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

Microsoft Academic Search

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

Kang Fu Gu; Thomas Ming Swi Chang



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


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

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



Development of micellar electro kinetic chromatography for the separation and quantitation of L-valine, L-leucine, L-isoleucin and L-phenylalanine in human plasma and comparison with HPLC.  


Phenylketonuria (PKU) and Maple Syrup Urine Disease (MSUD) are two inborn metabolic diseases which are carried by autosomal recessive genes in man. These genetic errors result in accumulation of phenylalanine (in PKU) or valine, leucine and isoluecin (in MSUD). At high concentrations, amongst other problems, these amino acids cause mental retardation. However if detected early after birth, using special diets and other forms of therapy, mental abnormalities can be prevented. As a result in many countries screening of infants for MSUD and PKU, by measuring plasma amino acids has become a routine neonatal test. Capillary Electrophoresis (CE) assays have a number of advantages over the traditional chromatography techniques (such as GC or HPLC). These include low cost, high speed of analysis and high resolution. These characteristics, make CE an ideal method for the screening of inborn errors of metabolism. We developed a CE assay based on pre-column derivatisation of amino acids with phenylisothiocyanate. This conjugate has strong absorbance at 254 nm. CE was carried out using a Spectraphoresis 1000 instrument, fitted with 40 cm of a 25 microm capillary, at 17 degrees C. A running voltage of 18KV was used to separate the amino acid mixture in an electrophoretic buffer containing 45 mM imidazole, 6 mM borate and 208 mM SDS, fixed at pH 9 with 2-N-morpholino ethane sulfonic acid. The assay was calibrated using various concentrations of amino acid standards. LOD, LOQ, recovery, inter-day and intra-day variations of the assay were determined. Also, levels of the 4 amino acids in normal and abnormal plasma were determined and compared with HPLC. PMID:19070110

Darvish, M; Ebrahimi, S A; Ghadam, P



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

PubMed Central

Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of Propionibacterium freudenreichii, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of l-leucine, ?-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced ?-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal nor?-hydroxyisocaproic acid was detected. l-Isoleucine and l-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24°C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening. PMID:11823198

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



Molecular Structure of L-Isoleucine  

NSDL National Science Digital Library

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



Characterization of Bacillus thuringiensis l-Isoleucine Dioxygenase for Production of Useful Amino Acids?†  

PubMed Central

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

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



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:; Romanyuk, N. A.; Kiryk, Yu. I. [Franko National University (Ukraine)




E-print Network

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

Holler, E.



Jasmonoyl-L-isoleucine hydrolase 1 (JIH1) regulates jasmonoyl-L-isoleucine levels and attenuates plant defenses against herbivores.  


For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA-Ile permanently inactivates JA-Ilemediated signaling in plants, the alternative deactivation pathway of JA-Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl-L-isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA-Ile and IAA-Ala in vitro. When the herbivory-inducible NaJIH1 gene was silenced by RNA interference, JA-Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild-type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild-type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA-Ile-dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory-elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA-Ilehydroxylation and carboxylation of JA-Ile, rapidly attenuates the JA-Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature. PMID:22860609

Woldemariam, Melkamu G; Onkokesung, Nawaporn; Baldwin, Ian T; Galis, Ivan



Absence d'effets de l'injection de surcharges de L-valine et L-leucine sur les teneurs plasmatiques en insuline  

E-print Network

en insuline et en glucagon de l'agneau préruminant D. ATTAIX Isabelle PAPET J. GRIZARD M. ARNAL.) had no effect on insulin and glucagon levels in plasma from fed or 10-hour starved preruminant lambs-leucine sur les teneurs plasmatiques en insuline et en glucagon de l'agneau préruminant. De telles injections

Boyer, Edmond


Neomycin inhibition of (+)-7-iso-jasmonoyl-L-isoleucine accumulation and signaling.  


The majority of plant defenses against insect herbivores are coordinated by jasmonate (jasmonic acid, JA; (+)-7-iso-jasmonoyl-L-isoleucine, JA-Ile)-dependent signaling cascades. Insect feeding and mimicking herbivory by application of oral secretions (OS) from the insect induced both cytosolic Ca(2+) and jasmonate-phytohormone elevation in plants. Here it is shown that in Arabidopsis thaliana upon treatment with OS from lepidopteran Spodoptera littoralis larvae, the antibiotic neomycin selectively blocked the accumulation of OS-induced Ca(2+) elevation and level of the bioactive JA-Ile, in contrast to JA level. Furthermore, neomycin treatment affected the downstream expression of JA-Ile-responsive genes, VSP2 and LOX2, in Arabidopsis. The neomycin-dependent reduced JA-Ile level is partially due to increased CYP94B3 expression and subsequent JA-Ile turn-over to12-hydroxy-JA-Ile. It is neither due to the inhibition of the enzymatic conjugation process nor to substrate availability. Thus, blocking Ca(2+) elevation specifically controls JA-Ile accumulation and signaling, offering an insight into role of calcium in defense against insect herbivory. PMID:24859518

Vadassery, Jyothilakshmi; Reichelt, Michael; Jimenez-Aleman, Guillermo H; Boland, Wilhelm; Mithöfer, Axel



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



d- and l-Isoleucine Metabolism and Regulation of Their Pathways in Pseudomonas putida  

PubMed Central

Pseudomonas putida oxidized isoleucine to acetyl-coenzyme A (CoA) and propionyl-CoA by a pathway which involved deamination of d-isoleucine by oxidation and l-isoleucine by transamination, oxidative decarboxylation, and beta oxidation at the ethyl side chain. At least three separate inductive events were required to form all of the enzymes of the pathway: d-amino acid dehydrogenase was induced during growth in the presence of d-isoleucine; branched-chain keto dehydrogenase was induced during growth on 2-keto-3-methylvalerate and enzymes specific for isoleucine metabolism; tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were induced by growth on isoleucine, 2-keto-3-methylvalerate, 2-methylbutyrate, or tiglate. Tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were purified simultaneously by several enzyme concentration procedures, but were separated by isoelectric focusing. Isoelectric points, pH optima, substrate specificity, and requirements for enzyme action were determined for both enzymes. Evidence was obtained that the dehydrogenase catalyzed the oxidation of 2-methyl-3-hydroxybutyryl-CoA to 2-methylacetoacetyl-CoA. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase catalyzed the oxidation of 3-hydroxybutyryl-CoA, but l-3-hydroxyacyl-CoA dehydrogenase from pig heart did not catalyze the oxidation of 2-methyl-3-hydroxybutyryl-CoA; therefore, they appeared to be different dehydrogenases. Furthermore, growth on tiglate resulted in the induction of tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase, but these two enzymes were not induced during growth on crotonate or 3-hydroxybutyrate. PMID:4150713

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



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


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



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


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



Deuterium isotope effects on 13C chemical shifts of lithium salts of Schiff bases amino acids  

NASA Astrophysics Data System (ADS)

The deuterium isotope effects on 13C chemical shift of Schiff bases lithium salts, derivatives of amino acids (glycine, L-alanine, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-methionine) and 2-hydroxynaphthaldehyde in D 2O as well as UV-vis spectra in different solvents have been measured. The results have shown that the lithium salts of the Schiff bases exist in the proton transferred NH form with intramolecular hydrogen bond in water.

Rozwadowski, Z.



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



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


An L-leucine aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC, 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



Jasmonoyl-l-Isoleucine Coordinates Metabolic Networks Required for Anthesis and Floral Attractant Emission in Wild Tobacco (Nicotiana attenuata).  


Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-l-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study. PMID:25326292

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



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


The present study reports on the absorption and emission properties of the Schiff bases formed by pyridoxal and pyridoxal 5'-phosphate withL-isoleucine in aqueous solutions. Species protonated at the imine and ring nitrogen are the most fluorescent in both Schiff bases with a quantum yield of 0.02, i.e., 20-fold the value found for species in alkaline solutions. In agreement with other studies, species protonated at the imine nitrogen shows an emission around 500 nm upon excitation at 415 nm. In contrast to previous observations on other PLP Schiff bases, emissions at 560 nm (PL-Ile) and 540 nm (PLP-Ile) are observed upon excitation at 365 and 415 nm, respectively. The emission at 470 nm found in PLP-Ile Schiff base upon excitation at 355 nm is ascribed to a multipolar monoprotonated species. An estimation for the pK a of the imine in the excited state ( ? 8.5) for both Schiff bases is also reached. Our results suggest that fast protonation reactions on the excited state are responsible for the observed fluorescence. These effects, in which the hydrogen bond and the phosphate group seem to play a role, could be extended to understanding coenzyme environments in proteins. PMID:24226991

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



Central administration of dipeptides, beta-alanyl-BCAAs, induces hyperactivity in chicks  

PubMed Central

Background Carnosine (?-alanyl-L-histidine) is a putative neurotransmitter and has a possible role in neuron-glia cell interactions. Previously, we reported that carnosine induced hyperactivity in chicks when intracerebroventricularly (i.c.v.) administered. In the present study, we focused on other ?-alanyl dipeptides to determine if they have novel functions. Results In Experiment 1, i.c.v. injection of ?-alanyl-L-leucine, but not ?-alanyl-glycine, induced hyperactivity behavior as observed with carnosine. Both carnosine and ?-alanyl-L-leucine stimulated corticosterone release. Thus, dipeptides of ?-alanyl-branched chain amino acids were compared in Experiment 2. The i.c.v. injection of ?-alanyl-L-isoleucine caused a similar response as ?-alanyl-L-leucine, but ?-alanyl-L-valine was somewhat less effective than the other two dipeptides. ?-Alanyl-L-leucine strongly stimulated, and the other two dipeptides tended to stimulate, corticosterone release. Conclusion These results suggest that central ?-alanyl-branched chain amino acid stimulates activity in chicks through the hypothalamus-pituitary-adrenal axis. We named ?-alanyl-L-leucine, ?-alanyl-L-isoleucine and ?-alanyl-L-valine as Excitin-1, Excitin-2 and Excitin-3, respectively. PMID:17537271

Tsuneyoshi, Yousuke; Tomonaga, Shozo; Asechi, Mari; Morishita, Koji; Denbow, D Michael; Furuse, Mitsuhiro



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

NASA Astrophysics Data System (ADS)

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

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



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



Volumetric properties of l-alanine, and l-valine in aqueous sucrose solutions at T = (288.15 and 308.15) K  

Microsoft Academic Search

Densities of l-alanine, and l-valine have been measured at T=(288.15 and 308.15) K in aqueous sucrose solutions ranging from pure water to 25 mass% of sucrose. From these densities, apparent molar volumes (V?) and limiting partial molar volumes (V??) of each amino acid in various aqueous sucrose solutions have been evaluated. These data were combined with the earlier reported V??

Amalendu Pal; Suresh Kumar



Synthesis and Toxicological Evaluation of a Chitosan-l-Leucine Conjugate for Pulmonary Drug Delivery Applications.  


Herein are reported the synthesis of a conjugate of chitosan with l-leucine, the preparation of nanoparticles from both chitosan and the conjugate for use in pulmonary drug delivery, and the in vitro evaluation of toxicity and inflammatory effects of both the polymers and their nanoparticles on the bronchial epithelial cell line, BEAS-2B. The nanoparticles, successfully prepared both from chitosan and the conjugate, had a diameter in the range of 10-30 nm. The polymers and their nanoparticles were tested for their effects on cell viability by MTT assay, on trans-epithelial permeability by using sodium fluorescein as a fluid phase marker, and on IL-8 secretion by ELISA. The conjugate nanoparticles had a low overall toxicity (IC50 = 2 mg/mL following 48 h exposure; no induction of IL-8 release at 0.5 mg/mL concentration), suggesting that they may be safe for pulmonary drug delivery applications. PMID:25191851

Muhsin, Mohammad D A; George, Graeme; Beagley, Kenneth; Ferro, Vito; Armitage, Charles; Islam, Nazrul



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

PubMed Central

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.



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

NASA Astrophysics Data System (ADS)

Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM K55.c, was coordinated by the Bureau International des Poids et Mesures (BIPM) in 2012. Twenty National Measurement Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of valine present as the main component in the comparison sample for CCQM-K55.c. The comparison samples were prepared from analytical grade L-valine purchased from a commercial supplier and used as provided without further treatment or purification. Valine was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of organic compounds of low structural complexity [molecular weight range 100-300] and high polarity (pKOW > -2). The KCRV for the valine content of the material was 992.0 mg/g with a combined standard uncertainty of 0.3 mg/g. The key comparison reference value (KCRV) was assigned by combination of KCRVs assigned from participant results for each orthogonal impurity class. The relative expanded uncertainties reported by laboratories having results consistent with the KCRV ranged from 1 mg/g to 6 mg/g when using mass balance based approaches alone, 2 mg/g to 7 mg/g using quantitative 1H NMR (qNMR) based approaches and from 1 mg/g to 2.5 mg/g when a result obtained by a mass balance method was combined with a separate qNMR result. The material provided several analytical challenges. In addition to the need to identify and quantify various related amino acid impurities including leucine, isoleucine, alanine and ?-amino butyrate, care was required to select appropriate conditions for performing Karl Fischer titration assay for water content to avoid bias due to in situ formation of water by self-condensation under the assay conditions. It also proved to be a challenging compound for purity assignment by qNMR techniques. There was overall excellent agreement between participants in the identification and the quantification of the total and individual related structure impurities, water content, residual solvent and total non-volatile content of the sample. Appropriate technical justifications were developed to rationalise observed discrepancies in the limited cases where methodology differences led to inconsistent results. The comparison demonstrated that to perform a qNMR purity assignment the selection of appropriate parameters and an understanding of their potential influence on the assigned value is critical for reliable implementation of the method, particularly when one or more of the peaks to be quantified consist of complex multiplet signals. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

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



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


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

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



Jasmonoyl-l-Isoleucine Coordinates Metabolic Networks Required for Anthesis and Floral Attractant Emission in Wild Tobacco (Nicotiana attenuata)[C][W][OPEN  

PubMed Central

Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-l-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study. PMID:25326292

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



Functional Heterogeneity of Single Pancreatic ?-Cells Stimulated by L-Leucine and the Methyl Ester of Succinic or Glutamic Acid  

Microsoft Academic Search

Single pancreatic ?-cells exposed to D-glucose in the absence or presence of L-leucine and to the amino acid in the absence or presence of either the monomethyl ester of succinic acid (SME) or the dimethyl ester of glutamic acid (SME) were examined in a reverse hemolytic plaque assay for insulin release. At a D-glucose concentration of 11.1 mM, plaque-forming cells

M. Hiriart; M. C. Sanchezsoto; M. C. Ramirezmedeles; W. J. Malaisse



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. PMID:24098154

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



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


Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner. PMID:25098371

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



Structure of aureobasidin A.  


Aureobasidin A, a new antifungal antibiotic, was isolated from the culture medium of Aureobasidium pullulans R106. Aureobasidin A was a cyclic depsipeptide consisting of eight alpha-amino acid units and one hydroxy acid unit. The structures of the units were found by acid hydrolysis of the antibiotic to be 2(R)-hydroxy-3(R)-methylpentanoic acid, beta-hydroxy-N-methyl-L-valine, N-methyl-L-valine, L-proline, allo-L-isoleucine, N-methyl-L-phenylalanine, L-leucine, and L-phenyl-alanine. The sequence of the units was identified by NMR and FAB-MS of the products from the alkaline hydrolysis of aureobasidin A. PMID:1938614

Ikai, K; Takesako, K; Shiomi, K; Moriguchi, M; Umeda, Y; Yamamoto, J; Kato, I; Naganawa, H



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



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


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

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



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

NASA Astrophysics Data System (ADS)

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

Tamasi, Gabriella; Casolaro, Mario; Cini, Renzo



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

PubMed Central

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

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



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


The mononuclear Schiff base complexes of the type, [ML(CH(3)OH)(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, (1)H NMR and (13)C 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. PMID:21807556

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



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

Microsoft Academic Search

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

Shadpour Mallakpour; Fatemeh Rafiemanzelat



Effects of L-leucine on the insulin production, oxidative metabolism and mitochondrial ultrastructure of isolated mouse pancreatic islets in tissue culture  

Microsoft Academic Search

Summary  This study was performed to evaluate whether L-leucine is able to relieve the structural and functional alterations previously described in pancreatic islets exposed in vitro for a prolonged time to a subnormal glucose concentration (3.3 mM). It was found that both the impairment of secretion and the decreased rate of biosynthesis of insulin characteristic, of islets cultured for one week

A. Andersson; J. Höiriis-Nielsen; L. A. H. Borg



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


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

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



Reduction of large neutral amino acid levels in plasma and brain of hyperleucinemic rats.  


Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly known. In the present study we investigated the effect of acute hyperleucinemia on plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected subcutaneously with 6 micromol L-leucine per gram body weight. Controls received saline in the same volumes. The animals were sacrificed 30--120 min after injection, blood was collected and their brain rapidly removed and homogenized. The amino acid concentrations were determined by HPLC using orthophtaldialdehyde for derivatization and fluorescence for detection. The results showed significant reductions of the large neutral amino acids (LNAA) L-phenylalanine, L-tyrosine, L-isoleucine, L-valine and L-methionine, as well as L-alanine, L-serine and L-histidine in plasma and of L-phenylalanine, L-isoleucine, L-valine and L-methionine in brain, as compared to controls. In vitro experiments using brain slices to study the influence of leucine on amino acid transport and protein synthesis were also carried out. L-Leucine strongly inhibited [14C]-L-phenylalanine transport into brain, as well as the incorporation of the [14C]-amino acid mixture, [14C]-L-phenylalanine and [14C]-L-lysine into the brain proteins. Although additional studies are necessary to evaluate the importance of these effects for MSUD, considering previous findings of reduced levels of LNAA in plasma and CSF of MSUD patients during crises, it may be speculated that a decrease of essential amino acids in brain may lead to reduction of protein and neurotransmiter synthesis in this disorder. PMID:11248401

Araújo, P; Wassermann, G F; Tallini, K; Furlanetto, V; Vargas, C R; Wannmacher, C M; Dutra-Filho, C S; Wyse, A T; Wajner, M



Proton-symport of L-valine in plasma membrane vesicles isolated from leaves of the wild-type and the Val(r)-2 mutant of Nicotiana tabacum L.  


Transport of amino acids across the plasma membranes of various cell types is a key process in controlling the nitrogen balance of leaves. We studied the transport of the neutral amino acid L-valine into plasma membrane vesicles obtained by aqueous polymer two-phase partitioning of a microsomal fraction isolated from leaves of the wild-type and the Val(r)-2 mutant of tobacco (Nicotiana tabacum L.). Initial influxes were determined after the imposition of a pH-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. The initial magnitudes of the imposed gradients were DeltapH=2 and Deltapsi=-68 mV. In vesicles from the wild-type, the DeltapH-dependent valine influx could be analysed into a high-affinity (Km approximately 20 microM) and a low-affinity (Km approximately 3 mM) component. The influx of valine by the low-affinity system was stimulated about twofold, and that by the high-affinity system more than sixfold by the imposition of Deltapsi. This strong stimulation of the high-affinity system may indicate that it transports 2H+/amino acid. In the Val(r)-2 mutant the high-affinity component appeared to be completely absent. PMID:11092905

Borstlap, A C; Schuurmans, J A



A study of androgen-stimulated L-leucine transport by the intestine of rainbow trout (Salmo gairdneri Richardson) in vitro.  


The intestinal transport of L-leucine and fluid was studied in response to in vitro administration of 17 alpha-methyltestosterone (MT; 50 micrograms/ml) and 11-ketotestosterone (KT; 10 micrograms/ml), using strips of rainbow trout intestine incubated for 60 min in vitro. Both MT and KT increased the intestinal absorption of the amino acid without significantly affecting fluid transport. Addition of ouabain, 2,4-dinitrophenol (DNP) and sodium fluoride (NaF) reduced the intestinal transport of leucine. It would appear that ouabain and DNP significantly impair the action of MT, although their effect on KT is uncertain. It is suggested that the steroid-induced leucine transport during the latter 40 min of incubation might be, in part, the result of an increased activity of Na+,K+-ATPase. PMID:6148177

Habibi, H R; Ince, B W



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

Microsoft Academic Search

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

Fatemeh Rafiemanzelat; Elahe Abdollahi


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


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

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



Lattice resolution and solution kinetics on surfaces of amino acid crystals: an atomic force microscope study  

NASA Astrophysics Data System (ADS)

We report atomic force microscopy (AFM) results on six amino acid crystal surfaces: glycine, L-aspartic acid, L-valine, L-isoleucine, L-leucine, and L-phenylalanine. Samples were grown by slow evaporation of concentrated aqueous solutions. All samples contained crystalline areas where the AFM showed extended molecularly flat sheets (up to hundreds of nm in size) separated by steps a single molecule thick. The ordered lattice of each amino acid could be imaged on the sheets. Images revealed periodicities corresponding to bulk terminations in most cases, as well as other periodicities which probably correspond to molecular structure within the unit cell. Step motion kinetics were also imaged in situ during dissolution of L-leucine in flowing propanol. Steps oriented along the <010> direction traveled with speeds that were independent of both interstep distance and solvent flow rate for flow rates above 20 ?l/s, indicating a reaction rate limited process. Orthogonal bends along the <001> direction moved at speeds one to ten times that of steps, with narrow bends moving faster than wide. We speculate that these speed differences were caused by anisotropy in reaction kinetics coupled with partially saturated boundary layers near wide bends.

Manne, S.; Cleveland, J. P.; Stucky, G. D.; Hansma, P. K.



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



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


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

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



Behavioral responses of crayfish (Orconectes virilis andOrconectes rusticus) to chemical feeding stimulants.  


We conducted two experiments to assess how chemical stimuli affect feeding behavior, grooming, and walking in the crayfishesOrconectes virilis andOrconectes rusticus. In the first experiment,O. virilis was tested with 29 amino acids; in the second experiment,0. rusticus was tested with 12 amino acids, 13 additional single compounds, and two six-compound mixtures. InO. virilis, the following amino acids, in order of potency, elicited feeding movements:L-isoleucine, glycine, hydroxy-L-proline,L-glutamate,L-valine, and B-alanine. Grooming increased in response toL-phenylalanine,L-tryptophan,L-tyrosine,L-leucine,L-methionine, and D-aspartate. InO. rusticus, both mixtures and the following single compounds, in order of potency, elicited feeding movements: cellobiose, sucrose, glycine, maltose, glycogen, nicotinic acid methyl ester, putrescine, andL-glutamate. Grooming increased in response to putrescine only, and walking increased in response to glycogen only. The responsiveness of these crayfishes to a wide variety of chemicals may reflect the omnivorous foraging habits of these crustaceans. PMID:24276999

Tierney, A J; Atema, J



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


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



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


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

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



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. PMID:19537965

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



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


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



Dissecting Complex Metabolic Integration Provides Direct Genetic Evidence for CodY Activation by Guanine Nucleotides?  

PubMed Central

The global regulator CodY controls the expression of dozens of metabolic genes and genes mediating adaptation to nutrient availability in many low-G+C Gram-positive bacteria. Branched-chain amino acids l-isoleucine, l-leucine, and l-valine (ILV) activate CodY both in vivo and in vitro, and genes that direct their synthesis (ilv, ybgE, and ywaA) are highly repressed by CodY, creating a potential negative feedback loop. The nucleoside triphosphate GTP also activates CodY in vitro, but the evidence for activation by GTP in vivo is limited and indirect. We constructed a Bacillus subtilis strain (ybgE bcd ywaA) that is unable to convert branched-chain ?-keto acids to ILV or to use ILV as a precursor for branched-chain fatty acid synthesis. Unexpectedly, the strain was not viable on rich medium. Supplementing rich medium with short, branched-chain fatty acids or derepressing expression of genes for de novo ILV synthesis bypassed the original lethality, restoring growth and showing that the lack of viability was due to insufficient intracellular production of the precursors of branched-chain fatty acids. Spontaneous extragenic suppressor mutants that arose in the triple mutant population proved to have additional mutations in guaA or guaB or codY. Expression of ILV biosynthetic genes in codY mutants was increased. The gua mutations caused guanine/guanosine auxotrophy and led to partial derepression of direct CodY-repressed targets, including ILV biosynthetic genes, under conditions similar to those that caused the original lethality. We conclude that a guanine derivative, most likely GTP, controls CodY activity in vivo. PMID:21856856

Brinsmade, Shaun R.; Sonenshein, Abraham L.



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


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

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



2-Methylbutyryl-coenzyme A dehydrogenase deficiency: a new inborn error of L-isoleucine metabolism.  


An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcamitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans. PMID:10832746

Gibson, K M; Burlingame, T G; Hogema, B; Jakobs, C; Schutgens, R B; Millington, D; Roe, C R; Roe, D S; Sweetman, L; Steiner, R D; Linck, L; Pohowalla, P; Sacks, M; Kiss, D; Rinaldo, P; Vockley, J



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

Microsoft Academic Search

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

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



Solubilities of amino acids in water at various pH values under 298.15 K  

Microsoft Academic Search

In this study the solubility of amino acids in water for various pH values at 298.15K is investigated. A colorimetric method using a spectrophotometer was employed to measure solubility data for the five amino acids, dl-alanine, l-leucine, l-isoleucine, l-serine and dl-phenylalanine, in water. The pH effect, changing from 2 to 10 adjusted by the addition of HCl or NaOH, has

Hsieng-Cheng Tseng; Ching-Yi Lee; Wen-Lu Weng; I-Min Shiah



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. PMID:17263313

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



Effect of glycyl dipeptides on the micellar behavior of gemini surfactant: A conductometric and fluorescence spectroscopic study  

Microsoft Academic Search

The effect of glycyl dipeptides (glycylglycine, glycyl-L-valine, and glycyl-L-leucine) on the micellar properties of gemini surfactant pentamethylene-1,5-bis(dodecyldimethylammonium bromide) (12-5-12) has been investigated by means of conductivity and fluorescence spectroscopy. The results obtained from conductivity show that the effect of glycyl dipeptides depends upon their nature and concentration, as well as the temperature. The values of critical micelle concentration (cmc) of

Zhenning Yan; Yu Li; Xiaolan Wang; Junying Dan; Jianji Wang



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


This work reports the preparation of water-soluble leucine capped gold nanoparticles by two single-step synthesis methods. The first procedure involves a citrate reduction approach where the citrate is used as reducing agent and leucine as capping/stabilizing agent. Different sizes of gold nanoparticles, citrate reduced and stabilized by leucine, Leu-AuNPs-C, with the mean diameters in the range of 21-56 nm, were obtained by varying the macroscopic parameters such as: concentration of the gold precursor solution, Au (III):citrate molar ratio and leucine pH. In the second procedure, leucine acts both as reducing and stabilizing agent, allowing us to obtain spherical gold nanoparticles, Leu-AuNPs, with a majority of 80 % (with the mean diameter of 63 nm). This proves that leucine is an appropriate reductant for the formation of water-soluble and stable gold nanoparticles colloids. The characterization of the leucine coated gold nanoparticles was carried out by TEM, UV-Vis and FT-IR analysis. The cytotoxic effect of Leu-AuNPs-C and Leu-AuNPs was also evaluated. PMID:25092048

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



Combination of amino acids reduces pigmentation in B16F0 melanoma cells.  


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

Ishikawa, Masago; Kawase, Ichiro; Ishii, Fumio



Isolation, purification, and characterization of phenylpyruvate transaminating enzymes of Erwinia carotovora.  


Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively. PMID:22339639

Paloyan, A M; Hambardzumyan, A A; Halebyan, Gh P



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



N-(tert-butoxycarbonyl)-O-allyl-L-seryl-?-aminoisobutyryl-L-valine methyl ester: a protected tripeptide with an allylated serine residue.  


The title compound [systematic name (6S,12S)-methyl 6-(allyloxymethyl)-12-isopropyl-2,2,9,9-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oate], C(21)H(37)N(3)O(7), containing the little studied O-allyl-L-serine residue [Ser(All)], crystallizes in the monoclinic space group C2 with one molecule in the asymmetric unit. The compound is an analogue of the Ser140-Val142 segment of the water channel aquaporin-4 (AQP4). It forms a distorted type-II ?-turn with a P(II)-3(10L)-P(II) backbone conformation (P(II) is polyproline II). The overall backbone conformation is markedly different from that of the CO(Pro139)-Val142 stretch of rat AQP4, but is quite similar to the corresponding segment of human AQP4, despite significant differences at the level of the individual residues. The side chain of the Ser(All) residue adopts a gauche conformation relative to the backbone CO-C(?) and C(?)-N bonds. The H atoms of the two CH(2) groups in the Ser(All) side chain are almost eclipsed. The crystal packing of the title compound is divided into one-molecule-thick layers, each layer having a hydrophilic core and distinct hydrophobic interfaces on either side. PMID:21881187

Gebreslasie, Hadgu Girmay; Jacobsen, Øyvind; Görbitz, Carl Henrik



Stereo and regioselectivity in ''Activated'' tritium reactions  

SciTech Connect

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

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



Effect of Cationic Micelles on the Kinetics of Interaction of Ninhydrin with l Leucine and l Phenylalanine  

Microsoft Academic Search

The effect of cationic micelles of cetyltrimethylammonium bromide (CTAB) andN-cetylpyridinium bromide (CPB) on the interaction ofl-leucine andl-phenylalanine with ninhydrin have been studied at 70°C. Both surfactants strongly catalyze the reactions. The reaction rates are higher in CTAB micelles than in CPB micelles. Quantitative kinetic analysis has been performed on the basis of a pseudo-phase model. The influence of different salts

Jamil K. J. Salem; Sanjeev Kumar; Zaheer Khan



L-Leucine and l-phenylalanine induced insulin release and the influence of D-glucose  

Microsoft Academic Search

Summary  The perfused pancreas was used for kinetic studies of leucine and phenylalanine induced insulin secretion. In a glucose-free medium, leucine stimulated insulin release over a wide range (5–80 mM). Substimulatory levels of glucose (3 mM) potentiated the leucine induced insulin release. At higher glucose concentrations (20 mM), the rate of leucine-provoked insulin release was not further enhanced. Phenylalanine (5–80 mM)

R. Landgraf; M. M. C. Landgraf-Leurs; R. Hörl



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


Leucine/isoleucine side chain polymers are of interest due to their hydrophobicity and reported role in the formation of ?-helical structures. The synthesis and reversible addition-fragmentation chain transfer (RAFT) polymerization of amino acid-based chiral monomers, namely Boc-L-leucine methacryloyloxyethyl ester (Boc-L-Leu-HEMA, 1a), Boc-L-leucine acryloyloxyethyl ester (Boc-L-Leu-HEA, 1b), Boc-L-isoleucine methacryloyloxyethyl ester (Boc-L-Ile-HEMA, 1c), and Boc-L-isoleucine acryloyloxyethyl ester (Boc-L-Ile-HEA, 1d), are reported. The controlled nature of the polymerization of the said chiral monomers in N, N-dimethylformamide (DMF) at 70 °C is evident from the formation of narrow polydisperse polymers, the molecular weight controlled by the monomer/chain transfer agent (CTA) molar ratio and the linear relationship between molecular weight and monomer conversion. The resulting well-defined polymers were used as macro-CTAs to prepare corresponding diblock copolymers by RAFT polymerization of methyl (meth)acrylate monomers. Deprotection of Boc groups in the homopolymers and block copolymers under acidic conditions produced cationic, pH-responsive polymers with primary amine moieties at the side chains. The optical activity of the homopolymers and block copolymers were studied using circular dichroism (CD) spectroscopy and specific rotation measurements. The self-assembling nature of the block copolymers to produce highly ordered structures was illustrated through dynamic light scattering (DLS) and atomic force microscopy (AFM) studies. The side chain amine functionality instills pH-responsive behavior, which makes these cationic polymers attractive candidates for drug delivery applications, as well as for conjugation of biomolecules. PMID:23346856

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



New salts of amino acids with dimeric cations  

NASA Astrophysics Data System (ADS)

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

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



Age-related increase in alanine aminotransferase correlates with elevated levels of plasma amino acids, decanoylcarnitine, Lp-PLA2 Activity, oxidative stress, and arterial stiffness.  


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

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



Precursor directed biosynthesis of aureobasidins.  


The antifungal antibiotic aureobasidin A (AbA) is a cyclic depsipeptide composed of eight amino acids and a hydroxy acid. New Ab analogs were produced by feeding various amino acids to Aureobasidium pullulans R 106 c-712 in a chemically-defined medium containing glucose and ammonium sulfate. The constituent amino acids of AbA at positions 3 (L-phenylalanine), 4 (N-methyl-L-phenylalanine), 5 (L-proline), 6 (L-allo-isoleucine) and 8 (L-leucine) were replaced by respective analogous amino acids such as o-fluoro-L-phenylalanine, 4-hydroxy-L-proline, L-norleucine and L-norvaline, resulting in the production of eight new Ab analogs. This is the first paper to describe amino acid replacements at positions 3, 5 and 8. L-[1-13C]-Valine exogenously added was incorporated into the three valine-related moieties of AbA at positions 2, 7 (both N-methyl-L-valine) and 9 (beta-hydroxy-N-methyl-L-valine), but these moieties were never replaced by exogenous amino acid analogs. The comparative antifungal activities of AbA and the eight new Ab analogs were determined. PMID:8784430

Takesako, K; Mizutani, S; Sakakibara, H; Endo, M; Yoshikawa, Y; Masuda, T; Sono-Koyama, E; Kato, I



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. PMID:16957230

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



Characterization of (R)-2-hydroxyisocaproate dehydrogenase and a family III coenzyme A transferase involved in reduction of L-leucine to isocaproate by Clostridium difficile.  


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 CO(2) 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 (K(m) = 68 muM, 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. PMID:16957230

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



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

PubMed Central

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

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



Effect of phosphate and amino acids on echinomycin biosynthesis by Streptomyces echinatus.  

PubMed Central

Streptomyces echinatus produces only echinomycin (quinomycin A), in contrast to other streptomycetes, which produce families of quinoxaline antibiotics differing in the amino acid composition of the oligopeptide (quinomycins A, B, B0, C, D, and E) or the structure of the sulfur-containing cross bridge (triostins A, B, and C). Attempts were made to establish conditions for directed biosynthesis with S. echinatus. The lability of the peptide lactone to alkaline pH was obviated by using high levels of phosphate or HEPES [4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid] buffer in the production medium. Maintaining the pH below 7.5 resulted in an apparent stimulation of production. Amino acids which serve as structural components or as precursors of echinomycin were employed singly or in combination with nitrate in a chemically defined medium. Based on specific yield (micrograms of echinomycin per milligram of mycelia [dry weight]), D- and L-serine, D-alanine, L-valine, and L-phenylalanine produced equivalent yields of antibiotic which were approximately twofold greater than yields obtained with nitrate alone. In contrast, L-alanine, beta-alanine, and L-threonine produced a three- to fourfold stimulation of production. Although the other amino acids diminished antibiotic production, L-isoleucine, which ostensibly was inhibitory to production, supported the accumulation of a quinoxaline antibiotic in which the cross-bridge sulfur lacked a methyl group. PMID:6660849

Formica, J V; Waring, M J



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


The leucine-to-glutamate (Leu?Glu) pathway, which metabolizes the carbon atoms of l-leucine to form l-glutamate, was studied by incubation of rat tissue segments with l-[U-(14)C]leucine and estimation of the [(14)C]glutamate formed. Metabolism of the leucine carbon chain occurs in most rat tissues, but maximal activity of the Leu?Glu pathway for glutamate formation is limited to the thoracic aorta and pancreas. In rat aorta, the Leu?Glu pathway functions to relax the underlying smooth muscle; its functions in the pancreas are unknown. This report characterizes the Leu?Glu pathway of rat pancreas and develops methods to examine its functions. Pancreatic segments effect net formation of glutamate on incubation with l-leucine, l-glutamine, or a mix of 18 other plasma amino acids at their concentrations in normal rat plasma. Glutamate formed from leucine remains mainly in the tissue, whereas that from glutamine enters the medium. The pancreatic Leu?Glu pathway uses the leucine carbons for net glutamate formation; the ?-amino group is not used; the stoichiometry is as follows: 1 mol of leucine yields 2 mol of glutamate (2 leucine carbons per glutamate) plus 2 mol of CO2. Comparison of the Leu?Glu pathway in preparations of whole pancreatic segments, isolated acini, and islets of Langerhans localizes it in the acini; relatively high activity is found in cultures of the AR42J cell line and very little in the INS-1 832/13 cell line. Pancreatic tissue glutamate concentration is homeostatically regulated in the range of ?1-3 ?mol/g wet wt. l-Valine and leucine ethyl, benzyl, and tert-butyl esters inhibit the Leu?Glu pathway without decreasing tissue total glutamate. PMID:24699330

Schachter, David; Buteau, Jean



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. PMID:23354716

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



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


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

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



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

NASA Astrophysics Data System (ADS)

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

Boghaei, Davar M.; Gharagozlou, Mehrnaz



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



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. PMID:23991355

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



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


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

Chatonnet, Pascal; Fleury, Antoine; Boutou, Stéphane



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


The idea of extraterrestrial delivery of organic matter to the early Earth is especially attractive at present and is strongly supported by the detection of a large variety of organic compounds, including amino acids and nucleobases, in carbonaceous chondrites. Whether these compounds can be delivered by other space bodies is unclear and depends primarily on capability of the biomolecules to survive high temperatures during atmospheric deceleration and impacts to the terrestrial surface. In the present study we estimated survivability of simple amino acids (alpha-aminoisobutyric acid, L-alanine, L-valine and L-leucine), purines (adenine and guanine) and pyrimidines (uracil and cytosine) under rapid heating to temperatures of 400 to 1000 degrees C under N2 or CO2 atmosphere. We have found that most of the compounds studied cannot survive the temperatures substantially higher than 700 degrees C; however at 500-600 degrees C, the recovery can be at a per cent level (or even 10%-level for adenine, uracil, alanine, and valine). Implications of the data for extraterrestrial delivery of the biomolecules are discussed. PMID:11543338

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



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


In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, L-alanine-ACV (AACV), L-serine-ACV (SACV), L-isoleucine-ACV (IACV), gamma-glutamate-ACV (EACV) and L-valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants (lambda(z)). SACV and VACV exhibited approximately five-fold increase in area under the curve (AUC) values relative to ACV (p<0.05). C(max(T)) (maximum concentration) of SACV was observed to be 39+/-22 microM in plasma which is 2 times better than VACV and 15 times better than ACV. C(last(T)) (concentration at the last time point) of SACV was observed to be 0.18+/-0.06 microM in plasma which is two times better than VACV and three times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (C(max)) and eliminated at varying rates (lambda(z)) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections. PMID:18638532

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



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

PubMed Central

Purpose To evaluate in vivo corneal absorption of the amino acid prodrugs of acyclovir (ACV) using a topical well model and microdialysis in rabbits. Methods Stability of L-Alanine-ACV (AACV), L-Serine-ACV (SACV), L-Isoleucine-ACV (IACV), ?-Glutamate-ACV (EACV) and L-Valine-ACV (VACV) prodrugs was evaluated in various ocular tissues. Dose dependent toxicity of these prodrugs was also examined in rabbit primary corneal epithelial cell culture (rPCEC) using 96-well based cell proliferation assay. In vivo ocular bioavailability of these compounds was also evaluated with a combination of topical well infusion and aqueous humor microdialysis techniques. Results Among the amino acid ester prodrugs, SACV was most stable in aqueous humor. Enzymatic degradation of EACV was the least compared to all other prodrugs. Cellular toxicity of all the prodrugs was significantly less compared to trifluorothymidine (TFT) at 5mM. Absorption rate constants of all the compounds were found to be lower than the elimination rate constants. All the prodrugs showed similar terminal elimination rate constants (?z). SACV and VACV exhibited approximately two fold increase in area under the curve (AUC) relative to ACV (p < 0.05). Clast (concentration at the last time point) of SACV was observed to be 8 ± 2.6µM in aqueous humor which is two and three times higher than VACV and ACV, respectively. Conclusions Amino acid ester prodrugs of ACV were absorbed through the cornea at varying rates (ka) thereby leading to varying extents (AUC). The amino acid ester prodrug, SACV owing to its enhanced stability, comparable AUC, and high concentration at last time point (Clast) seems to be a promising candidate for the treatment of ocular HSV infections. PMID:18472234

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



Effect of the headgroup structure on the aggregation behavior and stability of self-assemblies of sodium N-[4-(n-dodecyloxy)benzoyl]-l-aminoacidates in water.  


Three amino acid-derived chiral surfactants, sodium N-[4-(n-dodecyloxy)benzoyl]-L-leucinate (SDBL), sodium N-[4-(n-dodecyloxy)benzoyl]-L-isoleucinate (SDBIL), and sodium N-[4-(n-dodecyloxy)benzoyl]-L-threoninate (SDBT), were synthesized, and their aggregation behavior was studied in aqueous solution. Surface tension, fluorescence probe, dynamic light scattering, nuclear magnetic resonance (NMR), gel permeation chromatography, circular dichroism, and optical as well as transmission electron microscopic techniques were utilized to characterize the self-assemblies formed by the amphiphiles. Results of these studies reveal that the surfactants have a very low critical aggregation concentration (cac) and they form spherical vesicles spontaneously in dilute aqueous solution. The mean diameters of the vesicles were measured to be in the range of 130-190 nm. 1H NMR spectra indicated hydrogen bonding between the amide groups near the surfactant headgroup, which is one of the driving forces for vesicle formation. The vesicle formation is more favored at a pH of about 7.0. The amphiphiles also form chiral helical aggregates at relatively higher concentrations as indicated by circular dichroism spectra. The stability of the vesicles was also evaluated with respect to the surfactant concentration, pH, temperature, and aging. The vesicles have a tendency to transform into elongated vesicles (closed tubules) or rodlike micelles with an increase of the surfactant concentration and/or pH. On the basis of the results obtained from different studies, phase diagrams for all three water/amphiphile systems have been constructed. The studies have further shown that the stereogenic center at the amino acid side chain has a significant effect on the aggregation properties of the amphiphiles and on the stability of the self-assemblies. PMID:17241010

Mohanty, Ashok; Dey, Joykrishna



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

PubMed Central

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

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



Self-Assembly Thermodynamics of pH-Responsive Amino-Acid-Based Polymers with a Nonionic Surfactant.  


The behavior of pH-responsive polymers poly(N-methacryloyl-l-valine) (P1), poly(N-methacryloyl-l-phenylalanine) (P2), and poly(N-methacryloylglycyne-l-leucine) (P3) has been studied in the presence of the nonionic surfactant Brij98. The pure polymers phase-separate in an acidic medium with critical pHtr values of 3.7, 5.5, and 3.4, respectively. The addition of the surfactant prevents phase separation and promotes reorganization of polymer molecules. The nature of the interaction between polymer and surfactant depends on the amino acid structure in the side chain of the polymer. This effect was investigated by dynamic light scattering, isothermal titration calorimetry, electrophoretic measurements, small-angle neutron scattering, and infrared spectroscopy. Thermodynamic analysis revealed an endothermic association reaction in P1/Brij98 mixture, whereas a strong exothermic effect was observed for P2/Brij98 and P3/Brij98. Application of regular solution theory for the analysis of experimental enthalpograms indicated dominant hydrophobic interactions between P1 and Brij98 and specific interactions for the P2/Brij98 system. Electrophoretic and dynamic light scattering measurements support the applicability of the theory to these cases. The specific interactions can be ascribed to hydrogen bonds formed between the carboxylic groups of the polymer and the oligo(ethylene oxide) head groups of the surfactant. Thus, differences in polymer-surfactant interactions between P1 and P2 polymers result in different structures of polymer-surfactant complexes. Specifically, small-angle neutron scattering revealed pearl-necklace complexes and "core-shell" structures for P1/Brij98 and P2/Brij98 systems, respectively. These results may help in the design of new pH-responsive site-specific micellar drug delivery systems or pH-responsive membrane-disrupting agents. PMID:25192406

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



The role of bulk and surface properties of selected amino acid copolymers on blood coagulation  

Microsoft Academic Search

Copolymers formed from 20%-?-L-benzylglutamate-80%-L-leucine (GL-14), 50%-?-L-benzylglutamate-50%-L-leucine (GL-11), 80%-?-L-benzylglutamate-20%-L-leucine\\u000a (GL-41), 80%-?-L-benzylglutamate-20%-L-phenylalanine (GP-41), and 100%-?-L-benzylglutamate (G-1), where all percentages are\\u000a in mole percent, were cast as thin films and tested for their ability to cause platelet retention in columns and to initiate\\u000a coagulation, as measured by the partial thromboplastin times and Factor VII assays. Benzene, dichloroethane, and dioxane were\\u000a used as casting

P. J. Parks; D. F. Gibbons; O. P. Malhotra



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

NASA Technical Reports Server (NTRS)

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

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



Ultrastructural cytochemistry of atrial muscle cells  

Microsoft Academic Search

Sections of atrial cardiocytes from young rats were subjected to radioautography after a single intravenous injection of L-leucine-4,5 3H to identify the sites of synthesis and to follow the migration of newly-formed proteins. As early as 2 min after injection of L-leucine 3H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of

L. Yunge; S. Benchimol; M. Cantin



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


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

Kaneko, Daisuke; Igarashi, Toshinori; Aoyama, Kenji



Regulation of transmural transport of amino acid/metal conjugates by dietary calcium in crustacean digestive tract.  


Effects of luminal Ca(2+) and Mn(2+) on transmural mucosal to serosal (MS) transport of (3) H-L-leucine were characterized in the isolated and perfused intestine of the American lobster, Homarus americanus. (3) H-L-leucine MS transport in the presence of 20 µM Mn(2+) was a sigmoidal function of luminal amino acid concentration, following the Hill equation for multisite cooperative, carrier-mediated, transport. Luminal Ca(2+) was a non-competitive inhibitor of Mn(2+) -stimulated (3) H-L-leucine MS flux. Amino acid transport was hyperbolically stimulated by luminal Ca(2+) or Mn(2+). During 20 µM Mn(2+) -stimulation of (3) H-L-leucine MS flux, addition of 25 mM Ca(2+) strongly reduced amino acid transport Jmax , without affecting amino acid binding properties. Hyperbolic luminal Mn(2+) stimulation of 20 µM (3) H-L-leucine MS flux was also strongly inhibited by 25 mM luminal Ca(2+) , significantly reducing 20 µM (3) H-L-leucine Jmax . Increasing the luminal concentration of verapamil, a calcium channel blocker, significantly increased MS transport of 20 µM (3) H-L-leucine in the presence of 100 nM Mn(2+) by reducing diffusional Ca(2+) uptake into intestinal epithelial cells through verapamil-sensitive channels. A model is proposed supporting the concept of molecular mimicry, whereby (3) H-L-leucine enters lobster intestinal epithelial cells by one or more amino acid-specific transporters and by a dipeptide-like transporter that is capable of binding and transporting peptide molecular mimics (bis-complexes) between Ca(2+) or Mn(2+) and (3) H-L-leucine using the membrane potential as a major driving force for the transport event. According to the model, Ca(2+) entry through apical Ca(2+) channels regulates the magnitude of the membrane potential and therefore the size of the driving force for bis-complex uptake. PMID:24254522

Abdel-Malak, Rania; Ahearn, Gregory A



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

PubMed Central

The enzyme ?-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been identified as a possible target for design of new anti-tubercular therapeutics. Recently it was shown that MtIPMS is subject to slow-onset, feedback inhibition by L-leucine, the first instance of an allosteric regulator utilizing this mechanism. Structural studies are inconsistent with allosteric mechanisms including changes to the quarternary structure or large, rigid-body conformational changes to the enzyme upon L-leucine binding. Thus, the allosteric regulation may result from a discrete inhibitory signal transmitted to the active site upon L-leucine binding in the regulatory domain, a distance of over 50 Å In order to test this mechanism, site-directed mutagenesis was employed to construct enzymes with substitutions at phylogenetically conserved active site residues near the interface of the catalytic and linker domains. The substitutions had wide ranging effects on the kinetics of L-leucine inhibition, with some modest effects on the kinetic parameters of catalysis. The most dramatic result was the finding that the Y410F mutant form of MtIPMS is insensitive to L-leucine inhibition, suggesting that this residue has completely uncoupled the inhibitory signal to the active site. Overall, the data are consistent with a mechanism of allosteric regulation described by the inter-domain communication of the inhibitory signal from the regulatory to catalytic domain and implicate the interaction between the linker and catalytic domains as critical determinants of inhibitory signal transmission. PMID:19166329

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



Product Equivalence Study Comparing the Tolerability, Pharmacokinetics, and Pharmacodynamics of Various Human Immunoglobulin-G Formulations  

Microsoft Academic Search

In this randomized, double-blind, parallel-group study, a commercially available human immunoglobulin-G product, IVIG, was compared with two test formulations: (1) IVIG-N, which is a nanofiltered formulation of IVIG, and (2) IVIG-L, which is a nanofiltered, liquid, ready-for-use IgG formulation containing nicotinamide, L-proline, and L-isoleucine as stabilizers. Three groups of 10 healthy subjects each received a single 0.6 g\\/kg dose of

Irmgard Andresen; John M. Kovarik; Martin Spycher; Reinhard Bolli



Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris  

Microsoft Academic Search

The four amino acids of the “aspartate family” (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure “balanced” synthesis of the several

Huei-che Yen; Howard Gest



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

Microsoft Academic Search

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

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



Purification and properties of alanine racemase from crayfish Procambarus clarkii  

Microsoft Academic Search

Fresh water crayfish Procambarusclarkii is known to accumulate d-alanine remarkably in muscle after seawater acclimation, accompanied by an increase in alanine racemase activity. We have purified alanine racemase from crayfish muscle to homogeneity. The enzyme is a monomeric protein with a molecular mass of 58 kDa. It is highly specific to alanine and does not racemize l-serine, l-aspartate, l-glutamate, l-valine

Kimihiko Shibata; Katsushi Shirasuna; Kengo Motegi; Yoshio Kera; Hiroki Abe; Ryo-hei Yamada



Enthalpies of Dilution and Enthalpies of Mixing of Amino Acids with Sucrose in Aqueous Solutions at 298.15 K  

Microsoft Academic Search

The enthalpies of mixing of aqueous solutions have been determined for sucrose with six different amino acids (glycine, l-alanine, l-serine, l-valine, l-proline and l-threonine) at 298.15 K, by using a LKB-2277 flow microcalorimetric system. These results, along with the enthalpies of dilution of these solutes for the initial solutions, were used to determine the enthalpic interaction coefficients (h\\u000a xy, h

Huaji Liu; Ruisen Lin; Honglin Zhang




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




eschweizerbartxxx Verh. Internat. Verein. Limnol.  

E-print Network

proteolytic activities in lake sediments, and (c) compare the temporal pro- files of enzyme activity proteolytic enzyme. Key words: L-Leucine-4-methylcoumarinyl-7-amide, lake sediments, proteolytic activity, January 2008 © by E. Schweizerbart'sche Verlagsbuchhandlung 2008 Aminopeptidase activity in lake sediments

Mazumder, Asit


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

E-print Network

Sciences, Chinese Academy of Sciences, Shanghai, China Abstract The biosynthesis of neuroendocrine peptidesExamination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d10

Tian, Weidong


21 CFR 172.829 - Neotame.  

Code of Federal Regulations, 2011 CFR

...AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD...required to accomplish the intended technical effect, in foods for which standards of identity...use. (d) When neotame is used as a sugar substitute tablet, L-leucine may...



21 CFR 172.829 - Neotame.  

Code of Federal Regulations, 2010 CFR

...AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD...required to accomplish the intended technical effect, in foods for which standards of identity...use. (d) When neotame is used as a sugar substitute tablet, L-leucine may...



21 CFR 172.829 - Neotame.  

Code of Federal Regulations, 2012 CFR

...AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD...required to accomplish the intended technical effect, in foods for which standards of identity...use. (d) When neotame is used as a sugar substitute tablet, L-leucine may...



21 CFR 172.829 - Neotame.  

Code of Federal Regulations, 2013 CFR

...AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD...required to accomplish the intended technical effect, in foods for which standards of identity...use. (d) When neotame is used as a sugar substitute tablet, L-leucine may...



21 CFR 172.829 - Neotame.  

...AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD...required to accomplish the intended technical effect, in foods for which standards of identity...use. (d) When neotame is used as a sugar substitute tablet, L-leucine may...



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


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

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



Possible selective adsorption of enantiomers by Na-montmorillonite  

NASA Technical Reports Server (NTRS)

Racemic amino acids including (D,L) alpha-alamine, (D,L) alpha-aminobutyric acid, (D,L) valine, and (D,L) norvaline were incubated with Na-montmorillonite at 100% CEC at three hydrogen ion concentrations, and amino acid adsorption was determined by ion exchange chromatography. Enantiomers were analyzed by gas chromatography. Differences in the quantities of D and L enantiomers in any of the fractions was no larger than a few percent. Although a large difference in the adsorption of the amino acid enantiomers was not observed, the analysis may indicate a small preferential adsorption (0.5-2%) of L-amino acids by Na-montmorillonite.

Friebele, E.; Shimoyama, A.; Ponnamperuma, C.



Amino acid transport inhibition: brain and behavioral correlates.  


In vivo inhibition of uptake 14C-L-valine by brain following subcutaneous administration of either of two gamma-glutamyl cycle enzyme inhibitors, 2-imidazolidone-4-carboxylic acid (ICA), or, L-methionine-S-sulfoximine (MSO) is documented in C57BL/6J mice. Dose related decrease in exploratory activity, impairment of memory for foot shock, and reduced operant responding for food reinforcement parallels the time course for interference with uptake of a large neutral amino acid by these two compounds previously shown to inhibit different enzymes in the gamma-glutamyl cycle subserving active amino acid transport. PMID:981286

Randt, C T; Samuels, S; Fish, I



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



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


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

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



Synthesis of 6-substituted 1-oxoindanoyl isoleucine conjugates and modeling studies with the COI1-JAZ co-receptor complex of lima bean.  


The conjugates of 6-substituted 1-oxoindanoyl carboxylic acids with L-isoleucine are mimics of the plant hormone (+)-7-iso-JA-L-Ile (3) that controls and regulates secondary metabolism and stress responses. In order to generate ligands that can be used as hormone-like compounds possessing different biological activities, an efficient and short synthesis of 6-bromo-1-oxoindane-4-carboxylic acid opens a general route to 6-Br-1-oxoindanoyl L-isoleucine conjugate (Br-In-L-Ile) (9a) as a key intermediate for several bioactive 6-halogen-In-L-Ile analogs (7a, 8a, 10a). The 6-ethynyl-In-L-Ile analog (11a) might be a valuable tool to localize macromolecular receptor molecules by click-chemistry. The activities of In-Ile derivatives were evaluated by assays inducing the release of volatile organic compounds (VOCs) in lima bean (Phaseolus lunatus). Each compound showed slightly different VOC induction patterns. To correlate such differences with structural features, modeling studies of In-Ile derivatives with COI-JAZa/b/c co-receptors of P. lunatus were performed. The modeling profits from the rigid backbone of the 1-oxoindanonoyl conjugates, which allows only well defined interactions with the receptor complex. PMID:25008776

Nakamura, Yoko; Paetz, Christian; Brandt, Wolfgang; David, Anja; Rendón-Anaya, Martha; Herrera-Estrella, Alfredo; Mithöfer, Axel; Boland, Wilhelm



Control of calcium carbonate crystallization utilizing amphiphilic block copolypeptides  

NASA Astrophysics Data System (ADS)

Control over the morphology of calcium carbonate (CaCO 3) crystals has been achieved through the use of anionic, amphiphilic block copolypeptides during crystallization. Microspheres of CaCO 3 can be synthesized by the introduction of preformed organic, amphiphilic block copolypeptide templates, poly( L-aspartate sodium salt) 100- block-[poly( L-phenylalanine) 25- random-( L-leucine) 25] ( 1) and poly( L-glutamate sodium salt) 100- block-[poly( L-phenylalanine) 25- random-( L-leucine) 25] ( 2). When cationic amphiphiles are used in lieu of the anionic amphiphiles, only CaCO 3 rhombohedra are produced. The self-assembling amphiphile controls the precipitation of the microspheres by acting as a template to form giant CaCO 3 microspheres. These microspheres are composed of nanocrystals ranging in size from 10 to 60 nm using 1 and 20 to 100 nm using 2.

Euliss, Larken E.; Bartl, Michael H.; Stucky, Galen D.



Brush-border-enzyme-mediated intestine-specific drug delivery. Amino acid prodrugs of 5-aminosalicylic acid.  


5-Aminosalicylic acid (5-ASA) is the active principle of a number of preparations aimed at the treatment of inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, but its efficacy is limited by early absorption and metabolism. The possibility to exploit the selective hydrolytic activity of brush border enzymes such as aminopeptidase A and carboxypeptidases was studied by preparing the following four amino acid prodrugs of 5-ASA: 5-(N-L-aspartylamino)-2-salicylic acid, disodium salt (18), 5-(N-L-glutamylamino)-2-salicylic acid, disodium salt (19), [(5-aminosalicyl)-L-prolyl]-L-leucine, sodium salt (25), and [[5-(N-L-glutamylamino)salicyl]-L-prolyl]-L-leucine, disodium salt (28). In these compounds, the peptide bond is selectively split by the intestinal brush border aminopeptidase A (compounds 18, 19, and 28) and carboxypeptidases (compounds 25 and 28). PMID:8277502

Pellicciari, R; Garzon-Aburbeh, A; Natalini, B; Marinozzi, M; Clerici, C; Gentili, G; Morelli, A



Reduction of large neutral amino acid levels in plasma and brain of hyperleucinemic rats  

Microsoft Academic Search

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly known. In the present study we investigated the effect of acute hyperleucinemia on plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected subcutaneously with 6 ?mol l-leucine per gram body weight. Controls received saline in

P. Araújo; G. F. Wassermann; K. Tallini; V. Furlanetto; C. R. Vargas; C. M. D. Wannmacher; C. S. Dutra-Filho; A. T. S. Wyse; M. Wajner




Microsoft Academic Search

The development of unit-dose, inhalable, antibiotic microparticles for use in primary and combined therapy approaches to treating tuberculosis (TB), multi-drug-resistant (MDR-TB) and extensively drug-resistant TB is explored using the gentle drying process of Carbon-dioxide Assisted Nebulization with a Bubble Dryer® (CAN-BD). The microparticles produced using this method contain capreomycin, kanamycin, and isoniazid, respectively, imbedded in L-leucine. Antibioticswere developed into inhalable

J. R. Manion; S. P. Cape; D. H. McAdams; L. G. Rebits; Sarah Evans; R. E. Sievers



Overexpression of Non-Functional LAT1\\/4F2hc in Renal Proximal Tubular Epithelial Cells from the Spontaneous Hypertensive Rat  

Microsoft Academic Search

The present study examined the expression of type 1 L-amino acid transporter (LAT1) and its associated glycoprotein 4F2hc in freshly isolated renal proximal tubules and immortalized renal proximal tubular epithelial (PTE) cells from spontaneously hypertensive (SHR) and normotensive (WKY) rats. The study also examined the inward and outward transport of [14C]-L-leucine, the preferred substrate of LAT1. The abundance of LAT1

Maria Pinho; Maria Serrao; Pedro Jose; Patrício Soares-da-Silva



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


Microbial uptake of dissolved organic matter in Mcmurdo Sound, Antarctica  

Microsoft Academic Search

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

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



Aminoacid transport in suspension-cultured plant cells  

Microsoft Academic Search

The carrier system which transports L-leucine (L-leu) into suspension-culturedNicotiana tabacum L. cv. Wisconsin 38 cells appeared to be constitutive since it was always present and was not induced by L-leu even in nitrogen-starved cells. However, L-leu uptake rates for cells grown in medium containing L-leu were transiently reduced as a result of either transinhibition or repression. Growth-phase cells appeared to

Carl N. McDaniel; Paul M. Wozniak



Tumor Growth Inhibitory and Natural Suppressor Activities of Murine Bone Marrow Cells: A Comparative Study  

Microsoft Academic Search

The murine bone marrow (BM) cells having a certain phenotypic similarity to null natural suppressor (NS) cells have been previously established to be able to inhibitin vitroleukemic cell growth in a genetically unrestricted manner. In this study we found that the treatment of normal (C57BL\\/6 × DBA)F1 BM cells with a lysosomotropic agent,l-leucine methyl ester (LME), largely abrogated their ability

Victor I. Seledtsov; Vadim Y. Taraban; Galina V. Seledtsova; Denis M. Samarin; Ilias V. Avdeev; Vladimir V. Senyukov; Vladimir A. Kozlov



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

PubMed Central

Background The aim of this work was to develop dry powder inhaler (DPI) formulations of salbutamol sulfate (SS) by the aid of solid lipid microparticles (SLmPs), composed of biocompatible phospholipids or cholesterol. Methods The SLmPs were prepared by using two different solvent systems (ethanol and water-ethanol) and lipid carriers (dipalmitoylphosphatidylcholine (DPPC) and cholesterol) with/without L-leucine in the spray drying process. The spray-dried microparticles were physically-mixed with coarse lactose monohydrate in order to make our final DPI formulations and were investigated in terms of physical characteristics as well as in vitro drug release profile and aerosolization behavior. Results We observed significant differences in the sizes, morphologies, and in vitro pulmonary depositions between the formulations. In particular, the SS-containing SLmPs prepared with water-ethanol (30:70 v/v) solution of DPPC and L-leucine which had then been blended with coarse lactose (1:9 w/w) exhibited the highest emitted dose (87.9%) and fine particle fraction (42.7%) among the formulations. In vitro drug release study indicated that despite of having a significant initial burst release for both cholesterol and DPPC-based microparticles, the remained drug released more slowly than the pure drug. Conclusion This study demonstrated the potential of using lipid carriers as well as L-leucine in DPI formulations of SS to improve its aerosolization behavior and retard the release profile of the drug. PMID:24919924



beta-Adrenergics enhance brain extraction of levodopa.  


We sought to determine whether beta-adrenergic agonists enhance the brain extraction of L-dopa and L-leucine. Systemic administration of beta-adrenergic agonists increase brain concentrations of L-dopa and other large neutral amino acids (LNAA) in rats and monkeys and may improve symptoms and reduce daily L-dopa requirement in patients with Parkinson's disease. Cerebral blood flow (CBF) using [3H]nicotine and the extraction fraction of 14C-labeled L-dopa or L-leucine were measured simultaneously in various brain regions of conscious rats using the dual-isotope indicator fractionation technique after intraperitoneal administration of isoproterenol (a peripheral nonselective beta-adrenergic agonist), or clenbuterol (a beta2-adrenergic agonist that crosses the blood-brain barrier), or beta-adrenergic agonist preceded by nadolol (a peripheral nonselective beta-adrenergic antagonist), or saline vehicle. Both beta-adrenergic agonists increased regional brain extraction fraction of L-dopa and L-leucine tracers by 35-45%, without altering regional CBF. These changes were accompanied by about a 30% decrease in plasma branched chain LNAA concentrations. Nadolol blocked all these effects. beta-Adrenergic agonists increase the brain extraction of L-dopa and leucine, mainly by peripheral mechanisms that reduce the levels of other competing plasma LNAAs for transport. Thus, beta-adrenergic agonists might be useful in the treatment of patients with Parkinson's disease by enhancing delivery of L-dopa to the brain. PMID:11835439

Uc, Ergun Y; Dienel, Gerald A; Cruz, Nancy F; Harik, Sami I



The radiolysis and radioracemization of amino acids on silica surfaces  

NASA Technical Reports Server (NTRS)

Results are presented of experiments on the radioracemization of amino acids in the presence of silica surfaces such as may have been found on the prebiotic earth. L-leucine and a DL-leucine mixture deposited on samples of 1-quartz and an amorphous silica preparation (Syloid 63) was subjected to Co-60 gamma-ray irradiation, then analyzed by gas chromatography to determine the radiolysis and racemization rates. The quartz surface is found to have a marginal efficacy in enhancing radiolysis when compared with a crystalline L-leucine control, although enhancing radioracemization symmetrically by a factor of two. Both the radiolysis and radioracemization of L-leucine and DL-leucine on a Syloid-63 silica surface are observed to increase with increasing radiation dose, and to be substantially greater than in the crystalline controls. Additional experiments with the nonprotein amino acid isovaline deposited on Syloid 63 confirm the greater radiolysis susceptibility of amino acids deposited on silica with respect to the crystalline state, although racemization is not observed. The observations suggest that the presence of a silica surface would have a deleterious effect on any mechanism for the origin of molecular chirality relying on stereoselective beta-radiolysis.

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



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

NASA Technical Reports Server (NTRS)

Experiments are described to test a theory for the origin of optical activity wherein the longitudinally polarized electrons resulting from parity violation during radioactive beta decay, and their resulting circularly polarized Bremsstrahlung, might interact asymmetrically with organic matter to yield optically active products. Experiments involve subjecting a number of racemic and optically active amino acid samples to irradiation in a 61700 Ci90SR-90Y beta radiation source for a period of 1.34 years, then examining them for any asymmetric effects by means of optical rotatory dispersion and analytical gas chromatography. In the cases of D,L-leucine, norleucine, norvaline and proline as solids, of D,L-leucine in solution and of D,L-tyrosine in alkaline solution no optical rotation was observed during CRD measurements in the 250-630 nm spectral region. While slight differences were noted in the percent radiolysis of solid D- (12.7%) and L-leucine (16.2%) as determined by GC, no enrichment of either enantiomer was found.

Bonner, W. A.



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.



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). PMID:20690642

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



Modulation of P-glycoprotein-mediated efflux by prodrug derivatization: an approach involving peptide transporter-mediated influx across rabbit cornea.  


The aim of this study was to investigate the modulation of efflux mechanisms using transporter- targeted prodrug derivatization of a model P-gp substrate, quinidine. The L-valine, L-valine-valine esters of quinidine, val-quinidine (VQ), and val-val-quinidine (VVQ) were synthesized in our laboratory, respectively. [(14)C] erythromycin was chosen to delineate the affinity of quinidine (Q) toward P-gp. [(3)H] glycylsarcosine (GS, or glysar) was chosen as a model peptide transporter (PEPT) substrate. Uptake studies were performed on rPCEC (rabbit primary corneal epithelial culture) using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Efflux of [(14)C] erythromycin was significantly increased in the presence of quinidine, whereas it was unaltered in the presence of VQ and VVQ. VVQ was more stable, both in buffers and tissue homogenate. Transport of VQ and VVQ was inhibited with GS, and their permeability values were 1.5 and 3 times higher than the permeability of quinidine, respectively. Results from this study clearly indicate that prodrug derivatization of quinidine can modulate P-gp-mediated efflux. These prodrugs have a reduced or diminished affinity toward P-gp and were further recognized by the peptide transporter- mediated process. Enhanced permeabilities of the prodrugs indicate that drug derivatization can be a viable strategy for overcoming P-gp-mediated efflux. PMID:16722797

Katragadda, Suresh; Talluri, Ravi S; Mitra, Ashim K



Metabolism of Valine by the Filamentous Fungus Arthrobotrys conoides1  

PubMed Central

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

Gupta, Rishab K.; Pramer, David



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. PMID:22123250

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



Effects of cortex peptidoglycan structure and cortex hydrolysis on the kinetics of Ca(2+)-dipicolinic acid release during Bacillus subtilis spore germination.  


The kinetic parameters of the release of Ca(2+)-dipicolinic acid (CaDPA) during germination of spore populations and multiple individual spores of Bacillus subtilis strains with major alterations in the structure of the spore peptidoglycan (PG) cortex or lacking one or both of the two redundant enzymes involved in cortex hydrolysis (cortex-lytic enzymes [CLEs]) were determined. The lack of the CLE CwlJ greatly slowed CaDPA release with a germinant receptor (GR)-dependent germinant, l-valine, or a non-GR-dependent germinant, dodecylamine. The absence of the cortex-specific PG modification muramic acid-?-lactam also increased the time needed for full CaDPA release during germination with both types of germinants. In contrast, increased cortex PG cross-linking was associated with faster times for initiation of CaDPA release with both l-valine and dodecylamine but not with faster CaDPA release once this release had been initiated. These data suggest that the precise structure of the spore cortex plays a significant role in determining the timing and the rate of CaDPA release during B. subtilis spore germination and, further, that this effect is independent of effects of GRs. PMID:22123250

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



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

SciTech Connect

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

Mallakpour, Shadpour, E-mail: [Organic Polymer Chemistry Research Laboratory, Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of) [Organic Polymer Chemistry Research Laboratory, Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of); Nanotechnology and Advanced Materials Institute, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of); Dinari, Mohammad [Organic Polymer Chemistry Research Laboratory, Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of)] [Organic Polymer Chemistry Research Laboratory, Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of)



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

PubMed Central

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

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



A jasmonate ZIM-domain protein NaJAZd regulates floral jasmonic acid levels and counteracts flower abscission in Nicotiana attenuata plants.  


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

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



Activities of fluoroquinolone, macrolide, and aminoglycoside drugs combined with inhibitors of glycosylation and fatty acid and peptide biosynthesis against Mycobacterium avium.  

PubMed Central

Smooth- and rough-colony variants of Mycobacterium avium serovar 4 were treated with three classes of drugs. The drugs were chosen for their potential inhibitory effects on the biosynthesis of the cell envelope-associated serovar-specific glycopeptidolipid antigens. Growth was monitored radiometrically with a BACTEC 460-TB instrument, and MICs were determined for each drug. Both variants were then treated with inhibitory drugs in combination with antimicrobial agents that have demonstrated effectiveness against M. avium. No growth inhibition was observed with 6-fluoro-6-deoxy-D-glucose or avidin. Inhibitors of glycosylation, i.e., 2-deoxy-D-glucose, bacitracin, and ethambutol, were inhibitory to smooth- and rough-colony variants, whereas drugs that inhibit peptide synthesis, i.e., N-carbamyl-L-isoleucine and m-fluoro-phenylalanine, were more inhibitory for the rough-colony variant. Cerulenin, which affects fatty acid synthesis, was inhibitory for both variants, but it appeared to be more effective at inhibiting the growth of the smooth-colony variant at equivalent concentrations. Generally, when inhibitors of glycosylation were used with sparfloxacin and amikacin, a synergistic effect was observed for only the smooth variant. When drugs that affect peptide synthesis were used in combination with amikacin, a synergistic effect was observed for the rough variant, and when cerulenin was used in combination with sparfloxacin or amikacin, a synergistic effect was observed for both variants. Lipid analysis revealed that although the rough variant lacks the serovar-specific glycopeptidolipid antigens, it does possess a group of phenylalanine-isoleucine-containing lipopeptides that may explain its different susceptibility patterns to m-fluoro-phenylalanine and N-carbamyl-L-isoleucine. The significance of these results is discussed with reference to various components in the cell envelope and their importance in cell wall permeability. Images PMID:8494359

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



Cation-dependent nutrient transport in shrimp digestive tract.  


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



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


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

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



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 Central

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-?-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



An organotrifluoroborate-based convergent total synthesis of the potent cancer cell growth inhibitory depsipeptides kitastatin and respirantin.  


The total syntheses of the highly cytotoxic neo-antimycin macrocyclic depsipeptide natural products kitastatin and respirantin have been accomplished in a convergent manner using MNBA promoted esterifications and an efficient C- and N-terminus bis-deprotection/HATU promoted macrolactamization. The first examples of using a prenyltrifluoroborate reagent in additions to carbonyl groups are disclosed including a diastereoselective multigram scale montmorillonite K10 catalyzed prenylation of N-Boc-l-leucinal to install the structurally unique gem-dimethyl-?-keto-ester fragment. PMID:24730576

Beveridge, Ramsay E; Batey, Robert A



Separation of enantiomeric iodinated thyronines by liquid chromatography of diastereomers.  


A method for the separation of D,L-triiodothyronine and D,L-tetraiodothyronine optical isomers is described. The iodinated thyronines are coupled with L-leucine and the resulting diastereomers are separated by reversed-phase liquid chromatography. The derivatives are detected in the UV region at 230 nm. The technique can be used for the determination of the optical purity of thyroid hormones. It is possible to determine 0.2% of the L-isomer in D-tetraiodothyronine with a relative standard deviation of 8%. One complete analysis takes about 2 h. The results are in good agreement with those of an enzymatic method. PMID:7440681

Lankmayr, E P; Budna, K W; Nachtmann, F



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

Microsoft Academic Search

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

Shadpour Mallakpour; Samaneh Soltanian; Mohammad Reza Sabzalian


Distribution of the Isopropylmalate Pathway to Leucine Among Diverse Bacteria  

PubMed Central

?-Isopropylmalate synthase and ?-isopropylmalate dehydrogenase activities were detected in extracts of the following organisms: Chromatium D, Rhodopseudomonas spheroides, Hydrogenomonas H16, Pseudomonas aeruginosa, Pseudomonas fluorescens, Vibrio extorquens, Rhizobium japonicum, Alcaligenes viscolactis, Escherichia coli B, Proteus vulgaris, Aerobacter aerogenes, Salmonella typhimurium, Micrococcus sp., Micrococcus lysodeikticus, Bacillus polymyxa, Bacillus subtilis, and Nocardia opaca. The ?-isopropylmalate synthase activity in these extracts was inhibited by low concentrations of l-leucine. Taken together with other data, these results suggest that the isopropylmalate pathway is widespread among organisms that can synthesize leucine. PMID:4829932

Stieglitz, B. I.; Calvo, J. M.



Distribution of the isopropylmalate pathway to leucine among diverse bacteria.  


alpha-Isopropylmalate synthase and beta-isopropylmalate dehydrogenase activities were detected in extracts of the following organisms: Chromatium D, Rhodopseudomonas spheroides, Hydrogenomonas H16, Pseudomonas aeruginosa, Pseudomonas fluorescens, Vibrio extorquens, Rhizobium japonicum, Alcaligenes viscolactis, Escherichia coli B, Proteus vulgaris, Aerobacter aerogenes, Salmonella typhimurium, Micrococcus sp., Micrococcus lysodeikticus, Bacillus polymyxa, Bacillus subtilis, and Nocardia opaca. The alpha-isopropylmalate synthase activity in these extracts was inhibited by low concentrations of l-leucine. Taken together with other data, these results suggest that the isopropylmalate pathway is widespread among organisms that can synthesize leucine. PMID:4829932

Stieglitz, B I; Calvo, J M



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

Microsoft Academic Search

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

Tian Wu; Zhendong Tian; Jun Liu; Conghua Xie



Actions of GIP.  


Two structurally similar peptides were isolated from a preparation of GIP using an HPLC system. The major peptide corresponds to GIP1-42 and the minor has the sequence GIP3-42. GIP1-42 has both insulinotropic and somatostatinotropic activities, whereas GIP3-42 has only insignificant activity. GIP was also shown to potentiate insulin release initiated by D-glyceraldehyde, L-leucine/L-glutamine and 2-keto-isocaproic acid. No potentiation was observed with 2-ketocaproate. The 4 substrates studied are all metabolized but via different mechanisms. PMID:7045829

Brown, J C; Dahl, M; Kwauk, S; McIntosh, C H; Otte, S C; Pederson, R A



The genetic control of growth rate: a systems biology study in yeast.  

E-print Network

disappears as there are too few cells in the culture to utilize the available nutrients. Those cells remaining in the culture vessel grow at their maximum growth rate since even the limiting nutrient is present in excess. Finally, at D > (max , a steady state... (0.105g/l), threonine (0.119g/l), tryptophan (0.204g/l), valine (0.117g/l), citrate (0.210g/l), fumarate (0.160g/l), malate (0.134g/l), pyruvate(0.110g/l), succinate (0.270g/l), cytosine (0.111g/l), uracil (0.112g/l), glucose (20g/l) N-lim F1: KH2PO4...

Pir, Pinar; Gutteridge, Alex; Wu, Jian; Rash, Bharat; Kell, Douglas B; Zhang, Nianshu; Oliver, Stephen G



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

SciTech Connect

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

Reizer, J.; Panos, C.



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


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



The radiolysis and radioracemization of amino acids on clays  

NASA Technical Reports Server (NTRS)

The effects of the surfaces of kaolinite and bentonite clays on the radiolysis and radioracemization of L-leucine and its hydrochloride salt have been investigated experimentally. L-leucine and its hydrochloride salt were deposited on the clays and the amino acid/clay preparations were irradiated by a Co-60 gamma-ray source which induced 2-89 percent radiolysis. The efficiency of radiolysis and radioracemization were measured using gas chromatography. Results were obtained for leucine in 0.1 M aqueous solution for comparison with the clay-deposted leucine and leucine hydrochloride. It is found that radiolysis and radioracemization in the samples occurred according to a pseudo-first-order rate law. Comparison of the specific rate constants showed that leucine and its hydrochloride salt were the most resistant to both radiolysis and radioracemization, followed by leucine and its hydrochloride salt on kaolin. Leucine and its HCl salt on bentonite, and leucine in aqueous solution were found to be the least resistant to radiolysis and radioracemization. The experimental results are intepreted with respect to the Vester-Ulbricht mechanism for the origin of optical activity.

Bonner, W. A.; Hall, H.; Chow, G.; Yi, L.; Lemmon, R. M.



Proteases and Peptidases of Castor Bean Endosperm  

PubMed Central

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

Tully, Raymond E.; Beevers, Harry



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

PubMed Central

The aim of this study was to develop a spray dried submicrometer powder formulation suitable for the excipient enhanced growth (EEG) application. Combination particles were prepared using the Buchi Nano spray dryer B-90. A number of spray drying and formulation variables were investigated with the aims of producing dry powder formulations that were readily dispersed upon aerosolization and maximizing the fraction of submicrometer particles. Albuterol sulfate, mannitol, L-leucine, and poloxamer 188 were selected as a model drug, hygroscopic excipient, dispersibility enhancer and surfactant, respectively. Formulations were assessed by scanning electron microscopy and aerosol performance following aerosolization using an Aerolizer® dry powder inhaler (DPI). In vitro drug deposition was studied using a realistic mouth-throat (MT) model. Based on the in vitro aerosolization results, the best performing submicrometer powder formulation consisted of albuterol sulfate, mannitol, L-leucine and poloxamer 188 in a ratio of 30:48:20:2, containing 0.5% solids in a water:ethanol (80:20% v/v) solution which was spray dried at 70 °C. The submicrometer particle fraction (FPF1?m/ED) of this final formulation was 28.3% with more than 80% of the capsule contents being emitted during aerosolization. This formulation also showed 4.1% MT deposition. The developed combination formulation delivered a powder aerosol developed for the EEG application with high dispersion efficiency and low MT deposition from a convenient DPI device platform. PMID:23313343

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



Evaluation of the RBE4 cell line to explore carrier-mediated drug delivery to the CNS via the L-system amino acid transporter at the blood-brain barrier.  


The L-system amino acid transporter on the RBE4 cell line, a well established in vitro model of the blood-brain barrier (BBB), was characterised with the aim to evaluate this in vitro BBB model as tool for the systematic exploration of this endogenous carrier system for drug delivery to the CNS. Transport of L-[3H]-leucine in RBE4 cells was rapid, Na+-independent, bidirectional and followed the principles of trans-stimulation. The inhibition profile of L-leucine uptake was consistent with transport mediated by the L-system amino acid carrier with strong inhibition by large neutral amino acids (LNAA) such as L-phenylalanine and 2-aminobicyclo-heptanecarboxylic acid (BCH), whereas small neutral, basic and acidic amino acids had no significant effect. The transport of L-leucine into the RBE4 cells was saturable and followed single carrier Michaelis-Menten kinetics with Km 107 +/- 10 microM. Vmax 9.13 +/- 0.45 nmol/min/mg protein and KD 1.36 +/- 0.13 microl/min/mg protein. The kinetic constants of L-leucine transport, as well as the ranking of the kinetic constants of the transport of other LNAA investigated, correspond to those of the BBB in vivo. The characteristics of the LNAA transport in RBE4 cells suggest that transport is mediated by a system with characteristics similar to the L1 subtype of amino acid transporter, with carrier specificity equivalent to the L1 carrier system at the BBB in vivo. The study shows that the RBE4 cell line is a very suitable tool for the detailed examination of structure-transport relationships with respect to carrier-mediated drug delivery to the CNS via the L-system amino acid carrier at the BBB. The strength of this in vitro BBB model lies in the combination of the advantages of a cell line, being inexpensive, reproducible and easy to maintain, with the brain endothelium-specific expression of transport systems, to produce an efficient assay for the screening of potential neuropharmaceuticals targeted to specific transport routes to enhance CNS drug delivery. PMID:12164376

Reichel, Andreas; Abbott, N Joan; Begley, David J



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

PubMed Central

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

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



Rational design of a ligand-based antagonist of jasmonate perception.  


(+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile) regulates developmental and stress responses in plants. Its perception involves the formation of a ternary complex with the F-box COI1 and a member of the JAZ family of co-repressors and leads to JAZ degradation. Coronatine (COR) is a bacterial phytotoxin that functionally mimics JA-Ile and interacts with the COI1-JAZ co-receptor with higher affinity than JA-Ile. On the basis of the co-receptor structure, we designed ligand derivatives that spatially impede the interaction of the co-receptor proteins and, therefore, should act as competitive antagonists. One derivative, coronatine-O-methyloxime (COR-MO), has strong activity in preventing the COI1-JAZ interaction, JAZ degradation and the effects of JA-Ile or COR on several JA-mediated responses in Arabidopsis thaliana. Moreover, it potentiates plant resistance, preventing the effect of bacterially produced COR during Pseudomonas syringae infections in different plant species. In addition to the utility of COR-MO for plant biology research, our results underscore its biotechnological potential for safer and sustainable agriculture. PMID:24997606

Monte, Isabel; Hamberg, Mats; Chini, Andrea; Gimenez-Ibanez, Selena; García-Casado, Gloria; Porzel, Andrea; Pazos, Florencio; Boter, Marta; Solano, Roberto



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


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

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



Synthesis of 5?-androstane-17-spiro-?-lactones with a 3-keto, 3-hydroxy, 3-spirocarbamate or 3-spiromorpholinone as inhibitors of 17?-hydroxysteroid dehydrogenases.  


We synthesized two series of androstane derivatives as inhibitors of type 3 and type 5 17?-hydroxysteroid dehydrogenases (17?-HSDs). In the first series, four monospiro derivatives at position C17 were prepared from androsterone (ADT) or epi-ADT. After the protection of the alcohol at C3, the C17-ketone was alkylated with the lithium acetylide of tetrahydro-2-(but-3-ynyl)-2-H-pyran, the triple bond was hydrogenated, the protecting groups hydrolysed and the alcohols oxidized to give the corresponding 3-keto-17-spiro-lactone derivative. The other three compounds were generated from this keto-lactone by reducing the ketone at C3, or by introducing one or two methyl groups. In the second series, two dispiro derivatives at C3 and C17 were prepared from epi-ADT. After introducing a spiro-?-lactone at C17 and an oxirane at C3, an aminolysis of the oxirane with L-isoleucine methyl ester provided an amino alcohol, which was treated with triphosgene or sodium methylate to afford a carbamate- or a morpholinone-androstane derivative, respectively. These steroid derivatives inhibited 17?-HSD3 (14-88% at 1 ?M; 46-94% at 10 ?M) and 17?-HSD5 (54-73% at 0.3 ?M; 91-92% at 3 ?M). They did not produce any androgenic activity and did not bind steroid (androgen, estrogen, glucocorticoid and progestin) receptors, suggesting a good profile for prostate cancer therapy. PMID:23344201

Djigoué, Guy Bertrand; Ngatcha, Béatrice Tchédam; Roy, Jenny; Poirier, Donald



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


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



Structure-toxicity relationships in the amatoxin series. Structural variations of side chain 3 and inhibition of RNA polymerase II.  


The amatoxins, highly toxic components of death cap Amanita mushrooms, bind strongly to RNA polymerase II (or B) in cell nuclei thus preventing the transcription of DNAs to hn-RNAs (Pre-mRNAs), the precursors of messenger RNAs. Three of the binding sites of the bicyclic octapeptides have been identified: an isoleucine side chain in position 6, a trans-4-hydroxyl group at proline in position 2 and a hydroxylated L-isoleucine side chain in position 3. No information exists about the stereochemical conditions at the beta-C-atom (C-atom 3) of this side chain. We have now synthesized the diastereomeric S-deoxo-amaninamides (Fig. 1) containing, in position 3, L-allo-isoleucine (analog 1), (2S, 3R)-2-amino-4-hydroxy-3-methyl butyric acid (analog 2), the diastereomer (2S, 3S)-2-amino-4-hydroxy-3-methylbutyric acid (analog 3) and D-isoleucine (analog 4). In the last synthesis, besides the "normal" bicyclic octapeptide 4, an isomeric Iso-4 was formed. The affinities for Drosophila RNA polymerase II were 100 times weaker as compared to gamma-amanitin for 1, 10 times weaker for 2, 200 times weaker for 3, 100 times weaker for 4, and more than 1000 times weaker for Iso-4. The results point to the importance of a methyl group in (R)-configuration at the beta-C atom of side chain 3. PMID:1286940

Zanotti, G; Petersen, G; Wieland, T



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 PMID:1548227

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



Amino acid geochronology of raised beaches in south west Britain  

NASA Astrophysics Data System (ADS)

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

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


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


Application of sets of (13)C-(13)C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important (13)C-(13)C distances in uniformly (13)C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ((13)C') and aliphatic ((13)C(aliphatic)) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly (13)C,(15)N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of (13)C'-(13)C(aliphatic) distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform (13)C,(15)N-labeling on the FGAIL fragment. PMID:25240350

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



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

NASA Astrophysics Data System (ADS)

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

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



UVB radiation and 17-hydroxygeranyllinalool diterpene glycosides provide durable resistance against mirid (Tupiocoris notatus) attack in field-grown Nicotiana attenuata plants.  


Depending on geographical location, plants are exposed to variable amounts of UVB radiation and herbivore attack. Because the role(s) of UVB in the priming and/or accumulation of plant defence metabolites against herbivores are not well understood, we used field-grown Nicotiana attenuata plants to explore the effects of UVB on herbivore performance. Consistent with previous reports, UVB-exposed plants accumulated higher levels of ultraviolet (UV)-absorbing compounds (rutin, chlorogenic acid, crypto-chlorogenic acid and dicaffeoylspermidine). Furthermore, UVB increased the accumulation of jasmonic acid, jasmonoyl-L-isoleucine and abscisic acid, all phytohormones which regulate plant defence against biotic and abiotic stress. In herbivore bioassays, N.?attenuata plants experimentally protected from UVB were more infested by mirids in three consecutive field seasons. Among defence metabolites measured, 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) showed strongly altered accumulation patterns. While constitutive HGL-DTGs levels were higher under UVB, N.?attenuata plants exposed to mirid bugs (Tupiocoris notatus) had still more HGL-DTGs under UVB, and mirids preferred to feed on HGL-DTGs-silenced plants when other UVB protecting factors were eliminated by UVB filters. We conclude that UVB exposure not only stimulates UV protective screens but also affects plant defence mechanisms, such as HGL-DTGs accumulation, and modulates ecological interactions of N.?attenuata with its herbivores in nature. PMID:22897424

Ðinh, So'n Tru'ò'ng; Gális, Ivan; Baldwin, Ian T



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


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

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



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

NASA Astrophysics Data System (ADS)

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

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



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

SciTech Connect

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

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



Stimulation of amino acid transport in Saccharomyces cerevisiae by metabolic inhibitors.  


Inhibitors of energy metabolism (3-chlorophenylhydrazonomalononitrile, antimycin A, iodoacetamide, dicyclohexylcarbodiimide) but not of transport (uranyl ions) stimulate at low concentrations the uptake of L-leucine, L-glutamic acid, L-arginine and, to a lesser degree, of 2-aminoisobutyric acid in Saccharomyces cerevisiae. The effect is apparent only after augmenting the energy reserves of cells by preincubation with D-glucose or, more strikingly, with ethanol. It is absent in a mutant (op1) lacking the translocation system for ADP--ATP in mitochondria. The presence of two different energy reserves for amino acid transport is indicated (one in energy-poor, the other in energy-rich cells). The stimulating effect appears to be caused by a retarded degradation of the transport proteins as occurs at a lowered level of mitochondria-produced ATP. PMID:357269

Horák, J; Kotyk, A; Ríhová, L



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


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

NASA Technical Reports Server (NTRS)

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

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



Effect of vilon and epithalon on activity of enzymes in epithelial and subepithelial layers in small intestine of old rats.  


Per os administration of Vilon (Lys-Glu) or Epithalon (Ala-Glu-Asp-Gly) to aged Wistar rats for 1 month significantly increased activity of membrane enzymes maltase and alkaline phosphatase in epithelial layer of the small intestine. In addition, Vilon significantly increased activity of cytosolic glycyl-L-leucine dipeptidase in the stromal and seromuscular layers of the small intestine in comparison with the control rats not treated with this agent. These findings suggest improvement of trophic and barrier functions of the small intestine and corroborate the hypothesis on the existence of not only epithelial, but also subepithelial enzymatic barrier supporting the enzyme system in the small intestine, especially in aged animals. PMID:12660839

Khavinson, V Kh; Timofeeva, N M; Malinin, V V; Gordova, L A; Nikitina, A A



Effect of TiO2 Nanoparticles and UV Radiation on Extracellular Enzyme Activity of Intact Heterotrophic Biofilms.  


When introduced into the aquatic environment, TiO2 NP are likely to settle from the water column, which results in increased exposure of benthic communities. Here, we show that the activity of two extracellular enzymes of intact heterotrophic biofilms, ?-glucosidase (carbon-cycling) and l-leucin aminopeptidase (nitrogen-cycling), was reduced following exposure to surface functionalized TiO2 NP and UV radiation, depending on the particles' coating. This reduction was partially linked to ROS production. Alkaline phosphatase (phosphorus-cycling) activity was not affected, however in contrast, an alkaline phosphatase isolated from E. coli was strongly inhibited at lower concentrations of TiO2 NP than the intact biofilms. These results indicate that enzymes present in the biofilm matrix are partly protected against exposure to TiO2 NP and UV radiation. Impairment of extracellular enzymes which mediate the uptake of nutrients from water may affect ecosystem function. PMID:25208344

Schug, Hannah; Isaacson, Carl W; Sigg, Laura; Ammann, Adrian A; Schirmer, Kristin



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

PubMed Central

Microbial poly(?, l-malic acid) was modified with either l-leucine ethyl ester (L) or l-phenylalanine methyl ester (F) to produce amphiphylic copolymers. The degradation of these copolymers in aqueous buffer took place under physiological conditions in a few weeks by hydrolysis of the side chain ester group followed by cleavage of the main chain. Spherical nanoparticles with diameters ranging between 70 and 230 nm were prepared from these copolymers by the dialysis-precipitation method. No alteration of the cell viability was observed after incubation of these nanoparticles in different cell lines. Anticancer drugs temozolomide and doxorubicin were encapsulated in the nanoparticles. Temozolomide was released within several hours whereas doxorubicin took several weeks to be completely liberated. PMID:24954994

Lanz-Landázuri, Alberto; Portilla-Arias, José; de Ilarduya, Antxon Martínez; Holler, Eggehard; Ljubimova, Julia; Muñoz-Guerra, Sebastián



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

PubMed Central

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

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



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

NASA Technical Reports Server (NTRS)

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

Holstege, Gert; Blok, Bertil F.



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


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

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



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.  

PubMed Central

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. Images PMID:2107210

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



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. PMID:17007537

Rizvi, Syed Asad Ali; Shamsi, Shahab A.



Metabolic response to nonglucidic nutrient secretagogues and enzymatic activities in pancreatic islets of adult rats after neonatal streptozotocin administration.  


In islets from adult rats injected with streptozotocin during the neonatal period, both a nonmetabolized analog of L-leucine and 3-phenylpyruvate augmented 14CO2 output from islets either prelabeled with L-[U-14C]glutamine or exposed to D-[2-14C]glucose and D-[6-14C]glucose, in a manner qualitatively comparable to that found in islets from control rats. The islets of diabetic rats differed, however, from those of control rats by their unresponsiveness to both the L-leucine analog and a high concentration of D-glucose in terms of increasing 3HOH generation from [2-3H]glycerol, an impaired sparing action of the hexose upon 14CO2 output from islets prelabeled with [U-14C]palmitate, and, most importantly, by a decreased rate of D-[2-14C]glucose and D-[6-14C]glucose oxidation when either incubated at a high concentration of the hexose (16.7 mM) or stimulated by nonglucidic nutrient secretagogues at a low concentration of D-glucose (2.8 mM). In islet homogenates, the activity of glyceraldehyde phosphate dehydrogenase, glutamate decarboxylase, and NADP-malate dehydrogenase was lower in diabetic than control islets. Such was not the case for glutamate-alanine transaminase, glutamate-aspartate transaminase, or glutamate dehydrogenase. The neonatal injection of streptozotocin thus affected, in the adult rats, the activity of several islet enzymes. Nevertheless, the metabolic data suggest that an impaired circulation in the glycerol phosphate shuttle, as observed in response to stimulation of the islets by either a high concentration of D-glucose or nonglucidic nutrient secretagogues, represents an essential determinant of the preferential impairment of glucose-induced insulin release in this model of non-insulin-dependent diabetes. PMID:8484960

Sener, A; Giroix, M H; Malaisse-Lagae, F; Bailbe, D; Leclercq-Meyer, V; Portha, B; Malaisse, W J



In vivo ocular pharmacokinetics of acyclovir dipeptide ester prodrugs by microdialysis in rabbits.  


In vivo corneal absorption of the dipeptide prodrugs of acyclovir (ACV) was evaluated using microdialysis in rabbits. A corneal well was placed on the cornea of the anesthetized New Zealand White rabbits with implanted linear probes into the aqueous humor. Two hundred microliters of a 1% solution of L-valine-ACV (VACV), glycine-valine-ACV (GVACV), valine-valine-ACV (VVACV), and valine-tyrosine-ACV (VYACV) was placed in the corneal well and was allowed to diffuse for a period of 2 h, following which the drug solution was aspirated and well removed. Samples were collected every 20 min throughout the infusion and postinfusion phases and were analyzed by HPLC to obtain the aqueous humor concentrations. Absorption rate constants of all the compounds were found to be lower than the elimination rate constants. GVACV exhibited highest absorption rate (ka) compared with other prodrugs, but all the prodrugs showed similar terminal elimination rate (lambda(z)). The time of maximum absorption (Tmax) of ACV after administration of VACV and the dipeptide prodrugs did not vary significantly (p < 0.05). GVACV exhibited the highest concentration (Cmax) and area under curve (AUC) upon absorption (p < 0.05) compared to VACV, VVACV, and VYACV. Dipeptide prodrugs of ACV were absorbed through the cornea at similar rates but to varying extents. The dipeptide prodrug GVACV owing to its enhanced absorption of ACV seems to be a promising candidate for the treatment of ocular HSV infections. PMID:16889437

Anand, Banmeet S; Katragadda, Suresh; Gunda, Sriram; Mitra, Ashim K



Production of valine by a Bacillus sp.  


A bacterium isolated from Burdwan (India) soil was found to accumulate L-valine in the growth medium and identified to be a strain of Bacillus subtilis. The strain is able to grow and accumulate valine in a purely synthetic medium, but supplementation of the synthetic medium with either Casamino acids or yeast extract or with both, significantly improves the yield. The entire fermentation period can be divided into a growth phase and a production phase, which can be prolonged by adjustment of pH to the neutral range. Among the different carbon and nitrogen sources tested glucose at 8.5% and L-glutamic acid at 0.8%, respectively, were found most suitable. Cane sugar molasses tested as a substitute for glucose significantly stimulated growth but valine production was less. Different vitamins tested stimulated growth and valine yield and an inoculum level of 10% (v/v) of the medium was found to be optimal. The yield of valine under optimal conditions was found to be 4.53 g per litre of the medium. Valine has been isolated in crystalline form from the fermented broth by ion exchange resin chromatography and found to be a pure sample of the L-isomer. PMID:27903

Chattopadhyay, S P; Banerjee, A K



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


Capillary electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE-C(4)D) has been employed for the determination of the ?-blocker drugs (atenolol and amiloride) in pharmaceutical formulations. 150 mM acetic acid was used as background electrolyte. The influence of several factors (detector excitation voltage and frequency, buffer concentration, applied voltage, capillary temperature, and injection time) was studied. Non-UV absorbing L-valine was used as an internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28 °C, 25 kV, and using hydrodynamic injection (25 s). The separation was effected in a bare fused-silica capillary 75 ?m × 52 cm. The CE-C(4)D method was validated with respect to linearity, limit of detection and quantification, accuracy, precision, and selectivity. Calibration curves were linear over the range 5-250 ?g mL(-1) for the studied analytes. The relative standard deviations of intra- and inter-day precisions of migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of the ?-blocker drugs in different pharmaceutical tablets. PMID:22976091

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



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

PubMed Central

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

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



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


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

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



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


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 of 98.7% 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



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



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


Eleven laboratories collaboratively studied a liquid chromatographic (LC) method for determination of D-malic acid in apple juice. The mobile phase consisted of mM L-valine and 8 mM copper acetate adjusted to pH 5.5 with NaOH. The UV detector was set at 330 nm, and a single reversed-phase LC column was used. Seven paired samples containing various amounts of D-malic acid ranging from 0 to 188 mg/100 mL of 12 Brix pasteurized apple juice were tested by each collaborator. Repeatability and reproducibility coefficients of variation ranged from 1.0 to 3.5% and 7.7 to 11.7%, respectively, within the range of 26 to 188 mg D-malic acid/100 mL of 12 Brix apple juice. The collaborative study results demonstrated that the method could quantitate the economic adulteration of apple juice with DL-malic acid at lower levels than those reported with previous methods. The LC method for determination of D-malic acid in apple juice has been adopted first action by AOAC INTERNATIONAL. PMID:8620111

Eisele, T A




PubMed Central

Autoradiographs were prepared from frozen sections of everted sacs of hamster jejunum which had been incubated in vitro with C14- or H3-labeled sugars and amino acids. When such tissue was incubated in 1 mM solutions of L-valine or L-methionine, columnar absorptive cells at tips of villi accumulated these amino acids to concentrations ranging from 5 to 50 millimoles per liter of cells. Quantitative data were obtained by microdensitometry of C14 autoradiographs. Similar, though less striking, results were obtained with the sugars: galactose, 3-0-methylglucose, ?-methylglucoside, and 6-deoxyglucose. In all cases the marked "step-up" in concentration occurred near the brush border of the cell, and a "step-down" in concentration occurred at the basal pole of the cell. Known inhibitors of intestinal absorption, e.g., phlorizin in the case of sugars, blocked the concentrative step at the luminal border of the absorptive cell. It is inferred from these data that active transport systems for sugars and amino acids reside in the brush border region of the cell. Additional evidence suggests that the basal membrane of the cell may be the site of both a diffusion barrier and a weak transport system directed into the cell. PMID:19866662

Kinter, William B.; Wilson, T. Hastings



Biochemical characterization of Santalum album (Chandan) leaf peroxidase.  


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

Kumar, Pradeep; Kamle, Madhu; Singh, Jagtar



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

NASA Astrophysics Data System (ADS)

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

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



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. PMID:19557155

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



Barrier repair therapy for facial atopic eczema with a non-steroidal emollient cream containing rhamnosoft, ceramides and iso-leucine. A six-case report series.  


Atopic eczema (AE) is a skin disease very common in paediatric population and face region is commonly involved. AE of the face represents a therapeutic challenge limiting the use, especially for long periods, of corticosteroid topical products due to the high risk of atrophic skin changes. Skin barrier alterations and reduction of innate immune mechanisms (reduced levels of anti-microbial peptides) are now considered the hallmarks of AE. Therefore emollient and barrier repair therapies with topical steroid-free substances could be an alternative or an adjuvant strategy in managing AE especially for the face. A non-steroidal, anti-inflammatory moisturizing cream with barrier repair actions, containing rhamnosoft, ceramides and L-isoleucine (ILE) (Nutratopic pro-AMP) has been recently developed for the specific treatment of AE of the face. We report a series of 6 pediatric cases (2 female and 4 male, age from 6 months to 4 years) with facial eczema in children treated with pro-AMP cream for two/four weeks as single treatment, applied twice daily in the affected area with photograph documentation (baseline and after treatment). Pictures of the skin lesions at baseline and after treatment were taken in all cases using a high-definition digital camera. Pro-AMP cream use was associated with a clinical relevant improvement of all signs of eczema. The product was well tolerated. This case series document the clinical efficacy of a barrier repair therapy cream containing rhamnosoft, ceramides and iso-leucine in the treatment of atopic eczema of the face. PMID:25198568

Puviani, M; Agostinis, F; Milani, M



Ion and pH effect on the lower critical solution temperature phase behavior in neutral and acidic poly(organophosphazene) counterparts.  


Ion and pH effects on the phase transition behaviors are studied with a series of thermosensitive neutral and acidic poly(organophosphazene) counterparts. Poly(organophosphazenes) are substituted by hydrophobic L-isoleucine ethyl ester (IleOEt) and hydrophilic alpha-amino-omega-methoxy-poly(ethylene glycol) 550 Da (PEG550) together with a relatively small amount of glycylglycine ally ester (GlyGlyOALL). After deprotection, GlyGlyOALL changes into glycylglycine (GlyGlyOH), and neutral GlyGlyOALL and acidic GlyGlyOH polymers with same substituent ratios are compared as counterparts. All the synthesized poly(organophosphazenes) in this work exhibit lower critical solution temperature (LCST) for which sequential phase transitions are suggested: (i) homogeneous solution, (ii) homogeneous gel, (iii) heterogeneous gel, to (iv) heterogeneous solution as hydrophobicity increases either driven by temperature or substituent composition. Ions act on the hydrophobicity modification of the polymers where the polymers with lower hydrophobic/hydrophilic ratios are more sensitively salted-out by NaCl, while those with higher ratios are more effectively salted-in by NaI. At higher concentration of the added ions, the acid group effect on the cloud point becomes deactivated. Meanwhile, because of the conflicting role of amine and carboxylic acid in pH-responsiveness, neutral and acidic polymer counterparts exhibit opposite tendencies in the cloud points. Systematically controlled responsiveness to temperature, ion, and pH changes are created in random amphiphilic graft copolymers, poly(organophosphazenes). The results highlight the importance of the cooperative function of the dominant components in the poly(organophosphazenes) and also expand the general understanding in designing stimuli-responsive smart materials especially useful for various biomedical applications. PMID:19140714

Ahn, Sungsook; Monge, Estela C; Song, Soo-Chang



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

NASA Astrophysics Data System (ADS)

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

Meijer, T.; Cleveringa, P.



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

PubMed Central

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

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



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

PubMed Central

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

Arai, Toshinobu; Arimura, Yasuhiro; Ishikura, Shun



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


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

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



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. PMID:21217810

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



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.



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

PubMed Central

Acetate-mediated growth inhibition of Escherichia coli has been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance of E. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in the E. coli K-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treated E. coli cells among three mutant MetE proteins. E. coli WE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine in l-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45°C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (Tm) but an enhanced in vivo stability. Thus, we demonstrate improved E. coli growth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when an E. coli WE strain carrying the MetE mutant was combined with a previously found MetA (homoserine o-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45°C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by l-methionine. PMID:24123739

Mordukhova, Elena A.



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


Fetal growth is dependent on both the quantity and relative composition of amino acids delivered to the fetal circulation, and impaired placental amino acid supply is associated with restricted fetal growth. Amino acid exchangers can alter the composition, but not the quantity, of amino acids in the intra- and extracellular amino acid pools. In the placenta, exchangers may be important determinants of the amino acid composition in the fetal circulation. This study investigates the substrate specificity of exchange between the placenta and the feto-placental circulation. Maternal-fetal transfer of radiolabelled amino acids and creatinine were measured in the isolated perfused human placental cotyledon. Transfer of L-[14C]serine or L-[14C]leucine, and [3H]glycine, were measured in the absence of amino acids in the fetal circulation (transfer by non-exchange mechanisms) and following 10-20 micromol boluses of unlabelled amino acids into the fetal circulation to provide substrates for exchange (transfer by exchange and non-exchange mechanisms). The ability of fetal arterial boluses of L-alanine and L-leucine to stimulate release of amino acids from the placenta was also determined using HPLC in order to demonstrate the overall pattern of amino acid release. Experiments with radiolabelled amino acids demonstrated increased maternal-fetal transfer of L-serine and L-leucine, but not glycine, following boluses of specific amino acids into the fetal circulation. L-[14C]Leucine, but not L-[14C]serine or [3H]glycine, was transferred from the maternal to the fetal circulation by non-exchange mechanisms also (P<0.01). HPLC analysis demonstrated that fetal amino acid boluses stimulated increased transport of a range of different amino acids by 4-7 micromol l(-1) (P<0.05). Amino acid exchange provides a mechanism to supply the fetus with amino acids that it requires for fetal growth. This study demonstrates that these transporters have the capacity to exchange micromolar amounts of specific amino acids, and suggests that they play an important role in regulating fetal plasma amino acid composition. PMID:17478537

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



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


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

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



Human Lung Angiotensin Converting Enzyme  

PubMed Central

To enable its immunohistologic localization, angiotensin converting enzyme (EC 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 PMID:6259212

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



Regulation and compartmentalization of ?-lactam biosynthesis  

PubMed Central

Summary Penicillins and cephalosporins are ??lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l???aminoadipic acid, l?cysteine and l?valine to form the tripeptide ??l???aminoadipyl?l?cysteinyl?d?valine catalysed by the non?ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl?CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two?component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains. PMID:21255328

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



Simultaneous modulation of transport and metabolism of acyclovir prodrugs across rabbit cornea: An approach involving enzyme inhibitors.  


The aim of this study is to identify the class of enzymes responsible for the hydrolysis of amino acid and dipeptide prodrugs of acyclovir (ACV) and to modulate transport and metabolism of amino acid and dipeptide prodrugs of acyclovir by enzyme inhibitors across rabbit cornea. l-Valine ester of acyclovir, valacyclovir (VACV) and l-glycine-valine ester of acyclovir, gly-val-acyclovir (GVACV) were used as model compounds. Hydrolysis studies of VACV and GVACV in corneal homogenate were conducted in presence of various enzyme inhibitors. IC(50) values were determined for the enzyme inhibitors. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Complete inhibition of VACV hydrolysis was observed in the presence of Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl-fluoride) and PCMB (p-chloromercuribenzoic acid). Similar trend was also observed with GVACV in the presence of bestatin. IC(50) values of PCMB and bestatin for VACV and GVACV were found to be 3.81+/-0.94 and 0.34+/-0.08muM respectively. Eserine, tetraethyl pyrophosphate (TEPP) and diisopropyl fluorophosphate (DFP) also produced significant inhibition of VACV hydrolysis. Transport of VACV and GVACV across cornea showed decreased metabolic rate and modulation of transport in presence of PCMB and bestain respectively. The principle enzyme classes responsible for the hydrolysis of VACV and GVACV were carboxylesterases and aminopeptidases respectively. Enzyme inhibitors modulated the transport and metabolism of prodrugs simultaneously even though their affinity towards prodrugs was distinct. In conclusion, utility of enzyme inhibitors to modulate transport and metabolism of prodrugs appears to be promising strategy for enhancing drug transport across cornea. PMID:16720085

Katragadda, Suresh; Talluri, Ravi S; Mitra, Ashim K



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

PubMed Central

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

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



Impact of different CO2/HCO3- levels on metabolism and regulation in Corynebacterium glutamicum.  


We investigated the growth kinetics and transcriptional responses of Corynebacterium glutamicum in environments with low (pCO2<40 mbar) and high (pCO2 ? 300 mbar) CO2/HCO3(-) levels compared to standard conditions. When cultivated at high CO2/HCO3(-)-levels, C. glutamicum showed increased (63%) biomass to substrate yields during the initial growth phase. Other kinetic parameters such as growth rate (?), specific glucose consumption rate (qS), and selected enzymatic activities of anaplerotic reactions, the pentose phosphate pathway and the tricarboxylic acid cycle were similar to standard conditions. However, microarray hybridization disclosed a complex transcriptional response involving 117 differentially expressed genes. Among those, 60 genes were assigned to the complete DtxR/RipA regulon controlling iron homeostasis in C. glutamicum. Impaired growth of a ?dtxR mutant at high CO2/HCO3(-) levels validated the relevance of this master regulator to cope with excessive CO2/HCO3(-) availability. At low CO2/HCO3(-) levels, C. glutamicum grew in a bi-level manner with three distinct growth phases. Differential analyses revealed approximately doubled activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase accompanied by the formation of L-alanine and L-valine during the lowest ? occurring in mid-phase of the cultivation. DNA microarray analysis revealed more than 100 differentially expressed genes in growth phase II compared to phase I including almost all thiamin pyrophosphate (TPP) biosynthesis genes, which were significantly up regulated. Concluding, we hypothesize that C. glutamicum counteracts the lack of CO2/HCO3(-) by triggering TPP biosynthesis for increasing the activities of TPP-dependent enzymes involved in CO2 formation. PMID:24140290

Blombach, Bastian; Buchholz, Jens; Busche, Tobias; Kalinowski, Jörn; Takors, Ralf



Synthesis, structural elucidation, biological, antioxidant and nuclease activities of some 5-Fluorouracil-amino acid mixed ligand complexes.  


Some biologically active mixed ligand complexes (1-9) have been synthesized from 5-Fluorouracil (5-FU; A) and amino acids (B) such as glycine (gly), L-alanine (ala) and L-valine (val) with Ni(II), Cu(II) and Zn(II) ions. The synthesized mixed ligand complexes (1-9) were characterized by various physico-chemical, spectral, thermal and morphological studies. 5-Fluorouracil and its mixed ligand complexes have been tested for their in vitro biological activities against some pathogenic bacterial and fungal species by the agar well diffusion method. The in vitro antioxidant activities of 5-Fluorouracil and its complexes have also been investigated by using the DPPH assay method. The results demonstrate that Cu(II) mixed ligand complexes (4-6) exhibit potent biological as well as antioxidant activities compared to 5-Fluorouracil and Ni(II) (1-3) and Zn(II) (7-9) mixed ligand complexes. Further, the cleaving activities of CT DNA under aerobic conditions show moderate activity with the synthesized Cu(II) and Ni(II) mixed ligand complexes (1-6) while no activity is seen with Zn(II) complexes (7-9). Binding studies of CT DNA with these complexes show a decrease in intensity of the charge transfer band to the extent of 5-15% along with a minor red shift. The free energy change values (?(‡)G) calculated from intrinsic binding constants indicate that the interaction between mixed ligand complex and DNA is spontaneous. PMID:25022506

Shobana, Sutha; Subramaniam, Perumal; Mitu, Liviu; Dharmaraja, Jeyaprakash; Arvind Narayan, Sundaram



Integrative analysis of transcriptomic and metabolomic profiling of ascites syndrome in broiler chickens induced by low temperature.  


Ascites syndrome (AS) still has an unacceptably high incidence rate in both humans and animals although there have been many studies on AS. To continue our previous pathological and biochemical investigation on the underlying mechanisms of AS incidence in broiler chickens, cutting-edge technologies including RNA-seq and metabolimics were used by directly comparing AS chickens and healthy controls. The RNA-seq analysis in the liver identified 390 differentially expressed genes (DEGs), among which 212 genes were up-regulated and 178 genes were down-regulated in the AS group compared to the control. For the down-regulated DEGs, further gene ontology (GO) analysis suggested that lipid metabolism, cell differentiation, enzyme linked receptor protein signaling pathway and steroid biosynthesis pathway were significantly enriched. For up-regulated DEGs, the cholesterol metabolic process has the lowest p value (0.000966) of fold enrichment while the cholesterol biosynthetic process has the highest fold enrichment (46.67). The metabolomic analysis of serum revealed statistically significant changes in the concentrations of LysoPC(20?:?4), LysoPC(16?:?0), LysoPC(18?:?0), LysoPC(18?:?1), LysoPC(18?:?2), PC(14?:?1/20?:?1), PC(20?:?4/18?:?0), PC(14?:?1/22?:?1), dihydroxyacetone, indoleacrylic acid, ursodeoxycholic acid, l-valine, and l-tryptophan. The integrative analysis of transcriptome and metabolome indicated that two biological pathways of tryptophan biosynthesis and metabolism, and glycerophospholipid metabolism may contribute to the induction of AS in broilers. These findings have provided novel insights into our understanding of molecular mechanisms of AS incidence in broilers. PMID:25178933

Shi, Shourong; Shen, Yiru; Zhao, Zhenhua; Hou, Zhuocheng; Yang, Ying; Zhou, Huaijun; Zou, Jianmin; Guo, Yuming



Puromycin-Sensitive Aminopeptidase: An Antiviral Prodrug Activating Enzyme  

PubMed Central

Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models (Eriksson et al. Molecular Pharmaceutics, 2008 vol 5 598-609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the l-Valine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycin-sensitive aminopeptidase was generated and its substrate specificity investigated. The kcat for Val-pNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher kcat for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design. PMID:19969024

Tehler, Ulrika; Nelson, Cara H.; Peterson, Larryn W.; Provoda, Chester J.; Hilfinger, John M.; Lee, Kyung-Dall; McKenna, Charles E.; Amidon, Gordon L.



Improvement of Aspergillus nidulans penicillin production by targeting AcvA to peroxisomes.  


Aspergillus nidulans is able to synthesize penicillin and serves as a model to study the regulation of its biosynthesis. Only three enzymes are required to form the beta lactam ring tripeptide, which is comprised of l-cysteine, l-valine and l-aminoadipic acid. Whereas two enzymes, AcvA and IpnA localize to the cytoplasm, AatA resides in peroxisomes. Here, we tested a novel strategy to improve penicillin production, namely the change of the residence of the enzymes involved in the biosynthesis. We tested if targeting of AcvA or IpnA (or both) to peroxisomes would increase the penicillin yield. Indeed, AcvA peroxisomal targeting led to a 3.2-fold increase. In contrast, targeting IpnA to peroxisomes caused a complete loss of penicillin production. Overexpression of acvA, ipnA or aatA resulted in 1.4, 2.8 and 3.1-fold more penicillin, respectively in comparison to wildtype. Simultaneous overexpression of all three enzymes resulted even in 6-fold more penicillin. Combination of acvA peroxisomal targeting and overexpression of the gene led to 5-fold increase of the penicillin titer. At last, the number of peroxisomes was increased through overexpression of pexK. A strain with the double number of peroxisomes produced 2.3 times more penicillin. These results show that penicillin production can be triggered at several levels of regulation, one of which is the subcellular localization of the enzymes. PMID:25043338

Herr, Andreas; Fischer, Reinhard



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


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

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



Bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores.  


Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3xEGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed. PMID:15331749

Belov, George A; Lidsky, Peter V; Mikitas, Olga V; Egger, Denise; Lukyanov, Konstantin A; Bienz, Kurt; Agol, Vadim I



Bidirectional Increase in Permeability of Nuclear Envelope upon Poliovirus Infection and Accompanying Alterations of Nuclear Pores  

PubMed Central

Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3×EGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-l-alanyl-l-alanyl-l-prolyl-l-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed. PMID:15331749

Belov, George A.; Lidsky, Peter V.; Mikitas, Olga V.; Egger, Denise; Lukyanov, Konstantin A.; Bienz, Kurt; Agol, Vadim I.



Unique molecular conformation of aureobasidin A, a highly amide N-methylated cyclic depsipeptide with potent antifungal activity: X-ray crystal structure and molecular modeling studies.  


A structural feature of aureobasidins, cyclic depsipeptide antibiotics produced by Aureobasidium pullulans R106, is the N-methylation of four out of seven amide bonds. In order to investigate possible relationship between the molecular conformation and the amide N-methylation, aureobasidin A (AbA), which exhibits the potent antifungal activity, was subjected to X-ray crystal analysis. The crystal, recrystallized from ether (orthorhombic, space group P2(1)2(1)2(1), a = 21.643 (3) A, b = 49.865(10) A, c = 12.427 (1) A, z= 8), contained two independent conformers per asymmetric unit and they took on a similar arrowhead-like conformation. The conformation consisted of three secondary structures of antiparallel beta-sheet, and beta- and gamma-turns, and was stabilized by three intramolecular and transannular N-H O=C hydrogen bonds. The beta-hydroxy-N-methyl-l-valine residue, which is indispensable for its bioactivity, was located at the tip of the corner. Since a nearly identical conformation has been observed for aureobasidin E, a related cyclic depsipeptide, this arrowhead-like conformation may be energetically stable and important for biological activity. The contribution of the amide N-methylation to the conformation was investigated by model building and energy calculations. The energy-minimizations of AbA analogs, in which some (one to four) of four N-methylated amide bonds were replaced with usual amide bond, led to some conformers which are fairly different from the arrowhead form of AbA, although they are stabilized by three intramolecular N-H...O=C hydrogen bonds. This result explains the reason why four out of the seven amide bonds have to be methylated to manifest biological activity, i.e. the high N-methylation of aureobasidin is necessary to form only one well-defined conformation. PMID:10424343

In, Y; Ishida, T; Takesako, K



Anharmonicity of Low Frequency Modes in Protein Model Compounds Observed by Synchrotron Far-Infrared Absorption Spectroscopy  

NASA Astrophysics Data System (ADS)

Many computational and indirect experimental studies suggest that anharmonic low frequency modes are essential for large atomic displacements in proteins, and therefore functionally important. Here we report direct experimental evidence on anharmonicity of low frequency modes in protein model compounds (homopolymers of L-phenylalanine, L-alanine, L-leucine,and L-tryptophan) from the temperature dependence of their far-infrared absorption. For a low frequency mode, the occupancies in its vibrationally excited states may be significantly increased as the temperature is raised, leading to no shift in peak frequency if it is a harmonic oscillator, or to a down-shift in peak frequency if the potential is significantly anharmonic. Using a bright synchrotron light source, we have collected high signal-to-noise far-infrared absorption data (20 - 400 cm-1) for all four polymers at 100 K and 295 K, and for poly-L-Phe at 11 temperatures from 10 K to 290 K. The data show that (i) each of the homopolymers exhibits a small number of strong absorption bands; (ii) their well-resolved bands display down-shifts in frequencies as the temperature is elevated from 100 K to 295 K; (iii) the extent of the peak shifts with temperature for poly-L-phe are found qualitatively consistent with increases in excited state populations. These results

Xie, Aihua; He, Qin; Sclavi, Bianca; Chance, Mark



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

PubMed Central

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

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



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


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

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



Impact of Core-Forming Segment Structure on Drug Loading in Biodegradable Polymeric Micelles Using PEG-b-Poly(lactide-co-depsipeptide) Block Copolymers  

PubMed Central

We synthesized series of amphiphilic AB-type block copolymers having systematic variation in the core-forming segments using poly(lactide-co-depsipeptide)s as a hydrophobic segment and prepared polymeric micelles using the block copolymers, PEG-b-poly(lactide-co-depsipeptide). We then discussed the relationship between the core-forming segment structure and drug loading efficiency for the polymeric micelles. PEG-b-poly(lactide-co-depsipeptide)s, PEG-b-PLGL containing l-leucine (Leu), and PEG-b-PLGF containing l-phenylalanine (Phe), with similar molecular weights and various mole fractions of depsipeptide units, were synthesized. Polymeric micelles entrapping model drug (fluorescein, FL) were prepared using these copolymers. As a result, PEG-b-poly(lactide-co-depsipeptide) micelles showed higher drug loading compared with PEG-b-PLLA and PEG-b-PDLLA as controls. The drug loading increased with increase in the mole fraction of depsipeptide unit in the hydrophobic segments. The introduction of aliphatic and aromatic depsipeptide units was effective to achieve higher FL loading into the micelles. PEG-b-PLGL micelle showed higher drug loading than PEG-b-PLGF micelle when the amount of FL in feed was high. These results obtained in this study should be useful for strategic design of polymeric micelle-type drug delivery carrier with high drug loading efficiency. PMID:24696855

Takahashi, Akihiro; Ozaki, Yuta; Kuzuya, Akinori



Properties of Bacillus megaterium temperature-sensitive germination mutants.  

PubMed Central

Bacillus megaterium mutants JV-9 and JV-10 are temperature sensitive for initiation of spore germination. At 46 C, they did not lose heat resistance, dipicolinic acid, or absorbance, indicating that the temperature-sensitive blocks are very early in the sequence of initiation reactions. Strain JV-9 was temperature sensitive for initiation by glucose alone, and strain JV-10 was temperature sensitive for initiation by glucose, L-leucine, L-proline, KBr, or calcium dipicolinate. The kinetics of initiation were followed after two kinds of temperature change (shift-up and shift-down) experiments. Mutant spores incubated for different times at 46 C and then shifted down to 30 C showed no significant differences in the rates of absorbance decrease, i.e., no stimulation or inhibition. Conversely, when mutant spores were incubated for different times at 30 C, a fraction of the population initiated germination, and after shift-up to 46 C an additional fraction continued initiation while a third fraction stopped. This latter fraction did initiate germination when the temperature was lowered to 30 C. The kinetics of initiation after shift-up and shift-down in temperature suggest that the early events in initiation reagents, whereas the other four initiated sensitivity for all of the above initiation reagents, whereas the other four initiated very poorly. It was suggested that the lesion in strain JV-10 may result in the formation of one temperature-sensitive protein. Revertants of strain JV-9 could not be isolated. PMID:803950

Vary, J C



Effects of methionine supplement on methionine incorporation in rat embryos cultured in vitro.  


The effect of supplementary L-methionine (Met) on the incorporation of methionine was evaluated in 9.5-day rat conceptuses cultured in vitro. Parallel experiments with L-leucine (Leu) were performed for comparison. Conceptuses were cultured for 24 hr in the presence of 3H-labeled Met or Leu, and the incorporation of radiolabel into the embryo and visceral yolk sac was measured. Supplementary Met proportionately increased the incorporation of Met, but supplementary Leu did not have as great an effect on the incorporation of Leu. A hypothesis is presented to explain these findings. It is proposed that Met, but not Leu, is a rate-limiting nutrient for organogenesis-stage rat embryos cultured in rat serum. The results are also discussed with reference to the established efficacy of supplementary folic acid in decreasing the incidence of neural tube defects in human populations and to claims that Met reverses certain teratogenic phenomena, both in vitro and in vivo. PMID:10413332

Pugarelli, J E; Brent, R L; Lloyd, J B



Beta-decay, Bremsstrahlen, and the origin of molecular chirality  

NASA Technical Reports Server (NTRS)

A brief review is presented of the Vester-Ulbricht beta-decay Bremsstrahlen hypothesis for the origin of optical activity, and of subsequent experiments designed to test it. Certain experiments along these lines, begun in 1974 and involving the irradiation of racemic and optically active amino acids in a 61.7 KCi Sr-90-Y-90 Bremsstrahlen source, have now been completed and are described. After 10.89 years of irradiation with a total Bremsstrahlen dose of 2.5 x 10 to the 9th rads, crystalline DL-leucine, norleucine, and norvaline suffered 47.2, 33.6, and 27.4 percent radiolysis, respectively, but showed no evidence whatsoever of asymmetric degradation. Dand L-Leucine underwent about 48 percent radiolysis and showed 2.4-2.9 percent radioracemization. Other samples in solution were too severely degraded to analyze. Probable intrinsic reasons for the failure of the Vester-Ulbricht mechanism to afford asymmetric radiolysis in the present and related experiments involving beta-decay Bremsstrahlen are enumerated.

Bonner, W. A.; Yi, L.



Deuterium NMR study of amino acid coordination to chromium(III)  

SciTech Connect

A series of bis(ethylenediamine)Cr(III)-amino acid complexes, synthesized with deuterium-labeled alanine, glycine, homoserine, leucine, methionine, phenylalanine, serine, and threonine, was characterized by /sup 2/H NMR spectroscopy. The spectra show that these bidentate-coordinated amino acid complexes decompose via monodentate species. In addition, the diastereomeric isomers of alanine and leucine can be distinguished in the spectra. This was confirmed by the isolation of one of the L-leucine isomers. The bis(1,3-propanediamine)Cr(III) complexes of glycine and alanine were also synthesized, and the NMR spectra of these complexes and ..cap alpha..-cis-(Cr(ethylenediaminediacetate)(glycinate)) show changes due to variation in the ligand complement. The crystal structure of ..cap alpha..-cis(Cr(ethylenediaminediacetate)(glycinate)) x 2H/sub 2/O (CrO/sub 6/N/sub 3/C/sub 8/H/sub 14/ x 2H/sub 2/O) was determined and supports the NMR observations. This complex crystallizes in the space group P2/sub 1//c of the monoclinic crystal system with a = 8.9231 (19) A, b = 10.1889 (22) A, c = 15.4180 (30) A, ..beta.. = 102.657 (17)/sup 0/, and Z = 4. An improved method for the synthesis of bis(diamine)Cr(III)-amino acid complexes is also reported. 24 references, 3 figures, 7 tables.

Green, C.A.; Place, H.; Willett, R.D.; Legg, J.I.



Comparison of Alkalizing Solutions Used in Extracellular Enzyme Assays  

NASA Astrophysics Data System (ADS)

Extracellular enzymatic activity plays an important role in microbial nutrient cycling, and is commonly studied through the use of fluorogenic substrates. Samples are generally alkalized to maximize fluorescence yield and to prevent among sample variability in pH from affecting sample fluorescence. It is also frequently assumed that the alkaline solutions stop enzymatic reactions. Different alkalizing agents (pH 10 sodium carbonate-bicarbonate buffer, 0.05M glycine-ammonium hydroxide buffer, 40mM sodium hydroxide solution, and 2M sodium hydroxide solution) were explored for their ability to halt periphytic enzymatic activity without degrading the fluorescent product. L-Leucine-7-amido-4-methylcoumarin hydrochloride (LAMC) and 4-Methylumbeliferyl phosphate disodium salt (MUF-P) were used as fluorogenic substrates. Although 2M NaOH solution appeared to stop enzymatic reactions, it completely degraded both the AMC and MUF flurochromes, rendering it unsuitable for use as an alkalizing agent. The remaining three buffers did not greatly erode either fluorochrome, however, they were unable to halt enzymatic activity reliably. This brings into question the method of using alkalizing agents as a means of halting enzymatic reactions for later analysis.

Johnson, A. C.; Kuehn, K. A.; Francoeur, S. N.



Specific Counterion Repercussions on the Thermal, pH-Response, and Electrochemical Properties of Side-Chain Leucine Based Chiral Polyelectrolytes.  


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

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



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


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



Ribosomal protein deficiency causes Tp53-independent erythropoiesis failure in zebrafish.  


Diamond-Blackfan anemia is an inherited genetic disease caused by mutations in ribosomal protein genes. The disease is characterized by bone marrow failure, congenital anomalies, and a severe erythroid defect. The activation of the TP53 pathway has been suggested to be critical for the pathophysiology of Diamond-Blackfan anemia. While this pathway plays a role in the morphological defects that associate with ribosomal protein loss-of-function in animal models, its role in the erythroid defects has not been clearly established. To understand the specificity of erythroid defects in Diamond-Blackfan anemia, we knocked down five RP genes (two Diamond-Blackfan anemia-associated and three non-Diamond-Blackfan anemia-associated) in zebrafish and analyzed the effects on the developmental and erythroid phenotypes in the presence and absence of Tp53. The co-inhibition of Tp53 activity rescued the morphological deformities but did not alleviate the erythroid aplasia indicating that ribosomal protein deficiency causes erythroid failure in a Tp53-independent manner. Interestingly, treatment with L-Leucine or L-Arginine, amino acids that augment mRNA translation via mTOR pathway, rescued the morphological defects and resulted in a substantial recovery of erythroid cells. Our results suggest that altered translation because of impaired ribosome function could be responsible for the morphological and erythroid defects in ribosomal protein-deficient zebrafish. PMID:24417973

Yadav, Gnaneshwar V; Chakraborty, Anirban; Uechi, Tamayo; Kenmochi, Naoya



Immunogenicity of fully defined lysine LAG dodecapeptides in guinea-pigs: with a note on an antigenic cooperation effect in the delayed immune response  

PubMed Central

The immunogenicity of a series of dodecapeptides was evaluated in random-bred guinea-pigs. The peptides were synthesized by an economical two-phase approach specifically developed for the production of gram-amounts of peptide antigens. The peptides contain L-leucine, L-alanine, glycine and L-lysine and differ from each other by number and distribution of lysine residues. Some of the peptides carry DNP groups at their N-terminus. With the exception of one (5,11-Lys2-LAG12-OEt), all peptides induced a state of cellular immunity as evaluated by delayed dermal reactions and macrophage migration inhibition. Some peptides also induced the formation of circulating antibodies. Immunogenicity appears to increase with the number of basic groups and is more pronounced with DNP peptides. Delayed dermal responses are highly specific since even peptides closely related to the immunogen did not elicit reactions. Reactions were however observed when some mixtures of closely related peptides not used for immunizing the animal were administered. This `antigenic cooperation' effect is discussed briefly. PMID:4119344

Schneider, C. H.; De Weck, A. L.; Schenkel, E.; Wirz, W.; Lazary, S.



Suppression of mTORC1 activation in acid-?-glucosidase-deficient cells and mice is ameliorated by leucine supplementation.  


Pompe disease is due to a deficiency in acid-?-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog l-leucyl-l-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model. PMID:25231351

Shemesh, Adi; Wang, Yichen; Yang, Yingjuan; Yang, Gong-She; Johnson, Danielle E; Backer, Jonathan M; Pessin, Jeffrey E; Zong, Haihong



Peptide Coupling between Amino Acids and the Carboxylic Acid of a Functionalized Chlorido-gold(I)-phosphane.  


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

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



Myocardial Reloading after Extracorporeal Membrane Oxygenation Alters Substrate Metabolism While Promoting Protein Synthesis  

SciTech Connect

Extracorporeal membrane oxygenation (ECMO) unloads the heart providing a bridge to recovery in children after myocardial stunning. Mortality after ECMO remains high.Cardiac substrate and amino acid requirements upon weaning are unknown and may impact recovery. We assessed the hypothesis that ventricular reloading modulates both substrate entry into the citric acid cycle (CAC) and myocardial protein synthesis. Fourteen immature piglets (7.8-15.6 kg) were separated into 2 groups based on ventricular loading status: 8 hour-ECMO (UNLOAD) and post-wean from ECMO (RELOAD). We infused [2-13C]-pyruvate as an oxidative substrate and [13C6]-L-leucine, as a tracer of amino acid oxidation and protein synthesis into the coronary artery. RELOAD showed marked elevations in myocardial oxygen consumption above baseline and UNLOAD. Pyruvate uptake was markedly increased though RELOAD decreased pyruvate contribution to oxidative CAC metabolism.RELOAD also increased absolute concentrations of all CAC intermediates, while maintaining or increasing 13C-molar percent enrichment. RELOAD also significantly increased cardiac fractional protein synthesis rates by >70% over UNLOAD. Conclusions: RELOAD produced high energy metabolic requirement and rebound protein synthesis. Relative pyruvate decarboxylation decreased with RELOAD while promoting anaplerotic pyruvate carboxylation and amino acid incorporation into protein rather than to the CAC for oxidation. These perturbations may serve as therapeutic targets to improve contractile function after ECMO.

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



Characterization of angiotensin-converting enzyme in the gills of rainbow trout,Salmo gairdneri (Richardson).  


Several physical and chemical parameters of angiotensin-converting enzyme (ACE) were determined using a spectrophotometric assay of gill tissue homogenates from rainbow trout. This assay is based on the evolution of free hippuric acid via enzymatic cleavage of histidyl-leucine from the synthetic substrate hippuryl-l-histidyl-l-leucine (HHL). Piscine ACE exhibited enzymatic and kinetic properties similar to those reported for the partially purified mammalian enzyme. Proteolytic activity was both temperature and pH dependent and demonstrated hyperbolic kinetics with an apparent Km of 2.5 mM. Hydrolysis of HHL was activated by Cl(-) at concentrations between 20 mM and 100 mM. Captopril (1 × 10(-6) M) and MK-422 (1 × 10(-6) M) blocked trout gill ACE activity, however, EDTA was inhibitory only at high concentrations (1 × 10(-3) M). These results demonstrate that trout ACE is functionally similar to mammalian ACE and that the spectro-photometric assay for ACE developed by Cushman and Cheung can be applied to analysis of converting enzyme activity in fish tissue homogenates. PMID:24233338

Lipke, D W; Thomas, R L; Olson, K R



Purification, characterization and antibacterial activity of L-amino acid oxidase from Cerastes cerastes.  


Antibiotic resistance presents a real problem in which new antibacterial molecules from natural secretions could be beneficial in the development of new drugs. In this study, Cerastes cerastes venom was investigated for its antibacterial activity against Gram-positive and Gram-negative bacteria. The antibacterial activity was evaluated by measuring the halo inhibition and minimum inhibitory concentration (MIC). An L-amino acid oxidase (CcLAAO) was purified from this venom using three chromatographic steps; its homogeneity (60 kDa) was confirmed by SDS-PAGE. LC-MS/MS analysis of CcLAAO showed similarities with other LAAO enzymes from Echis ocellatus and Viridovipera stejnegeri venoms. CcLAAO presents an antibacterial activity against three bacterial strains (Staphylococcus aureus, Methicillin-resistant S. aureus, and Pseudomonas aeruginosa) with MIC values of 10, 10, and 20 ?g/mL, respectively. However, no effect was observed against Escherichia coli and yeast strains. Kinetic parameters of CcLAAO evaluated on L-leucine at pH 8.0 and 20°C were Km = 0.06 mmol and Vmax = 164 mmol/min. PMID:24817275

Hanane-Fadila, Ziad-Meziane; Fatima, Laraba-Djebari



Influence of different cultural conditions on cellulase production by Nectria catalinensis.  


The production of the extracellular cellulolytic enzyme system (endoglucanase, exoglucanase and cellobiase) of N. catalinensis was tested with different nitrogen sources, inorganic and organic ones, in liquid culture medium with microcrystalline cellulose. The nitrogen compounds used were: potassium nitrate, sodium nitrate, ammonium nitrate, ammonium phosphate, ammonium sulphate, ammonium chloride, ammonium carbonate, ammonium acetate, ammonium tartrate, urea, casamino acids, glycine, L-alanine, L-leucine, L-proline, L-lysine, L-aspartic acid, L-glutamic acid, L-asparagine, L-glutamine, L-phenylalanine, L-tyrosine, L-tryptophan, L-methionine and L-cysteine. Among these, ammonium nitrate and ammonium tartrate gave the highest yields of cellulases in 20-day-old cultures at a concentration equivalent to 0.75 g N/l in both cases. Optimal temperature for cellulase production, growth and cellulose degradation was 23 degrees C. On the other hand, an initial pH of 6.5 gave the highest yields of endoglucanase and cellobiase. In the same way, at pH 6.5, maximal growth and cellulose degradation were achieved. However, maximal exoglucanase production and glycogen content were reached at pH 7.5. PMID:9629604

Pardo, A G; Forchiassin, F



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


In this study, an optically active diamine, N,N'-(pyromellitoyl)-bis{N-[4(4-aminophenoxy)phenyl]-2-(4-methyl)pentanamide} (1) containing amino acid L-leucine was prepared in three steps. The step-growth polymerization of this chiral diamine with several diisocyanates in room temperature ionic liquid (IL), 1,3-dipropylimidazolium bromide as an environmentally friendly solvent and in a volatile organic solvent, is investigated. The polymerization yields and inherent viscosities of the resulting poly(amide-ether-imide-urea)s are compared in both solvents. The results show that the IL to be the superior polymerization media. All of the obtained polymers exhibited good solubility in some polar aprotic organic solvents such as N,N-dimethyacetamide, N,N-dimethyformamide, dimethyl sulfoxide while thermal stability was not disturbed based on thermogravimetric analysis and differential scanning calorimetry experiments. X-ray diffraction analysis of polymers shows that they are amorphous. The observation of optical rotation confirms the optical activity of prepared polymers. PMID:20571840

Mallakpour, Shadpour



Kinetics and conformational stability studies of recombinant leucine aminopeptidase.  


Leucine aminopeptidase from Vibrio proteolyticus is a broad specificity N-terminal aminopeptidase that is widely used in pharmaceutical processes where the removal of N-terminal residues in recombinant proteins is required. We previously reported the expression of a heterologous construction of the mature protein fused to a 6-histidine tag that presents a reasonable refolding rate for its use at industrial level. Here, we investigate this recombinant leucine aminopeptidase (rLAP) to explain the gain of activity observed when incubated at 37 °C after its production. Unfolding transitions of rLAP as a function of urea concentration were monitored by circular dichroism (CD) and fluorescence (FL) spectroscopy exhibiting single transitions by both techniques. Free energy change for unfolding measured by CD and FL spectroscopy are 2.8 ± 0.4 and 3.7 ± 0.4 kcal mol(-1), respectively. Thermal stability conformation of rLAP is 2.6 ± 0.1 and 6.1 kcal mol(-1) for CD and Nano-Differential Scanning Calorimetry (Nano-DSC), respectively. Enzyme activity was assessed with L-leucine-p-nitroanilide (L-pNA) as substrate. The catalytic efficiency was 3.87 ± 0.10 min(-1) ?M(-1) at 37 °C and pH 8.0. Kinetic and conformation studies show differences between the enzyme native and rLAP; however rLAP is selective and specific to remove N-terminal groups from amino acids. PMID:24368112

Hernández-Moreno, Ana V; Villaseñor, Francisco; Medina-Rivero, Emilio; Pérez, Néstor O; Flores-Ortiz, Luis F; Saab-Rincón, Gloria; Luna-Bárcenas, Gabriel



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

NASA Astrophysics Data System (ADS)

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

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



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

SciTech Connect

Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 Angstroms resolution reveals a substrate-binding pocket with composite features of the distinct {beta}1, {beta}2 and {beta}5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the a-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapeptides of the a-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-{beta}-(1-naphthyl)-l-alanine-l-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

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



Kokumi-active glutamyl peptides in cheeses and their biogeneration by Penicillium roquefortii.  


Recently, a group of gamma-glutamyl dipeptides, but not the alpha-glutamyl dipeptides, were found to induce the attractive kokumi flavor of matured Gouda cheese. In the present investigation, the spatial distribution of alpha- and gamma-glutamyl dipeptides in Gouda cheese wheels and the concentration of these peptides in other cheese types were determined by means of HPLC-MS/MS. Among all cheeses investigated, by far the highest gamma-glutamyl peptide concentration (3590 mumol/kg) was found for Blue Shropshire, a blue-veined cheese. To check whether the gamma-glutamyl transferase (GGT) from Penicillium roquefortii is involved in gamma-glutamyl peptide production in this cheese, the GGT activity was measured and gamma-glutamyl peptides were analyzed in liquid cultures of mold isolated from Blue Shropshire as well as single P. roquefortiii strains incubated with the gamma-glutamyl donor l-glutamine and the candidate substrates l-glutamic acid, l-histidine, l-leucine, and l-methionine. Being well in line with the GGT activity found in Blue Shropshire, P. roquefortii was found for the first time to produce and secrete gamma-glutamyl peptides. Among the amino acids tested, l-methionine was found as a preferred gamma-glutamyl acceptor; for example, gamma-Glu-Met was produced in yields of about 50 mmol/mol and [(2)H(3)]-gamma-Glu-Met was obtained when [(2)H(3)]-l-methionine was used as substrate amino acid. PMID:19338275

Toelstede, Simone; Hofmann, Thomas



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. PMID:23226342

Dutoit, Raphael; Brandt, Nathalie; Legrain, Christianne; Bauvois, Cedric



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

PubMed Central

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

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



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


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

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



Bacterial Metabolism of Arylsulfonates: Role of meta Cleavage in Benzene Sulfonate Oxidation by Pseudomonas testosteroni  

PubMed Central

Pseudomonas testosteroni H-8 oxidizes certain lower alkylbenzene sulfonates at rates inversely related to the length of the alkyl group. Appreciable Q(O)2 values were observed for benzene sulfonate (BS), toluene sulfonate (TS), and ethylbenzene sulfonate (EBS), but not for propylbenzene sulfonate (PS) and higher homologues. Catechol oxidation was catalyzed by a constitutive catechol-2,3-oxygenase (EC 1.99.2.a). Yellow meta cleavage products accumulated when BS-grown cells were exposed to catechol, 4-methylcatechol, 3-methylcatechol, EBS and PS, but not BS or TS. Traces of a yellow metabolite (probably 2-hydroxymuconic semialdehyde) were detectable during growth on BS. PS completely inhibited growth on BS, but not on L-leucine or nutrient broth. Also, PS antagonized respiration on BS and catechol, but not glutamate, the extent of inhibition being directly related to PS concentration. Formation of a meta cleavage product from PS, and inhibition of catechol oxidation by PS, suggested that the actual inhibitor may not be PS itself, but a metabolite. PMID:163618

Ripin, Marilyn J.; Cook, Thomas M.; Noon, Kerry F.; Stark, Leonard E.



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


This study was focused on investigating the role of the initial residue community, i.e. microorganisms and enzymes from the epiphytic and endophytic compartments, in soil decomposition processes. Aerial and underground parts (leaves and roots) of maize (Zea mays L.) plants were ?-irradiated, surface-sterilized with sodium hypochlorite (NaOCl)/ethanol or non-sterilized (controls), while the outer surface morphology of maize leaves and roots was examined by scanning electron microscopy (SEM). Non-sterilized and sterilized maize leaves and roots were incubated in soil to study carbon (C) mineralization kinetics and enzyme dynamics (L-leucine aminopeptidase, CBH-1, xylanase, cellulase and laccase). SEM results showed that initial microbial colonization was more pronounced on non-sterilized leaf and root surfaces than on sterilized samples. The hypochlorite treatment removed a part of the soluble components of leaves by washing and no specific effect of any type of colonizing microorganisms was observed on C mineralization. In contrast, ? irradiation and hypochlorite treatments did not affect root chemical characteristics and the quantitative effect of initial residue-colonizing microorganisms on C mineralization was demonstrated. The variations in C mineralization and enzyme dynamics between non-sterilized and sterilized roots suggested that activities of epiphytic and endogenic microorganisms were of the same order of magnitude. PMID:22078739

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



Improved pulse sequences for pure exchange solid-state NMR spectroscopy.  


Spin-exchange experiments are useful for improving the resolution and establishment of sequential assignments in solid-state NMR spectra of uniformly (15)N-labeled proteins oriented macroscopically in phospholipid bilayers. To exploit this advantage fully, it is crucial that the diagonal peaks in the two-dimensional exchange spectra are suppressed. This may be accomplished using the recent pure-exchange (PUREX) experiments, which, however, suffer from up to a threefold reduction of the cross-peak intensity relative to experiments without diagonal-peak suppression. This loss in sensitivity may severely hamper the applicability for the study of membrane proteins. In this paper, we present a two-dimensional exchange experiment (iPUREX) which improves the PUREX sensitivity by 50%. The performance of iPUREX is demonstrated experimentally by proton-mediated (15)N-(15)N spin-exchange experiments for a (15)N-labeled N-acetyl-L-valyl-L-leucine dipeptide. The relevance of exchange experiments with diagonal-peak suppression for large, uniformly (15)N-labeled membrane proteins in oriented phospholipid bilayers is demonstrated numerically for the G-protein coupled receptor rhodopsin. PMID:14745809

Vosegaard, Thomas; Nielsen, Niels C



AFRRI (Armed Forces Radiobiology Research Institute) reports, July, August, September 1989. Technical report  

SciTech Connect

This volume contains AFRRI Scientific Reports SR 89-26 through SR89-39 and Technical Report TR89-1 for Jul-Sep 1989. Partial contents include: Induction of marrow hypoxia by radioprotective agents; Cell-cycle radiation response: Role of intracellular factors; Characteristics of radiation-induced performance changes in bar-press avoidance with and without a preshock warning cue; Norepinephrine-induced phosphorylation of a 25 kd phosphoprotein in rat aorta is altered in intraperitoneal sepsis; Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry; Tropism of canine neutrophils to xanthine oxidase; Effects of acute sublethal gamma radiation exposure on aggressive behavior in male mice: A dose-response study; Progressive behavioral changes during the maturation of rats with early radiation-induced hypoplasia of fascia dentata granule cells; Stomach nodules in pigeons; An assessment of the behavioral toxicity of high-energy iron particles compared to other qualities of radiation; L-leucyl-L-leucine methyl ester treatment of canine marrow and peripheral blood cells; Localization of cyclo-oxygenase and prostaglandin E2 in the secretory granule of the mast cell; Radioprotection of mice with interleukin-1: Relationship to the number of spleen colony-forming units; Survival after total-body irradiation. I. Effects of partial small-bowel shielding; Laboratory x-ray irradiator for cellular radiobiology research studies: Dosimetry report.

Not Available



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 PMID:169297

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



Tumour-associated macrophages secrete IL-6 and MCP-1 in head and neck squamous cell carcinoma tissue.  


Conclusion. Tumour-associated macrophages (TAMs) in head and neck squamous cell carcinomas (HNSCCs) secrete interleukin 6 (IL-6) and monocyte chemotactic protein (MCP-1) that can be down-regulated by L-leucine-methylester (LLME); however, there is no qualitative difference between function of TAMs and tissue macrophages in mucosa as measured by IL-6 and MCP-1 secretion. Objectives. TAMs play an important role in the interaction with tumour cells in malignant tumours. The cells in the tumours that are the main sources of the various signal substances need to be further elucidated. The aim of this investigation was to reveal whether TAMs in HNSCCs secrete IL-6 and MCP-1. These cytokines influence tumour cell growth and macrophage influx in tumours, respectively. Materials and methods. In order to inhibit macrophage function in F-spheroids, in some experiments the tissue fragments were initially incubated with LLME, a substance that selectively inhibits function of phagocytes. IL-6 and MCP-1 secretion from untreated F-spheroids was compared to cytokine secretion from LLME-treated F-spheroids as measured by ELISA. Results. LLME did not affect the viability of F-spheroids and reduced IL-6 and MCP-1 secretion from monocyte-derived macrophages in vitro. F-spheroids from LLME-treated tissue fragments showed lower IL-6 and MCP-1 secretion compared with F-spheroids from tissue fragment untreated with LLME. PMID:17453481

Kross, Kenneth W; Heimdal, John-Helge; Olsnes, Carla; Olofson, Jan; Aarstad, Hans Jørgen



Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light  

SciTech Connect

Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a 13C15N-L-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 422 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly and degradation showed higher levels of isotope incorporation suggesting that these biochemical pathways are important for growth under non-diazotrophic conditions. Calculation of relative isotope abundances (RIA) values allowed to measure actual active protein synthesis over time for different biochemical pathways under non-diazotrophic conditions. Overall results demonstrated the utility of 'non-steady state' pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

Aryal, Uma K.; Stockel, Jana; Welsh, Eric A.; Gritsenko, Marina A.; Nicora, Carrie D.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.



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

PubMed Central

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

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



Rapid chiral separation and impurity determination of levofloxacin by ligand-exchange chromatography.  


A sensitive, simple, and accurate method for determination of levofloxacin and its (R)-enantiomer was developed to determine the chiral impurity of levofloxacin in Cravit Tablets material by ligand-exchange high performance liquid chromatography. The effects of different kinds of ligands, concentration of ligands in mobile phase, organic modifier, pH of mobile phase, and temperature on enantioseparation were investigated and evaluated. Chiral separation was performed on a conventional C(18) column, where the mobile phase consisted of a methanol-water solution (containing 10 mmol L(-1)l-leucine and 5 mmol L(-1) copper sulfate) (88:12, v/v) and its flow-rate was set at 1.0 mL min(-1). The conventional C(18) column offers baseline separation of two enantiomers with a resolution of 2.4 in less than 20 min. Thermodynamic data (DeltaDeltaH and DeltaDeltaS) obtained by Van't Hoff plots revealed the chiral separation is an enthalpy-controlled process. The standard curves showed excellent linearity over the concentration range from 0.5 to 400 mg L(-1) for levofloxacin and its (R)-enantiomer. The linear correlation equations are: y=1.33 x 10(5)x+6297 (r=0.9991) and y=1.34 x 10(5)x+3565 (r=0.9997), respectively. The relative standard deviation (RSD) of the method was below 2.3% (n=3). PMID:17386599

Yan, Hongyuan; Row, Kyung Ho



Proposed involvement of an internal promoter in regulation and synthesis of mitochondrial and cytoplasmic leucyl-tRNA synthetases of Neurospora.  

PubMed Central

Genetic analysis of an electrophoretic variant of the mitochondrial leucyl-tRNA synthetase [L-leucine:tRNALeu ligase (AMP-forming), EC] indicates that it is either an allele of or linked closely to leu-5ts, a mutant that is known to produce a cytoplasmic leucyl-tRNA synthetase with an altered affinity for leucine as well as being deficient in the production of the mitochondrial enzyme. Immunological analysis indicates that the two synthetases have little, if any, structural homology. The pattern of synthesis of the enzymes in leu-5ts revertants, the reciprocal relationship of the production of the two enzymes in response to a negative regulatory element, presumably of mitochondrial origin, as well as the lack of detectable structural homology, led to the proposal that the phenotype of leu-5ts results from a mutational alteration in the structural gene for the cytoplasmic enzyme in a region involved in the initiation of transcription of the adjacent gene for the mitochondrial enzyme. PMID:139609

Beauchamp, P M; Horn, E W; Gross, S R



Molecular Dynamics of Peptide Folding at Aqueous Interfaces  

NASA Technical Reports Server (NTRS)

Even though most monomeric peptides are disordered in water they can adopt sequence-dependent, ordered structures, such as a-helices, at aqueous interfaces. This property is relevant to cellular signaling, membrane fusion, and the action of toxins and antibiotics. The mechanism of folding nonpolar peptides at the water-hexane interface was studied in the example of an 11-mer, of poly-L-leucine. Initially placed as a random coil on the water side of the interface, the peptide folded into an a-helix in 36 ns. Simultaneously, the peptide translocated into the hexane side of the interface. Folding was not sequential and involved a 3/10-helix as an intermediate. The folded peptide was either parallel to the interface or had its C-terminus exposed to water. An 11-mer, LQQLLQQLLQL, composed of leucine (L) and glutamine (G), was taken as a model amphiphilic peptide. It rapidly adopted an amphiphilic, disordered structure at the interface. Further folding proceeded through a series of amphiphilic intermediates.

Pohorille, Andrew; Chipot, Christophe; Chang, Sherwood (Technical Monitor)



Human monoclonal antibodies produced by primary in vitro immunization of peripheral blood lymphocytes.  

PubMed Central

A general procedure is described for the production of human monoclonal antibodies from peripheral blood lymphocytes immunized in vitro against T-cell-dependent antigens. These lymphocytes immunized in culture were used to produce human-human or human-mouse hybridomas secreting monoclonal antibodies specific for digoxin, hemocyanin, a recombinant fragment of the gp120 envelope glycoprotein of human immunodeficiency virus (PB1), or a melanoma-associated antigen (p97). Depletion of a lysosome-rich cell population, containing large granular lymphocytes, monocytes, cytotoxic T cells, and a subset of CD8-positive T cells, was shown to be crucial before the cells could be immunized in vitro. This depletion was accomplished by treating the peripheral blood lymphocytes with the lysosomotropic agent L-leucine methyl ester. In addition, the in vitro immunization had to be supported by interleukin 2, gamma-interferon, and B-cell growth and differentiation factors, derived from irradiated, pokeweed-mitogen-stimulated human T cells. The production of human monoclonal antibodies from primary, antigen-specifically activated peripheral lymphocytes might obviate the need to immunize volunteers or patients. PMID:3131770

Borrebaeck, C A; Danielsson, L; Moller, S A



Long polypeptide 310-helices at atomic resolution  

PubMed Central

The crystal-state preferred conformation of the terminally blocked homooctapeptide from the C?,?-dimethylated ?-aminoisobutyric acid (Aib) residue, pBrBz-(Aib)8-OBut, in which pBrBz is para-bromobenzoyl and OBut is tert-butoxy, determined by x-ray diffraction analysis using direct methods, was found to be a 310-helix stabilized by six consecutive intramolecular N—H....O=C hydrogen bonds of the C10-III (or III?) type. This is the first observation at atomic resolution of a regular 310-helix longer than two complete turns. The solid-state structural analysis was extended to the terminally blocked, ?-aminoisobutyric acid-rich octapeptide corresponding to the 2-9 sequence of the peptaibol antibiotics emerimicins III and IV, pBrBz-Aib3-L-Val-Gly-L-Leu-Aib2-OMe. Again, this peptide adopts a (right-handed) 310-helical structure, although slightly distorted at the level of the L-leucine residue. The role of specific amino acid sequence and peptide main-chain length in stabilizing either the 310- or the ?-helical conformation and their possible implications on the nature of the channel formed by peptaibol antibiotics in the membrane are also briefly discussed. PMID:16593674

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



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

NASA Astrophysics Data System (ADS)

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

Hearty, Paul J.; Vacher, H. Leonard


Photoacoustic spectroscopy study of neodymium complexes with alanine, valine, phenylalanine and tryptophan  

NASA Astrophysics Data System (ADS)

Neodymium complexes with amino acids: Nd(Ala) 3Cl 3?·?3H 2O, Nd(Val) 3Cl 3?·?3H 2O, Nd(Phe)Cl 3?·?5H 2O and Nd(Trp) 3Cl 3?·?3H 2O (Ala: L-alanine, Val: L-valine, Phe: L-phenylalanine, Trp: L-tryptophan) are synthesized and their photoacoustic (PA) spectra are reported. The nephelauxetic ratio ?, bonding parameter b1/2 and Sinha parameter ? are calculated based on their PA spectra. The variation of these parameters and correlation of them with the nature of metal-ligand bonding are discussed. The PA intensity analysis of the f-f transitions of neodymium ion is carried out by calculating the intensity branching vector. The environmental effect on the f-f transitions of neodymium ion is also studied. The branching vectors of the f-f transitions of Nd(Ala) 3Cl 3?·?3H 2O and Nd(Val) 3Cl 3?·?3H 2O are similar, which indicates the perturbation of the two ligand fields is similar. The branching vectors of energy levels 4G 5/2+ 2G 7/2 of Nd(Phe)Cl 3?·?5H 2O and Nd(Trp) 3Cl 3?·?3H 2O increase remarkably compared with those of Nd(Ala) 3Cl 3?·?3H 2O and Nd(Val) 3Cl 3?·?3H 2O. As the degree of covalency increases, the oscillator strength of the hypersensitive transition exhibits a corresponding increase. The relaxation process of Nd(Ala) 3Cl 3?·?3H 2O is established through its PA and electron absorption spectroscopy (EAS). A method used to resolve the PA amplitude spectrum is suggested. With the phase spectrum, PA absorption bands of Nd(Trp) 3Cl 3?·?3H 2O are resolved well in the region of ligand absorption.

Yang, Yuetao; Zhang, Shuyi



Complexes of 5,5'-aminoacido-substituted 2,2'-bipyridyl ligands: control of diastereoselectivity with a pH switch and a chloride-responsive combinatorial library.  


The synthesis and coordination chemistry of a new chiral ligand, 2,2'-bipyridine substituted at the 5 and 5' positions by N-methyl-L-valine methyl ester (5), is presented. The ligand readily forms complexes [M(5)3]2+ where M = Co(II) and Fe(II) in CH3CN, and the complexation reaction is slightly diastereoselective (d.e. =ca. 20%) in favour of the Delta diastereomer. The addition of six equivalents of HCl to these complexes [M(II)(5)3]2+ leads to formation of Delta-[M(II)(5H2)3]8+ with a d.e. of 100%. This high diastereoselectivity can be reversed by the addition of base i.e. the diastereoselectivity can be controlled by the pH. Delta-[Fe(5H2)3]8+ was found to bind chloride ions in CD3OD-CD3CN (6:1) with a binding constant of 260 M(-1). [Co(II)(5)3]2+ can be oxidised to Delta-[Co(III)(5H2)3]9+. Formation constants for both [Co(II)(5)3]2+ and [Co(II)(5H2)3]8+ in acetonitrile were obtained by spectrophotometric titrations. In the former case, the stability constant, log beta3 = 19.5(8), is very similar to that measured for [Co(II)(bipy)3]2+ (log beta3 = 19.3(7)) but this drops significantly when the amine groups of are protonated (log beta3 = 16.5(2)). A dynamic combinatorial library was prepared by mixing three equivalents of, three equivalents of bipy, and two equivalents of Co(II) in CD3CN. The presence of all possible Delta- and Lambda-[Co(II)(5)x(bipy)(3-x)]2+ complexes was inferred from 1H NMR and ES-MS spectra. Addition of protons to this library reduced the number of components by inducing diastereoselectivity, and presence of chloride further simplified the 1H NMR spectrum, indicating that [Cl2 ligand Delta-[Co(II)(5H2)3

Telfer, Shane G; Yang, Xiao-Juan; Williams, Alan F



Significance of peptide transporter 1 in the intestinal permeability of valacyclovir in wild-type and PepT1 knockout mice.  


The purpose of this study was to quantitatively determine the contribution of PepT1 [peptide transporter 1 (SLC15A1)] to the intestinal permeability of valacyclovir, an ester prodrug of the antiviral drug acyclovir. In situ single-pass intestinal perfusions were employed (pH 6.5 × 90 minutes) to assess the effective permeability (P(eff)) of 100 ?M [(3)H]valacyclovir in wild-type and PepT1 knockout mice. Acyclovir pharmacokinetics was also evaluated after oral administration of 25 nmol/g valacyclovir. In wild-type mice, jejunal uptake of valacyclovir was best described by both saturable (K(m) = 10.2 mM) and nonsaturable components where the saturable pathway accounted for 82% of total transport. Valacyclovir P(eff) was 2.4 × 10(-4) cm/s in duodenum, 1.7 × 10(-4) cm/s in jejunum, 2.1 × 10(-4) cm/s in ileum, and 0.27 × 10(-4) cm/s in colon. In Pept1 knockout mice, P(eff) values were about 10% of that in wild-type animals for these small intestinal segments. Valacyclovir P(eff) was similar in the colon of both genotypes. There were no differences in valacyclovir P(eff) between any of the intestinal segments of PepT1 knockout mice. Valacyclovir P(eff) was significantly reduced by the dipeptide glycylsarcosine and the aminocephalosporin cefadroxil, but not by the amino acids l-valine or l-histidine, the organic acid p-aminohippurate, or the organic base tetraethylammonium (all at 25 mM). PepT1 ablation resulted in 3- to 5-fold reductions in the in vivo rate and extent of valacyclovir absorption. Our findings conclusively demonstrate, using in situ and in vivo validations in genetically modified mice, that PepT1 has a major influence in improving the oral absorption of valacyclovir. PMID:23264448

Yang, Bei; Smith, David E



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



Solid state radiolysis of amino acids in an astrochemical perspective  

NASA Astrophysics Data System (ADS)

The aliphatic amino acids L-alanine and L-leucine and the aromatic amino acids L-phenylalanine, L-tyrosine and L-tryptophan were irradiated in the solid state to a dose of 3.2 MGy. The degree of decomposition was measured by differential scanning calorimetry (DSC). Furthermore the degree of radioracemization was measured by optical rotatory dispersion (ORD) spectroscopy. From the DSC measurement a radiolysis rate constant k and the half life T1/2 for each amino acid have been determined and extrapolated to a dose of 14 MGy, which corresponds to the expected total dose delivered by the decay of radionuclides to the organic molecules present in comets and asteroids in 4.6×109 years, the age of the Solar System. It is shown that all the amino acids studied can survive a radiation dose of 14 MGy although they are reduced to 1/4-1/5 of their original value they had at the beginning of the history of the Solar System. Consequently, the amount of alanine or leucine found today in the meteorites known as carbonaceous chondrites is just 1/4-1/5 of the amount originally present at the epoch of the formation of the Solar System 4.6×109 years ago. Among the amino acids studied, tyrosine shows the highest radiation resistance while tryptophan does not combine its relatively high radiation resistance with an elevated level of radioracemization resistance. Apart from the exception of tryptophan, it is shown that the radiolysis rate constants k of all the amino acids studied are in reasonable agreement with the radioracemization rate constant krac.

Cataldo, Franco; Angelini, Giancarlo; Iglesias-Groth, Susana; Manchado, Arturo



Obesity-related metabolomic analysis of human subjects in black soybean peptide intervention study by ultraperformance liquid chromatography and quadrupole-time-of-flight mass spectrometry.  


The present study aimed to identify key metabolites related to weight reduction in humans by studying the metabolic profiles of sera obtained from 34 participants who underwent dietary intervention with black soybean peptides (BSP) for 12 weeks. This research is a sequel to our previous work in which the effects of BSP on BMI and blood composition of lipid were investigated. Sera of the study were subjected to ultra performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and the data were analyzed using partial least-squares discriminate analysis (PLS-DA) score plots. Body mass index and percent body fat of the test group were reduced. Levels of betaine, benzoic acid, pyroglutamic acid, pipecolic acid, N-phenylacetamide, uric acid, l-aspartyl-l-phenylalanine, and lysophosphatidyl cholines (lysoPCs) (C18:1, C18:2, C20:1, and C20:4) showed significant increases. Levels of l-proline, valine, l-leucine/isoleucine, hypoxanthine, glutamine, l-methionine, phenylpyruvic acid, several carnitine derivatives, and lysoPCs (C14:0, PC16:0, C15:0, C16:0, C17:1, C18:0, and C22:0) were significantly decreased. In particular, lysoPC 16:0 with a VIP value of 12.02 is esteemed to be the most important metabolite for evaluating the differences between the 2 serum samples. Our result confirmed weight-lowering effects of BSP, accompanied by favorable changes in metabolites in the subjects' blood. Therefore, this research enables us to better understand obesity and increases the predictability of the obesity-related risk by studying metabolites present in the blood. PMID:23862058

Kim, Min Jung; Yang, Hye Jeong; Kim, Jin Hee; Ahn, Chang-Won; Lee, Jong Ho; Kim, Kang Sung; Kwon, Dae Young



Saccharomyces boulardii enhances N-terminal peptide hydrolysis in suckling rat small intestine by endoluminal release of a zinc-binding metalloprotease.  


Saccharomyces boulardii (S. boulardii), a biotherapeutic agent effective in acute and chronic enterocolopathies, produces trophic intestinal effects at least in part mediated by the endoluminal release of polyamines. However, the effects of the yeast on peptide hydrolysis have not yet been studied. The objectives of this study were to assess in suckling rats the endoluminal and mucosal aminopeptidase activities in response to S. boulardii treatment and to analyze their related mechanisms. Peptidase activities were assayed on yeast cells by using several L-amino acid-p-nitroanilide substrates in the pH range of 2 to 10. A marked hydrolytic activity was found for L-leucine-p-nitroanilide that peaked at pH = 8 (K(m) = 0.334 mM, V(max) = 44.7 micromol.min(-1).g(-1) protein). N-terminal peptide hydrolysis was confirmed using as substrate L-Leu-Gly-Gly (K(m) = 4.71 mM, V(max) = 18.08 micromol.min(-1).g(-1) protein). Enzyme reactions were inhibited in the presence of 1 mM Zn(2+). Oral treatment of sucklings with S. boulardii significantly enhanced jejunal and ileal mucosal leucine-aminopeptidase activities by 24 and 34%, respectively, over controls. In concordance, aminopeptidase activity was enhanced in jejunal and ileal endoluminal fluid samples by 47 and 105%, respectively. By use of an IgG-purified antibody raised against the zinc-binding domain common to metalloproteases, the yeast aminopeptidase was immunoprecipitated and detected as an heteromeric enzyme of 108 and 87-kD subunits. S. boulardii, when given orally to suckling rats, is able to significantly enhance hydrolysis of N-terminal oligopeptides in both endoluminal fluid and intestinal mucosa by the endoluminal release of a leucine aminopeptidase that appears to be a zinc-binding metalloprotease belonging to the M1 family of peptidases. PMID:11919341

Buts, Jean-Paul; De Keyser, Nadine; Stilmant, Catherine; Sokal, Etienne; Marandi, Soheila



Obesity-Related Metabolomic Analysis of Human Subjects in Black Soybean Peptide Intervention Study by Ultraperformance Liquid Chromatography and Quadrupole-Time-of-Flight Mass Spectrometry  

PubMed Central

The present study aimed to identify key metabolites related to weight reduction in humans by studying the metabolic profiles of sera obtained from 34 participants who underwent dietary intervention with black soybean peptides (BSP) for 12 weeks. This research is a sequel to our previous work in which the effects of BSP on BMI and blood composition of lipid were investigated. Sera of the study were subjected to ultra performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and the data were analyzed using partial least-squares discriminate analysis (PLS-DA) score plots. Body mass index and percent body fat of the test group were reduced. Levels of betaine, benzoic acid, pyroglutamic acid, pipecolic acid, N-phenylacetamide, uric acid, l-aspartyl-l-phenylalanine, and lysophosphatidyl cholines (lysoPCs) (C18:1, C18:2, C20:1, and C20:4) showed significant increases. Levels of l-proline, valine, l-leucine/isoleucine, hypoxanthine, glutamine, l-methionine, phenylpyruvic acid, several carnitine derivatives, and lysoPCs (C14:0, PC16:0, C15:0, C16:0, C17:1, C18:0, and C22:0) were significantly decreased. In particular, lysoPC 16:0 with a VIP value of 12.02 is esteemed to be the most important metabolite for evaluating the differences between the 2 serum samples. Our result confirmed weight-lowering effects of BSP, accompanied by favorable changes in metabolites in the subjects' blood. Therefore, this research enables us to better understand obesity and increases the predictability of the obesity-related risk by studying metabolites present in the blood. PMID:23862058

Kim, Min Jung; Yang, Hye Jeong; Kim, Jin Hee; Ahn, Chang-Won; Lee, Jong Ho; Kim, Kang Sung; Kwon, Dae Young



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


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



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


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

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



Hormonal regulation of leucine catabolism in mammary epithelial cells.  


Branched-chain amino acids (BCAA) are actively taken up and catabolized by the mammary gland during lactation for syntheses of glutamate, glutamine and aspartate. Available evidence shows that the onset of lactation is associated with increases in circulating levels of cortisol, prolactin and glucagon, but decreases in insulin and growth hormone. This study determined the effects of physiological concentrations of these hormones on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 3 mM D-glucose, 0.5 mM L-leucine, L-[1-14C]leucine or L-[U-14C]leucine, and 0-50 ?U/mL insulin, 0-20 ng/mL growth hormone 0-200 ng/mL prolactin, 0-150 nM cortisol or 0-300 pg/mL glucagon. Increasing extracellular concentrations of insulin did not affect leucine transamination or oxidative decarboxylation, but decreased the rate of oxidation of leucine carbons 2-6. Elevated levels of growth hormone dose dependently inhibited leucine catabolism, ?-ketoisocaproate (KIC) production and the syntheses of glutamate plus glutamine. In contrast, cortisol and glucagon increased leucine transamination, leucine oxidative decarboxylation, KIC production, the oxidation of leucine 2-6 carbons and the syntheses of glutamate plus glutamine. Prolactin did not affect leucine catabolism in the cells. The changes in leucine degradation were consistent with alterations in abundances of BCAA transaminase and phosphorylated levels of branched-chain ?-ketoacid dehydrogenase. Reductions in insulin and growth hormone but increases in cortisol and glucagon with lactation act in concert to stimulate BCAA catabolism for glutamate and glutamine syntheses. These coordinated changes in hormones may facilitate milk production in lactating mammals. PMID:22707151

Lei, Jian; Feng, Dingyuan; Zhang, Yongliang; Dahanayaka, Sudath; Li, Xilong; Yao, Kang; Wang, Junjun; Wu, Zhenlong; Dai, Zhaolai; Wu, Guoyao



Regulation of leucine catabolism by metabolic fuels in mammary epithelial cells.  


Lactation is associated with elevated catabolism of branched-chain amino acids (BCAA) in mammary glands to produce glutamate, glutamine, alanine, aspartate, and asparagine. This study determined effects of metabolic fuels on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 0.5 mM L-leucine and either L-[1-(14)C]leucine or L-[U-(14)C]leucine. The medium also contained 0-5 mM D-glucose, 0-2 mM L-glutamine, 0-4 mM DL-?-hydroxybutyrate, or 0-2 mM oleic acid. Rates of leucine decarboxylation were 60 % lower, but rates of ?-ketoisocaproate production were 34 % higher, in the presence of 2 mM glucose than in its absence. All variables of leucine catabolism did not differ between 2 and 5 mM glucose or between 0 and 4 mM DL-?-hydroxybutyrate. Compared with 0-0.25 mM glutamine, 0.5 and 2 mM L-glutamine reduced leucine transport, transamination, and decarboxylation. In contrast, increasing the concentration of oleic acid from 0 to 2 mM dose-dependently stimulated leucine transamination, decarboxylation, and oxidation of carbons 2-6. Oleic acid also enhanced the abundance of cytosolic BCAA transaminase, while reducing the phosphorylated level (inactive state) of the E1? subunit of the mitochondrial branched-chain ?-ketoacid dehydrogenase complex. Thus, hypoglycemia or ketosis in early lactation does not likely affect BCAA metabolism in mammary epithelial cells. Increasing circulating levels of BCAA and oleic acid may have great potential to increase the syntheses of glutamate, glutamine, aspartate, alanine, and asparagine by lactating mammary glands, thereby leading to enhanced production of milk for suckling neonates. PMID:22543725

Lei, Jian; Feng, Dingyuan; Zhang, Yongliang; Dahanayaka, Sudath; Li, Xilong; Yao, Kang; Wang, Junjun; Wu, Zhenlong; Dai, Zhaolai; Wu, Guoyao



Metabolism of branched-chain amino acids in maple syrup urine disease.  


Maple syrup urine disease (MSUD) is an autosomal recessive disorder. Impaired activity of the branched-chain 2-oxo acid dehydrogenase complex (BCOA-DH) causes accumulation of branched-chain L-amino (BCAA) and 2-oxo acids (BCOA) that can exert neurotoxic effects. MSUD presents as a heterogeneous clinical and molecular phenotype. Severity of the disease, ranging from classical to mild variant types, is commonly classified on the basis of indirect parameters, e.g. onset, leucine tolerance and/or residual enzyme activity in cells. Available information on BCAA turnover in vivo suggests that renal clearance is low and that the main route of BCAA disposal in MSUD is via protein synthesis, similar to healthy subjects. Information on BCAA oxidation is poor. In vivo oxidation rates have been assessed in a few studies in patients with claimed classical form of MSUD, using (stable) isotopically labelled L-leucine and both the (primed) continuous infusion and the oral bolus test approach. However, highly variable results have been obtained with both methods not only with respect to the number of patients exhibiting measurable leucine oxidation (range: 0%-100%; two to seven patients investigated) but also considering the extent of residual whole body leucine oxidation (range: < or = 2%-43% of control). Whether the different findings on whole body leucine oxidation actually reflect the variability of in vivo severity in classical MSUD as opposed to the measurements in cultured cells (generally < or = 2% of control), alternative pathways of leucine oxidation in some patients or were rather attributable to inadequate classification of patients or/and to inherent methodological problems remains to be clarified. PMID:9266218

Schadewaldt, P; Wendel, U



Proteasome activity is altered in skeletal muscle tissue of tumour-bearing rats a leucine-rich diet.  


Leucine can modulate skeletal muscle metabolism by enhancing protein synthesis and decreasing proteolysis. In this study, we investigated the effects of leucine on the ubiquitin-proteasome system in skeletal muscle of pregnant tumour-bearing rats fed a leucine-rich diet. Pregnant Wistar rats were distributed into three groups that were fed a semi-purified control diet (C, control; W, Walker tumour-bearing; P, pair-fed) and three other groups of pregnant rats fed a semi-purified leucine-rich diet (L, leucine; WL, Walker tumour-bearing; PL, pair-fed). The tumour-bearing rats were injected subcutaneously with a suspension of Walker 256 tumour cells. Protein synthesis and degradation were measured in gastrocnemius muscle; the total protein content and tissue chymotrypsin-like and alkaline phosphatase enzyme activities were also determined. Muscle protein extracts were run on SDS-PAGE to assess the expression of the myosin heavy chain (MHC), 20S alpha proteasome subunit, 19S MSSI ATPase regulator subunit and 11S alpha subunit. Although tumour growth decreased the incorporation of [3H]-Phe, the concomitant feeding of a leucine-rich diet increased the rate of protein synthesis. Muscle proteolysis in both tumour-bearing groups was increased more than in the respective control groups. Conversely, the leucine-rich diet caused less protein breakdown in the WL group than in the W group. Only the W group showed a significant reduction (71%) in the myosin content. In WL rats, the 20S proteasome content (32 kDa band) was reduced, while the expression of the 19S subunit was 3-fold less than in the W group and the 11S proteasome subunit reduced, to around 32% less than in the W group. These findings clearly indicate that leucine can stimulate protein synthesis and inhibit protein breakdown in pregnant rats, probably by modulating the activation of the ubiquitin-proteasome system during tumour growth. PMID:15613461

Ventrucci, G; Mello, M A R; Gomes-Marcondes, M C C



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

PubMed Central

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

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



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

PubMed Central

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

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



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

SciTech Connect

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

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



Non-adiabatic tapered optical fiber sensor for measuring the interaction between ?-amino acids in aqueous carbohydrate solution  

NASA Astrophysics Data System (ADS)

A single-mode non-adiabatic tapered optical fiber (NATOF) sensor was utilized for sensing the variation in refractive index (RI) with concentration of d-glucose in deionized water and measurement of the RI of amino acids (AAs) in carbohydrate solutions. This method showed a rewarding ability in understanding the basis of biomolecular interactions in biological systems. Due to high sensitivity, ease of application, low cost and real-time measurement, this method is more efficient in comparison with other techniques, such as calorimetric titration, NMR, UV absorption spectroscopy, x-ray crystallography, computer calculations, kinetic studies and chromatography data. The NATOF is fabricated by the heat pulling method, utilizing a CO2 laser. The limit of detection of the NATOF was 55 ppm for a d-glucose concentration ranging from 0 to 80 mg ml-1, and the limit of detection of the RI measurement corresponding to these concentrations in the range from 1.3330 to 1.3447 was 8.2 × 10-6 as a refractometer sensor. The response of the NATOF shows that different kinds of interactions of various groups of AAs, such as l-alanine, l-leucine and l-cysteine with d-glucose, sucrose and water molecules, depend on functional groups in AAs such as OH, SH, CH2, NH+3 and COO-. These results can be interpreted in terms of solute-solute and solute-solvent interactions and the structure making/breaking ability of solutes in the given solution. Such a study helps in the better understanding of the interactions occurring between AA molecules and entities present in biological matrices.

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



Molecular imaging of urogenital diseases.  


There is an expanding and exciting repertoire of PET imaging radiotracers for urogenital diseases, particularly in prostate cancer, renal cell cancer, and renal function. Prostate cancer is the most commonly diagnosed cancer in men. With growing therapeutic options for the treatment of metastatic and advanced prostate cancer, improved functional imaging of prostate cancer beyond the limitations of conventional CT and bone scan is becoming increasingly important for both clinical management and drug development. PET radiotracers, apart from ¹?F-FDG, for prostate cancer are ¹?F-sodium fluoride, ¹¹C-choline, and ¹?F-fluorocholine, and (¹¹C-acetate. Other emerging and promising PET radiotracers include a synthetic l-leucine amino acid analogue (anti-¹?F-fluorocyclobutane-1-carboxylic acid), dihydrotestosterone analogue (¹?F-fluoro-5?-dihydrotestosterone), and prostate-specific membrane antigen-based PET radiotracers (eg, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-¹?F-fluorobenzyl-l-cysteine, ??Zr-DFO-J591, and ??Ga [HBED-CC]). Larger prospective and comparison trials of these PET radiotracers are needed to establish the role of PET/CT in prostate cancer. Although renal cell cancer imaging with FDG-PET/CT is available, it can be limited, especially for detection of the primary tumor. Improved renal cell cancer detection with carbonic anhydrase IX (CAIX)-based antibody (¹²?I-girentuximab) and radioimmunotherapy targeting with ¹??Lu-cG250 appear promising. Evaluation of renal injury by imaging renal perfusion and function with novel PET radiotracers include p-¹?F-fluorohippurate, hippurate m-cyano-p-¹?F-fluorohippurate, and rubidium-82 chloride (typically used for myocardial perfusion imaging). Renal receptor imaging of the renal renin-angiotensin system with a variety of selective PET radioligands is also becoming available for clinical translation. PMID:24484747

Cho, Steve Y; Szabo, Zsolt



Bacterial proteolytic activity in sediments of the Subantarctic Indian Ocean sector  

NASA Astrophysics Data System (ADS)

Organic material entering the oceanic benthic zone can be permanently buried or recycled to CO 2 in the sediment. Therefore it is important to know the carbon flux across the sediment-water interface to determine the initial and rate-limiting step in carbon oxidation, a bacterial enzymatic activity. Bacterial density and ectoproteolytic activity, determined using a fluorogenic substrate analog ( L-leucine-7-amino-4-methyl coumarin, Leu-MCA) were investigated in the water column and in sediments during the ANTARES 1 JGOFS cruise in the Indian sector of the Southern Ocean. A strong decrease in ectoproteolytic activity was observed with increasing water depth. Peak activity in surface water was three orders of magnitude less than in surface sediment. Analysis of experimental data revealed, for most sediment bacterial communities, the existence of a biphasic mechanism with different velocities for organic matter degradation and different affinities of enzymatic systems for substrates. To explain this, we hypothesize a strategy of bacterial communities that use the episodic supplies of organic matter reaching the sediment. Microbial ectoproteolytic activities were highest in surface sediment horizons and decreased progressively with depth. As benthic microbial activities reflect the quantity and quality of organic matter reaching the sea floor, high potential ectoproteolytic activities (PEA) measured in the sediment of the Polar Front Zone could indicate a direct and rapid coupling of relatively high surface productivity and deep ocean water by sinking particle fluxes. Lower values of PEA were found in sediment in the northern study area where lateral processes associated with ocean circulation (Antarctic Circumpolar Current) have an important influence on settling particles. The lowest sediment PEA values were measured at 52°S, a region in which low primary production provides a poor supply of organic matter for the sea floor.

Talbot, Vincent; Bianchi, Micheline


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

PubMed Central

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

Jabeen, Qaiser; Aslam, Naveed



Pulmonary immunization of guinea pigs with diphtheria CRM-197 antigen as nanoparticle aggregate dry powders enhance local and systemic immune responses.  


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. PMID:20878294

Muttil, Pavan; Pulliam, Brian; Garcia-Contreras, Lucila; Fallon, John Kevin; Wang, Chenchen; Hickey, Anthony James; Edwards, David A




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. PMID:4353639

Corradino, R. A.



Genotype to phenotype: identification of diagnostic vibrio phenotypes using whole genome sequences.  


Vibrios are ubiquitous in the aquatic environment and can be found in association with animal or plant hosts. The range of ecological relationships includes pathogenic and mutualistic associations. To gain a better understanding of the ecology of these microbes, it is important to determine their phenotypic features. However, the traditional phenotypic characterization of vibrios has been expensive, time-consuming and restricted in scope to a limited number of features. In addition, most of the commercial systems applied for phenotypic characterization cannot characterize the broad spectrum of environmental strains. A reliable and possible alternative is to obtain phenotypic information directly from whole genome sequences. The aim of the present study was to evaluate the usefulness of whole genome sequences as a source of phenotypic information. We performed a comparison of the vibrio phenotypes obtained from the literature with the phenotypes obtained from whole genome sequences. We observed a significant correlation between the previously published phenotypic data and the phenotypic data retrieved from whole genome sequences of vibrios. Analysis of 26 vibrio genomes revealed that all genes coding for the specific proteins involved in the metabolic pathways responsible for positive phenotypes of the 14 diagnostic features (Voges-Proskauer reaction, indole production, arginine dihydrolase, ornithine decarboxylase, utilization of myo-inositol, sucrose and L-leucine, and fermentation of D-mannitol, D-sorbitol, L-arabinose, trehalose, cellobiose, D-mannose and D-galactose) were found in the majority of the vibrios genomes. Vibrio species that were negative for a given phenotype revealed the absence of all or several genes involved in the respective biochemical pathways, indicating the utility of this approach to characterize the phenotypes of vibrios. The absence of the global regulation and regulatory proteins in the Vibrio parahaemolyticus genome indicated a non-vibrio phenotype. Whole genome sequences represent an important source for the phenotypic identification of vibrios. PMID:24505074

Amaral, Gilda Rose S; Dias, Graciela M; Wellington-Oguri, Michiyo; Chimetto, Luciane; Campeão, Mariana E; Thompson, Fabiano L; Thompson, Cristiane C



Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1.  


We successfully cloned a novel branched-chain amino acid aminotransferase (Ts-BcAT; EC gene from the Thermococcus sp. CKU-1 genome and expressed it in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Ts-BcAT is a homodimer with an apparent molecular mass of approximately 92 kDa. The primary structure of Ts-BcAT showed high homology with the fold-type I, subgroup I aminotransferases, but showed little homology with BcATs known to date, i.e., those of Escherichia coli and Salmonella typhimurium, which belong to the fold-type IV, subgroup III aminotransferases. The maximum enzyme activity of Ts-BcAT was detected at 95 °C, and Ts-BcAT did not lose any enzyme activity, even after incubation at 90 °C for 5 h. Ts-BcAT was active in the pH range from 4.0 to 11.0, the optimum pH was 9.5, and the enzyme was stable between pH 6 and 7. The exceptionally low pK a of the nitrogen atom in the Lys258 ?-amino group in the internal aldimine bond of Ts-BcAT was determined to be 5.52 ± 0.05. Ts-BcAT used 21 natural and unnatural amino acids as a substrate in the overall transamination reaction. L-Leucine and other aliphatic amino acids are efficient substrates, while polar amino acids except glutamate were weak substrates. Phylogenetic analysis revealed that Ts-BcAT is a novel fold-type I, subgroup I branched-chain aminotransferase. PMID:24687296

Uchida, Yuki; Hayashi, Hideyuki; Washio, Tsubasa; Yamasaki, Ryo; Kato, Shiro; Oikawa, Tadao



The TonB3 System in the Human Pathogen Vibrio vulnificus Is under the Control of the Global Regulators Lrp and Cyclic AMP Receptor Protein  

PubMed Central

TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position ?59 to ?509, cAMP-CRP binds exclusively in a region centered at position ?122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between “feast” and “famine”). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms. PMID:22307757

Crosa, Jorge H.



Defects of protein production in erythroid cells revealed in a zebrafish Diamond-Blackfan anemia model for mutation in RPS19  

PubMed Central

Diamond–Blackfan anemia (DBA) is a rare congenital red cell aplasia that classically presents during early infancy in DBA patients. Approximately, 25% of patients carry a mutation in the ribosomal protein (RP) S19 gene; mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 have been reported. How ribosome protein deficiency causes defects specifically to red blood cells in DBA has not been well elucidated. To genetically model the predominant ribosome defect in DBA, we generated an rps19 null mutant through the use of TALEN-mediated gene targeting in zebrafish. Molecular characterization of this mutant line demonstrated that rps19 deficiency reproduced the erythroid defects of DBA, including a lack of mature red blood cells and p53 activation. Notably, we found that rps19 mutants' production of globin proteins was significantly inhibited; however, globin transcript level was either increased or unaffected in rps19 mutant embryos. This dissociation of RNA/protein levels of globin genes was confirmed in another zebrafish DBA model with defects in rpl11. Using transgenic zebrafish with specific expression of mCherry in erythroid cells, we showed that protein production in erythroid cells was decreased when either rps19 or rpl11 was mutated. L-Leucine treatment alleviated the defects of protein production in erythroid cells and partially rescued the anemic phenotype in both rps19 and rpl11 mutants. Analysis of this model suggests that the decreased protein production in erythroid cells likely contributes to the blood-specific phenotype of DBA. Furthermore, the newly generated rps19 zebrafish mutant should serve as a useful animal model to study DBA. Our in vivo findings may provide clues for the future therapy strategy for DBA. PMID:25058426

Zhang, Y; Ear, J; Yang, Z; Morimoto, K; Zhang, B; Lin, S



Alteration of the catalytic efficiency of penicillin amidase from Escherichia coli.  


Ampicillin and cephalexin are beta-lactam antibiotics that are synthesized by the condensation of D-(-)-alpha-aminophenylacetic acid with 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, respectively. The rates at which the penicillin amidase of Escherichia coli catalyzes these reactions are too low to be of practical use. The objective of this study was to determine whether it is possible to alter the substrate specificity of penicillin amidase and select enzymes that efficiently hydrolyze substrates with alpha-aminophenylacetyl moieties at low pH, at which the alpha-amino group is nearly completely protonated. In this study, D-(-)-alpha-aminophenylacetyl-(L)-leucine (APAL) was used as a substrate analog of ampicillin and cephalexin. The gene for the penicillin amidase of E. coli ATCC 11105 was cloned and transferred to a leucine auxotroph of E. coli; numerous amidase mutants were selected by their ability to cleave APAL and provide leucine for growth in low-pH medium. The plasmid encoding one of the mutant amidases (pA135) was used to transform naive cells, and transformants that expressed the mutant amidase were shown to grow more rapidly in medium at pH 6.5 containing 0.1 mM APAL as the sole leucine source than did cells with the wild-type amidase. The mutant amidase was purified, and the second-order rate constant (kcat/Km) for APAL hydrolysis at pH 6.5 was found to be 10-fold greater than the rate observed with the wild-type enzyme. The difference between the rates of APAL hydrolysis by the mutant and wild-type amidases increased as the pH of the reactions decreased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2690734

Forney, L J; Wong, D C



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

PubMed Central

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



(19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution.  


The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein. PMID:12956607

Salopek-Sondi, Branka; Vaughan, Mark D; Skeels, Matthew C; Honek, John F; Luck, Linda A



Multiphasic control of hepatic protein degradation by regulatory amino acids. General features and hormonal modulation.  


Previous studies with livers from fed rats perfused in the single-pass mode have shown that regulatory amino acids (Leu, Tyr, Gln, Pro, Met, His, and Trp) as a group as well as leucine alone inhibit deprivation-induced protein degradation optimally at 0.5 and 4 times (X) normal plasma amino acid concentrations. However, they lose inhibitory effectiveness almost completely within a narrow zone centered at normal (1 X) levels (Pösö, A. R., Wert, J. J., Jr., and Mortimore, G.E. (1982) J. Biol. Chem. 257, 12114-12120; Pösö, A. R., and Mortimore, G. E. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4270-4274). We now report similar effects for tyrosine and glutamine and suggest that this multiphasic dose response is a general feature of the regulatory group. Insulin (2.4 micrograms h-1) selectively modulated the response by abolishing the zonal loss, whereas glucagon (10 micrograms h-1) blocked the initial inhibition (0.5 X); proteolytic suppression was restored at 4 X normal plasma levels. Although the zonal loss of inhibition at 1 X was associated with a near maximal increase in the volume density of macroautophagy, the vacuoles differed from those induced by stringent amino acid deprivation in containing 4.5-fold more smooth than rough endoplasmic reticulum and thus represented a separate population. Surprisingly, the leucine analog, L-alpha-hydroxyisocaproate, elicited multiphasic responses identical to those of L-leucine, including inhibition at 0.1 mM (equivalent to 0.5 X Leu). Inasmuch as alpha-ketoisocaproate is not effective at this concentration, the initial suppression of protein degradation could be mediated from a site that recognizes structural features common to leucine and its hydroxyl analog. PMID:3316218

Mortimore, G E; Pösö, A R; Kadowaki, M; Wert, J J



Stereospecificity of amino acid hydroxamate inhibition of aminopeptidases.  


Hydroxamates of amino acids and aliphatic acids are effective inhibitors of Aeromonas proteolytica amino-peptidase (EC and of both the cytosolic (EC and microsomal (EC aminopeptidases of swine kidney. Cytosolic leucine aminopeptidase and the Aeromonas enzyme were inhibited to a greater extent by D isomers than by the L enantiomorphs, manganese-activated kidney cytosolic leucine aminopeptidase being inhibited 10 times more effectively by D-leucine and D-valine hydroxamic acids than by the L isomers. The D isomers of these two compounds inhibited Aeromonas aminopeptidase to an even greater extent with Ki values of 2 X 10(-9) and 5 X 10(-9), respectively, whereas the corresponding L isomers were bound 150 times less tightly. With the Aeromonas enzyme, a comparison of inhibition by racemic mixtures with that of the corresponding L isomers indicated that in all cases the contribution of the D isomer was predominant. Isocaproic hydroxamic acid inhibited this enzyme equally well as L-leucine hydroxamic acid, indicating that the amino group orientation in the D isomer contributes to the binding efficacy. Swine kidney microsomal aminopeptidase was also inhibited by D isomers of leucine and valine hydroxamic acids but in contrast to the other two enzymes, the inhibition was 10-fold less than that observed for the corresponding L isomers. Cytosolic leucine aminopeptidase with either 6 g atoms of zinc per mol or 12 g atoms of zinc per mol was inhibited only slightly by any of the hydroxamic acid compounds; evidently enzyme-bound manganese (or magnesium) is specific for hydroxamate binding to this aminopeptidase. PMID:6643439

Wilkes, S H; Prescott, J M



Allocation of endogenous and dietary protein in the reconstitution of the gastrointestinal tract in migratory blackcaps at stopover sites.  


During migratory flight, the mass of the gastrointestinal tract (GIT) and its associated organs in small birds decreases in size by as much as 40%, compared with the preflight condition because of the catabolism of protein. At stopover sites, birds need 2-3 days to rebuild their GIT so that they can restore body mass and fat reserves to continue migration. The source of protein used to rebuild the GIT may be exogenous (from food ingested) or endogenous (reallocated from other organs) or both. Because the relative contribution of these sources to rebuild the GIT of migratory birds is not yet known, we mimicked in-flight fasting and then re-feeding in two groups of blackcaps (Sylvia atricapilla), a Palearctic migratory passerine. The birds were fed a diet containing either 3% or 20% protein to simulate different refueling scenarios. During re-feeding, birds received known doses of (15)N-(l)-leucine before we measured the isotope concentrations in GIT and associated digestive organs and in locomotory muscles. We then quantified the extent to which blackcaps rebuilt their GIT with endogenous and/or dietary protein while refeeding after a fast. Our results indicate that blackcaps fed the low-protein diet incorporated less exogenous nitrogen into their tissues than birds fed the 20% protein diet. They also allocated relatively more exogenous protein to the GIT than to pectoral muscle than those birds re-fed with the high-protein diet. However, this compensation was not sufficient for birds eating the low-protein diet to rebuild their intestine at the same rate as the birds re-fed the high-protein diet. We concluded that blackcaps must choose stopover sites at which they can maximize protein intake to minimize the time it takes to rebuild their GIT and, thus, resume migration as soon as possible. PMID:22399651

Muñoz-Garcia, Agustí; Aamidor, Sarah E; McCue, Marshall D; McWilliams, Scott R; Pinshow, Berry



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


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

Viana, Tiago; Loureiro-Dias, Maria C; Prista, Catarina



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.



Prevalence and mutation analysis of short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) detected on newborn screening in Wisconsin.  


Short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD), also called 2-methylbutyryl CoA dehydrogenase deficiency (2-MBCDD), is a disorder of l-isoleucine metabolism of uncertain clinical significance. SBCADD is inadvertently detected on expanded newborn screening by elevated 2-methylbutyrylcarnitine (C5), which has the same mass to charge (m/s) on tandem mass spectrometry (MS/MS) as isovalerylcarnitine (C5), an analyte that is elevated in isovaleric acidemia (IVA), a disorder in leucine metabolism. SBCADD cases identified in the Hmong-American population have been found in association with the c.1165 A>G mutation in the ACADSB gene. The purposes of this study were to: (a) estimate the prevalence of SBCADD and carrier frequency of the c.1165 A>G mutation in the Hmong ethnic group; (b) determine whether the c.1165 A>G mutation is common to all Hmong newborns screening positive for SBCADD; and (c) evaluate C5 acylcarnitine cut-off values to detect and distinguish between SBCADD and IVA diagnoses. During the first 10years of expanded newborn screening using MS/MS in Wisconsin (2001-2011), 97 infants had elevated C5 values (?0.44?mol/L), of whom five were Caucasian infants confirmed to have IVA. Of the remaining 92 confirmed SBCADD cases, 90 were of Hmong descent. Mutation analysis was completed on an anonymous, random sample of newborn screening cards (n=1139) from Hmong infants. Fifteen infants, including nine who had screened positive for SBCADD based on a C5 acylcarnitine concentration ?0.44?mol/L, were homozygous for the c.1165 A>G mutation. This corresponds to a prevalence in this ethnic group of being homozygous for the mutation of 1.3% (95% confidence interval 0.8-2.2%) and of being heterozygous for the mutation of 21.8% (95% confidence interval 19.4-24.3%), which is consistent with the Hardy-Weinberg equilibrium. Detection of homozygous individuals who were not identified on newborn screening suggests that the C5 screening cut-off would need to be as low as 0.20?mol/L to detect all infants homozygous for the ACADSB c.1165 A>G mutation. However, lowering the screening cut-off to 0.20 would also result in five "false positive" (non-homozygous) screening results in the Hmong population for every c.1165 A>G homozygote detected. Increasing the cut-off to 0.60?mol/L and requiring elevated C5/C2 (acetylcarnitine) and C5/C3 (propionylcarnitine) ratios to flag a screen as abnormal would reduce the number of infants screening positive, but would still result in an estimated 5 infants with SBCADD per year who would require follow-up and additional biochemical testing to distinguish between SBCADD and IVA diagnoses. Further research is needed to determine the clinical outcomes of SBCADD detected on newborn screening and the c.1165 A>G mutation before knowing whether the optimal screening cut-off would minimize true positives or false negatives for SBCADD associated with this mutation. PMID:23712021

Van Calcar, Sandra C; Baker, Mei W; Williams, Phillip; Jones, Susan A; Xiong, Blia; Thao, Mai Choua; Lee, Sheng; Yang, Mai Khou; Rice, Greg M; Rhead, William; Vockley, Jerry; Hoffman, Gary; Durkin, Maureen S



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

NASA Astrophysics Data System (ADS)

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

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



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

SciTech Connect

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

Husain, Z.



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

SciTech Connect

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

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



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

PubMed Central

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



Measurements of substrate oxidation using (13)CO 2-breath testing reveals shifts in fuel mix during starvation.  


Most fasting animals are believed to sequentially switch from predominantly utilizing one metabolic substrate to another from carbohydrates, to lipids, then to proteins. The timing of these physiological transitions has been estimated using measures of substrate oxidation including changes in respiratory exchange ratios, blood metabolites, nitrogen excretion, or enzyme activities in tissues. Here, we demonstrate how (13)CO2-breath testing can be used to partition among the oxidation of distinct nutrient pools in the body (i.e., carbohydrates, lipids, and proteins) that have become artificially enriched in (13)C. Seventy-two Swiss Webster mice were raised to adulthood on diets supplemented with (13)C-1-L-leucine, (13)C-1-palmitic acid, (13)C-1-D-glucose, or no tracer. Mice were then fasted for 72 h during which [Formula: see text], [Formula: see text], ?(13)C of exhaled CO2, body temperature, body mass, and blood metabolites (i.e., glucose, ketone bodies, and triacylglycerols) were measured. The fasting mice exhibited reductions in body mass (29 %), body temperature (3.3 °C), minimum observed metabolic rates (24 %), and respiratory exchange ratio (0.18), as well as significant changes in blood metabolites; but these responses were not particularly indicative of changes in oxidative fuel mixture. Measurements of endogenous nutrient oxidation by way of (13)CO2-breath testing revealed a decrease in the rate of oxidation of carbohydrates from 61 to 10 % of the total energy expenditure during the first 6 h without food. This response was mirrored by a coincidental increase in rate of endogenous lipid oxidation from 18 to 64 %. A transient peak in carbohydrate oxidation occurred between 8 and 14 h, presumably during increased glycogen mobilization. A well-defined period of protein sparing between 8 and 12 h was observed where endogenous protein oxidation accounted for as little as 8 % of the total energy expenditure. Thereafter, protein oxidation continually increased accounting for as much as 24 % of the total energy expenditure by 72 h. This study demonstrates that (13)CO2-breath testing may provide a complementary approach to characterizing the timing and magnitude of sequential changes in substrate oxidation that occur during prolonged fasting and starvation. PMID:23925409

McCue, Marshall D; Pollock, Erik D



Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance.  


Resistance to QoI fungicides in Pyrenophora teres (Dreschsler) and P. tritici-repentis (Died.) Dreschsler was detected in 2003 in France and in Sweden and Denmark respectively. Molecular analysis revealed the presence of the F129L mutation in resistant isolates of both pathogens. In 2004, the frequency of the F129L mutation in populations of both pathogens further increased. The G143A mutation was also detected in a few isolates of P. tritici-repentis from Denmark and Germany. In 2005, the F129L mutation in P. teres increased in frequency and geographical distribution in France and the UK but remained below 2% in Germany, Switzerland, Belgium and Ireland. In P. tritici-repentis, both mutations were found in a significant proportion of the isolates from Sweden, Denmark and Germany. The G143A mutation conferred a significantly higher level of resistance (higher EC50 values) to Qo inhibitors (QoIs) than did the F129L mutation. In greenhouse trials, resistant isolates with G143A were not well controlled on plants sprayed with recommended field rates, whereas satisfactory control of isolates with F129L was achieved. For the F129L mutation, three different single nucleotide polymorphisms (SNPs), TTA, TTG and CTC, can code for L (leucine) in P. teres, whereas only the CTC codon was detected in P. tritici-repentis isolates. In two out of 250 isolates of P. tritici-repentis from 2005, a mutation at position 137 (G137R) was detected at very low frequency. This mutation conferred similar resistance levels to F129L. The structure of the cytochrome b gene of P. tritici-repentis is significantly different from that of P. teres: an intron directly after amino acid position 143 was detected in P. teres which is not present in P. tritici-repentis. This gene structure suggests that resistance based on the G143A mutation may not occur in P. teres because it is lethal. No G143A isolates were found in any P. teres populations. Although different mutations may evolve in P. tritici-repentis, the G143A mutation will have the strongest impact on field performance of QoI fungicides. PMID:17212344

Sierotzki, Helge; Frey, Regula; Wullschleger, Jürg; Palermo, Simona; Karlin, Serge; Godwin, Jeremy; Gisi, Ulrich



Finite-pulse radio frequency driven recoupling with phase cycling for 2D (1)H/(1)H correlation at ultrafast MAS frequencies.  


The first-order recoupling sequence radio frequency driven dipolar recoupling (RFDR) is commonly used in single-quantum/single-quantum homonuclear correlation 2D experiments under magic angle spinning (MAS) to determine homonuclear proximities. From previously reported analysis of the use of XY-based super-cycling schemes to enhance the efficiency of the finite-pulse-RFDR (fp-RFDR) pulse sequence, XY8(1)4 phase cycling was found to provide the optimum performance for 2D correlation experiments on low-? nuclei. In this study, we analyze the efficiency of different phase cycling schemes for proton-based fp-RFDR experiments. We demonstrate the advantages of using a short phase cycle, XY4, and its super-cycle XY4(1)4 that only recouples the zero-quantum homonuclear dipolar coupling, for the fp-RFDR sequence in 2D (1)H/(1)H correlation experiments at ultrafast MAS frequencies. The dipolar recoupling efficiencies of XY4, XY4(1)4 and XY8(1)4 phase cycling schemes are compared based on results obtained from 2D (1)H/(1)H correlation experiments, utilizing the fp-RFDR pulse sequence, on powder samples of U-(13)C,(15)N-l-alanine, N-acetyl-(15)N-l-valyl-(15)N-l-leucine, and glycine. Experimental results and spin dynamics simulations show that XY4(1)4 performs the best when a high RF power is used for the 180° pulse, whereas XY4 renders the best performance when a low RF power is used. The effects of RF field inhomogeneity and chemical shift offsets are also examined. Overall, our results suggest that a combination of fp-RFDR-XY4(1)4 employed in the recycle delay with a large RF-field to decrease the recycle delay, and fp-RFDR-XY4 in the mixing period with a moderate RF-field, is a robust and efficient method for 2D single-quantum/single-quantum (1)H/(1)H correlation experiments at ultrafast MAS frequencies. PMID:24713171

Nishiyama, Yusuke; Zhang, Rongchun; Ramamoorthy, Ayyalusamy



Experiments of the origins of optical activity.  


Two recent reports claim that (1) aqueous L-aspartic acid polymerizes faster than D-Asp in the presence of kaolin at 90 degrees, and (2) L-phenylalanine is adsorbed by kaolin more extensively than D-Phe at pH 4(the reverse being true at pH2). The novelty of these observations and their potential significance for the origin of optical activity has prompted us to duplicate these experiments using more sensitive methods. L- and D, L-Asp in 0.01 M solution were incubated with kaolin at 90 degrees for 8 days. Careful examination of the aqueous residues from such experiments failed to demonstrate any preferential polymerization of L-Asp over D-Asp, or indeed any significant gross polymerization of Asp at all. In other experiments 0.001 M solutions of D, L-Phe at pH 6 and pH 2 were stirred with large excesses of kaolin for 24 hr, and the aqueous extracts from these mixtures were examined for gross adsorption using the amino acid analyzer. No significant gross adsorption was noted. We then looked for asymmetric adsorption in the aqueous residues using optical rotatory dispersion, gas chromatography and thin layer chromatography. By none of these analytical criteria could we find any evidence whatsoever for the preferential adsorption of D- versus L-Phe from either pH 6 or pH 2 solutions. Finally, in experiments bearing on the origin of optical activity by parity violation during beta-decay, we have irradiated solid samples of D-, L- and D,L-leucine in a 61700 Ci Sr-90 source at Oak Ridge National Lab. for 1.34 yr (total dose: 4.2 x 10(8) rad). Gas chromatographic examination of the (appropriately derivitized) recovered samples showed that the L-Leu was 16.7% decomposed, the D-Leu 11.4% and theD,L-Leu 13.8% decomposed. The recovered D,L-Leu sample had a gas-chromatographically determined enantiomeric composition of 50.8% D-leu and 49.2% L-Leu. These data, though very close to experimental error, may indicate a slight preferential radiolysis of L-Leu compared to D-Leu by the Bremsstrahlung from Sr-90 beta-decay. These high intensity irradiation experiments are being continued on a prolonged basis in order to reach more definitive conclusions. PMID:239374

Bonner, W A; Flores, J J



The estimation of antistress properties of peat degradation products  

NASA Astrophysics Data System (ADS)

Introduction. It is known that polyphenol preparations, produced from peat, represent adaptogens, immunomodulators and can participate in regulation of genetic informational realization as triggers of nonspecific nature. These compounds promote launching of protein-synthesizing system that is very important under unfavorable influence on organism. The experimental data of last years confirmed doth therapeutic value of humic acids as adaptogenes and their antioxidant, anti-inflammatory, antimutogenic, radioprotective and other properties. Lysosomes take the key positions in many physiological and pathological processes of organism owing to their unique structural-functional properties, reactivity and plasticity. These organelles take especial meaning in increased functional activity under stress factors influence. In this way lysosomes become modulators of intracellular processes. It is known that under chronic stress, the systems of neurohumoral regulation and adaptation gradually run out, the function of brain cellular membrane structures disturbs. Understanding of stress developing mechanisms is necessary condition for means development of operative avoiding of the harmful consequences. Purpose. The aim of the work was to investigate corrective influence of hydrohumates on compartmentalization changing of lysosomal cysteine cathepsin H (EC in different rat brain structures. The experiment was held on Wistar's rats (160-200 g weight) which were divided into 4 groups: 1 - the control group; 2 - the animals which were received the hydrohumate with water (10 mg hydrohumate (0,1% solution) per 1 kg of weight) during 3 weeks; 3 - the group of stressed rats (test "forced swimming" for 2 hours); 4 - the stressed rats which received the hydrohumate. The activity of lysosomal cysteine cathepsin H was determined spectrophotometerically by hydrolysis of 2-naphthyl-amid L-leucine (Koch-Light Lab., England). It was found out that intracellular compartmentalization changes of lysosomal cysteine protease (cathepsin H) occur as a result of increasing its free activity in 1,6 times in neocortex and 1,8 times in cerebellum, which testify to stress-induced disruption of architectonics and stability of brain cellular lysosomal membranes. These changes could be considered as important biochemical indicators of chronic stress severity. Besides, they could be interpreted as trigger switching over to another functional condition, when power of system reparation becomes not enough for effective removal of disorders. Conclusions. The hydrohumates make corrective action on activity indices of researched enzymes by decreasing it on 45%. Such influence testifies to its membranotrophic properties. It could be suggested that hydrohumates stimulate the reparative processes because of its high antioxidant activity and levels sharp fluctuation of physiological state.

Chorna, V. I.; Lyanna, O. L.



Relationship of whole body nitrogen utilization to urea kinetics in growing steers.  


Urea kinetics were measured in 2 experiments, with treatments designed to change protein deposition by the animal. Our hypothesis was that increased protein deposition by cattle (Bos taurus) would reduce urea production and recycling to the gastrointestinal tract. Urea kinetics were measured by continuous intravenous infusion of (15)N(15)N-urea followed by measurement of enrichment in urinary urea at plateau. In Exp. 1, 6 steers (139 kg) were maintained in a model in which leucine was the most limiting AA. Treatments were arranged as a 2 × 3 factorial and were provided to steers in a 6 × 6 Latin square design. Leucine treatments included 0 or 4 g/d of abomasally supplemented L-leucine, and energy treatments included control, abomasal glucose infusion (382 g DM/d), or ruminal VFA infusion (150 g/d of acetic acid, 150 g/d of propionic acid, and 50 g/d of butyric acid). Leucine supplementation increased (P < 0.01) N retention, and energy supplementation tended to increase (P = 0.09) N retention without differences between glucose and VFA supplements (P = 0.86). Energy supplementation did not strikingly improve the efficiency of leucine utilization. Although both leucine and energy supplementation reduced urinary urea excretion (P ? 0.02), treatments did not affect urea production (P ? 0.34) or urea recycling to the gut (P ? 0.30). The magnitude of change in protein deposition may have been too small to significantly affect urea kinetics. In Exp. 2, 6 steers (168 kg) were maintained in a model wherein methionine was the most limiting AA. Steers were placed in 2 concurrent 3 × 3 Latin squares. Steers in one square were implanted with 24 mg of estradiol and 120 mg trenbolone acetate, and steers in the other square were not implanted. Treatments in each square were 0, 3, or 10 g/d of L-methionine. Implantation numerically improved N retention (P = 0.13) and reduced urea production rate (P = 0.03), urinary urea excretion (P < 0.01), and urea recycling to the gastrointestinal tract (P = 0.14). Effects of methionine were similar to implantation, but smaller in magnitude. When protein deposition by the body is increased markedly, ruminally available N in the diet may need to be increased to offset reductions in urea recycling. PMID:22851238

Titgemeyer, E C; Spivey, K S; Parr, S L; Brake, D W; Jones, M L



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

PubMed Central

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



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

PubMed Central

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

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



Effect of an isoenergetic traditional Mediterranean diet on apolipoprotein A-I kinetic in men with metabolic syndrome  

PubMed Central

Background The impact of the Mediterranean diet (MedDiet) on high-density lipoprotein (HDL) kinetics has not been studied to date. The objective of this study was therefore to investigate the effect of the MedDiet in the absence of changes in body weight on apolipoprotein (apo) A-I kinetic in men with metabolic syndrome (MetS). Methods Twenty-six men with MetS (NCEP-ATP III) were recruited from the general community. In this fixed sequence study, participants’ diet was first standardized to a control diet reflecting current averages in macronutrient intake in North American men, with all foods and beverages provided under isoenergetic conditions for 5 weeks. Participants were then fed an isoenergetic MedDiet over a subsequent period of 5 weeks to maintain their weight constant. During the last week of each diet, participants received a single bolus dose of [5,5,5-2H3] L-leucine and fasting blood samples were collected at predetermined time points. ApoA-I kinetic was determined by multicompartmental modeling using isotopic enrichment data over time. Data were analyses using MIXED models. Results The response of HDL-cholesterol (C) to MedDiet was heterogeneous, such that there was no mean change compared with the control diet. Plasma apoA-I concentration (?3.9%) and pool size (?5.3%, both P?