. However, effect of divalent salts on thermolysin activity has not been investigated systematically. In this study, effect of Co(2+) ion on thermolysin activity in the hydrolysis of FAGLA was examined. Thermolysin activity increased 3-4 times with increasing the Co(2+) concentration to 2 mM, but the enhanced activity was considerably reduced with higher Co(2+) concentration (2-18 mM). The activation-and-inhibition dual effects of Co(2+) ion were analysed kinetically. Release of the catalytic Zn(2+) ion from thermolysin, concomitantly occurred with the Co(2+)-dependent activation, was measured with a Zn(2+)-specific fluorescent probe. This indicates that the activation is caused by substituting Co(2+) ion for the catalytic Zn(2+) ion. Meanwhile, the Co(2+)-dependent activation was inhibited competitively by Zn(2+) ion (0.1-1.0 muM) added, similarly to that it is inhibited by higher concentration of Co(2+) ion. These lines of evidence provide a strategy for regulating thermolysin activity with Co(2+) and Zn(2+) ions. PMID:17405798

Hashida, Yasuhiko; Inouye, Kuniyo

2007-04-03

321

Peptidase-deficient mutants of Escherichia coli.  

PubMed Central

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides. Images

Miller, C G; Schwartz, G

1978-01-01

322

Cloning, sequencing, expression, and characterization of protealysin, a novel neutral proteinase from Serratia proteamaculans representing a new group of thermolysin-like proteases with short N-terminal region of precursor.  

PubMed

The gene of Serratia proteamaculans proteinase, protealysin, was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a precursor of 341 amino acids (AAs) with a significant homology to thermolysin-like proteinases (TLPs). The molecular weight of the purified mature active recombinant protein was 32 kDa, the N-terminal amino acid sequence was AKTSTGGEVI. The optimum pH for azocasein hydrolysis by protealysin was seven and it was completely inhibited by o-phenanthroline. The enzyme hydrolyzed 3-(2-furyl)acryloyl-glycyl-L-leucine amide, the standard substrate for TLPs, with k(cat)/K(m) ratio of (2.52 +/- 0.02) x 10(2) M(-1) s(-1). Protealysin maturation removes 50 AA from the N-terminus of the precursor. The removed region had no similarity with the preprosequence of thermolysin (232 AA) but was homologous to some other TLPs. These proteins shared a conserved 7-AA motif near the initial methionine. Such motif was also found in some nonproteolytic putative proteins; two of them were eukaryotic. PMID:16442309

Demidyuk, Ilya V; Kalashnikov, Alexander E; Gromova, Tatiana Yu; Gasanov, Eugene V; Safina, Dina R; Zabolotskaya, Maria V; Rudenskaya, Galina N; Kostrov, Sergey V

2006-01-05

323

Effect of amino acid residues at the cleavable site of substrates on the remarkable activation of thermolysin by salts.  

PubMed Central

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced in the presence of high concentrations (1-5 M) of neutral salts [Inouye (1992) J. Biochem. (Tokyo) 112, 335-340]. In this study, the effect of salts on such activity has been examined using a series of substrates, furylacryloyl dipeptide amides, which have various hydrophobic amino acids at the cleavable bond. Although the enzyme activity varies widely depending on the substrate employed, the degree of activation at a given concentration of NaCl is considerably similar. This indicates that the degree of activation is not dependent on the hydrophobicity of the amino acid side chains at the scissile bond of the substrates. The molecular activity, kcat, and Michaelis constant, Km, were evaluated separately for substrates N[3-(2-furyl)acryloyl]-L-leucyl-L-alanine amide and N-[3-(2-furyl)acryloyl]L-phenyl-alanyl-L-alanine amide, and the activation was found to be brought about only by an increase in k(cat'). The effectiveness of monovalent cations on the increase of k(cat) was determined to follow the order of Na(+)>K(+)>Li(+).

Inouye, K; Lee, S B; Tonomura, B

1996-01-01

324

[A method for the determination of cytosolic aminopeptidase in serum and its usefulness for clinical application. Selection of inhibitors for membrane-bound aminopeptidase and cystyl aminopeptidase].  

PubMed

A method for the determination of cytosolic aminopeptidase (EC 3.4.11.1; C-AP) in serum was developed. At first, more specific and adequate inhibitor for other serum peptidases, such as membrane-bound aminopeptidase (EC 3.4.11.2; arylamidase, AA) and cystyl aminopeptidase (EC 3.4.11.3; CAP) was selected from 1,10-phenanthroline derivatives. The compound, 4,7-dimethyl-1,10-phenanthroline (4,7-DMP) is one of the most effective inhibitor for AA and CAP, and it inhibits completely these enzymes at the concentration of less than 0.4 mmol/l. C-AP in serum at an optimum pH of 8.0 in the assay using L-leucine amide (LA) as the substrate had no inhibitory effects at the concentration of more than 0.4 mmol/l of 4,7-DMP. The results with the proposed method correlated with those with a conventional electrophoretic method. The proposed method hence is a specific and easily available assay for C-AP in serum. The further analysis of C-AP using this method would promise the establishment of clinical assessment of the enzyme. PMID:2607648

Taniguchi, K; Sugiyama, M; Shimizu, H; Azuma, Y; Kanno, T

1989-07-01

325

Equilibrium study on the binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI).  

PubMed

The binding between thermolysin and its specific inhibitor, talopeptin (MKI), was found to show a fluorescence increase when excited at 280 nm and 295 nm, and a difference spectrum characterized by two peaks at 294 nm and 285 nm with a shoulder around 278 nm, indicating a microenvironmental change in tryptophan residue(s) of thermolysin and/or talopeptin. The inhibitor constant of talopeptin against thermolysin, Ki, was determined over the pH range 5-9 from the inhibition of the enzyme activity towards 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) as a substrate. The dissociation constant of thermolysin-talopeptin complex, Kd, determined directly from fluorometric titration was in good agreement with the inhibitor constant, Ki, between pH 6 and 8.5. The pH dependence of Ki and Kd suggested that at least two ionizable groups of thermolysin in their protonated forms are essential for the binding between thermolysin and talopeptin. The temperature dependence of K1 at pH 5.5 indicated that the binding is largely exothermic (delta H degree = -12 kcal/mol) and essentially enthalpy-driven. PMID:6341369

Kitagishi, K; Hiromi, K; Oda, K; Murao, S

1983-01-01

326

Leucine aminopeptidase in intracytoplasmic membranes of Acinetobacter calcoaceticus.  

PubMed

Cells of Acinetobacter calcoaceticus strain 69-V contain an aminopeptidase that cleaves L-leucine amide, leucylglycine or leucine hydrazide with high efficiency. Leucine 4-nitroanilide and hydrazide are hydrolyzed to less than 0.1% and 1%, resp. of leucine amide. Grown on acetate-NH4+ medium the activity of the enzyme in the cytoplasm is increased 5-fold compared with cells grown on a casamino acid medium or on yeast extract. In these cases the specific activity of the unpurified enzyme is about 5 nkat/mg for the cytoplasmic and membrane-bound enzyme species as well. Up to 30% of the aminopeptidase activity were found mainly in intracytoplasmic membranes, less in cytoplasmic membranes and only traces in outer membranes, presumably as contaminations. It is solubilized by detergents but not by high salt concentrations. An addition of antipain or Z-Ala2-Phe-CH3 before cell rupture did not change the distribution of the enzyme. A mixture of EDTA and 1.10-phenanthroline diminished the membrane-bound enzyme from 11.4% to 4.3% and leupeptin or E-64 increased it to 20%. The enzyme is regarded as leucine aminopeptidase (LAP) bound to intracytoplasmic membranes. PMID:3503925

Ludewig, M; Fricke, B; Aurich, H

1987-01-01

327

A new antiinflammatory compound, leumedin, inhibits modification of low density lipoprotein and the resulting monocyte transmigration into the subendothelial space of cocultures of human aortic wall cells.  

PubMed Central

Addition of leumedin, N-[9H-(2,7-dimethylfluorenyl-9-methoxy) carbon]-L-leucine at 30-60 microM together with LDL almost completely prevented the induction of monocyte chemotactic protein mRNA, reduced monocyte chemotactic protein 1 levels by 84%, and inhibited monocyte migration into the subendothelial space of cocultures of human aortic wall cells by < or = 98%. LDL incubated with leumedin formed a stable complex that remained intact even after refloating in an ultracentrifuge. Leumedin at 50 microM did not change conjugated diene formation during coculture modification of LDL or Cu++ catalyzed oxidation of LDL. Unlike LDL from control rabbits, LDL isolated from rabbits that were injected with 20 mg/kg leumedin was remarkably resistant to modification by the coculture and did not induce monocyte migration to a significant degree. Moreover, HDL isolated from rabbits injected with leumedin was far more effective in protecting against LDL modification by the artery wall cocultures than HDL from control rabbits. We conclude that leumedins can associate with lipoproteins in vivo, rendering LDL resistant to biological modification and markedly amplifying the protective capacity of HDL against in vitro LDL oxidation by artery wall cells. Images

Navab, M; Hama, S Y; Van Lenten, B J; Drinkwater, D C; Laks, H; Fogelman, A M

1993-01-01

328

Multivariate approach for the enantioselective analysis in MEKC-MS: II. Optimization of 1,1'-binaphthyl-2,2'-diamine in positive ion mode.  

PubMed

Enantiomeric separation and detection of 1,1'-binaphthyl-2,2'-diamine (BNA) has been successfully optimized by MEKC-ESI-MS using a polymeric surfactant polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) as a pseudostationary phase. In the first step, MEKC conditions were optimized by a five-factor three-level central composite design (CCD) of experiment. All five MEKC factors (buffer pH, percentage of ACN in the running buffer, concentration of surfactant, concentration of ammonium acetate (NH4OAc), and voltage) were found significant to the responses (measured as the chiral resolution and analysis time). The interactions between MEKC factors were further evaluated using a quadratic model equation which allowed the generation of 3-D response surface image to reach the optimum conditions. To obtain the best S/N, sheath liquid composition and spray chamber parameters were successfully optimized using the same strategy. Baseline enantiomeric resolution in less than 20 min and optimum MS signal of BNA enantiomers (S/N = 45 at 0.4 mg/mL) were ultimately achieved at the optimized conditions. The adequacy of the model was validated by experimental runs at the optimal predicted conditions. The predicted results were found to be in good agreement with the experimental data. PMID:19479771

He, Jun; Shamsi, Shahab A

2009-06-01

329

Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells.  

PubMed

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens. PMID:18071242

Tamura, Takashi; Tomimatsu, Kosuke; Katakura, Yoshinori; Yamashita, Makiko; Matsumoto, Shin-ei; Aiba, Yoshihiro; Jung, Yeon Suk; Abe, Yoshiichi; Fujiki, Tsukasa; Teruya, Kiichiro; Shirahata, Sanetaka

2007-12-07

330

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

PubMed

An l-amino amidase from Mycobacterium neoaurum ATCC 25795 responsible for the enantioselective resolution of dl-alpha-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 degrees 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 alpha-H- and alpha-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 (k(cat)/K(m) ratio) was measured with dl-alpha-allyl alanine amide, dl-alpha-methyl phenylalanine amide, and dl-alpha-methyl leucine amide. PMID:16349147

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

1994-01-01

331

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

PubMed Central

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

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

1996-01-01

332

Biochemical characterization of the soluble alkaline phosphatase isolated from the venomous snake W. aegyptia.  

PubMed

A soluble form of alkaline phosphatase (ALP) has been identified and purified from Walterinnesia aegyptia venom using an HPLC system Gold 126/1667 equipped with Protein PAK 125 and Protein PAK 60 columns. The enzyme was purified 3.4 fold over crude venom with a yield of 37.3%. On SDS-PAGE under non-reduced conditions the purified enzyme showed three bands of 212 kD, 80 kD, and 55 kD. However, under reducing conditions, the enzyme showed two bands of 80 kD and 55 kD. The specific activity of ALP was 24 U/mg with p-nitrophenylephosphate as the substrate. During isoelectric focusing experiments the ALP exhibited two bands focused at pH 6.2 and 6.8, which suggests that either the enzyme exists as two different isoforms or the two bands in IEF may be two subunits of 80 kD and 55 kD. The kinetic parameters (Km and Vmax) and IC50 of ALP inhibition by L-phenylalanine, L-leucine, imidazole, caffeine, orthophosphate and permanganate were also investigated in the present study. Zinc and cyanide ions at a concentration of 15 mM and 10 mM, respectively, completely inhibited the activity of W. aegyptia ALP. PMID:12503880

Al-Saleh, Saad S M

2002-12-01

333

Increased production of HDL ApoA-I in homozygous familial defective ApoB-100.  

PubMed

Familial defective apolipoprotein (apo) B-100 (FDB) is a frequent cause of hypercholesterolemia. Hypercholesterolemia in homozygous FDB is less severe than in homozygotes for familial hypercholesterolemia. Recently, we showed decreased low density lipoprotein (LDL) apoB-100 fractional catabolism and decreased production of LDL due to an enhanced removal of apoE-containing precursors in a patient with homozygous FDB. The effects of defective apoB-100 on high density lipoprotein (HDL) metabolism are unknown. We studied HDL apoA-I metabolism in this FDB patient and in 6 control subjects by using (2)H(3)-L-leucine as a tracer. ApoA-I levels were normal in all study subjects. However, the fractional catabolic rate and the production rate of apoA-I were increased, by 79% and 70%, respectively, in FDB; the fractional catabolic rate of apoA-I in FDB was 0.34 day(-1) compared with 0.19+/-0.03 day(-1) in normal controls. The production rate of apoA-I in FDB was 18.4 mg. kg(-1). d(-1) compared with 10.8+/-2.3 mg. kg(-1). d(-1) in controls. Thus, we have shown for the first time that defective apoB-100 may influence HDL kinetics. The increase in total HDL turnover might enhance reverse cholesterol transport and could contribute to the seemingly benign clinical course of FDB compared with that of familial hypercholesterolemia. PMID:10894819

Schaefer, J R; Winkler, K; Schweer, H; Hoffmann, M M; Soufi, M; Scharnagl, H; Maisch, B; Wieland, H; Steinmetz, A; März, W

2000-07-01

334

Size and shape of two intestinal dipeptidases.  

PubMed

Physicochemical parameters were determined on glycyl-L-leucine hydrolase (glycy-leucine dipeptidase, EC 3.4.13.2) and aminoacyl-L-proline hydrolase (proline dipeptidase, EC 3.4.13.9), purified from pig small intestine. The native molecular weights were found to be 115,000 and 113,000, respectively, as determined by a sedimentation equilibrium technique. Under denaturing conditions the molecular weights were found to be 51,000 and 63,200, respectively, using the same technique. It is concluded that each dipeptidase is composed of two subunits of equal molecular weight. The two dipeptidases have the same Stokes radius, 4.2 nm, analysed by gel chromatography. The sedimentation coefficients were found to be 5.8. S and 6.5 S and the intrinsic viscosities 5.4 ml/g and 5.8 ml/g, respectively. For both dipeptidases the measured physicochemical parameters are in accordance with the model of a prolate ellipsoid of revolution, having an axial ratio of about 5. PMID:640775

Sjöström, H; Norén, O

1978-02-01

335

A high-throughput assay for screening l- or d-amino acid specific aminotransferase mutant libraries.  

PubMed

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 kcat/KM 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

2013-07-16

336

Changes in the composition of free amino acids and sugars of leaf sheath and culm of wheat during uredospore and teleutospore formation of Puccinia graminis tritici.  

PubMed

There were altogether fourteen amino acids in leaf sheath and culm of wheat infected with Puccinia graminis tritici, especially in and around uredial and telial pustules. Valine, tyrosine, and proline, due to their exclusive presence in uredial pustules on leaf sheath and culm of wheat, were involved in the eruption of uredospores of P. graminis tritici. Glutamic acid and dl-threonine were, however, involved in a different manner during uredospore differentiation; their amounts diminishesd parallel to sporulation. The other amino compounds, detected in and around uredial and telial pustules on leaf sheaths and culms, were l-leucine/isoleucine, beta-phenylalanine, beta-alanine, glycine, serine, aspartic acid, homoserine, and glutamine. The amounts of these amino acids either remained the same or were lowered during uredo- and teluto-spores formation, except for serine which increased in its amount. The depletion of these amino compounds indicated their metabolic activity and utilization for uredo- and teleuto-spores differentiation of P. graminis tritici. Sucrose, glucose, and fructose, among sugars, were also utilized as their amounts diminished, for uredo- and teleuto-spores formation. PMID:7424230

Pandey, P K; Prasad, M; Bhushan, A

1980-01-01

337

RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs  

PubMed Central

Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the Schistosoma mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP 1 and 2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP 2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate L-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by ? 80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases.

Rinaldi, Gabriel; Morales, Maria E.; Alrefaei, Yousef N.; Cancela, Martin; Castillo, Estela; Dalton, John P.; Tort, Jose F.; Brindley, Paul J.

2009-01-01

338

Effect of weight loss, independent of change in diet composition, on apolipoprotein AI kinetic in men with metabolic syndrome.  

PubMed

We investigated the effect of weight loss, independent of change in diet composition, on HDL and apoAI metabolism in men with metabolic syndrome (MetS). Subjects (19 men with MetS [NCEP-ATPIII]) were fed an isoenergetic Mediterranean-style diet for 5 weeks (all foods provided). Participants then underwent a 20-week free-living period during which they were counseled to restrict energy intake, after which they were again fed an isoenergetic Mediterranean-style diet for 5 weeks. At the end of the two controlled diets, participants received a single bolus of [5,5,5-(2)H(3)] (L)-leucine, and fasting blood samples were collected over a 96 h period. ApoAI kinetic was assessed using multicompartmental modeling of the tracer enrichment data. Participants achieved a 9.1 ± 2.8% reduction in body weight (P < 0.001). Weight loss resulted in an increase in plasma HDL-cholesterol (HDL-C) concentrations of 6.0% (P = 0.059) and HDL(3)-C of 7.9% (P = 0.045), attributable to a reduction in apoAI fractional catabolic rate (-7.8%; P = 0.046) with no change in apoAI production rate (2.2%; P = 0.58). These data indicate that weight loss, independent of variation in diet composition, increases plasma HDL primarily by delaying the catabolism of apoAI. PMID:23125458

Richard, Caroline; Couture, Patrick; Desroches, Sophie; Lichtenstein, Alice H; Lamarche, Benoît

2012-11-02

339

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

PubMed

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

2012-09-27

340

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.  

PubMed

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

2013-09-07

341

Microbial decomposition in aquatic environments: combined process of extracellular enzyme activity and substrate uptake  

SciTech Connect

The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with (/sup 3/H) leucine as a marker for the leucine pool and L-leucine-4-methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. Calculated on an annual average basis, turnover rates were ca. nine times higher than hydrolysis rates at the polluted station and ca., five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station.

Hoppe, H.G.; Kim, S.J.; Gocke, K.

1988-03-01

342

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

PubMed

N,N'-pyromelliticdiimido-di-L-alanine (1), N,N'-Pyromelliticdiimido-di-L-phenylalanine (2), and N,N'-Pyromelliticdiimido-di-L-leucine (3) were prepared from the reaction of Pyromellitic dianhydride with corresponding L-amino acids in a mixture of glacial acetic acid and pyridine solution (3/2 ratio) under refluxing conditions. A series of poly (amide-imide)s containing L-amino acids were prepared from the synthesized dicarboxylic acids with two synthetic aromatic diamines in an ionic liquid (IL) as a green, safe and eco-friendly medium and also reactions catalysis agent. Evaluation of data shows that IL is the better polyamidation medium than the reported method and the catalysis stand on the higher inherent viscosities of the obtained PAIs and the rate of polymerizations beyond the greener reaction conditions and deletion of some essential reagents in conventional manners. Characterization were performs by means of IR, MS and (1)H NMR spectroscopy, elemental analysis, specific rotation, thermogravimetric analysis and differential scanning calorimetric techniques. Molecular weights of the obtained polymers were evaluated viscometrically, and the measured inherent viscosities were in the range 0.43-0.85 dL/g. These polymers were readily soluble in many organic solvents. These polymers still kept good thermal stability with glass transition temperatures in the range of 94-154°C, and the decomposition temperature under the nitrogen atmosphere for 10% weight-loss temperatures in excess of 308°C. PMID:20607323

Zahmatkesh, Saeed

2010-07-07

343

Immobilization of the Aminopeptidase from Aeromonas Proteolytica on Mg2+/Al3+ Layered Double Hydroxide Particles  

PubMed Central

A novel biomaterial formed by the immobilization of the Aminopeptidase from Aeromonas Proteolytica (AAP) on synthetic Mg2+ and Al3+ ion-containing layered double hydroxide (LDH) particles was prepared. Immobilization of AAP on the LDH particles in a buffered, aqueous mixture is rapid such that the maximum loading capacity, 1 × 10-9 moles of AAP/mg LDH, is achieved in a few minutes. X-ray powder diffraction of LDH samples before and after treatment with AAP indicates that the enzyme does not intercalate between the layers of LDH, but instead binds to the surface. Treatment of AAP/LDH with various amounts of salt in a buffered mixture demonstrates that between 15 and 20% of AAP can be removed from the LDH by washing the composite material in 0.2 M NaCl. However, the residual AAP remains bound to the LDH even at 1 M salt concentrations. A suspension of the AAP/LDH biomaterial in 10 mM Tricine buffered, aqueous solution (pH 8.0 and 25 ° C) catalyzes the hydrolysis of l-leucine-p-nitroanilide demonstrating that immobilized AAP remains available to substrate and retains its catalytic activity. Recycling experiments reveal that the AAP/LDH particles can be recovered and reused multiple times without appreciable loss of activity. This work provides the foundation for the development of materials that will function in the degradation or detection of peptide hormones or neurotoxins.

Frey, Steven T.; Guilmet, Stephanie L.; Egan, Richard G.; Bennett, Alyssa; Soltau, Sarah R.; Holz, Richard C.

2010-01-01

344

Purification and properties of an aminopeptidase from seeds of Japanese apricot.  

PubMed

An aminopeptidase was purified about 1,700-fold from seeds of Japanese apricot (Prunus mume Sieb.) by a seven-step procedure comprising extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, first and second DEAE-Sepharose chromatography, hydroxyapatite chromatography, and Sephadex G-200 gel filtration. The purified enzyme was shown to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56,000 by gel filtration on Sephadex G-200 and the isoelectric point was 4.9. The pH optimum for L-leucine beta-naphthylamide was between pH 6.5 and 7.0, and the enzyme was stable in the pH 5.0 to 8.3 region and up to 50 degrees C. The enzyme hydrolyzed a variety of aminopeptidase substrates with a free alpha-amino group. Of the amino acid beta-naphthylamides, the enzyme was highly specific for substrates with a hydrophobic side chain in the amino terminal residue. The enzyme was strongly inhibited by p-chloromercuribenzoate, heavy metals, diethyl pyrocarbonate, and photooxidation with methylene blue, but was not affected by thiol compounds, peptidase inhibitors of microbial origin, such as bestatin and puromycin, or metal chelating agents. No activation of the enzyme by metal ions was observed. These results suggest that the enzyme is a true aminopeptidase in which cysteine and histidine residues participate in the catalytic process, and is not classifiable as a metalloenzyme. PMID:7217032

Ninomiya, K; Tanaka, S; Kawata, S; Makisumi, S

1981-01-01

345

Effects of histamine and activators of the cyclic AMP system on protein synthesis in and release of high molecular weight glycoproteins from isolated gastric non-parietal cells.  

PubMed Central

1. Glycoprotein and protein synthesis in and release from pig isolated, enriched gastric mucous cells were measured by the incorporation of N-acetyl-[14C]-D-glucosamine and [3H]-L-leucine, respectively, into cellular and released acid precipitable material. 2. Histamine and activators of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) system maximally stimulated total protein and glycoprotein synthesis in and release from the cells at concentrations of histamine (10 microM), forskolin (10-100 microM), 3-isobutyl-1-methylxanthine (100 microM), and dibutyryl cyclic AMP (1-3 mM), respectively. In the presence of 3-isobutyl-1-methylxanthine (30 microM) histamine stimulation was enhanced. 3. As shown by gel chromatography, stimulation by histamine (100 microM), forskolin (10 microM), 3-isobutyl-1-methylxanthine (100 microM) and dibutyryl cyclic AMP (1 mM) resulted in a release of high molecular weight (approximately 2 x 10(6) daltons) glycoproteins from the cells. The histamine H2-receptor antagonist, ranitidine (100 microM), blocked the effect of histamine. 4. We conclude that cyclic AMP-dependent processes are involved in the regulation of protein and glycoprotein synthesis in and the release of high molecular weight (mucous) glycoproteins from pig gastric non-parietal cells and that histamine may be a physiological activator of this system.

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

1991-01-01

346

Effects of histamine and activators of the cyclic AMP system on protein synthesis in and release of high molecular weight glycoproteins from isolated gastric non-parietal cells.  

PubMed

1. Glycoprotein and protein synthesis in and release from pig isolated, enriched gastric mucous cells were measured by the incorporation of N-acetyl-[14C]-D-glucosamine and [3H]-L-leucine, respectively, into cellular and released acid precipitable material. 2. Histamine and activators of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) system maximally stimulated total protein and glycoprotein synthesis in and release from the cells at concentrations of histamine (10 microM), forskolin (10-100 microM), 3-isobutyl-1-methylxanthine (100 microM), and dibutyryl cyclic AMP (1-3 mM), respectively. In the presence of 3-isobutyl-1-methylxanthine (30 microM) histamine stimulation was enhanced. 3. As shown by gel chromatography, stimulation by histamine (100 microM), forskolin (10 microM), 3-isobutyl-1-methylxanthine (100 microM) and dibutyryl cyclic AMP (1 mM) resulted in a release of high molecular weight (approximately 2 x 10(6) daltons) glycoproteins from the cells. The histamine H2-receptor antagonist, ranitidine (100 microM), blocked the effect of histamine. 4. We conclude that cyclic AMP-dependent processes are involved in the regulation of protein and glycoprotein synthesis in and the release of high molecular weight (mucous) glycoproteins from pig gastric non-parietal cells and that histamine may be a physiological activator of this system. PMID:1724626

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

1991-10-01

347

Effect of Klebsiella pneumoniae enterotoxin on intestinal transport in the rat.  

PubMed Central

The effects on intestinal transport of either a semipurified preparation of enterotoxin elaborated by Klebsiella pneumoniae or similaryly prepared control material were tested by marker perfusion studies in the small intestine of rats. At a concentration of 2 mg/ml, the enterotoxin produced net secretion of water, Na, and Cl in both jejunal and ileal segments; HCO3 transport was not affected. Net secretion was evident within 30 min after intorduction of the toxin and was maximal after 90 min. The addition of 56 mM glucose to the enterotoxin-containing perfusion fluid resulted in reversal of water and Na transport to net absorption in both intestinal segments. The enterotoxin also produced a significant depression of xylose absorption in both the jejunum and ileum but did not affect the absorption of either glucose or L-leucine. Intestinal structure was not altered after perfusion of the toxin but insillation of approximately one-quarter of the total perfusion dose into a ligated jejunal loop for 18 h produced fluid secretion and structural abnormalities. These observations confirm the fact that other species of coliform bacteria in addition to tescherichia coli are capable of elaborating an enterotoxin. Such species commonly contaminate the small intestine of persons with tropical sprue and it is suggested that chronic exposure of the intestinal mucosa to the enterotoxin elaborated by these bacteria may be a factor in the pathogenesis of intestinal abnormalities in thid disorder. Images

Klipstein, F A; Horowitz, I R; Engert, R F; Schnenk, E A

1975-01-01

348

Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling  

SciTech Connect

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4/sup 0/C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodiumdodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with (/sup 3/H)D-glucosamine or (/sup 3/H)L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecule haptenized with trinitrophenol (TNP) groups were followed radio-chemically by means of (/sup 125/I)anti-DNP antibodies. Within 15 min at 37/sup 0/C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t/sub 1/2/ approx. 5 min). Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.

Raub, T.J.; Denny, J.B.; Roberts, R.M.

1986-01-01

349

Rapid liquid chromatographic method to distinguish wild salmon from aquacultured salmon fed synthetic astaxanthin.  

PubMed

Analytical methods are needed to determine the presence of color additives in fish. We report a liquid chromatographic (LC) method developed to identify the synthetic form of the color additive astaxanthin in salmon, based on differences in the relative ratios of the configurational isomers of astaxanthin. The distributions of configurational isomers of astaxanthin in the flesh of wild Atlantic and wild Pacific salmon are similar, but significantly different from that in aquacultured salmon. Astaxanthin is extracted from the flesh of salmon, passed through a silica gel Sep-Pak cartridge, and analyzed directly by LC on a Pirkle covalent L-leucine column. No derivatization of the astaxanthin is required-an important advantage of our approach, which is a modification of our previously described method. This method can be used to distinguish between aquacultured and wild salmon. The method has general applicability and can also be used to identify astaxanthins derived from other sources such as Phaffia yeast and Haematococcus pluvialis algae. PMID:9170658

Turujman, S A; Wamer, W G; Wei, R R; Albert, R H

350

Enantioselective separation of all-E-astaxanthin and its determination in microbial sources.  

PubMed

A method for the enantioselective separation of all-E-astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione), an important colorant in the feed industry, was developed. Different chiral stationary phases (CSPs) such as Pirkle phases (R,R Ulmo and l-leucine), modified polysaccharides and a beta-cyclodextrin have been investigated on their separation performance of astaxanthin enantiomers. Direct resolution was only achieved employing the Chiralcel OD-RH (cellulose-tris-3,5-dimethylphenyl-carbamate) under reversed phase conditions. The chiral separation of the enantiomeric forms of astaxanthin produced in microalgae and yeasts was reported. The yeast Xanthophyllomyces sp. produces astaxanthin predominantly in the R,R configuration, whereas in the green microalgae Scenedesmus sp. astaxanthin is built primarily in the S,S form. The separation method for the identification of astaxanthin enantiomers is of great interest since astaxanthin is used as functional food additive in human nutrition. Moreover the method may be used as a food chain indicator in farmed salmon. PMID:17727867

Grewe, Claudia; Menge, Sieglinde; Griehl, Carola

2007-08-06

351

Metabolic and K(ATP) channel-independent actions of keto acid initiators of insulin secretion.  

PubMed

Insulin-releasing effects of 2-ketobutyric acid (KB), 2-ketoisocaproic acid (KIC), 2-keto-3-methylvaleric acid (KMV), and 3-phenylpyruvic acid (PP) were examined by using clonal beta cells. Whereas KIC, KMV, and PP dose-dependently initiated insulin secretion and potentiated the effects of 4.2-16.7 mM glucose, equimolar KB was without effect. Transport inhibition by using 10 mM valine, isoleucine, 2-cyano-3 hydroxycinnamate or 2-cyano-4 hydroxycinnamate, or metabolic inhibition by 15 mM mannoheptulose, 5 mM sodium azide, 5 mM sodium cyanide, or removal of HCO3 reduced the secretory effects of KIC, KMV, and PP. Whereas K+ depletion reduced keto acid-induced insulin output, depolarizing concentrations of L-leucine and L-arginine potentiated the keto acid-induced effects. Under depolarizing conditions (25 mM KCI and 16.7 mM glucose), 10 mM KIC, KMV, or PP induced insulin secretion, suggesting K(ATP) channel-independent actions. Furthermore, the K(ATP) channel opener diazoxide reduced, but did not abolish, the keto acid-induced effects. However, voltage-dependent Ca2+ channel blockade with verapamil or removal of extracellular Ca2+ abolished keto acid-induced insulin release. Collectively, these results indicate that KIC, KMV, and PP initiate insulin secretion at least partially independently of K(ATP) channel activity, through both mitochondrial metabolism and regulation of Ca2+ influx. PMID:10630382

McClenaghan, N H; Flatt, P R

2000-01-01

352

Glucose and non-glucidic nutrients exert permissive effects on 2-keto acid regulation of pancreatic beta-cell function.  

PubMed

Insulin-releasing effects of straight and branched chain 2-keto acids were assessed using clonal glucose-responsive beta-cells. Pyruvic acid (PA), 2-ketovaleric acid (KV), 2-ketoisovaleric acid (KIV) or 2-keto-3-methylvaleric acid (KMV) dose-dependently promoted the stimulatory effects of D-glucose, whereas 2-ketobutyric acid (KB) did not affect insulin release. The stimulatory 2-keto acids also promoted the stimulatory activity of D-glyceraldehyde, L-leucine or L-arginine. Responses to PA, KV, KIV or KMV were significantly reduced by transport inhibition with 2-cyano-3 hydroxycinnamate, glucokinase inhibition with mannoheptulose or metabolic inhibition with sodium azide or sodium cyanide. Membrane hyperpolarisation with K+ depletion or diazoxide reduced insulin output, but failed to abolish secretory responses to KV, KIV and KMV. Secretory effects of these 2-keto acids also persisted in beta-cells depolarised with high KCl and glucose. Voltage-dependent Ca2+ channel blockade, with verapamil, or depletion of extracellular Ca2+ abolished the secretory activity of 2-keto acids. Collectively, these results indicate that glucose and metabolisable nutrients exert permissive effects on 2-keto acid-induced insulin release. In addition, KV, KIV and KMV can regulate beta-cell function at least partially independently of K+-ATP channel activity, both through their mitochondrial metabolism and regulation of Ca2+ influx. PMID:9878701

McClenaghan, N H; Flatt, P R

1999-01-01

353

Ligand-specific allosteric regulation of coactivator functions of androgen receptor in prostate cancer cells.  

PubMed

The androgen receptor not only mediates prostate development but also serves as a key regulator of primary prostatic cancer growth. Although initially responsive to selective androgen receptor modulators (SARMs), which cause recruitment of the nuclear receptor-corepressor (N-CoR) complex, resistance invariably occurs, perhaps in response to inflammatory signals. Here we report that dismissal of nuclear receptor-corepressor complexes by specific signals or androgen receptor overexpression results in recruitment of many of the cohorts of coactivator complexes that permits SARMs and natural ligands to function as agonists. SARM-bound androgen receptors appear to exhibit failure to recruit specific components of the coactivators generally bound by liganded nuclear receptors, including cAMP response element-binding protein (CBP)/p300 or coactivator-associated arginine methyltransferase 1 (CARM1) to the SARM-bound androgen receptor, although still causing transcriptional activation of androgen receptor target genes. SARM-bound androgen receptors use distinct LXXLL (L, leucine; X, any amino acid) helices in the p160 nuclear receptor interaction domains that may impose selective allosteric effects, providing a component of the molecular basis of differential responses to different classes of ligands by androgen receptor. PMID:16492776

Baek, Sung Hee; Ohgi, Kenneth A; Nelson, Charles A; Welsbie, Derek; Chen, Charlie; Sawyers, Charles L; Rose, David W; Rosenfeld, Michael G

2006-02-21

354

Direct spectrophotometric measurement of angiotensin I-converting enzyme inhibitory activity for screening bioactive peptides.  

PubMed

A direct, extraction-free spectrophotometric assay was developed for determination of angiotensin I-converting enzyme activity (ACE) in the presence of ACE inhibitors using hippuryl-l-histidyl-l-leucine (HHL) as the ACE-specific substrate. This method relies on previously published spectrophotometric determination of hippuric acid (HA) content in the urine, the method of which was based on the specific colorimetric reaction of HA with benzene sulfonyl chloride (BSC) in the presence of quinoline. The proposed ACE inhibition assay was applied to the measurement of the ACE inhibitory activity of Captopril. IC(50) value of Captopril corresponded well with literature data. Furthermore, Alcalase hydrolysates of mung bean and rice protein isolates were assessed for ACE inhibitory activity by this method. These two hydrolysates showed high ACE inhibitory activity. This method proposed here was shown to be direct, sensitive, accurate, reproducible, and less expensive without separation of HA from ACE reaction mixture, and can be used for the screening of ACE inhibitory peptides derived from food proteins. PMID:15708660

Li, Guan-Hong; Liu, Huan; Shi, Yong-Hui; Le, Guo-Wei

2005-02-23

355

SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G.  

PubMed

Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1(-/-)) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia of sB1(-/-) mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1(-/-) mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1(-/-) PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent l-leucyl-l-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity. PMID:23532733

Baumann, Mathias; Pham, Christine T N; Benarafa, Charaf

2013-03-26

356

Molecular structure of bis(L-leucinato)zinc(II) and single-crystal EPR spectra of the substitutionally sup 63 Cu(II)-doped complex  

SciTech Connect

The title compound, Zn(H{sub 2}NCHCO{sub 2}CH{sub 2}CH(CH{sub 3}){sub 2}){sub 2} (abbreviated as Zn(L-Leu){sub 2}), crystallizes in the monoclinic system, space group P2{sub 1}, with a = 9.584 (4) {angstrom}, b = 5.389 (2) {angstrom}, c = 14.866 (3) {angstrom}, {beta} = 106.84 (3){degree}, and Z = 2. The two L-leucine molecules per formula unit act as bidentate ligands of the Zn(II) ion, forming a N{sub 2}O{sub 2} squashed tetrahedral configuration. The carboxyl oxygen of a third amino acid molecule completes a pentagonal coordination around Zn(II). The crystal structure of Zn(L-Leu){sub 2} was compared to the quasi-isomorphous structure of the Cu(L-Leu){sub 2} complex. Room-temperature EPR data of substitutional {sup 63}Cu(II) impurities in single crystals of Zn(L-Leu){sub 2} were taken at 34 GHz. The EPR results are discussed in terms of the structure of Zn(L-Leu){sub 2} and compared with EPR data in the structurally related Cu(L-Leu){sub 2} complex. Lattice distortions at the impurity sites of Zn(L-Leu){sub 2} are detected and discussed. 29 refs., 6 figs., 5 tabs.

Steren, C.A.; Calvo, R. (Instituto de Desarrollo Tecnologico para la Industia Quimica (INTEC), Santa Fe (Argentina)); Piro, O.E.; Rivero, B.E. (Universidad Nacional de La Plata (Argentina))

1989-05-17

357

Choline transport in Saccharomyces cerevisiae.  

PubMed Central

Choline transport of Saccharomyces cerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N,N-dimethylethanolamine, and beta-methylcholine were competitive inhibitors of choline transport, with Ki values of 40.1, 3.1, and 6.9 microM, respectively. Ethanolamine, phosphorylcholine, and various amino acids examined had no effect. Choline transport required metabolic energy; removal of glucose resulted in a great loss of transport activity, and the remaining activity was abolished by 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, arsenate, and cyanide. External Na+ was not required, and the transport was not effected by ionophores, valinomycin, and gramicidin D. These results indicate that S. cerevisiae possess an active choline transport system mediated by a specific carrier. This view is further supported by the isolation and characterization of a choline transport mutant. The choline transport activity in this mutant was very low, whereas the transport of L-leucine, L-methionine, D-glucose, and myo-inositol was normal. Together with the choline transport mutant, mutants defective in choline kinase were also isolated.

Hosaka, K; Yamashita, S

1980-01-01

358

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

PubMed

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

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

2013-04-17

359

Effects of lead on viability and intracellular metal content of C6 rat glioma cells  

SciTech Connect

Cultured C6 rat glioma cells were exposed to lead (Pb) acetate (0, 1, 10, or 100 ..mu..M) for 3-4 d. Cells were analyzed for changes in viability and intracellular lead, iron, and copper concentrations after Pb treatment was discontinued. The results were compared with previous findings on astroglia and oligodendroglia in culture in order to evaluate C6 cultures as a model for Pb toxicity in glia. Viability was measured by three methods on the day Pb was removed from the cells (designated d 0), and 2 and 9 d after Pb treatment was discontinued (designated d 2 and 9). The methods used were trypan blue dye exclusion, total cell counts, and incorporation of (/sup 3/H)-L-leucine into proteins. With respect to Pb and Fe uptake, C6 cells closely resembled immature astroglia in culture. Unlike C6 cells, however, astroglia showed elevations of intracellular Fe and Cu after treatment. Thus, Pb effects on C6 cells resembled those on cultured oligodendroglia and astroglia in some respects but not in others. C6 cells appear to be an adequate model for selected events in glial toxicosis, such as PB-stimulated protein synthesis in oligodendroglia and Pb uptake in astroglia, but not Pb-induced alterations of intracellular Cu and Fe in astroglia. Their use as a model for glial progenitor cells in Pb toxicity studies remains to be determined.

Tiffany-Castiglioni, E.; Garcia, D.M.; Wu, J.N.; Zmudzki, J.; Bratton, G.R.

1988-01-01

360

Effects of taxifolin on the activity of angiotensin-converting enzyme and reactive oxygen and nitrogen species in the aorta of aging rats and rats treated with the nitric oxide synthase inhibitor and dexamethasone.  

PubMed

The action of taxifolin on the angiotensin-converting enzyme (ACE) and the formation of reactive oxygen and nitrogen species (ROS/RNS) in the aorta of aging rats and rats treated with nitric oxide synthase inhibitor (N ?-nitro-L-arginine methyl ester (L-NAME)) or dexamethasone have been studied. The ACE activity in aorta sections was determined by measuring the hydrolysis of hippuryl-L-histidyl-L-leucine, and the ROS/RNS production was measured by oxidation of dichlorodihydrofluorescein. It was shown that taxifolin at a dose of 30-100 ?g/kg/day decreases the ACE activity in the aorta of aging rats and of rats treated with L-NAME or dexamethasone to the level of the ACE activity in young control rats. Taxifolin (100 ?g/kg/day) was found to also reduce the amount of ROS/RNS in the aorta that increased as a result of L-NAME intake. L-NAME treatment increases the contribution of 5-lipoxygenase and NADPH oxidase to ROS/RNS production in the aorta, while taxifolin (100 ?g/kg/day) decreases the contribution of these enzymes to the normal level. PMID:23271616

Arutyunyan, Tamara V; Korystova, Antonina F; Kublik, Ludmila N; Levitman, Maria Kh; Shaposhnikova, Vera V; Korystov, Yuri N

2012-12-28

361

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.

2013-11-01

362

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

2012-01-01

363

Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases.  

PubMed

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. PMID:23226342

Dutoit, Raphaël; Brandt, Nathalie; Legrain, Christianne; Bauvois, Cédric

2012-11-30

364

S-Nitrosation of Cellular Proteins by NO Donors in Rat Embryonic Fibroblast 3Y1 Cells: Factors Affecting S-Nitrosation  

PubMed Central

The mechanism of protein S-nitrosation in cells is not fully understood. Using rat 3Y1 cells, we addressed this issue. Among S-nitrosothiols and NO donors tested, only S-nitrosocysteine (CysNO) induced S-nitrosation when exposed in Hanks' balanced salt solution (HBSS) and not in serum-containing general culture medium. In HBSS, NO release from CysNO was almost completely abolished by sequestering metal ions with a metal chelator without affecting cellular S-nitrosation. In contrast, L-leucine, a substrate of L-type amino acid transporters (LATs), significantly inhibited S-nitrosation. The absence of S-nitrosation with CysNO in general culture medium resulted not only from a competition with amino acids in the medium for LATs but also from transnitrosation of cysteine residues in serum albumin. Collectively, these results suggest that in simple buffered saline, CysNO-dependent S-nitrosation occurs through a cellular incorporation-dependent mechanism, but if it occurs in general culture media, it may be through an NO-dependent mechanism.

Ryuman, Norihiro; Watanabe, Nobuo; Arai, Takao

2011-01-01

365

Bidirectional transport of amino acids regulates mTOR and autophagy.  

PubMed

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. PMID:19203585

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

2009-02-01

366

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))

1990-07-01

367

Angiotensin converting enzyme activity and nitric oxide level in serum patients with dehydration.  

PubMed

Angiotensin converting enzyme (ACE) and nitric oxide (NO) have been suggested to be involved in the regulation of fluid homeostasis. In the present investigation, ACE activity and NO levels were determined in serum of 20 patients (10 men and 10 women) with dehydration caused by gastroenterocolitis and 20 healthy individuals (10 men and 10 women). Serum and tissue ACE activity was determined by spectrophotometric method using hippuryl-l-histidyl-l-leucine (Hip-His-Leu) as a substrate. NO synthesis was determined by measuring the products of NO, nitrite and nitrate. The concentration of nitrites was determined by classic colorimetric method using Griess reagent. Nitrate concentration was determined indirectly by their reduction with elementary zinc into nitrite. Results have shown that serum ACE activity in patients with dehydration (36,46+/-2,74 U/L) is statistically higher then in healthy individuals (28,71+/-1,77 U/L, p<0,05). The average level of nitrites/nitrates in serum of patients with dehydration (30,57+/-1,05 microM; mean +/- SEM) is also statistically higher then in healthy individuals (12,44+/-0,60 microM, p<0,0001). There was no correlation between ACE activity and NO production. The results indicate that ACE and NO may participate in the regulation of the alteration in blood flow and in the regulation of the water balance in patients with dehydration. PMID:17489765

Huski?, Jasminko; Culo, Filip; Dautovi?, Sajma; Mulabegovi?, Nedzad

2007-02-01

368

Acetylene Reduction (Nitrogen Fixation) by Enterobacteriaceae Isolated from Paper Mill Process Waters  

PubMed Central

Using selective media containing galactitol, over 130 Enterobacteriaceae have been isolated from paper mill process waters collected from different localities. These bacteria were extensively characterized and tested for acetylene-reducing (nitrogen-fixing) activity under anaerobic conditions. High activity was found in representatives of Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Erwinia herbicola, Citrobacter freundii, Citrobacter intermedius, and Escherichia coli. Under argon, nitrogenase synthesis was generally not repressed by 5 mM l-glutamate, l-aspartate, l-leucine or Casamino Acids (0.5 g/liter). In many strains, both the specific activities (nanomoles of C2H4 per minute per milligram of protein) and the activities (nanomoles of C2H4 per minute) had considerably declined after 24 h. In three selected strains, activity in intact cells grown under nitrogen was unaffected by the presence during assay of 10 mM l-amino acids or ammonium acetate. All of the strains examined were tolerant towards inactivation of nitrogen-fixing activity by 1.8% (vol/vol) oxygen during assay, and inactivation by up to 10% oxygen was partly reversible. Representatives of the six taxa synthesized nitrogenase in stirred aerobic cultures, though the protein concentrations attained were lower than under anaerobic conditions. It seems reasonable to suggest that under natural conditions, nitrogen fixation is able to contribute significantly to the nitrogen economy of the cells.

Neilson, A. H.; Sparell, L.

1976-01-01

369

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

2006-05-01

370

Calcium triggers beta-defensin (hBD-2 and hBD-3) and chemokine macrophage inflammatory protein-3 alpha (MIP-3alpha/CCL20) expression in monolayers of activated human keratinocytes.  

PubMed

The inducible epidermal beta-defensins and the chemokine macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20) are important mediators involved in innate and adaptive immunity and in the recruitment of immune cells. The aim of our study was to determine whether calcium could trigger the induction of beta-defensins (hBD-2 and hBD-3) mRNA and the release of MIP-3alpha by normal human keratinocyte monolayers. Epidermal cells derived from foreskin were cultured in defined medium supplemented with different calcium levels (0.09, 0.8 and 1.7 mM) and were stimulated or not with the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha 1-500 ng/ml) or interferon-gamma (INF-gamma 1-100 ng/ml). A high calcium concentration (1.7 mM) alone applied in culture medium for 4 days was sufficient to induce hBD-2 and hBD-3 mRNA expression. Whatever interindividual variability in the expression of hBD-2 and hBD-3 mRNA and MIP-3alpha secretion, the addition of TNF-alpha for a short duration (26h), initiated a dose-dependent and coordinated up-regulation of hBD-2 and hBD-3 mRNA and MIP-3alpha release in keratinocyte cultures. Unlike hBD-2 and hBD-3 mRNA was preferentially stimulated by IFN-gamma rather than TNF-alpha. In our experimental conditions, L-isoleucine, described to stimulate beta-defensin in bovine epithelial cells, did not exert any effect either on hBD-2 and hBD-3 transcripts or MIP-3alpha protein. Taken together, these results confirm the major role of the maturation/differentiation process of normal human keratinocytes in the induction of inducible beta-defensins and MIP-3alpha chemokine, which contribute in vivo to the immunosurveillance of the skin barrier function. PMID:14714554

Pernet, I; Reymermier, C; Guezennec, A; Branka, J-E; Guesnet, J; Perrier, E; Dezutter-Dambuyant, C; Schmitt, D; Viac, J

2003-12-01

371

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

2010-01-01

372

Mixed ligand complex formation of 2-aminobenzamide with Cu(II) in the presence of some amino acids: Synthesis, structural, biological, pH-metric, spectrophotometric and thermodynamic studies  

NASA Astrophysics Data System (ADS)

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 ? log K, 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.

Dharmaraja, Jeyaprakash; Esakkidurai, Thirugnanasamy; Subbaraj, Paramasivam; Shobana, Sutha

2013-10-01

373

Transport of L-asparagine in Tetrahymena pyriformis ecto-L-asparaginase is not related to L-asparagine-protein transport system.  

PubMed

L-Asparaginase of T. pyriformis is a membrane-bound enzyme with an active site situated on the outside surface of the membrane. When radioactive L-asparagine was incubated with T. pyriformis cells in the L-asparaginase assay medium, the hydrolysis was 240 higher than the uptake of this amino acid. In a similar experiment performed in salt medium (Wagner's solution), the hydrolysis was linearly increased and reached after one hour of incubation a value of 60 nmol/10(6) cells, while the uptake after 20 min of incubation reached a plateau with a value of 15 nmol/10(6) cells. The uptake of L-leucine under these conditions was 44 nmol/10(6) cells/hr, while no measurable transport of aspartic acid was observed. That L-aspartic acid is not migrated into T. pyriformis cells is in agreement with the finding that no efflux of this amino acid takes place as well. The uptake of L-asparagine is pH and K+ dependent, whereas Na+ ions strongly inhibit this uptake. The Km and Vmax values of L-asparagine uptake is 1.43 mM and 0.7 nmol/min, respectively. The half life of L-asparagine "protein transport system" was 40 min, a value which is very close to the half life of the membrane-bound L-asparaginase of this microorganism. Ouabain and vanadate inhibit the uptake of L-asparagine by more than 80%, while ouabain or vanadate inhibit in vivo 5% or 95% the activity of L-asparaginase, respectively. This indicates the lack of interrelationship between the L-asparagine "protein transport system" and the L-asparaginase protein molecule. PMID:1930247

Tsavdaridis, I K; Triantafillou, D J; Kyriakidis, D A

1991-05-01

374

A novel neutral protease from Thermoactinomyces species 27a: sequencing of the gene, purification, and characterization of the enzyme.  

PubMed

The nucleotide sequence of the previously cloned (Zabolotskaya, M. V., Nosovskaya, E. A., Kaplun, M. A., and Akimkina, T. V. (2001). Mol. Gen. Mikrobiol. Virusol. No 1, 32-34) DNA fragment from Thermoactinomyces sp. 27a (GenBank Accession No. AY280367) containing the metalloproteinase gene was determined. A continuous open reading frame encoding a polypeptide of 673 aa was revealed. Analysis of this sequence demonstrated that the metalloproteinase from Thermoactinomyces sp. 27a is synthesized as a preproprotein and includes a leader peptide (26 aa), N-terminal propeptide (215 aa), mature region (317 aa), and additional C-terminal domain (115 aa). The recombinant enzyme from Thermoactinomyces sp. 27a was expressed in Bacillus subtilis AJ73 cells and purified by anion exchange chromatography to an electrophoretically homogeneous state. The determined N-terminal amino acid sequence of the mature protein was identical to that deduced from the gene. The obtained data suggest that the mature protein should include 432 aa and have a calculated molecular weight of 46,262 Da. However, the molecular weight of the mature protein determined by mass spectrometry was 34,190+/-70 Da indicating a C-terminal processing. The proteinase was not inhibited by phenylmethyl sulfonyl fluoride but was inhibited by o-phenanthroline and ethylenediaminetetraacetic acid. The enzyme had maximum activity by azocasein hydrolysis at 55 degrees C and pH 6.5-7.5; it was stable at pH 7.5-8.5 and remained stable at 50 degrees C for several hours. The k(cat)/Km for 3-(2-furyl)acryloyl-glycyl-L-leucine amide hydrolysis was (2.8+/-0.1) x 10(3) M(-1) x s(-1). PMID:15635941

Zabolotskaya, Maria V; Demidyuk, Ilya V; Akimkina, Tatiana V; Kostrov, Sergey V

2004-10-01

375

Improving the activity and stability of thermolysin by site-directed mutagenesis.  

PubMed

In previous site-directed mutagenesis study on thermolysin, mutations which increase the catalytic activity or the thermal stability have been identified. In this study, we attempted to generate highly active and stable thermolysin by combining the mutations so far revealed to be effective. Three mutant enzymes, L144S (Leu144 in the central alpha-helix located at the bottom of the active site cleft is replaced with Ser), G8C/N60C/S65P (Gly8, Asn60, and Ser65 in the N-terminal region are replaced with Cys, Cys, and Pro, respectively, to introduce a disulfide bridge between the positions 8 and 60), and G8C/N60C/S65P/L144S, were constructed by site-directed mutagenesis. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM), the k(cat)/K(m) values of L144S and G8C/N60C/S65P/L144S were 5- to 10-fold higher than that of the wild-type enzyme. The rate constants for thermal inactivation at 70 degrees C and 80 degrees C of G8C/N60C/S65P and G8C/N60C/S65P/L144S decreased to 50% of that of the wild-type enzyme. These results indicate that G8C/N60C/S65P/L144S is more active and stable than the wild-type thermolysin. Thermodynamic analysis suggests that the single mutation of Leu144-->Ser and the triple mutation of Gly8-->Cys, Asn60-->Cys, and Ser65-->Pro are independent. PMID:17869197

Yasukawa, Kiyoshi; Inouye, Kuniyo

2007-08-14

376

Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin.  

PubMed

Thermolysin activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA) and FA-l-leucyl-l-alanine amide (FALAA) was examined at various Co(2+) and Ca(2+) concentrations. It decreased to 28% with increasing [Co(2+)] up to 18 mM. The Co(2+)-dependent inactivation was in part suppressed by adding Ca(2+) ion up to 0.5 mM, but 33% of the activity remained to be inactivated even with a sufficient concentration of Ca(2+) (>0.5 mM). The Co(2+)-dependent inactivation was shown to be composed of Ca(2+)-sensitive and Ca(2+)-insensitive parts. In the latter part which is observed at [Ca(2+)] >0.5 mM, Co(2+) plays as a competitive inhibitor. On the other hand, the Co(2+)-dependent inactivation in the Ca(2+)-sensitive part observed at [Ca(2+)] <0.5 mM proceeds time-dependently following second-order kinetics, and the time-course is in good agreement with that of decrease in the thermolysin band due to autolysis in SDS-PAGE. This indicates that Co(2+) accelerates the autolysis. Here, we describe the co-regulation of thermolysin activity by Co(2+) and Ca(2+) ions and propose a molecular mechanism for the inhibition of thermolysin by Co(2+) and suppressive effect of Ca(2+) on the Co(2+)-dependent inhibition. Co(2+) ion inhibits thermolysin activity not only as a competitive inhibitor but also promoting the autolysis. PMID:17405797

Hashida, Yasuhiko; Inouye, Kuniyo

2007-04-03

377

A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli.  

PubMed

Thermolysin, a representative zinc metalloproteinase from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of thermolysin containing an NH(2)-terminal pelB leader sequence and the pre-prosequence of thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes thermolysin. Unlike the conventional expression method, not only the wild-type thermolysin but also mutant thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in thermolysin. PMID:17616558

Yasukawa, Kiyoshi; Kusano, Masayuki; Inouye, Kuniyo

2007-07-06

378

Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability.  

PubMed

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures. PMID:18187054

Takita, Teisuke; Aono, Takahiro; Sakurama, Haruko; Itoh, Takafumi; Wada, Takumi; Minoda, Masashi; Yasukawa, Kiyoshi; Inouye, Kuniyo

2007-12-15

379

Effects of site-directed mutagenesis of Asn116 in the ?-hairpin of the N-terminal domain of thermolysin on its activity and stability.  

PubMed

In the N-terminal domain of thermolysin, two anti-parallel ?-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a ?-hairpin structure. In this study, we examined the role of Asn116 in the activity and stability of thermolysin by site-directed mutagenesis. Of the 19 Asn116 variants, four (N116A, N116D, N116T and N116Q) were produced in Escherichia coli, by co-expressing the mature and pro domains separately, while the other 15 were not. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) at 25°C, the intrinsic k(cat)/K(m) value of N116D was 320% of that of the wild-type thermolysin (WT), and in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM) at pH 7.5 at 25°C, the k(cat)/K(m) value of N116D was 140% of that of WT, indicating that N116D exhibited higher activity than WT. N116Q exhibited similar activity as WT, and N116A and N116T exhibited reduced activities. The first-order rate constants, k(obs), of the thermal inactivation at 80°C were in the order N116A, N116D, N116T > N116Q > WT at all CaCl(2) concentrations examined (1-100 mM), indicating that all variants exhibited reduced stabilities. These results suggest that Asn116 plays an important role in the activity and stability of thermolysin presumably by stabilizing this ?-hairpin structure. PMID:22648563

Menach, Evans; Yasukawa, Kiyoshi; Inouye, Kuniyo

2012-05-29

380

Effects of site-directed mutagenesis of the loop residue of the N-terminal domain Gly117 of thermolysin on its catalytic activity.  

PubMed

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

2010-12-07

381

Effects of pH, temperature, and alcohols on the remarkable activation of thermolysin by salts.  

PubMed

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

1997-08-01

382

Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.  

PubMed

The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis. PMID:9548762

Veltman, O R; Eijsink, V G; Vriend, G; de Kreij, A; Venema, G; Van den Burg, B

1998-04-14

383

Immunoaffinity purification and characterization of leucine aminopeptidase from human liver.  

PubMed

Leucine aminopeptidase was purified from human liver cytosol to homogeneity, 1538-fold, with a yield of 84.4% by immunoaffinity chromatography. Increases in the activity and the stability of the enzyme were simultaneously observed during the purification procedure, suggesting the presence of some endogenous inhibitor in cytosol. The specific activity and Km value of the enzyme for L-leucine amide were found to be 58.00 mumol/min/mg of protein and 4.02 mM, respectively, at pH 8.0. The molecular weight of the enzyme was determined to be 360,000 by both polyacrylamide gradient gel electrophoresis and Sephadex G-200 gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of native and dimethyl suberimidate cross-linked enzyme indicate that the native enzyme has two subunits of Mr 53,000 (a) and 65,000 (b) and is a hexamer arranged as a trimer of dimers (3 X (a X b)). The optimum pH was 10.5, and the enzyme was stable in the pH range from 7.5-8.5. The enzyme was activated by divalent metal ions, especially by Mg2+ and Mn2+, with no change in Km value. The enzyme was inhibited by metal-chelating agents, indicating it to be a metalloenzyme. Amastatin and bestatin strongly inhibited the enzyme, but leupeptin did not. The enzyme had a broad substrate specificity toward oligopeptides and amino acid amides but had little or no activity toward chromogenic substrates. The enzyme also could hydrolyze natural substrates contained in liver cytosol and accordingly produce many kinds of amino acids commonly found in proteins. PMID:3733731

Kohno, H; Kanda, S; Kanno, T

1986-08-15

384

Development of Spray Dried Liposomal Dry Powder Inhaler of Dapsone  

PubMed Central

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

Chougule, Mahavir; Padhi, Bijay

2008-01-01

385

Leptin and leucine synergistically regulate protein metabolism in C2C12 myotubes and mouse skeletal muscles.  

PubMed

Leucine and leptin play important roles in regulating protein synthesis and degradation in skeletal muscles in vitro and in vivo. However, the objective of the present study was to determine whether leptin and leucine function synergistically in regulating protein metabolism of skeletal muscles. In the in vitro experiment, C2C12 myotubes were cultured for 2 h in the presence of 5 mm-leucine and/or 50 ng/ml of leptin. In the in vivo experiment, C57BL/6 and ob/ob mice were randomly assigned to be fed a non-purified diet supplemented with 3 % L-leucine or 2·04 % L-alanine (isonitrogenous control) for 14 d. Ob/ob mice were injected intraperitoneally with sterile PBS or recombinant mouse leptin (0·1 ?g/g body weight) for 14 d. In C57BL/6 mice, dietary leucine supplementation increased (P< 0·05) plasma leptin, leptin receptor expression and protein synthesis in skeletal muscles, but reduced (P< 0·05) plasma urea and protein degradation in skeletal muscles. Dietary leucine supplementation and leptin injection increased the relative weight of the gastrocnemius and soleus muscles in ob/ob mice. Moreover, leucine and leptin treatments stimulated (P< 0·05) protein synthesis and inhibited (P< 0·05) protein degradation in C2C12 myotubes and skeletal muscles of ob/ob mice. There were interactions (P< 0·05) between the leucine and leptin treatments with regard to protein metabolism in C2C12 myotubes and soleus muscles of ob/ob mice but not in the gastrocnemius muscles of ob/ob mice. Collectively, these results suggest that leptin and leucine synergistically regulate protein metabolism in skeletal muscles both in vitro and in vivo. PMID:23211060

Mao, Xiangbing; Zeng, Xiangfang; Huang, Zhimin; Wang, Junjun; Qiao, Shiyan

2012-12-05

386

Dry powder inhalation of macromolecules using novel PEG-co-polyester microparticle carriers.  

PubMed

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

2012-11-01

387

The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb.  

PubMed

Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. To acquire new knowledge that will assist in the development of therapeutic strategies for minimizing disuse atrophy, the present study was undertaken with the aim of identifying molecular mechanisms that mediate control of protein synthesis and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Male Sprague-Dawley rats were subjected to unilateral hindlimb immobilization for 1, 2, 3, or 7 days or served as nonimmobilized controls. Following an overnight fast, rats received either saline or L-leucine by oral gavage as a nutrient stimulus. Hindlimb skeletal muscles were extracted 30 min postgavage and analyzed for the rate of protein synthesis, mRNA expression, phosphorylation state of key proteins in the mTORC1 signaling pathway, and mTORC1 signaling repressors. In the basal state, mTORC1 signaling and protein synthesis were repressed within 24 h in the soleus of an immobilized compared with a nonimmobilized hindlimb. These responses were accompanied by a concomitant induction in expression of the mTORC1 repressors regulated in development and DNA damage responses (REDD) 1/2. The nutrient stimulus produced an elevation of similar magnitude in mTORC1 signaling in both the immobilized and nonimmobilized muscle. In contrast, phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) on Thr(229) and Thr(389) in response to the nutrient stimulus was severely blunted. Phosphorylation of Thr(229) by PDK1 is a prerequisite for phosphorylation of Thr(389) by mTORC1, suggesting that signaling through PDK1 is impaired in response to immobilization. In conclusion, the results show an immobilization-induced attenuation of mTORC1 signaling mediated by induction of REDD1/2 and defective p70S6K1 phosphorylation. PMID:23193052

Kelleher, Andrew R; Kimball, Scot R; Dennis, Michael D; Schilder, Rudolf J; Jefferson, Leonard S

2012-11-27

388

Pulmonary Immunization of Guinea Pigs with Diphtheria CRM-197 Antigen as Nanoparticle Aggregate Dry Powders Enhance Local and Systemic Immune Responses  

PubMed Central

This study establishes the immune response elicited in guinea pigs after pulmonary and parenteral immunizations with diphtheria CRM-197 antigen (CrmAg). Several spray-dried powders of formalin-treated/untreated CrmAg nanoaggregates with L-leucine were delivered to the lungs of guinea pigs. A control group consisting of alum with adsorbed CrmAg in saline was administered by intramuscular injection. Animals received three doses of powder vaccines containing 20 or 40 ?g of CrmAg. The serum IgG titers were measured for 16 weeks after the initial immunization; IgA titers were measured at the time of sacrifice in the broncho-alveolar lavage fluid. Further, toxin neutralization tests in naïve guinea pigs were performed for a few select serum samples. Histopathology of the lung tissues was conducted to evaluate inflammation or injury to the lung tissues. While the highest titer of serum IgG antibody was observed in guinea pigs immunized by the intramuscular route, those animals immunized with dry powder formulation by the pulmonary route, and without the adjuvant alum, exhibited high IgA titers. A pulmonary administered dry powder, compared to parenteral immunization, conferred complete protection in the toxin neutralization test. Mild inflammation was observed in lung tissues of animals receiving dry powder vaccines by the pulmonary route. Thus, administering novel CrmAg as dry powders to the lungs may be able to overcome some of the disadvantages observed with the existing diphtheria vaccine which is administered by the parenteral route. In addition, these powders will have the advantage of eliciting a high mucosal immune response in the lungs without using traditional adjuvants.

Muttil, Pavan; Pulliam, Brian; Garcia-Contreras, Lucila; Fallon, John Kevin; Wang, Chenchen; Edwards, David A.

2010-01-01

389

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

PubMed

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

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

2012-01-23

390

EMBRYONIC CHICK INTESTINE IN ORGAN CULTURE  

PubMed Central

Duodena from 20-day-old chick embryos can be maintained in large scale organ culture on specially designed stainless-steel grids in contact with serum-free medium for 48 h with excellent preservation of mucosal structure at both the light and electron microscope levels. Although mitotic rate was subnormal, several other factors attest to the essential viability of the cultured intestine: L-leucine incorporation into protein, as well as the synthesis of a specific vitamin D3-induced calcium-binding protein (CaBP), increased over a 48-h culture period, and the electropotential gradient across the intestine was maintained throughout the culture period as was a concentration gradient for calcium. The tissue responded to vitamin D3 in the medium by synthesizing the calcium-binding protein within 6 h and by exhibiting enhanced 45Ca uptake within 12–24 h. Concentrations of vitamin D3, or its 25-hydroxylated derivative, higher than necessary for CaBP induction, also increased the activity of alkaline phosphatase. The 1,25-dihydroxylated derivative of vitamin D3, at a level extremely potent in CaBP induction, did not stimulate alkaline phosphatase. Mucosal to serosal transport of 45Ca could also be measured in everted duodenal sacs, subsequent to culture under similar conditions, and was also increased by vitamin D3 in the medium. Other embryonic organs, esophagus, stomach, liver, pancreas, lung, skin, and muscle, did not produce CaBP in response to vitamin D3 in the culture medium. However, CaBP-synthesizing capacity was present in the entire intestinal tract, exclusive of the rectum. 59Fe and 32P uptake by cultured duodenum were also stimulated by vitamin D3. The system has proven quite useful in the study of the vitamin D-mediated calcium absorptive mechanism but should be applicable to the study of the absorption of other nutrients, drugs, hormones, etc., as well as other studies of intestinal function.

Corradino, R. A.

1973-01-01

391

Late failure of autologous marrow grafts in lethally irradiated dogs given anti-class II monoclonal antibody.  

PubMed

We established a model of canine marrow autografts after 9.2 Gy total body irradiation (TBI) to study the role of class II antigens in hematopoietic stem cell growth and differentiation. Twenty dogs were given 9.2 Gy TBI, marrow, and intravenous (IV) murine anti-class II monoclonal antibody (MoAb). Infusion of 0.6 mg/kg/d of MoAb H81.98.21, an IgG2a reactive with HLA-DR, on days 0 to 4 after TBI did not prevent initial engraftment, but dogs died with late graft failure. MoAb B1F6, an IgG2a reactive with HLA-DR + DP, had no adverse effect on engraftment, although both MoAbs detect antigens on stem cells. The critical time for the effect of MoAbs is the first 4 days after transplantation. Our findings argue against several pathogenetic mechanisms, including removal of MoAb-coated stem cells by the reticuloendothelial system (RES), canine complement-mediated cytotoxic effects on stem cells, antibody-dependent cellular cytotoxicity, and inactivation of MoAb-coated cells by dog anti-mouse antibody. To distinguish between MoAb-induced damage to microenvironment (ME)/accessory cells (AC) and late graft failure from a lack of pluripotent stem cells, three dogs were given TBI, a marrow autograft, and MoAb H81.98.21 on days 0 to 4; one, given thoracic duct cells on day 6, developed graft failure; the other two, given marrow depleted of AC by L-leucyl L-leucine o-methyl ester (Leu-Leu-OMe), had sustained grafts. Findings support the notion that originally transplanted pluripotent stem cells are no longer present on day 6 and that the ME is functional and able to support newly injected stem cells. PMID:1912590

Greinix, H T; Ladiges, W C; Graham, T C; Maslan, S; Raff, R F; Sandmaier, B M; Appelbaum, F R; Schuening, F G; Deeg, H J; Storb, R

1991-10-15

392

Effects of single-walled carbon nanotubes on soil microorganisms  

NASA Astrophysics Data System (ADS)

Single-walled carbon nanotubes (SWCNTs) are novel materials that have the potential to be used in various commercial fields due to their unique physicochemical properties. As a result of commercial development of nanotechnology, SWCNTs may be discharged to the soil environment with unknown consequences. However, there are as yet no data in the scientific literature that demonstrate the effects of SWCNTs on microbial function in soils. Therefore, we aimed to determine the effects of SWCNTs on soil microbial activity through a 2-week incubation study on urban soils supplemented with different concentrations of SWCNTs ranging from 0 to 1000 ?g CNT/g soil. Fluorometric test using fluorogenic substrates were employed for the measurement of several enzyme activities in soil samples. More specifically, we determined the changes in the activities of cellobiohydrolase, ?-1,4-glucosidase, ?-1,4-xylosidase, ?-1,4-N-acetylglucosaminidase, L-leucine aminopeptidase and acid phosphatase which play important roles in the carbon, nitrogen, and phosphorus cycles in response to the addition of SWCNTs. We found that microbial enzyme activities decreased as the concentrations of SWCNT added increased. The lowest enzyme activities were observed under 1000 ?g CNT/g soil. The overall pattern shows that enzyme activities decreased slightly in the first 2-3 days and increased in the later stage of the incubation. Our results suggest that relatively high concentrations of SWCNTs can inhibit microbial activities, and this may be due to microbial cell membrane damage caused by SWCNTs. However, further study needs to be conducted to determine the mechanism responsible for inhibitory effect of SWCNTs on soil microbial activity. It can be concluded that changes in the activities of extracellular enzymes can indicate the effect of SWCNTs on soil microorganisms and nutrient cycling.

Jin, L.; Chung, H.; Son, Y.

2011-12-01

393

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.

1997-01-01

394

Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans.  

PubMed

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 mumol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM beta-mercaptoethanol. The purified aminopeptidase (Mr 300000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of L-alanyl-p-nitroanilide exhibited a bell-shaped pH dependence for log Vmax/Km (pK1 = 6.35; pK2 = 8.50) while the log Vmax versus pH profile showed only an acid limb (pK = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity. PMID:6430350

Machuga, E J

1984-07-30

395

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

PubMed Central

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

Gogerty, David S.; Bobik, Thomas A.

2010-01-01

396

Chiral resolution of a racemic macrocyclic complex by recognition of one enantiomer over the other: structures and DFT calculations.  

PubMed

The enantiopure agents d- and l-leucine, selectively bind RR and SS enantiomers from a racemate [Ni(alpha-rac-L)](2+) to give {[Ni(RR-L)(d-HLeu)](ClO(4))(2)}(n) (Delta-) and {[Ni(SS-L)(l-HLeu)](ClO(4))(2)}(n) (Lambda-), respectively, and leave the corresponding uninteracted SS and RR enantiomers of [Ni(alpha-SS-L)](ClO(4))(2) (S-) and [Ni(alpha-RR-L)](ClO(4))(2) (R-). Occasionally, a few crystals of {[Ni(RR-L)(l-HLeu)](ClO(4))(2)}(n) (Delta-) and {[Ni(SS-L)(d-HLeu)](ClO(4))(2)}(n) (Lambda-) were found to have accreted with the crystals of Lambda-/R-, and Delta-/S-, respectively (the yields are less than 2%). The results of X-ray crystal structural analysis reveal that Delta- and Lambda-, S- and R-, and Delta- and Lambda- are enantiomers, in which Delta- and Delta- possess 1D right-handed helical chains, while Lambda- and Lambda- exhibit a motif of 1D left-handed helical chains. The results of DFT calculations reveal that the single-point energies of [Ni(RR-L)(d-HLeu)](2+)/[Ni(SS-L)(l-HLeu)](2+) in Delta-/Lambda- are 582 kJ mol(-1) lower than those of [Ni(RR-L)(l-HLeu)](2+)/[Ni(SS-L)(d-HLeu)](2+) in Delta-/Lambda-, demonstrating the favorable stereo-coordination environments of [Ni(alpha-RR-L)](2+) and [Ni(alpha-SS-L)](2+) towards d and l-HLeu, respectively. PMID:20422084

Ou, Guang-Chuan; Wang, Zi-Zhou; Yang, Li-Zi; Zhao, Cun-Yuan; Lu, Tong-Bu

2010-03-24

397

NLRP3 Inflammasome Activation in Retinal Pigment Epithelial Cells by Lysosomal Destabilization: Implications for Age-Related Macular Degeneration  

PubMed Central

Purpose. To evaluate the effect of lysosomal destabilization on NLRP3 inflammasome activation in RPE cells and to investigate the mechanisms by which inflammasome activation may contribute to the pathogenesis of age-related macular degeneration (AMD). Methods. Human ocular tissue sections from patients with geographic atrophy or neovascular AMD were stained for NLRP3 and compared to tissues from age-matched controls. Expression of the IL-1? precursor, pro-IL-1?, was induced in ARPE-19 cells by IL-1? treatment. Immunoblotting was performed to assess expression of NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and pro-IL-1? in ARPE-19 cells. Lysosomes were destabilized using the lysosomotropic agent L-leucyl-L-leucine methyl ester (Leu-Leu-OMe). Active caspase-1 was detected using FAM-YVAD-FMK, a fluorescent-labeled inhibitor of caspases (FLICA) specific for caspase-1. IL-1? was detected by immunoblotting and ELISA, and cytotoxicity was evaluated by LDH quantification. Results. RPE of eyes affected by geographic atrophy or neovascular AMD exhibited NLRP3 staining at lesion sites. ARPE-19 cells were found to express NLRP3, ASC, and procaspase-1. IL-1? dose-dependently induced pro-IL-1? expression in ARPE-19 cells. Lysosomal destabilization induced by Leu-Leu-OMe triggered caspase-1 activation, IL-1? secretion, and ARPE-19 cell death. Blocking Leu-Leu-OMe–induced lysosomal disruption with the compound Gly-Phe-CHN2 or inhibiting caspase-1 with Z-YVAD-FMK abrogated IL-1? release and ARPE-19 cytotoxicity. Conclusions. NLRP3 upregulation occurs in the RPE during the pathogenesis of advanced AMD, in both geographic atrophy and neovascular AMD. Destabilization of RPE lysosomes induces NLRP3 inflammasome activation, which may contribute to AMD pathology through the release of the proinflammatory cytokine IL-1? and through caspase-1-mediated cell death, known as “pyroptosis.”

Tseng, Wen Allen; Thein, Thuzar; Kinnunen, Kati; Lashkari, Kameran; Gregory, Meredith S.; D'Amore, Patricia A.; Ksander, Bruce R.

2013-01-01

398

Long Distance Translocation of Sucrose, Serine, Leucine, Lysine, and Carbon Dioxide Assimilates  

PubMed Central

To establish whether several amino acids were equally able to enter the phloem of oat (Avena sativa L.) plants and be transported, several 14C-labeled amino acids were applied individually to an abraded spot on a fully expanded source leaf. The base of an immature sink leaf was monitored with a GM tube for time and rate of arrival of radioactivity. Transport of 14C-sucrose and 14CO2 assimilates was measured for a comparison. The applied l-serine, l-lysine, and l-leucine, as well as sucrose, entered the phloem and were transported to the sink leaf at rates between 1.16 and 1.83 cm/min. Transport velocity for CO2 assimilates was 1.57 cm/min. A heat girdle near the top of the source leaf sheath blocked most transport, which indicated that transport was primarily through the phloem. Mass transfer rates for amino acids were only 3% as great as that for sucrose, suggesting different mechanisms of entry for sucrose than for amino acids into the phloem. The higher percentage of CO2 assimilates mobilized to the sink leaf was attributed to the greater surface area of minor veins accessible to loading, as compared to those compounds supplied via an abraded spot. Serine was extensively metabolized in the source leaf, and radioactive products in the sink leaf mirrored those in the source leaf. Most radioactivity of lysine and leucine remained within these compounds in the source, path, and sink tissues. We concluded that there was no barrier to entry of amino acids into the phloem and transport therein. Data do not suggest a specific mechanism for entry of amino acids into the phloem.

Peterson, David M.; Housley, Thomas L.; Schrader, Larry E.

1977-01-01

399

Myocardial oxidative metabolism and protein synthesis during mechanical circulatory support by extracorporeal membrane oxygenation.  

PubMed

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 [(13)C(6),(15)N]-L-leucine (3.7 mM) alone or with [2-(13)C]-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. PMID:23203964

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

2012-11-30

400

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.

2009-01-01

401

Effect of a leucine-supplemented diet on body composition changes in pregnant rats bearing Walker 256 tumor.  

PubMed

Cancer patients present high mobilization of host protein, with a decrease in lean body mass and body fat depletion occurring in parallel to neoplastic growth. Since leucine is one of the principal amino acids used by skeletal muscle for energy, we investigated the changes in body composition of pregnant tumor-bearing rats after a leucine-supplemented diet. Sixty pregnant Wistar rats divided into six groups were fed a normal protein diet (18%, N) or a leucine-supplemented diet (3% L-leucine, L). The pregnant groups were: control (CN), Walker 256 carcinoma-bearing rats (WN), control rats pair-fed with tumor-bearing rats (pfN), leucine-supplemented (CL), leucine-supplemented tumor-bearing (WL), and leucine-supplemented rats pair-fed with tumor-bearing rats (pfL). At the end of pregnancy, all animals were sacrificed and body weight and tumor and fetal weight were determined. The carcasses were then analyzed for water, fat and total, collagen and non-collagen nitrogen content. Carcass weight was reduced in the WN, WL, pfN and pfL groups compared to control. The lean body mass and total carcass nitrogen were reduced in both tumor-bearing groups. Despite tumor growth and a decrease in fetal weight, there was a slight decrease in collagen (7%) and non-collagen nitrogen (8%) in the WL group compared with the WN group which showed a decrease of 8 and 12%, respectively. Although the WL group presented severe tumor growth effects, total carcass nitrogen and non-collagen nitrogen were particularly higher in this leucine-supplemented group compared to the WN group. These data suggest that the leucine-supplemented diet had a beneficial effect, probably attenuating body wasting. PMID:11262583

Ventrucci, G; Mello, M A; Gomes-Marcondes, M C

2001-03-01

402

Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.  

PubMed Central

We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene. Images

Perry, J R; Basrai, M A; Steiner, H Y; Naider, F; Becker, J M

1994-01-01

403

Leucine-Derived Cyano Glucosides in Barley1  

PubMed Central

Barley (Hordeum vulgare) seedlings contain five cyano glucosides derived from the amino acid l-leucine (Leu). The chemical structure and the relative abundance of the cyano glucosides were investigated by liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses using spring barley cultivars with high, medium, and low cyanide potential. The barley cultivars showed a 10-fold difference in their cyano glucoside content, but the relative content of the individual cyano glucosides remained constant. Epiheterodendrin, the only cyanogenic glucoside present, comprised 12% to 18% of the total content of cyano glucosides. It is proposed that the aglycones of all five cyano glucosides are formed by the initial action of a cytochrome P450 enzyme of the CYP79 family converting l-Leu into Z-3-methylbutanal oxime and subsequent action of a less specific CYP71E enzyme converting the oxime into 3-methylbutyro nitrile and mediating subsequent hydroxylations at the ?-, as well as ?- and ?-, carbon atoms. Presence of cyano glucosides in the barley seedlings was restricted to leaf tissue, with 99% confined to the epidermis cell layers of the leaf blade. Microsomal preparations from epidermal cells were not able to convert l-[14C]Leu into the biosynthetic intermediate, Z-3-methylbutanal-oxime. This was only achieved using microsomal preparations from other cell types in the basal leaf segment, demonstrating translocation of the cyano glucosides to the epidermal cell layers after biosynthesis. A ?-glucosidase able to degrade epiheterodendrin was detected exclusively in yet a third compartment, the endosperm of the germinating seed. Therefore, in barley, a putative function of cyano glucosides in plant defense is not linked to cyanide release.

Nielsen, Kirsten Annette; Olsen, Carl Erik; Pontoppidan, Katrine; M?ller, Birger Lindberg

2002-01-01

404

Comparison of high-protein diets and leucine supplementation in the prevention of metabolic syndrome and related disorders in mice.  

PubMed

High-protein diets have been shown to promote weight loss, to improve glucose homeostasis and to increase energy expenditure and fat oxidation. We aimed to study whether leucine supplementation is able to mimic the alleviating effects of high-protein diets on metabolic syndrome parameters in mice fed high-fat diet. Male C57BL/6 mice were fed for 20 weeks with semisynthetic high-fat diets (20% w/w of fat) containing either an adequate (10% protein, AP) or high (50% protein, HP) amount of whey protein, or an AP diet supplemented with L-leucine corresponding to the leucine content of the HP diet (6% leucine, AP+L). Body weight and composition, energy expenditure, glucose tolerance, hepatic triacylglycerols (TG), plasma parameters as well as expression levels of mRNA and proteins in different tissues were measured. HP feeding resulted in decreased body weight, body fat and hepatic TG accumulation, as well as increased insulin sensitivity compared to AP. This was linked to an increased total and resting energy expenditure (REE), decreased feed energy efficiency, increased skeletal muscle (SM) protein synthesis, reduced hepatic lipogenesis and increased white fat lipolysis. Leucine supplementation had effects that were intermediate between HP and AP with regard to body composition, liver TG content, insulin sensitivity, REE and feed energy efficiency, and similar effects as HP on SM protein synthesis. However, neither HP nor AP+L showed an activation of the mammalian target of rapamycin pathway in SM. Leucine supplementation had no effect on liver lipogenesis and white fat lipolysis compared to AP. It is concluded that the essential amino acid leucine is able to mimic part but not all beneficial metabolic effects of HP diets. PMID:22405695

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

2012-03-08

405

Influence of leucine infusion on intracellular amino acids in humans.  

PubMed

A continuous intravenous infusion of L-leucine (300 mumols min-1) was given to 12 healthy females over a 2 1/2 h period. Arterial plasma concentrations of amino acids and the keto acids of the branched-chain amino acids (BCAA) were measured. In six subjects muscle biopsies were taken before and at the end of the infusion for determination of intracellular (i.c.) free amino acid concentrations, and leg exchange of amino acids was measured. During infusion the plasma level of leucine rose sixfold. Approximately 40% of the infused amount was taken up by muscle. Of this, half was accumulated intracellularly, where the free leucine concentration increased from basal 190 +/- 22 to 580 +/- 110 mumols l-1 ICW (intracellular water) at the end of infusion. The concentrations of most other amino acids, above all the other BCAA and the aromatic amino acids, decreased, by 17-48% in the i.c. pool and by 17-79% in plasma. The plasma level of ketoisocaproic acid (KIC), the keto acid of leucine, increased in parallel with that of leucine. The concentration of keto valine, ketoisovaleric acid (KIV), decreased by 75%, whereas the keto acid of isoleucine, ketomethylvaleric acid (KMV), was unchanged. Leg release of alanine decreased significantly, whereas the exchange of other amino acids were unchanged. Taken together, decreased i.c. and plasma concentrations but unchanged leg exchange of tyrosine and phenylalanine suggest i.c. accumulation of protein. It can be calculated that approximately 40% of the leucine taken up by muscle was accumulated in the intracellular free pool, some 20% could have been incorporated into protein and 40% was probably oxidized. PMID:2114990

Alvestrand, A; Hagenfeldt, L; Merli, M; Oureshi, A; Eriksson, L S

1990-06-01

406

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

PubMed

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P<0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [(3)H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P<0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM. PMID:23954667

Fukumoto, Shinya; Hanazono, Kiwamu; Fu, Dah-Renn; Endo, Yoshifumi; Kadosawa, Tsuyoshi; Iwano, Hidetomo; Uchide, Tsuyoshi

2013-08-14

407

Microbial Decomposition in Aquatic Environments: Combined Process of Extracellular Enzyme Activity and Substrate Uptake  

PubMed Central

The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with [3H]leucine as a marker for the leucine pool and l-leucine-4 methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. It was found that turnover rates of leucine (Tr, percent per hour) and hydrolysis rates of Leu-MCA (Hr, percent per hour), as well as the quotient Tr/Hr, reflect the impact of environmental conditions on decomposition processes at both sampling sites. The quotient Tr/Hr is interpreted as an indirect measurement of the pool size ratio (polymers/monomers), which may serve as an index of hydrolysis-uptake coupling in bacterial utilization of dissolved protein. Calculated on an annual average basis, turnover rates are ca. nine times higher than hydrolysis rates at the polluted station and ca. five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station.

Hoppe, Hans-Georg; Kim, Sang-Jin; Gocke, Klaus

1988-01-01

408

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

PubMed

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 death was partially inhibited by pan-caspase inhibitor zAVD-fmk and translational inhibitor cy