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Sample records for alanine transaminase activity

  1. 3-Hydroxykynurenine transaminase identity with alanine glyoxylate transaminase. A probable detoxification protein in Aedes aegypti.

    PubMed

    Han, Qian; Fang, Jianmin; Li, Jianyong

    2002-05-03

    This study describes the functional characterization of a specific mosquito transaminase responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). The enzyme was purified from Aedes aegypti larvae by ammonium sulfate fractionation, heat treatment, and various chromatographic techniques, plus non-denaturing electrophoresis. The purified transaminase has a relative molecular mass of 42,500 by SDS-PAGE. N-terminal and internal sequencing of the purified protein and its tryptic fragments resolved a partial N-terminal sequence of 19 amino acid residues and 3 partial internal peptide sequences with 7, 10, and 7 amino acid residues. Using degenerate primers based on the partial internal sequences for PCR amplification and cDNA library screening, a full-length cDNA clone with a 1,167-bp open reading frame was isolated. Its deduced amino acid sequence consists of 389 amino acid residues with a predicted molecular mass of 43,239 and shares 45-46% sequence identity with mammalian alanine glyoxylate transaminases. Northern analysis shows the active transcription of the enzyme in larvae and developing eggs. Substrate specificity analysis of this mosquito transaminase demonstrates that the enzyme is active with 3-HK, kynurenine, or alanine substrates. The enzyme has greater affinity and catalytic efficiency for 3-HK than for kynurenine and alanine. The biochemical characteristics of the enzyme in conjunction with the profiles of 3-HK transaminase activity and XA accumulation during mosquito development clearly point out its physiological function in the 3-HK to XA pathway. Our data suggest that the mosquito transaminase was evolved in a manner precisely reflecting the physiological requirement of detoxifying 3-HK produced in the tryptophan oxidation pathway in the mosquito.

  2. Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate

    PubMed Central

    Barb, A.W.; Hekmatyar, S.K.; Glushka, J.N.; Prestegard, J.H.

    2013-01-01

    Hyperpolarized metabolites offer a tremendous sensitivity advantage (>104 fold) when measuring flux and enzyme activity in living tissues by magnetic resonance methods. These sensitivity gains can also be applied to mechanistic studies that impose time and metabolite concentration limitations. Here we explore the use of hyperpolarization by dissolution dynamic nuclear polarization (DNP) in mechanistic studies of alanine transaminase (ALT), a well-established biomarker of liver disease and cancer that converts pyruvate to alanine using glutamate as a nitrogen donor. A specific deuterated, 13C-enriched analog of pyruvic acid, 13C3D3-pyruvic acid, is demonstrated to have advantages in terms of detection by both direct 13C observation and indirect observation through methyl protons introduced by ALT-catalyzed H–D exchange. Exchange on injecting hyperpolarized 13C3D3-pyruvate into ALT dissolved in buffered 1H2O, combined with an experimental approach to measure proton incorporation, provided information on mechanistic details of transaminase action on a 1.5 s timescale. ALT introduced, on average, 0.8 new protons into the methyl group of the alanine produced, indicating the presence of an off-pathway enamine intermediate. The opportunities for exploiting mechanism-dependent molecular signatures as well as indirect detection of hyperpolarized 13C3-pyruvate and products in imaging applications are discussed. PMID:23357427

  3. In Vitro antioxidative activity of pumpkin seed (Cucurbita pepo) protein isolate and its In Vivo effect on alanine transaminase and aspartate transaminase in acetaminophen-induced liver injury in low protein fed rats.

    PubMed

    Nkosi, C Z; Opoku, A R; Terblanche, S E

    2006-09-01

    The antioxidative effects of pumpkin seed protein isolate (Cucurbita pepo) were investigated in vitro. The isolate exhibited about 80% radical scavenging activity, chelating activity of approximately 64% on Fe2+ ions and an inhibition of approximately 10% of xanthine oxidase. Subsequently the effects of the isolate on the plasma activity levels of alanine transaminase and aspartate transaminase against acetaminophen induced acute liver injury in low-protein fed male Sprague-Dawley rats were ascertained. The rats were maintained on a low-protein diet for 5 days and divided into three subgroups. Two subgroups were injected with acetaminophen and the other with an equivalent amount of polyethylene glycol 400. Two hours after intoxication one of the two subgroups was administered with the protein isolate. Rats from the different subgroups were killed at 24, 48 and 72 h after treatment. After 5 days on the low-protein diet the activity levels of the enzymes were significantly higher than their counterparts on a normal balanced diet. The administration of protein isolate after acetaminophen intoxication resulted in significantly reduced activity levels. It is concluded that the protein isolate has promising antioxidative properties. Furthermore, the isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition and acetaminophen intoxication.

  4. Porcine alanine transaminase after liver allo-and xenotransplantation

    PubMed Central

    Ekser, Burcin; Gridelli, Bruno; Cooper, David K.C.

    2013-01-01

    Aspartate transaminase (AST) and alanine transaminase (ALT) are measured following liver transplantation as indicators of hepatocellular injury. During a series of orthotopic liver allo-and xenotransplants, we observed that there was an increase in AST in all cases. The anticipated concomitant rise in ALT did not occur when a wild-type (WT) pig was the source of the liver graft, but did occur when a baboon or a genetically engineered (α1,3-galactosyltransferase gene-knockout [GTKO]) pig was the source of the graft. We hypothesized that the cience of Galα1,3 Gal in GTKO pig livers may render pig hepatocytes similar to human and baboon hepatocytes in their response to hepatocellular injury. Reviewing the literature, after WT pig liver allotransplantation or xenotransplantation, in the majority of reports, although changes in AST were reported, no mention was made of changes in ALT, suggesting that there was no change in ALT. However, Ramirez et al. reported two cases of liver xenotransplants from hCD55 pigs, following which there were increases in both AST and ALT, suggesting that it is not simply the cience of expression of Galα1,3 Gal that is the cause. We acknowledge that our observation is based on a small number of experiments, but we believe it is worth recording. PMID:22360753

  5. Porcine alanine transaminase after liver allo-and xenotransplantation.

    PubMed

    Ekser, Burcin; Gridelli, Bruno; Cooper, David K C

    2012-01-01

    Aspartate transaminase (AST) and alanine transaminase (ALT) are measured following liver transplantation as indicators of hepatocellular injury. During a series of orthotopic liver allo-and xenotransplants, we observed that there was an increase in AST in all cases. The anticipated concomitant rise in ALT did not occur when a wild-type (WT) pig was the source of the liver graft, but did occur when a baboon or a genetically engineered (α1,3-galactosyltransferase gene-knockout [GTKO]) pig was the source of the graft. We hypothesized that the cience of Galα1,3Gal in GTKO pig livers may render pig hepatocytes similar to human and baboon hepatocytes in their response to hepatocellular injury. Reviewing the literature, after WT pig liver allotransplantation or xenotransplantation, in the majority of reports, although changes in AST were reported, no mention was made of changes in ALT, suggesting that there was no change in ALT. However, Ramirez et al. reported two cases of liver xenotransplants from hCD55 pigs, following which there were increases in both AST and ALT, suggesting that it is not simply the cience of expression of Galα1,3Gal that is the cause. We acknowledge that our observation is based on a small number of experiments, but we believe it is worth recording.

  6. A β-Alanine Catabolism Pathway Containing a Highly Promiscuous ω-Transaminase in the 12-Aminododecanate-Degrading Pseudomonas sp. Strain AAC

    PubMed Central

    Wilding, Matthew; Peat, Thomas S.; Newman, Janet

    2016-01-01

    ABSTRACT We previously isolated the transaminase KES23458 from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. Here, we report the subsequent characterization of this transaminase. It exhibits activity with a broad substrate range which includes α-, β-, and ω-amino acids, as well as α,ω-diamines and a number of other industrially relevant compounds. It is therefore a prospective candidate for the biosynthesis of a range of polyamide monomers. The crystal structure of KES23458 revealed that the protein forms a dimer containing a large active site pocket and unusual phosphorylated histidine residues. To infer the physiological role of the transaminase, we expressed, purified, and characterized a dehydrogenase from the same operon, KES23460. Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from β-alanine transamination via KES23458. In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate. These findings support the original functional assignment of KES23458 as a β-alanine transaminase. However, a seemingly well-adapted active site and promiscuity toward unnatural compounds, such as 12-aminododecanoic acid, suggest that this enzyme could perform multiple functions for Pseudomonas sp. strain AAC. IMPORTANCE We describe the characterization of an industrially relevant transaminase able to metabolize 12-aminododecanoic acid, a constituent building block of the widely used polymer nylon-12, and we report the biochemical and structural characterization of the transaminase protein. A physiological role for this highly promiscuous enzyme is proposed based on the characterization of a related gene from the host organism. Molecular dynamics

  7. Enzymatic resolution for the preparation of enantiomerically enriched D-beta-heterocyclic alanine derivatives using Escherichia coli aromatic L-amino acid transaminase.

    PubMed

    Cho, Byung-Kwan; Park, Hyung-Yeon; Seo, Joo-Hyun; Kinnera, Koteshwar; Lee, Bon-Su; Kim, Byung-Gee

    2004-11-20

    An enzymatic resolution was carried out for the preparation of enriched beta-heterocyclic D-alanine derivatives using Escherichia coli aromatic L-amino acid transaminase. The excess of pyrazole, imidazole, or 1,2,4-triazole reacted with methyl-2-acetamidoacrylate in acetonitrile in the presence of potassium carbonate at 60 degrees C, directly leading to make the potassium salt of the corresponding N-acetyl-beta-heterocyclic alanine derivatives. After the acidic deprotection of the N-acetyl group, 10 mM of racemic pyrazolylalanine, triazolylalanine, and imidazolylalanine were resolved to D-pyrazolylalanine, D-triazolylalanine, and D-imidazolylalanine with 46% (85% ee), 42% (72% ee), and 48% (95% ee) conversion yield in 18 h, respectively, using E. coli aromatic L-amino acid transaminase (EC 2.6.1.5). Although the three beta-heterocyclic L-alanine derivatives have similar molecular structures, they showed different reaction rates and enantioselectivities. The relative reactivities of the transaminase toward the beta-heterocyclic L-alanine derivatives could be explained by the relationship between the substrate binding energy (E, kcal/mol) to the enzyme active site and the distance (delta, A) from the nitrogen of alpha-amino group of the substrates to the C4' carbon of PLP-Lys258 Schiff base. As the ratio of the substrate binding energy (E) to the distance (delta) becomes indicative value of k(cat)/K(M) of the enzyme to the substrate, the relative reactivities of the beta-heterocyclic L-alanine derivatives were successfully correlated with E/delta, and the relationship was confirmed by our experiments.

  8. Structural analysis and mutant growth properties reveal distinctive enzymatic and cellular roles for the three major L-alanine transaminases of Escherichia coli.

    PubMed

    Peña-Soler, Esther; Fernandez, Francisco J; López-Estepa, Miguel; Garces, Fernando; Richardson, Andrew J; Quintana, Juan F; Rudd, Kenneth E; Coll, Miquel; Vega, M Cristina

    2014-01-01

    In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.

  9. PNPLA3 I148M polymorphism is associated with elevated alanine transaminase levels in Mexican Indigenous and Mestizo populations.

    PubMed

    Larrieta-Carrasco, Elena; Acuña-Alonzo, Victor; Velázquez-Cruz, Rafael; Barquera-Lozano, Rodrigo; León-Mimila, Paola; Villamil-Ramírez, Hugo; Menjivar, Marta; Romero-Hidalgo, Sandra; Méndez-Sánchez, Nahúm; Cárdenas, Vanessa; Bañuelos-Moreno, Manuel; Flores, Yvonne N; Quiterio, Manuel; Salmerón, Jorge; Sánchez-Muñoz, Fausto; Villarreal-Molina, Teresa; Aguilar-Salinas, Carlos A; Canizales-Quinteros, Samuel

    2014-07-01

    The patatin like phospholipase domain-containing (PNPLA3) I148M variant is the strongest genetic factor associated with elevated alanine transaminase (ALT) levels in different populations, particularly in Hispanics who have the highest 148M risk allele frequency reported to date. It has been suggested that Indigenous ancestry is associated with higher ALT levels in Mexicans. The aim of the present study was to assess the frequency of the PNPLA3 148M risk allele in Mexican indigenous and Mestizo individuals, and to examine its association with serum ALT levels. The study included a total of 1624 Mexican individuals: 919 Indigenous subjects from five different native groups and 705 Mexican Mestizo individuals (141 cases with ALT levels ≥ 40 U/L and 564 controls with ALT <40 U/L). The I148M polymorphism was genotyped by TaqMan assays. The frequency of elevated ALT levels in Indigenous populations was 18.7%, and varied according to obesity status: 14.4% in normal weight, 19.9% in overweight and 24.5% in obese individuals. The Mexican indigenous populations showed the highest reported frequency of the PNPLA3 148M risk allele (mean 0.73). The M148M genotype was significantly associated with elevated ALT levels in indigenous individuals (OR = 3.15, 95 % CI 1.91-5.20; P = 7.1 × 10(-6)) and this association was confirmed in Mexican Mestizos (OR = 2.24, 95% CI 1.50-3.33; P = 8.1 × 10(-5)). This is the first study reporting the association between M148M genotype and elevated ALT levels in Indigenous Mexican populations. The 148M allele risk may be considered an important risk factor for liver damage in Mexican indigenous and Mestizo populations.

  10. A systems biology approach to understanding elevated serum alanine transaminase levels in a clinical trial with ximelagatran.

    PubMed

    Andersson, Ulf; Lindberg, Johan; Wang, Shunghuang; Balasubramanian, Raji; Marcusson-Ståhl, Maritha; Hannula, Mira; Zeng, Chenhui; Juhasz, Peter J; Kolmert, Johan; Bäckström, Jonas; Nord, Lars; Nilsson, Kerstin; Martin, Steve; Glinghammar, Björn; Cederbrant, Karin; Schuppe-Koistinen, Ina

    2009-12-01

    Ximelagatran was developed for the prevention and treatment of thromboembolic conditions. However, in long-term clinical trials with ximelagatran, the liver injury marker, alanine aminotransferase (ALT) increased in some patients. Analysis of plasma samples from 134 patients was carried out using proteomic and metabolomic platforms, with the aim of finding predictive biomarkers to explain the ALT elevation. Analytes that were changed after ximelagatran treatment included 3-hydroxybutyrate, pyruvic acid, CSF1R, Gc-globulin, L-glutamine, protein S and alanine, etc. Two of these analytes (pyruvic acid and CSF1R) were studied further in human cell cultures in vitro with ximelagatran. A systems biology approach applied in this study proved to be successful in generating new hypotheses for an unknown mechanism of toxicity.

  11. [Regulation of key enzymes of L-alanine biosynthesis by Brevibacterium flavum producer strains].

    PubMed

    Melkonian, L O; Avetisova, G E; Ambartsumian, A A; Chakhalian, A Kh; Sagian, A S

    2013-01-01

    The mechanisms of L-alanine overproduction by Brevibacterium flavum producer strains were studied. It was shown that beta-CI-L-alanine is an inhibitor of some key enzymes involved in the synthesis of L-alanine, including alanine transaminase and valine-pyruvate transaminase. Two highly active B. flavum GL1 and GL1 8 producer strains, which are resistant to the inhibitory effect of beta-Cl-L-alanine, were obtained using a parental B. flavum AA5 producer strain, characterized by a reduced activity of alanine racemase (>or=98%). It was demonstrated that the increased L-alanine synthesis efficiency observed in the producer strains developed in this work is associated with the absence of inhibition of alanine transaminase by the end product of the biosynthesis reaction, as well as with the effect of derepression of both alanine transaminase and valine-pyruvate transaminase synthesis by the studied compound.

  12. Central neural regulation by adrenergic nerves of the daily rhythm in hepatic tyrosine transaminase activity

    PubMed Central

    Black, Ira B.; Reis, Donald J.

    1971-01-01

    1. In adrenalectomized fasted rats transection of the spinal cord at C7-C8 or placement of bilateral electrolytic lesions in the lateral hypothalamus when performed in the morning interrupted the daily rhythm of hepatic tyrosine transaminase by elevating low (AM) enzyme activities to high (PM) levels; lesions placed in PM did not affect the late afternoon rise in enzyme activity. 2. Bilateral thalamic lesions had no affect on enzyme activity. 3. The activity of hepatic catechol-O-methyl transferase was unaffected by hypothalamic lesions. 4. The lesion-evoked rise of tyrosine transaminase activity was abolished by exogenously administered norepinephrine. 5. Cycloheximide blocked the rise of tyrosine transaminase activity caused by hypothalamic lesions. 6. The results suggest that rhythmic activity of sympathetic nerves governed by lateral hypothalamus contribute to regulation of the daily rhythm in tyrosine transaminase by regulating the release of norepinephrine peripherally; norepinephrine may block the daily rise of enzyme by interfering with protein synthesis, possibly of new enzyme, by competing with pyridoxal co-factor. 7. It is proposed that alternating activity of sympathetic-adrenergic and vagal-cholinergic nerves to liver, controlled by the C.N.S., contribute to rhythmic activity of hepatic tyrosine transaminase. ImagesFig. 2 PMID:4400586

  13. Alanine transaminase (ALT) blood test

    MedlinePlus

    ... liver damage. Normal Results The normal range is: Male: 10 to 40 U/L Female: 7 to 35 U/L Normal value ranges may vary slightly among different laboratories. Some labs use different measurements or may test different samples. Talk to your ...

  14. Factors Predicting HBsAg Seroclearance and Alanine Transaminase Elevation in HBeAg-Negative Hepatitis B Virus-Infected Patients with Persistently Normal Liver Function

    PubMed Central

    Chien, Tai-Long; Wang, Jing-Houng; Kee, Kwong-Ming; Chen, Chien-Hung; Hung, Chao-Hung; Lu, Sheng-Nan

    2016-01-01

    Background A certain proportion of hepatitis B virus (HBV)-infected patients with persistently normal alanine transaminase (ALT) levels have significant fibrosis. Using liver stiffness measurements (Fibroscan®) and laboratory data, including serum ALT, quantitative HBsAg (qHBsAg), and HBV DNA, we attempted to predict the natural histories of these patients. Methods Non-cirrhotic HBeAg-negative chronic hepatitis B patients with persistently normal ALT were followed up prospectively with the end points of HBsAg seroclearance and ALT elevation above the upper limit of normal. The factors that were predictive of the end points were identified. Results A total of 235 patients with an average age of 48.1 +/- 10.7 years were followed up for 7 years. Eight patients (3.4%) lost HBsAg, and 15 patients (6.4%) experienced ALT elevation. The overall cumulative HBsAg seroclearances were 0.4%, 1.3% and 2.3% at years 1, 3 and 5, respectively. Regarding HBsAg seroclearance, the qHBsAg (< 30 IU/ml) cutoff resulted in a hazard ratio (HR) of 19.6 with a 95% confidence interval (CI) of 2.2–166.7 (P = 0.008). The baseline ALT level (odd ratio (OR) 1.075, 95% CI 1.020–1.132, P = 0.006) and a qHBsAg above 1000 IU/ml (3.7, 1.1–12.4, P = 0.032) were associated with ALT elevation. Limited to men, the baseline liver stiffness (1.6, 1.0–2.5, P = 0.031) and a qHBsAg above 1000 IU/ml (10.4, 2.1–52.4, P = 0.004) were factors that were independently associated with ALT elevation. Conclusion A low qHBsAg level predicted HBsAg clearance. Baseline ALT and a qHBsAg above 1000 IU/ml were independent predictive factors for ALT elevation. Among the men, the independent predictive factors for ALT elevation were qHBsAg and liver stiffness. PMID:27935953

  15. Effect of maternal fasting on ovine fetal and maternal branched-chain amino acid transaminase activities.

    PubMed

    Liechty, E A; Barone, S; Nutt, M

    1987-01-01

    Activities of branched-chain amino acid transaminase were assayed in maternal skeletal muscle, liver and fetal skeletal muscle, cardiac muscle, liver, kidney and placenta obtained from fed and 5-day-fasted late gestation ewes. Very high activities were found in placenta; fetal skeletal muscle also had high activity. Fetal brain had intermediate activity, followed by cardiac muscle and kidney. Fetal liver possessed negligible activity. Activities were low in both maternal liver and skeletal muscle. Trends were seen for fasting to increase activities in fetal placenta, skeletal muscle, brain, kidney, heart and maternal liver, but these changes were statistically significant only for fetal brain and placental tissue. Fetal skeletal muscle activity was 100 times that of maternal skeletal muscle. These data imply differences in the metabolism of the branched-chain amino acids by fetal and adult ruminants and expand the thesis that branched-chain amino acids are important to the metabolism of the ovine fetus.

  16. Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

    PubMed Central

    Han, Sang-Woo; Park, Eul-Soo; Dong, Joo-Young

    2015-01-01

    ω-Transaminase (ω-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most ω-TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino acids. Here we report on the engineering of an (S)-selective ω-TA from Ochrobactrum anthropi (OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which l-alanine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for α-keto acids and α-amino acids carrying substituents whose bulk is up to that of an n-butyl substituent (e.g., 48- and 56-fold increases in activity for 2-oxopentanoic acid and l-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an α-carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically pure l- and d-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively. PMID:26231640

  17. Synchronization by Food Access Modifies the Daily Variations in Expression and Activity of Liver GABA Transaminase

    PubMed Central

    De Ita-Pérez, Dalia; Vázquez-Martínez, Olivia; Villalobos-Leal, Mónica

    2014-01-01

    Daytime restricted feeding (DRF) is an experimental protocol that influences the circadian timing system and underlies the expression of a biological clock known as the food entrained oscillator (FEO). Liver is the organ that reacts most rapidly to food restriction by adjusting the functional relationship between the molecular circadian clock and the metabolic networks. γ-Aminobutyric acid (GABA) is a signaling molecule in the liver, and able to modulate the cell cycle and apoptosis. This study was aimed at characterizing the expression and activity of the mostly mitochondrial enzyme GABA transaminase (GABA-T) during DRF/FEO expression. We found that DRF promotes a sustained increase of GABA-T in the liver homogenate and mitochondrial fraction throughout the entire day-night cycle. The higher amount of GABA-T promoted by DRF was not associated to changes in GABA-T mRNA or GABA-T activity. The GABA-T activity in the mitochondrial fraction even tended to decrease during the light period. We concluded that DRF influences the daily variations of GABA-T mRNA levels, stability, and catalytic activity of GABA-T. These data suggest that the liver GABAergic system responds to a metabolic challenge such as DRF and the concomitant appearance of the FEO. PMID:24809054

  18. [Activity of various oxidases and transaminases in the rat liver in the readaptation period after hypokinesia up to 30 days].

    PubMed

    Potapov, P P

    1990-01-01

    The activity of alpha-ketoglutarate dehydrogenase and succinic dehydrogenase in readaptation after 15-day hypokinesia was within normal limits, whereas following 30-day hypokinesia it was enhanced on days 11-15. Pyruvate dehydrogenase exhibited hyperactivity in the end of readaptation week 2 both in 15- and 30-day hypokinesia which resulted in rat liver hyperactivity of glutamate dehydrogenase and transaminases. Normal levels of the latter were recorded on readaptation day 12-19.

  19. Charge dependent photodynamic activity of alanine based zinc phthalocyanines.

    PubMed

    Wang, Ao; Li, Yejing; Zhou, Lin; Yuan, Linxin; Lu, Shan; Lin, Yun; Zhou, Jiahong; Wei, Shaohua

    2014-12-01

    In this paper, to minimize the effects of different structure, three alanine-based zinc phthalocyanines (Pcs) of differing charges were engineered and synthesized with the same basic structure. On this premise, the relationship between nature of charge and photodynamic activity was studied. Besides, further verification and explanation of some inconsistent results were also carried out. The results showed that charge can influence the aggregation state, singlet oxygen generation ability and cellular uptake of Pcs, thereby affecting their photodynamic activity. In addition, the biomolecules inside cells may interact with Pcs of differing charges, which can also influence the aggregation state and singlet oxygen generation of the Pcs, and then influence the relationship between nature of charge and photodynamic activity.

  20. Bioassay-guided fractionation of lemon balm (Melissa officinalis L.) using an in vitro measure of GABA transaminase activity.

    PubMed

    Awad, Rosalie; Muhammad, Asim; Durst, Tony; Trudeau, Vance L; Arnason, John T

    2009-08-01

    A novel pharmacological mechanism of action for the anxiolytic botanical Melissa officinalis L. (lemon balm) is reported. The methanol extract was identified as a potent in vitro inhibitor of rat brain GABA transaminase (GABA-T), an enzyme target in the therapy of anxiety, epilepsy and related neurological disorders. Bioassay-guided fractionation led to the identification and isolation of rosmarinic acid (RA) and the triterpenoids, ursolic acid (UA) and oleanolic acid (OA) as active principles. Phytochemical characterization of the crude extract determined RA as the major compound responsible for activity (40% inhibition at 100 microg/mL) since it represented approximately 1.5% of the dry mass of the leaves. Synergistic effects may also play a role.

  1. Antibacterial Activity of Alanine-Derived Gemini Quaternary Ammonium Compounds.

    PubMed

    Piecuch, Agata; Obłąk, Ewa; Guz-Regner, Katarzyna

    The antibacterial activity of alanine-derived gemini quaternary ammonium salts (chlorides and bromides) with various spacer and alkyl chain lengths was investigated. The studied compounds exhibited a strong bactericidal effect, especially bromides with 10 and 12 carbon alkyl chains and 3 carbon spacer groups (TMPAL-10 Br and TMPAL-12 Br), with a short contact time. Both salts dislodged biofilms of Pseudomonas aeruginosa and Staphylococcus epidermidis, and were lethal to adherent cells of S. epidermidis. Bromide with 2 carbon spacer groups and 12 carbon alkyl chains (TMEAL-12 Br) effectively reduced microbial adhesion by coating polystyrene and silicone surfaces. The results obtained suggest that, after further studies, gemini QAS might be considered as antimicrobial agents in medicine or industry.

  2. Transaminase Activity Predicts Survival in Patients with Head and Neck Cancer

    PubMed Central

    Takenaka, Yukinori; Takemoto, Norihiko; Yasui, Toshimichi; Yamamoto, Yoshifumi; Uno, Atsuhiko; Miyabe, Haruka; Ashida, Naoki; Shimizu, Kotaro; Nakahara, Susumu; Hanamoto, Atshushi; Fukusumi, Takahito; Michiba, Takahiro; Cho, Hironori; Yamamoto, Masashi; Inohara, Hidenori

    2016-01-01

    Various serum biomarkers have been developed for predicting head and neck squamous cell carcinoma (HNSCC) prognosis. However, none of them have been proven to be clinically significant. A recent study reported that the ratio of aspartate aminotransaminase (AST) to alanine aminotransaminase (ALT) had a prognostic effect on non-metastatic cancers. This study aimed to examine the effect of the AST/ALT ratio on the survival of patients with HNSCC. Clinical data of 356 patients with locoregionally advanced HNSCC were collected. The effect of the AST/ALT ratio on overall survival was analyzed using a Cox proportional hazard model. Moreover, recursive partitioning analysis (RPA) was used to divide the patients into groups on the basis of the clinical stage and AST/ALT ratio. The prognostic ability of this grouping was validated using an independent data set (N = 167). The AST/ALT ratio ranged from 0.42 to 4.30 (median, 1.42) and was a prognostic factor for overall survival that was independent of age, primary sites, and tumor stage (hazard ratio: 1.36, confidence interval: 1.08−1.68, P = 0.010). RPA divided patients with stage IVA into the following two subgroups: high AST/ALT (≥2.3) and low AST/ALT (<2.3) subgroups. The 5-year survival rate for patients with stage III, stage IVA with a low AST/ALT ratio, stage IVA with a high AST/ALT ratio, and stage IVB were 64.8%, 49.2%, 28.6%, and 33.3%, respectively (p < 0.001). Compared with the low AST/ALT group, the adjusted hazard ratio for death was 2.17 for high AST/ALT group (confidence interval: 1.02–.22 P = 0.045). The AST/ALT ratio was demonstrated to be a prognostic factor of HNSCC. The ratio subdivided patients with stage IVA into low- and high-risk groups. Moreover, intensified treatment for the high-risk group may be considered. PMID:27732629

  3. [Unexplained, subclinical chronically elevated transaminases].

    PubMed

    Vital Durand, D; Lega, J-C; Fassier, T; Zenone, T; Durieu, I

    2013-08-01

    Unexplained, subclinical chronically elevated transaminases is mainly a marker of non-alcoholic fatty liver disease, metabolic syndrome, alcoholism and diabetes, which are very common situations but viral hepatitis and iatrogenic origin must also be considered. Before looking for hepatic or genetic rare diseases, it is worth considering hypertransaminasemia as a clue for muscular disease, particularly in paediatric settings, and creatine phosphokinase is a specific marker. Then, patient history, examination and appropriate biologic requests can permit the identification of less frequent disorders where isolated hypertransaminasemia is possibly the unique marker of the disease for a long while: hemochromatosis, celiac disease, autoimmune hepatitis, Wilson's disease, α1-anti-trypsine deficiency, thyroid dysfunctions, Addison's disease. Liver biopsy should be performed only in patients with aspartate aminotransferases upper the normal range or alanine aminotransferases higher than twice the normal range after 6 months delay with dietetic corrections.

  4. Choledocholithiasis presenting with very high transaminase level

    PubMed Central

    Agahi, Amy; McNair, Alistair

    2012-01-01

    We present three cases of choledocholithiasis presenting with a rise in transaminase to levels normally associated with acute hepatitis (alanine aminotransferase in excess of 1000 IU/l). All three cases had repeated investigation for liver disease before identification of common bile duct stones with magnetic resonance cholangiopancreatogram, and removal at endoscopic retrograde cholangiopancreatogram. We discuss the existing literature and the potential mechanisms of hepatocyte injury in extrahepatic obstruction. PMID:23188856

  5. An Agrobacterium tumefaciens Strain with Gamma-Aminobutyric Acid Transaminase Activity Shows an Enhanced Genetic Transformation Ability in Plants

    PubMed Central

    Nonaka, Satoko; Someya, Tatsuhiko; Zhou, Sha; Takayama, Mariko; Nakamura, Kouji; Ezura, Hiroshi

    2017-01-01

    Agrobacterium tumefaciens has the unique ability to mediate inter-kingdom DNA transfer, and for this reason, it has been utilized for plant genetic engineering. To increase the transformation frequency in plant genetic engineering, we focused on gamma-aminobutyric acid (GABA), which is a negative factor in the Agrobacterium-plant interaction. Recent studies have shown contradictory results regarding the effects of GABA on vir gene expression, leading to the speculation that GABA inhibits T-DNA transfer. In this study, we examined the effect of GABA on T-DNA transfer using a tomato line with a low GABA content. Compared with the control, the T-DNA transfer frequency was increased in the low-GABA tomato line, indicating that GABA inhibits T-DNA transfer. Therefore, we bred a new A. tumefaciens strain with GABA transaminase activity and the ability to degrade GABA. The A. tumefaciens strain exhibited increased T-DNA transfer in two tomato cultivars and Erianthus arundinacues and an increased frequency of stable transformation in tomato. PMID:28220841

  6. Synthesis and GGCT Inhibitory Activity of N-Glutaryl-L-alanine Analogues.

    PubMed

    Ii, Hiromi; Yoshiki, Tatsuhiro; Hoshiya, Naoyuki; Uenishi, Jun'ichi

    2016-01-01

    γ-Glutamylcyclotransferase (GGCT) is an important enzyme that cleaves γ-glutamyl-amino acid in the γ-glutamyl cycle to release 5-oxoproline and amino acid. Eighteen N-acyl-L-alanine analogues including eleven new compounds have been synthesized and examined for their inhibitory activity against recombinant human GGCT protein. Simple N-glutaryl-L-alanine was found to be the most potent inhibitor for GGCT. Other N-glutaryl-L-alanine analogues having methyl and dimethyl substituents at the 2-position were moderately effective, while N-(3R-aminoglutary)-L-alanine, the substrate having an (R)-amino group at the 3-position or N-(N-methyl-3-azaglutaryl)-L-alanine, the substrate having an N-methyl substituent on the 3-azaglutaryl carbon, in constract, exhibited excellent inhibition properties.

  7. Antimicrobial activity of antihypertensive food-derived peptides and selected alanine analogues.

    PubMed

    McClean, Stephen; Beggs, Louise B; Welch, Robert W

    2014-03-01

    This study evaluated four food-derived peptides with known antihypertensive activities for antimicrobial activity against pathogenic microorganisms, and assessed structure-function relationships using alanine analogues. The peptides (EVSLNSGYY, barley; PGTAVFK, soybean; TTMPLW, α-casein; VHLPP, α-zein) and the six alanine substitution peptides of PGTAVFK were synthesised, characterised and evaluated for antimicrobial activity using the bacteria, Escherichia coli, Staphylococcus aureus, and Micrococcus luteus and the yeast, Candida albicans. The peptides TTMPLW and PGTAVFK inhibited growth of all four microorganisms tested, with activities of a similar order of magnitude to ampicillin and ethanol controls. EVSLNSGYY inhibited the growth of the bacteria, but VHLPP showed no antimicrobial activity. The alanine analogue, PGAAVFK showed the highest overall antimicrobial activity and PGTAVFA showed no activity; overall, the activities of the analogues were consistent with their structures. Some peptides with antihypertensive activity also show antimicrobial activity, suggesting that food-derived peptides may exert beneficial effects via a number of mechanisms.

  8. The enzyme 3-hydroxykynurenine transaminase as potential target for 1,2,4-oxadiazoles with larvicide activity against the dengue vector Aedes aegypti.

    PubMed

    Oliveira, Vanessa S; Pimenteira, Cecília; da Silva-Alves, Diana C B; Leal, Laylla L L; Neves-Filho, Ricardo A W; Navarro, Daniela M A F; Santos, Geanne K N; Dutra, Kamilla A; dos Anjos, Janaína V; Soares, Thereza A

    2013-11-15

    The mosquito Aedes aegypti is the vector agent responsible for the transmission of yellow fever and dengue fever viruses to over 80 million people in tropical and subtropical regions of the world. Exhaustive efforts have lead to a vaccine candidate with only 30% effectiveness against the dengue virus and failure to protect patients against the serotype 2. Hence, vector control remains the most viable route to dengue fever control programs. We have synthesized a class of 1,2,4-oxadiazole derivatives whose most biologically active compounds exhibit potent activity against Aedes aegypti larvae (ca. of 15 ppm) and low toxicity in mammals. Exposure to these larvicides results in larvae pigmentation in a manner correlated with the LC50 measurements. Structural comparisons of the 1,2,4-oxadiazole nucleus against known inhibitors of insect enzymes allowed the identification of 3-hydroxykynurenine transaminase as a potential target for these synthetic larvicides. Molecular docking calculations indicate that 1,2,4-oxadiazole compounds can bind to 3-hydroxykynurenine transaminase with similar conformation and binding energies as its crystallographic inhibitor 4-(2-aminophenyl)-4-oxobutanoic acid.

  9. [Temperature-dependent optical activity and birefringence study of D-alanine single crystal].

    PubMed

    Li, Zong-Sheng; Gong, Yan; Wang, Wen-Qing; Du, Wei-Min

    2006-02-01

    The measurement of the anisotropy of optical acitivity and birefringence is one of the most important clues to studying physical properties of a biaxial crystal of D-alanine. In order to investigate a second-order phase transition predicted by A. Salam between two states of D-alanine, the behavior of birefringence and optical activity is useful for the phenomenological approach to the transition mechanism. The optical activity as a peculiar quantity can respond to the modulation of the crystal lattice and to the change in the bonding nature of constituent atoms. In the present paper, the authors use the PEM-90 photoelastic modulator to study the conformation change of D-alanine at the temperature ranging from 220 to 290 K. The temperature dependence of I(2f)/I(dc) showed that the conformation of D-alanine molecule in single crystal changed around 250 K. The obtained results provide an obvious evidence of optical rotation phase transition predicted by Salam.

  10. Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine*

    PubMed Central

    Pinto, John T.; Krasnikov, Boris F.; Alcutt, Steven; Jones, Melanie E.; Dorai, Thambi; Villar, Maria T.; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J. L.

    2014-01-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. PMID:25231977

  11. Structure-function relationship in the antifreeze activity of synthetic alanine-lysine antifreeze polypeptides.

    PubMed

    Wierzbicki, A; Knight, C A; Rutland, T J; Muccio, D D; Pybus, B S; Sikes, C S

    2000-01-01

    Recently antifreeze proteins (AFP) have been the subject of many structure-function relationship studies regarding their antifreeze activity. Attempts have been made to elucidate the structure-function relationship by various amino acid substitutions, but to our knowledge there has been no successful from first principles design of a polypeptide that would bind to designated ice planes along a specific direction. In this paper we show the results of our first attempt on an entirely de novo design of an alanine-lysine-rich antifreeze polypeptide. This 43 residue alanine-lysine peptide exhibits characteristic nonequilibrium freezing point depression and binds to the designated (210) planes of ice along the [122] vector. The structural and thermodynamic properties of this polypeptide were determined using circular dichroism spectroscopy and its nonequilibrium antifreeze properties were investigated using an ice-etching method and nanoliter osmometry.

  12. Similarities between cysteinesulphinate transaminase and aspartate aminotransferase.

    PubMed

    Recasens, M; Mandel, P

    1979-01-01

    A method for the purification of two cysteinesulphinate transaminases, A and B (EC 2.6.1), is described. These enzymes catalyse the conversion of cysteinesulphinic acid to beta-sulphinyl pyruvate. The final preparations are homogeneous by polyacrylamide gel electrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectrofocusing. The molecular weight of the subunits is 41 000 for cysteinesulphinate transaminase A and 43 400 for B. Both enzymes are unspecific, as L-asparate, L-glutamate and L-cysteic acid serve as substrates in addition to L-cysteinesulphinic acid. Cysteinesulphinate transaminase A has a Km of 9.8 mM for cysteinesulphinic acid and 0.25 mM for aspartic acid, whereas the B enzyme has a Km of 6.5 mM for cysteinesulphinic acid and 1.4 mM for aspartic acid. The Vmax values of the A and B enzymes are respectively 7.1 and 6.2 mmol h-1 mg-1 protein for aspartic acid and 45 and 9.3 mmol h-1 mg-1 protein for cysteinesulphinic acid. Both enzymes exhibit maximum activity at pH 8.6. A high specific activity is found in optimal conditions for these two transaminases, the pI values being 9.06 and 5.70 for cysteinesulphinate transaminase A and B respectively. These results have been compared with those already obtained for purified aspartate aminotransferase. Similarities in the pathways of taurine and gamma-aminobutyric acid (GABA) metabolism are discussed.

  13. 13C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts.

    PubMed

    Tran-Dinh, S; Hoerter, J A; Mateo, P; Gyppaz, F; Herve, M

    1998-12-01

    A new mathematical model, based on the observation of 13C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-13C]Glc or [1,2-(13)C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the 13C-enrichment of acetyl-CoA can be deduced from 13C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-13C]glucose in the absence of insulin, was investigated by 13C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate <--> 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G0) or aspartate (A0) were approximately the same for all hearts and remained constant during perfusion: G0 = (0.74 +/- 0.03) micromol/g; A0 = (1.49 +/- 0.05) micromol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min(-1) and 0.72 min(-1), respectively; the flux of this cycle is about (1.07 +/- 0.02) micromol min(-1) g(-1). Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin

  14. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency.

  15. Attenuation of γ-aminobutyric acid (GABA) transaminase activity contributes to GABA increase in the cerebral cortex of mice exposed to β-cypermethrin.

    PubMed

    Han, Y; Cao, D; Li, X; Zhang, R; Yu, F; Ren, Y; An, L

    2014-03-01

    The current study investigated the γ-aminobutyric acid (GABA) levels and GABA metabolic enzymes (GABA transaminase (GABA(T)) and glutamate decarboxylase (GAD)) activities at 2 and 4 h after treatment, using a high-performance liquid chromatography with ultraviolet detectors and colorimetric assay, in the cerebral cortex of mice treated with 20, 40 or 80 mg/kg β-cypermethrin by a single oral gavage, with corn oil as vehicle control. In addition, GABA protein (4 h after treatment), GABA(T) protein (2 h after treatment) and GABA receptors messenger RNA (mRNA) expression were detected by immunohistochemistry, Western blot and real-time quantitative reverse transcriptase polymerase chain reaction, respectively. β-Cypermethrin (80 mg/kg) significantly increased GABA levels in the cerebral cortex of mice, at both 2 and 4 h after treatment, compared with the control. Also, GABA immunohistochemistry results suggested that the number of positive granules was increased in the cerebral cortex of mice 4 h after exposure to 80 mg/kg β-cypermethrin when compared with the control. Furthermore, the results also showed that GABA(T) activity detected was significantly decreased in the cerebral cortex of mice 2 h after β-cypermethrin administration (40 or 80 mg/kg). No significant changes were found in GAD activity, or the expression of GABA(T) protein and GABAB receptors mRNA, in the cerebral cortex of mice, except that 80 mg/kg β-cypermethrin caused a significant decrease, compared with the vehicle control, in GABAA receptors mRNA expression 4 h after administration. These results suggested that attenuated GABA(T) activity induced by β-cypermethrin contributed to increased GABA levels in the mouse brain. The downregulated GABAA receptors mRNA expression is most likely a downstream event.

  16. Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.

    PubMed

    McDonald, Charles J; Acheff, Eric; Kennedy, Ryan; Taylor, Lynn; Curthoys, Norman P

    2015-09-01

    The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors.

  17. Proteins with β-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids

    PubMed Central

    Budisa, Nediljko; Alefelder, Stefan; Bae, Jae Hyun; Golbik, Ralph; Minks, Caroline; Huber, Robert; Moroder, Luis

    2001-01-01

    L-β-(Thieno[3,2-b]pyrrolyl)alanine and L-β-(thieno[2,3-b]pyrrolyl)alanine are mutually isosteric and pharmaceutically active amino acids that mimic tryptophan with the benzene ring in the indole moiety replaced by thiophene. Sulfur as a heteroatom causes physicochemical changes in these tryptophan surrogates that bring about completely new properties not found in the indole moiety. These synthetic amino acids were incorporated into recombinant proteins in response to the Trp UGG codons by fermentation in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. Related protein mutants expectedly retain the secondary structure of the native proteins but show significantly changed optical and thermodynamic properties. In this way, new spectral windows, fluorescence, polarity, thermodynamics, or pharmacological properties are inserted into proteins. Such an engineering approach by translational integration of synthetic amino acids with a priori defined properties, as shown in this study, proved to be a novel and useful tool for protein rational design. PMID:11420430

  18. Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

    PubMed

    Peigneur, Steve; Yamaguchi, Yoko; Kawano, Chihiro; Nose, Takeru; Nirthanan, Selvanayagam; Gopalakrishnakone, Ponnampalam; Tytgat, Jan; Sato, Kazuki

    2016-05-31

    Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency.

  19. Microwave-assisted synthesis and characterization of optically active poly (ester-imide)s incorporating L-alanine.

    PubMed

    Zahmatkesh, Saeed; Hajipour, Abdol R

    2010-04-01

    Pyromellitic dianhydride (1) was reacted with L-alanine (2) to result [N,N'-(pyromellitoyl)-bis-L-alanine diacid] (3). This compound (3) was converted to N,N'-(pyromellitoyl)-bis-L-alanine diacyl chloride (4) by reaction with thionyl chloride. The microwave-assisted polycondensation of this diacyl chloride (4) with polyethyleneglycol-diol (PEG-200) and/or three synthetic aromatic diols furnish a series of new PEIs and Co-PEIs in a laboratory microwave oven (Milestone). The resulting polymers and copolymers have inherent viscosities in the range of 0.31-0.53 dl g(-1). These polymers are optically active, thermally stable and soluble in polar aprotic solvents such as DMF, DMSO, NMP, DMAc, and sulfuric acid. All of the above polymers were fully characterized by IR spectroscopy, (1)H NMR spectroscopy, elemental analyses, specific rotation and thermal analyses. Some structural characterizations and physical properties of these optically active PEIs and Co-PEIs have been reported.

  20. Activity of the lactate-alanine shuttle is independent of glutamate-glutamine cycle activity in cerebellar neuronal-astrocytic cultures.

    PubMed

    Bak, Lasse K; Sickmann, Helle M; Schousboe, Arne; Waagepetersen, Helle S

    The glutamate-glutamine cycle describes the neuronal release of glutamate into the synaptic cleft, astrocytic uptake, and conversion into glutamine, followed by release for use as a neuronal glutamate precursor. This only explains the fate of the carbon atoms, however, and not that of the ammonia. Recently, a role for alanine has been proposed in transfer of ammonia between glutamatergic neurons and astrocytes, denoted the lactate-alanine shuttle (Waagepetersen et al. [ 2000] J. Neurochem. 75:471-479). The role of alanine in this context has been studied further using cerebellar neuronal cultures and corresponding neuronal-astrocytic cocultures. A superfusion paradigm was used to induce repetitively vesicular glutamate release by N-methyl-D-aspartate (NMDA) in the neurons, allowing the relative activity dependency of the lactate-alanine shuttle to be assessed. [(15)N]Alanine (0.2 mM), [2-(15)N]/[5-(15)N]glutamine (0.25 mM), and [(15)N]ammonia (0.3 mM) were used as precursors and cell extracts were analyzed by mass spectrometry. Labeling from [(15)N]alanine in glutamine, aspartate, and glutamate in cerebellar cocultures was independent of depolarization of the neurons. Employing glutamine with the amino group labeled ([2-(15)N]glutamine) as the precursor, an activity-dependent increase in the labeling of both glutamate and aspartate (but not alanine) was observed in the cerebellar neurons. When the amide group of glutamine was labeled ([5-(15)N]glutamine), no labeling could be detected in the analyzed metabolites. Altogether, the results of this study support the existence of the lactate-alanine shuttle and the associated glutamate-glutamine cycle. No direct coupling of the two shuttles was observed, however, and only the glutamate-glutamine cycle seemed activity dependent.

  1. Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine.

    PubMed

    Ghanavatian, Parisa; Khalifeh, Khosrow; Jafarian, Vahab

    2016-12-01

    Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala(19) can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor.

  2. Transaminases for the synthesis of enantiopure beta-amino acids

    PubMed Central

    2012-01-01

    Optically pure β-amino acids constitute interesting building blocks for peptidomimetics and a great variety of pharmaceutically important compounds. Their efficient synthesis still poses a major challenge. Transaminases (also known as aminotransferases) possess a great potential for the synthesis of optically pure β-amino acids. These pyridoxal 5'-dependent enzymes catalyze the transfer of an amino group from a donor substrate to an acceptor, thus enabling the synthesis of a wide variety of chiral amines and amino acids. Transaminases can be applied either for the kinetic resolution of racemic compounds or the asymmetric synthesis starting from a prochiral substrate. This review gives an overview over microbial transaminases with activity towards β-amino acids and their substrate spectra. It also outlines current strategies for the screening of new biocatalysts. Particular emphasis is placed on activity assays which are applicable to high-throughput screening. PMID:22293122

  3. Characterization of lipoteichoic acid structures from three probiotic Bacillus strains: involvement of D-alanine in their biological activity.

    PubMed

    Villéger, Romain; Saad, Naima; Grenier, Karine; Falourd, Xavier; Foucat, Loïc; Urdaci, Maria C; Bressollier, Philippe; Ouk, Tan-Sothea

    2014-10-01

    Probiotics represent a potential strategy to influence the host's immune system thereby modulating immune response. Lipoteichoic Acid (LTA) is a major immune-stimulating component of Gram-positive cell envelopes. This amphiphilic polymer, anchored in the cytoplasmic membrane by means of its glycolipid component, typically consists of a poly (glycerol-phosphate) chain with D-alanine and/or glycosyl substitutions. LTA is known to stimulate macrophages in vitro, leading to secretion of inflammatory mediators such as Nitric Oxide (NO). This study investigates the structure-activity relationship of purified LTA from three probiotic Bacillus strains (Bacillus cereus CH, Bacillus subtilis CU1 and Bacillus clausii O/C). LTAs were extracted from bacterial cultures and purified. Chemical modification by means of hydrolysis at pH 8.5 was performed to remove D-alanine. The molecular structure of native and modified LTAs was determined by (1)H NMR and GC-MS, and their inflammatory potential investigated by measuring NO production by RAW 264.7 macrophages. Structural analysis revealed several differences between the newly characterized LTAs, mainly relating to their D-alanylation rates and poly (glycerol-phosphate) chain length. We observed induction of NO production by LTAs from B. subtilis and B. clausii, whereas weaker NO production was observed with B. cereus. LTA dealanylation abrogated NO production independently of the glycolipid component, suggesting that immunomodulatory potential depends on D-alanine substitutions. D-alanine may control the spatial configuration of LTAs and their recognition by cell receptors. Knowledge of molecular mechanisms behind the immunomodulatory abilities of probiotics is essential to optimize their use.

  4. The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates.

    PubMed

    Lilly, Mariska; Bauer, Florian F; Styger, Gustav; Lambrechts, Marius G; Pretorius, Isak S

    2006-08-01

    In Saccharomyces cerevisiae, branched-chain amino acid transaminases (BCAATases) are encoded by the BAT1 and BAT2 genes. BCAATases catalyse the transfer of amino groups between those amino acids and alpha-keto-acids. alpha-Keto-acids are precursors for the biosynthesis of higher alcohols, which significantly influence the aroma and flavour of yeast-derived fermentation products. The objective of this study was to investigate the influence of BAT-gene expression on general yeast physiology, on aroma and flavour compound formation and on the sensory characteristics of wines and distillates. For this purpose, the genes were overexpressed and deleted in a laboratory strain, BY4742, and overexpressed in an industrial wine yeast strain, VIN13. The data show that, with the exception of a slow growth phenotype observed for the BAT1 deletion strain, the fermentation behaviour of the strains was unaffected by the modifications. The chemical and sensory analysis of fermentation products revealed a strong correction between BAT gene expression and the formation of many aroma compounds. The data suggest that the adjustment of BAT gene expression could play an important role in assisting winemakers in their endeavour to produce wines with specific flavour profiles.

  5. Structural and biochemical characterization of the dual substrate recognition of the (R)-selective amine transaminase from Aspergillus fumigatus.

    PubMed

    Skalden, Lilly; Thomsen, Maren; Höhne, Matthias; Bornscheuer, Uwe T; Hinrichs, Winfried

    2015-01-01

    Chiral amines are important precursors for the pharmaceutical and fine-chemical industries. Because of this, the demand for enantiopure amines is currently increasing. Amine transaminases can produce a large spectrum of chiral amines in the (R)- or (S)-configuration, depending on their substrate scope and stereo-preference, by converting a prochiral ketone into the chiral amine while using alanine as the amine donor producing pyruvate as an α-keto acid product. In order to guide the protein engineering of transaminases to improve substrate specificity and enantioselectivity, we carried out a crystal structure analysis at 1.6 Å resolution of the (R)-amine transaminase from Aspergillus fumigatus with the bound inhibitor gabaculine. This revealed that Arg126 has an important role in the dual substrate recognition of this enzyme because mutating this residue to alanine reduced substantially the ability of the enzyme to use pyruvate as an amino acceptor.

  6. Selection and Characterization of Conditionally Active Promoters in Lactobacillus plantarum, Using Alanine Racemase as a Promoter Probe

    PubMed Central

    Bron, Peter A.; Hoffer, Sally M.; Van Swam, Iris I.; De Vos, Willem M.; Kleerebezem, Michiel

    2004-01-01

    This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum. Random fragments of the L. plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an L. plantarum Δalr strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the d-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 μg of the competitive Alr inhibitor d-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of d-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen. Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity. PMID:14711657

  7. Crystal Structures of Aedes Aegypt Alanine Glyoxylate Aminotransferase

    SciTech Connect

    Han,Q.; Robinson, H.; Gao, Y.; Vogelaar, N.; Wilson, S.; Rizzi, M.; Li, J.

    2006-01-01

    Mosquitoes are unique in having evolved two alanine glyoxylate aminotransferases (AGTs). One is 3-hydroxykynurenine transaminase (HKT), which is primarily responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). Interestingly, XA is used by malaria parasites as a chemical trigger for their development within the mosquito. This 3-HK to XA conversion is considered the major mechanism mosquitoes use to detoxify the chemically reactive and potentially toxic 3-HK. The other AGT is a typical dipteran insect AGT and is specific for converting glyoxylic acid to glycine. Here we report the 1.75{angstrom} high-resolution three-dimensional crystal structure of AGT from the mosquito Aedes aegypti (AeAGT) and structures of its complexes with reactants glyoxylic acid and alanine at 1.75 and 2.1{angstrom} resolution, respectively. This is the first time that the three-dimensional crystal structures of an AGT with its amino acceptor, glyoxylic acid, and amino donor, alanine, have been determined. The protein is dimeric and adopts the type I-fold of pyridoxal 5-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure, and its binding site is similar to those of other AGTs. The comparison of the AeAGT-glyoxylic acid structure with other AGT structures revealed that these glyoxylic acid binding residues are conserved in most AGTs. Comparison of the AeAGT-alanine structure with that of the Anopheles HKT-inhibitor complex suggests that a Ser-Asn-Phe motif in the latter may be responsible for the substrate specificity of HKT enzymes for 3-HK.

  8. Crystal structures of Aedes aegypti alanine glyoxylate aminotransferase.

    PubMed

    Han, Qian; Robinson, Howard; Gao, Yi Gui; Vogelaar, Nancy; Wilson, Scott R; Rizzi, Menico; Li, Jianyong

    2006-12-01

    Mosquitoes are unique in having evolved two alanine glyoxylate aminotransferases (AGTs). One is 3-hydroxykynurenine transaminase (HKT), which is primarily responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). Interestingly, XA is used by malaria parasites as a chemical trigger for their development within the mosquito. This 3-HK to XA conversion is considered the major mechanism mosquitoes use to detoxify the chemically reactive and potentially toxic 3-HK. The other AGT is a typical dipteran insect AGT and is specific for converting glyoxylic acid to glycine. Here we report the 1.75A high-resolution three-dimensional crystal structure of AGT from the mosquito Aedes aegypti (AeAGT) and structures of its complexes with reactants glyoxylic acid and alanine at 1.75 and 2.1A resolution, respectively. This is the first time that the three-dimensional crystal structures of an AGT with its amino acceptor, glyoxylic acid, and amino donor, alanine, have been determined. The protein is dimeric and adopts the type I-fold of pyridoxal 5-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure, and its binding site is similar to those of other AGTs. The comparison of the AeAGT-glyoxylic acid structure with other AGT structures revealed that these glyoxylic acid binding residues are conserved in most AGTs. Comparison of the AeAGT-alanine structure with that of the Anopheles HKT-inhibitor complex suggests that a Ser-Asn-Phe motif in the latter may be responsible for the substrate specificity of HKT enzymes for 3-HK.

  9. Abnormal serum transaminases following therapeutic doses of acetaminophen in the absence of known risk factors.

    PubMed

    Kwan, D; Bartle, W R; Walker, S E

    1995-09-01

    J.M., a healthy, 25-year-old male, volunteered for a study involving warfarin and acetaminophen. Acetaminophen 1 g four times a day was started for 21 days. Liver function tests taken at regular intervals for the first 12 days were unremarkable. On day 18, however, aspartate aminotransferase (AST) was 527 IU/liter and alanine aminotransferase (ALT) was 166 IU/liter. Acetaminophen was discontinued and serum transaminase levels returned to baseline levels two weeks later (AST = 26, ALT = 20). Analysis of J.M.'s urine samples over the first 18 days showed excretion patterns of glucuronide, sulfate, and glutathione derived cysteine and mercapturic acid conjugates were similar to the other subjects in the study. Acetaminophen causes hepatotoxicity in overdose or malnourished or alcoholic patients, none of which applied to our subject. Differences in metabolic activation and capacity for glutathione synthesis can predispose individuals given therapeutic doses of acetaminophen to adverse effects. Failure to detoxify a highly reactive metabolite, formed by P-450 metabolism, via glutathione conjugation is responsible for the development of acute hepatic necrosis. Accumulation of the toxic metabolite due to depleted glutathione stores may have occurred with prolonged high dosing in our subject and been responsible for his abnormal rise in liver enzymes.

  10. Serum γ-Glutamyltransferase, Alanine Aminotransferase and Aspartate Aminotransferase Activity in Healthy Blood Donor of Different Ethnic Groups in Gorgan

    PubMed Central

    Mehrpouya, Masoumeh; Pourhashem, Zeinab

    2016-01-01

    Introduction Measure of liver enzymes may help to increase safety of blood donation for both blood donor and recipient. Determination of liver enzymes may prepare valuable clinical information. Aim To assess serum γ-Glutamyltransferase (GGT), Alanine Aminotransferase (ALT), and Aspartate Aminotransferase (AST) activities in healthy blood donors in different ethnic groups in Gorgan. Materials and Methods This study was performed in 450 healthy male blood donors, in three ethnic groups (Fars, Sistanee and Turkman) who attended Gorgan blood transfusion center. Liver enzymes (GGT, ALT and AST) were determined. Results Serum AST and ALT in three ethnic groups were significant except for serum GGT levels. There was significant correlation between family histories of liver disease and systolic blood pressure and AST in Fars, and GGT in Sistanee ethnic groups. Conclusion Several factors, such as age, family history of diabetes mellitus, family history of liver disease and smoking habit had no effect on some liver enzymes in different ethnic groups in this area. Variation of AST, ALT, and GGT enzyme activities in healthy subjects was associated with some subjects in our study groups. According to our study, it suggests that screening of AST and GGT enzymes in subjects with family history of liver disease is necessary in different ethnic groups. PMID:27630834

  11. A new PEG-beta-alanine active derivative for releasable protein conjugation.

    PubMed

    Pasut, Gianfranco; Mero, Anna; Caboi, Francesca; Scaramuzza, Silvia; Sollai, Luigi; Veronese, Francesco M

    2008-12-01

    A new PEGylating agent, PEG-betaAla-NHCO-OSu, has been studied for protein amino conjugation using human growth hormone (hGH) and granulocyte colony stimulating factor (G-CSF) as model therapeutic proteins. This new activated PEG possesses a convenient property for protein modification when compared to other activated carboxylate PEGs, namely, lower reactivity. When this polymer reacts with a protein, its features lead to fewer PEG-protein conjugate isomers because it preferentially binds the most nucleophilic and exposed amines. Furthermore, the conjugates obtained with PEG-betaAla-NHCO-OSu showed an interesting slow release of polymer chains upon incubation under physiological conditions. Further investigations determined that the PEG chains released are those coupled to histidine residues, and this finally yields less PEGylated species as well as free protein. This release allows a partial recovery of protein activity that is often remarkably and permanently reduced after stable PEGylation, and it occurs in water or blood without the involvement of enzymes. On the other hand, the rate of PEG release, tuned by the chemical structure of this new PEGylating agent, is not too high, and therefore, the achievement of a desired prolongation of protein half-life in vivo is still feasible. The pharmacokinetics of hGH-PEG6k-betaAla conjugate was compared to that of native hGH in rats and monkeys, and the blood residence times were increased by 10- and 7-fold, respectively. The conjugate potency was evaluated in hypophysectomized rats demonstrating a superior pharmacodynamic profile with respect to native hGH.

  12. Ramachandran Plot for Alanine Dipeptide as Determined from Raman Optical Activity.

    PubMed

    Parchaňský, Václav; Kapitán, Josef; Kaminský, Jakub; Šebestík, Jaroslav; Bouř, Petr

    2013-08-15

    Accessible values of the φ and ψ torsional angles determining peptide main chain conformation are traditionally displayed in the form of Ramachandran plots. The number of experimental methods making it possible to determine such conformational distribution is limited. In the present study, Raman optical activity (ROA) spectra of Ac-Ala-NHMe were measured and fit by theoretical curves. This revealed the most favored conformers and a large part of the potential energy surface (PES) of this model dipeptide. Such experimental PES compares well to quantum chemical computations, whereas molecular dynamics (MD) modeling reproduces it less faithfully. The surface shape is consistent with the temperature dependence of the spectra, as observed experimentally and predicted by MD. Despite errors associated with spectral modeling and the measurement, the results are likely to facilitate future applications of ROA spectroscopy.

  13. E. coli histidine triad nucleotide binding protein 1 (ecHinT) is a catalytic regulator of D-alanine dehydrogenase (DadA) activity in vivo.

    PubMed

    Bardaweel, Sanaa; Ghosh, Brahma; Chou, Tsui-Fen; Sadowsky, Michael J; Wagner, Carston R

    2011-01-01

    Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both gram-negative and gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.

  14. Effect of substituting arginine and lysine with alanine on antimicrobial activity and the mechanism of action of a cationic dodecapeptide (CL(14-25)), a partial sequence of cyanate lyase from rice.

    PubMed

    Taniguchi, Masayuki; Takahashi, Nobuteru; Takayanagi, Tomohiro; Ikeda, Atsuo; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Ochiai, Akihito; Tanaka, Takaaki

    2014-01-01

    The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic α-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3 -5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

  15. Four cases of type 1 diabetes mellitus showing sharp serum transaminase increases and hepatomegaly due to glycogenic hepatopathy.

    PubMed

    Ikarashi, Yuichi; Kogiso, Tomomi; Hashimoto, Etsuko; Yamamoto, Kuniko; Kodama, Kazuhisa; Taniai, Makiko; Torii, Nobuyuki; Takaike, Hiroko; Uchigata, Yasuko; Tokushige, Katsutoshi

    2017-03-01

    Poorly controlled diabetes mellitus (DM) patients sometimes show serum transaminase elevations due to steatohepatitis. However, we experienced four cases with type 1 DM with sharp elevations in serum transaminases that could not be explained by steatohepatitis alone and showed bright liver. They were diagnosed with glycogenic hepatopathy (GH) clinicopathologically. The four patients had a median age of 22.5 years (range, 19-29 years) and 12.5 (4-15)-year histories of type 1 DM and showed marked increases in serum transaminases (aspartate aminotransferase, 698 U/L [469-2763 U/L]; alanine transaminase, 255 U/L [216-956 U/L]). Diabetes mellitus control was poor and hemoglobin A1c was 12.7% (11-16.5%). Three cases had a past history of diabetic ketoacidosis. Hepatomegaly and hyperdense liver were seen on computed tomography scans. Magnetic resonance imaging showed low intensity in T2-weighted images. The pathological findings revealed pale and swollen hepatocytes and glycogenated nuclei. The architecture of the liver was preserved, and steatosis and fibrosis were mild. The cytoplasm of hepatocytes stained densely positive with periodic acid-Schiff, and the positive staining disappeared after diastase digestion, suggesting glycogen deposition. No other cause of hepatitis was evident, and the diagnosis was GH. Elevated transaminases improved within 1 month with good glycemic control. Transaminase elevations were observed several times in three cases with poor glycemic control. Glycogenic hepatopathy is rare, but extremely high serum elevations of transaminases are important to identify clinically. Despite showing a good clinical course in general, GH sometimes recurs and requires strict glycemic control. Clinicians should be aware of and recognize GH when dealing with uncontrolled DM patients.

  16. Alanine water complexes.

    PubMed

    Vaquero, Vanesa; Sanz, M Eugenia; Peña, Isabel; Mata, Santiago; Cabezas, Carlos; López, Juan C; Alonso, José L

    2014-04-10

    Two complexes of alanine with water, alanine-(H2O)n (n = 1,2), have been generated by laser ablation of the amino acid in a supersonic jet containing water vapor and characterized using Fourier transform microwave spectroscopy. In the observed complexes, water molecules bind to the carboxylic group of alanine acting as both proton donors and acceptors. In alanine-H2O, the water molecule establishes two intermolecular hydrogen bonds forming a six-membered cycle, while in alanine-(H2O)2 the two water molecules establish three hydrogen bonds forming an eight-membered ring. In both complexes, the amino acid moiety is in its neutral form and shows the conformation observed to be the most stable for the bare molecule. The microsolvation study of alanine-(H2O)n (n = 1,2) can be taken as a first step toward understanding bulk properties at a microscopic level.

  17. Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines

    NASA Astrophysics Data System (ADS)

    Pavlidis, Ioannis V.; Weiß, Martin S.; Genz, Maika; Spurr, Paul; Hanlon, Steven P.; Wirz, Beat; Iding, Hans; Bornscheuer, Uwe T.

    2016-11-01

    The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.

  18. ACTION OF A HISTIDINE ANALOGUE, 1,2,4-TRIAZOLE-3-ALANINE, IN SALMONELLA TYPHIMURIUM

    PubMed Central

    Levin, Alfred P.; Hartman, Philip E.

    1963-01-01

    Levin, Alfred P. (The Johns Hopkins University, Baltimore, Md.), and Philip E. Hartman. Action of a histidine analogue, 1,2,4-triazole-3-alanine, in Salmonella typhimurium. J. Bacteriol. 86:820–828. 1963.—The effect of the histidine analogue, 1,2,4-triazole-3-alanine (TRA), on growth and enzyme synthesis in histidine auxotrophs of Salmonella typhimurium has been studied. TRA allows an increase of approximately 50% in the amount of protein in a culture but does not allow concomitant synthesis of ribonucleic acid and deoxyribonucleic acid. Although the analogue prevents the formation of active bacteriophage and of enzymatically active inosine 5′-phosphate dehydrogenase, it does not prevent the formation of enzymatically active l-histidinol phosphate phosphatase or of imidazoleacetol phosphate transaminase, two enzymes involved in the biosynthesis of histidine. Of the three known functions of histidine in the cell, TRA mimics two: it is incorporated into protein, and it acts as a repressor material for synthesis of enzymes involved in the formation of histidine. TRA fails to act as a feedback inhibitor of the first step in the formation of histidine. Images PMID:14066480

  19. FT-IR and Raman spectroscopic and DFT studies of anti-cancer active molecule N-{(meta-ferrocenyl) Benzoyl} - L-Alanine - Glycine ethyl ester

    NASA Astrophysics Data System (ADS)

    Xavier, T. S.; Kenny, Peter T. M.; Manimaran, D.; Joe, I. Hubert

    2015-06-01

    FT-Raman and FT-IR spectra of N-{(meta-ferrocenyl) Benzoyl} - L-alanine - glycine ethyl ester were recorded in solid phase. The optimized molecular geometry, the vibrational wavenumbers, the infrared intensities and the Raman scattering intensities were calculated by using density functional method(B3LYP) with 6-31G(d, p) basis set. Vibrational assignment of the molecule was done by using potential energy distribution analysis. Natural bond orbital analysis, Mulliken charge analysis and HOMO-LUMO energy were used to elucidate the reasons for intra molecular charge transfer. Docking studies were conducted to predict its anticancer activity.

  20. Serum Glutamic-Oxaloacetic Transaminase (GOT) and Glutamic-Pyruvic Transaminase (GPT) Levels in Children and Adolescents with Intellectual Disabilities

    ERIC Educational Resources Information Center

    Lin, Jin-Ding; Lin, Pei-Ying; Chen, Li-Mei; Fang, Wen-Hui; Lin, Lan-Ping; Loh, Ching-Hui

    2010-01-01

    The elevated serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) rate among people with intellectual disabilities (ID) is unknown and have not been sufficiently studies. The present paper aims to provide the profile of GOT and GPT, and their associated relationship with other biochemical levels of children or…

  1. Synthesis, structural characterization, antiradical and antidiabetic activities of copper(II) and zinc(II) Schiff base complexes derived from salicylaldehyde and beta-alanine.

    PubMed

    Vanco, Ján; Marek, Jaromír; Trávnícek, Zdenek; Racanská, Eva; Muselík, Jan; Svajlenová, Ol'ga

    2008-04-01

    A series of copper(II) and zinc(II) complexes involving a tridentate O,N,O'-donor Schiff base derived from salicylaldehyde and beta-alanine {i.e. N-salicylidene-beta-alanine(2-), (L)}, having the composition [Cu(2)(L)(2)(H(2)O)].H(2)O (1), [Cu(L)(H(2)O)](n) (2), and [Zn(L)(H(2)O)](n) (3), have been prepared and characterized by elemental analyses, UV-visible (UV-VIS), FT-IR and ESI-MS spectra, and thermal analyses. Complexes 1 and 2 have been investigated by single crystal X-ray analysis and also by temperature dependent magnetic susceptibility measurements (294-80K). All prepared complexes have been evaluated by the antiperoxynitrite activity assay and alloxan-induced diabetes model. The significant antioxidant and antidiabetic activities have been found in the case of both copper(II) complexes 1 and 2. In spite of first two complexes, the zinc(II) complex 3, as well as the potassium salt of the ligand (KHL) showed only insignificant protective effect against the tyrosine nitration in vitro.

  2. Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

    SciTech Connect

    Ruel, Nancy . E-mail: n-ruel@northwestern.edu; Zago, Anna . E-mail: anna_zago@acgtinc.com; Spear, Patricia G. . E-mail: p-spear@northwestern.edu

    2006-03-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.

  3. Simultaneous synthesis of 2-phenylethanol and L-homophenylalanine using aromatic transaminase with yeast Ehrlich pathway.

    PubMed

    Hwang, Joon-Young; Park, Jihyang; Seo, Joo-Hyun; Cha, Minho; Cho, Byung-Kwan; Kim, Juhan; Kim, Byung-Gee

    2009-04-01

    2-Phenylethanol is a widely used aroma compound with rose-like fragrance and L-homophenylalanine is a building block of angiotensin-converting enzyme (ACE) inhibitor. 2-phenylethanol and L-homophenylalanine were synthesized simultaneously with high yield from 2-oxo-4-phenylbutyric acid and L-phenylalanine, respectively. A recombinant Escherichia coli harboring a coupled reaction pathway comprising of aromatic transaminase, phenylpyruvate decarboxylase, carbonyl reductase, and glucose dehydrogenase (GDH) was constructed. In the coupled reaction pathway, the transaminase reaction was coupled with the Ehrlich pathway of yeast; (1) a phenylpyruvate decarboxylase (YDR380W) as the enzyme to generate the substrate for the carbonyl reductase from phenylpyruvate (i.e., byproduct of the transaminase reaction) and to shift the reaction equilibrium of the transaminase reaction, and (2) a carbonyl reductase (YGL157W) to produce the 2-phenylethanol. Selecting the right carbonyl reductase showing the highest activity on phenylacetaldehyde with narrow substrate specificity was the key to success of the constructing the coupling reaction. In addition, NADPH regeneration was achieved by incorporating the GDH from Bacillus subtilis in the coupled reaction pathway. Based on 40 mM of L-phenylalanine used, about 96% final product conversion yield of 2-phenylethanol was achieved using the recombinant E. coli.

  4. Disorders of GABA metabolism: SSADH and GABA-transaminase deficiencies.

    PubMed

    Parviz, Mahsa; Vogel, Kara; Gibson, K Michael; Pearl, Phillip L

    2014-11-25

    Clinical disorders known to affect inherited gamma-amino butyric acid (GABA) metabolism are autosomal recessively inherited succinic semialdehyde dehydrogenase and GABA-transaminase deficiency. The clinical presentation of succinic semialdehyde dehydrogenase deficiency includes intellectual disability, ataxia, obsessive-compulsive disorder and epilepsy with a nonprogressive course in typical cases, although a progressive form in early childhood as well as deterioration in adulthood with worsening epilepsy are reported. GABA-transaminase deficiency is associated with a severe neonatal-infantile epileptic encephalopathy.

  5. Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme.

    PubMed Central

    Heaton, M P; Neuhaus, F C

    1992-01-01

    The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacillus casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound lipoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the dae gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the dae gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% of the total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid. PMID:1385594

  6. Alanine 310 is important for the activity of 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02.

    PubMed

    Liu, Yiting; Li, Caiming; Gu, Zhengbiao; Xin, Chenhao; Cheng, Li; Hong, Yan; Li, Zhaofeng

    2017-04-01

    1,4-α-Glucan branching enzyme (GBE) catalyzes the formation of α-1,6 branch points in starch or glycogen by hydrolyzing α-1,4-glucosidic linkages and then synthesizing α-1,6-glucosidic linkages. In the GBE from Geobacillus thermoglucosidans STB02, alanine 310 (Ala310) is located in conserved region II. An analysis of the amino acid sequence shows that Ala310 is highly conserved in the prokaryotic GBE subfamily. Site-directed mutagenesis was used to determine the function of Ala310 in GBE. Replacement of Ala310 with glycine, aspartate, asparagine, isoleucine, glutamate, or glutamine resulted in mutant enzymes with less than 10% to 25% of wild-type activity when amylopectin or amylose was used as substrate. Studies using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) showed that A310G mutant had no effect on the transfer pattern, but the branching activity had been repressed to a large extent. Kinetic analysis also showed that mutations of Ala310 had an effect on the KM value that changed the preferred substrate from amylopectin to amylose. These results show that Ala310 is important for the catalytic activity of the GBE from G. thermoglucosidans STB02.

  7. Discovery and structural characterisation of new fold type IV-transaminases exemplify the diversity of this enzyme fold

    PubMed Central

    Pavkov-Keller, Tea; Strohmeier, Gernot A.; Diepold, Matthias; Peeters, Wilco; Smeets, Natascha; Schürmann, Martin; Gruber, Karl; Schwab, Helmut; Steiner, Kerstin

    2016-01-01

    Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%. PMID:27905516

  8. Discovery and structural characterisation of new fold type IV-transaminases exemplify the diversity of this enzyme fold.

    PubMed

    Pavkov-Keller, Tea; Strohmeier, Gernot A; Diepold, Matthias; Peeters, Wilco; Smeets, Natascha; Schürmann, Martin; Gruber, Karl; Schwab, Helmut; Steiner, Kerstin

    2016-12-01

    Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%.

  9. The effect of active-site isoleucine to alanine mutation on the DHFR catalyzed hydride-transfer

    PubMed Central

    Stojković, Vanja; Perissinotti, Laura L.; Lee, Jeeyeon; Benkovic, Stephen J.; Kohen, Amnon

    2015-01-01

    Comparison of the nature of hydride transfer in wild-type and active site mutant (I14A) of dihydrofolate reductase suggests that the size of this side chain at position 14 modulates H-tunneling. PMID:20972508

  10. A novel C-S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity.

    PubMed

    Munt, Oliver; Prüfer, Dirk; Schulze Gronover, Christian

    2013-01-01

    We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C-S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon-sulfur lyase (C-S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19nkatmg(-1) protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C-S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications.

  11. Transplantation of Deceased Donor Livers With Elevated Levels of Serum Transaminases at Shiraz Transplant Center

    PubMed Central

    Fakhar, Nasir; Nikeghbalian, Saman; Kazemi, Kourosh; Shamsayeefar, Ali Reza; Gholami, Siavash; Kasraianfard, Amir; Malek-Hosseini, Seyed Ali

    2016-01-01

    Background The current organ shortage has prompted the use of marginal organs. We conducted this retrospective study to present our experience with transplanting deceased donor livers with elevated levels of serum transaminases and to explain whether elevated levels of serum transaminases in donors affect allograft function and survival of the recipients. Methods Data of deceased donor livers and patients, who underwent liver transplantation from March 2013 to March 2015 at Shiraz center for organ transplantation, was reviewed. Liver donors with aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) level of more than 500 IU/l and their related recipients were considered as the case group (n = 24) and the others were considered as the control group (n = 834). Results In the case group, the medians of levels of serum AST and ALT of donors were 834 ± 425 IU/L (range: 250 - 2285) and 507 ± 367 IU/L (range: 100 - 1600), respectively. Recipients were followed for a median of 13.6 ± 9 months (range: 7 - 28.4). Post-transplant complications were acute rejection (n = 5), infection (n = 3), portal vein thrombosis (n = 3), bile duct stricture (n = 1), and hepatic artery stenosis (n = 1). The one-year survival rate of the patients was 91.7%. Demographics, post-transplant complications and one-year survival rates were not significantly different between the two study groups. Conclusions Transplanting deceased donor livers with markedly elevated liver enzymes may be an acceptable choice for expanding the donor pool. PMID:27882068

  12. A comprehensive alanine-scanning mutagenesis study reveals roles for salt bridges in the structure and activity of Pseudomonas aeruginosa elastase.

    PubMed

    Bian, Fei; Yue, Shousong; Peng, Zhenying; Zhang, Xiaowei; Chen, Gao; Yu, Jinhui; Xuan, Ning; Bi, Yuping

    2015-01-01

    The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (kcat/Km), half-lives (t1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.

  13. Enantioselective Collision-Activated Dissociation of Gas-Phase Tryptophan Induced by Chiral Recognition of Protonated uc(l)-Alanine Peptides

    NASA Astrophysics Data System (ADS)

    Fujihara, Akimasa; Matsuyama, Hiroki; Tajiri, Michiko; Wada, Yoshinao; Hayakawa, Shigeo

    2016-06-01

    Enantioselective dissociation in the gas phase is important for enantiomeric enrichment and chiral transmission processes in molecular clouds regarding the origin of homochirality in biomolecules. Enantioselective collision-activated dissociation (CAD) of tryptophan (Trp) and the chiral recognition ability of uc(l)-alanine peptides (uc(l)-Ala n ; n = 2-4) were examined using a linear ion trap mass spectrometer. CAD spectra of gas-phase heterochiral H+(uc(d)-Trp)(uc(l)-Ala n ) and homochiral H+(uc(l)-Trp)(uc(l)-Ala n ) noncovalent complexes were obtained as a function of the peptide size n. The H2O-elimination product was observed in CAD spectra of both heterochiral and homochiral complexes for n = 2 and 4, and in homochiral H+(uc(l)-Trp)(uc(l)-Ala3), indicating that the proton is attached to the uc(l)-alanine peptide, and H2O loss occurs from H+(uc(l)-Ala n ) in the noncovalent complexes. H2O loss did not occur in heterochiral H+(uc(d)-Trp)(uc(l)-Ala3), where NH3 loss and (H2O + CO) loss were the primary dissociation pathways. In heterochiral H+(uc(d)-Trp)(uc(l)-Ala3), the protonation site is the amino group of uc(d)-Trp, and NH3 loss and (H2O + CO) loss occur from H+(uc(d)-Trp). uc(l)-Ala peptides recognize uc(d)-Trp through protonation of the amino group for peptide size n = 3. NH3 loss and (H2O + CO) loss from H+(uc(d)-Trp) proceeds via enantioselective CAD in gas-phase heterochiral H+(uc(d)-Trp)(uc(l)-Ala3) at room temperature, whereas uc(l)-Trp dissociation was not observed in homochiral H+(uc(l)-Trp)(uc(l)-Ala3). These results suggest that enantioselective dissociation induced by chiral recognition of uc(l)-Ala peptides through protonation could play an important role in enantiomeric enrichment and chiral transmission processes of amino acids.

  14. Effect of cinnamon, clove and some of their constituents on the Na(+)-K(+)-ATPase activity and alanine absorption in the rat jejunum.

    PubMed

    Kreydiyyeh, S I; Usta, J; Copti, R

    2000-09-01

    The effect of a water extract of some spices on the in vitro activity of the rat jejunal Na(+)-K(+)-ATPase was investigated. Extracts of nutmeg, cinnamon, clove, cumin, coriander, turmeric and caraway all inhibited the ATPase, while anise seed and white pepper exerted no significant effects. The extracts of clove and cinnamon had the most potent inhibitory effect on the intestinal ATPase as compared to extracts of other spices. They also inhibited the in vitro Na(+)-K(+)-ATPase activity in a crude kidney homogenate and the activity of an isolated dog kidney Na(+)-K(+)-ATPase. The alcoholic extract of cinnamon, compared to the aqueous extract, had a stronger inhibitory action on the jejunal enzyme and a lower IC(50) value, which was not significantly different from the one observed with cinnamaldehyde, the major volatile oil present cinnamon, suggesting that in alcoholic extracts cinnamaldehyde is the major inhibitory component. The IC(50) values of eugenol, aqueous clove extract and ethanolic clove extract all fell within the same range and were not significantly different from each other, suggesting that eugenol is the major inhibitory component in both alcoholic and aqueous extracts. Based on the IC(50) values, the order of sensitivity of the enzyme to the spices extracts is as follows: isolated dog kidney ATPase>rat kidney ATPase>rat intestine ATPase. The aqueous extracts of clove and cinnamon also significantly lowered the absorption of alanine from the rat intestine. It was concluded that the active principle(s) in clove and cinnamon can permeate the membrane of the enterocytes and inhibit the Na(+)-K(+)-ATPase that provides the driving force for many transport processes.

  15. Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels.

    PubMed

    Corzo, Gerardo; Sabo, Jennifer K; Bosmans, Frank; Billen, Bert; Villegas, Elba; Tytgat, Jan; Norton, Raymond S

    2007-02-16

    Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion beta-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded beta-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion beta-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

  16. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    PubMed

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.

  17. Observed activities of serum creatine kinase: total and B subunit activity and other enzymes in young persons abusing solvents.

    PubMed Central

    Spooner, R J; Birrell, R C; McLelland, A S; Baird, J A; Sourindhrin, I

    1984-01-01

    Serum enzymes (aspartate transaminase, alanine transaminase, alkaline phosphatase (ALP), gamma-glutamyltransferase, and creatine kinase (CK] were measured in 296 young persons who admitted to recent inhalation of solvents, usually toluene based glues. In general, results fell within expected adult reference ranges except for ALP and CK. About 60% of subjects had CK activities above the upper reference limit and these activities were investigated in terms of their isoenzyme composition. CK B subunit activity was measured in 90 subjects with raised total CK activities. In five instances the CK B subunit activity was judged abnormal and in two subjects the presence of CK BB was confirmed. These two subjects were thought to have a circulating macro CK, type 1. It is concluded that the increased total CK activity found in this group of solvent abusers was due to physical activity, but a contribution from specific muscle toxicity by solvents cannot be excluded. PMID:6142904

  18. The ribavirin analog ICN 17261 demonstrates reduced toxicity and antiviral effects with retention of both immunomodulatory activity and reduction of hepatitis-induced serum alanine aminotransferase levels.

    PubMed

    Tam, R C; Ramasamy, K; Bard, J; Pai, B; Lim, C; Averett, D R

    2000-05-01

    The demonstrated utility of the nucleoside analog ribavirin in the treatment of certain viral diseases can be ascribed to its multiple distinct properties. These properties may vary in relative importance in differing viral disease conditions and include the direct inhibition of viral replication, the promotion of T-cell-mediated immune responses via an enhanced type 1 cytokine response, and a reduction of circulating alanine aminotransferase (ALT) levels associated with hepatic injury. Ribavirin also has certain known toxicities, including the induction of anemia upon chronic administration. To determine if all these properties are linked, we compared the D-nucleoside ribavirin to its L-enantiomer (ICN 17261) with regard to these properties. Strong similarities were seen for these two compounds with respect to induction of type 1 cytokine bias in vitro, enhancement of type 1 cytokine responses in vivo, and the reduction of serum ALT levels in a murine hepatitis model. In contrast, ICN 17261 had no in vitro antiviral activity against a panel of RNA and DNA viruses, while ribavirin exhibited its characteristic activity profile. Importantly, the preliminary in vivo toxicology profile of ICN 17261 is significantly more favorable than that of ribavirin. Administration of 180 mg of ICN 17261 per kg of body weight to rats by oral gavage for 4 weeks generated substantial serum levels of drug but no observable clinical pathology, whereas equivalent doses of ribavirin induced a significant anemia and leukopenia. Thus, structural modification of ribavirin can dissociate its immunomodulatory properties from its antiviral and toxicologic properties, resulting in a compound (ICN 17261) with interesting therapeutic potential.

  19. Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine.

    PubMed

    Montioli, Riccardo; Oppici, Elisa; Dindo, Mirco; Roncador, Alessandro; Gotte, Giovanni; Cellini, Barbara; Borri Voltattorni, Carla

    2015-10-01

    Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.

  20. Serum transaminase levels after experimental paracetamol-induced hepatic necrosis.

    PubMed Central

    Dixon, M F; Fulker, M J; Walker, B E; Kelleher, J; Losowsky, M S

    1975-01-01

    The relationship between serum transaminase levels and the extent of paracetamol-induced liver necrosis has been investigated in the rat. Three methods of histological quantitation were used to assess of necrosis--arbitrary grading, point counting, and the image-analysis computer. Highly significant correlations were obtained between the three methods and all were found to be reproducible. A close correlation was found between the extent of hepatic necrosis and the serum ASAT and ALAT 24 hours after a large dose (4 g/kg) of paracetamol. Likewise, the mean grade of necrosis correlated reasonably well with the serum enzyme levels in the recovery phase at 36 and 72 hours, although the transaminase level for a given degree of necrosis was considerably lower at 72 hours than at 24 hours. These findings suggest that serum transaminase levels gives a reliable indication of the severity of hepatic necrosis if the time of ingestion of the paracetamol is known and taken into account. Images Fig 1 Fig 2 PMID:1205274

  1. Synthesis, CMC Determination, Antimicrobial Activity and Nucleic Acid Binding of A Surfactant Copper(II) Complex Containing Phenanthroline and Alanine Schiff-Base.

    PubMed

    Nagaraj, Karuppiah; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2014-03-01

    A new water-soluble surfactant copper(II) complex [Cu(sal-ala)(phen)(DA)] (sal-ala = salicylalanine, phen = 1,10-phenanthroline, DA = dodecylamine), has been synthesized and characterized by physico-chemical and spectroscopic methods. The critical micelle concentration (CMC) values of this surfactant-copper(II) complex in aqueous solution were obtained from conductance measurements. Specific conductivity data (at 303, 308, 313. 318 and 323 K) served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (ΔG(0)m, ΔH(0)m and ΔS(0)m). The interaction of this complex with nucleic acids (DNA and RNA) has been explored by using electronic absorption spectral titration, competitive binding experiment, cyclic voltammetry, circular dichroism (CD) spectra, and viscosity measurements. Electronic absorption studies have revealed that the complex can bind to nucleic acids by the intercalative binding mode which has been verified by viscosity measurements. The DNA binding constants have also been calculated (Kb = 1.2 × 10(5) M(-1) for DNA and Kb = 1.6 × 10(5) M(-1) for RNA). Competitive binding study with ethidium bromide (EB) showed that the complex exhibits the ability to displace the DNA-bound-EB indicating that the complex binds to DNA in strong competition with EB for the intercalative binding site. The presence of hydrophobic ligands, alanine Schiff-base, phenanthroline and long aliphatic chain amine in the complex were responsible for this strong intercalative binding. The surfactant-copper (II) complex was screened for its antibacterial and antifungal activities against various microorganisms. The results were compared with the standard drugs, amikacin(antibacterial) and ketokonazole(antifungal).

  2. Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains: Evidence of a New Pathway of d-Alanine Biosynthesis.

    PubMed

    Marshall, Darrell D; Halouska, Steven; Zinniel, Denise K; Fenton, Robert J; Kenealy, Katie; Chahal, Harpreet K; Rathnaiah, Govardhan; Barletta, Raúl G; Powers, Robert

    2017-03-03

    In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc(2)155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc(2)155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.

  3. The structure of alanine racemase from Acinetobacter baumannii.

    PubMed

    Davis, Emily; Scaletti-Hutchinson, Emma; Opel-Reading, Helen; Nakatani, Yoshio; Krause, Kurt L

    2014-09-01

    Acinetobacter baumannii is an opportunistic Gram-negative bacterium which is a common cause of hospital-acquired infections. Numerous antibiotic-resistant strains exist, emphasizing the need for the development of new antimicrobials. Alanine racemase (Alr) is a pyridoxal 5'-phosphate dependent enzyme that is responsible for racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell wall, its inhibition is lethal to prokaryotes, making it an excellent antibiotic drug target. The crystal structure of A. baumannii alanine racemase (AlrAba) from the highly antibiotic-resistant NCTC13302 strain has been solved to 1.9 Å resolution. Comparison of AlrAba with alanine racemases from closely related bacteria demonstrates a conserved overall fold. The substrate entryway and active site of the enzymes were shown to be highly conserved. The structure of AlrAba will provide the template required for future structure-based drug-design studies.

  4. Nucleic acids encoding plant glutamine phenylpyruvate transaminase (GPT) and uses thereof

    DOEpatents

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2016-03-29

    Glutamine phenylpyruvate transaminase (GPT) proteins, nucleic acid molecules encoding GPT proteins, and uses thereof are disclosed. Provided herein are various GPT proteins and GPT gene coding sequences isolated from a number of plant species. As disclosed herein, GPT proteins share remarkable structural similarity within plant species, and are active in catalyzing the synthesis of 2-hydroxy-5-oxoproline (2-oxoglutaramate), a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism.

  5. Function of the D-alanine:D-alanine ligase lid loop: a molecular modeling and bioactivity study.

    PubMed

    Hrast, Martina; Vehar, Blaž; Turk, Samo; Konc, Janez; Gobec, Stanislav; Janežič, Dušanka

    2012-08-09

    D-Alanine:D-alanine ligase (Ddl) is an essential ATP-dependent bacterial enzyme involved in peptidoglycan biosynthesis. Discovery of Ddl inhibitors not competitive with ATP has proven to be difficult because the Ddl bimolecular d-alanine binding pocket is very restricted, as is accessibility to the active site for larger molecules in the catalytically active closed conformation of Ddl. A molecular dynamics study of the opening and closing of the Ddl lid loop informs future structure-based design efforts that allow for the flexibility of Ddl. A virtual screen on generated enzyme conformations yielded some hit inhibitors whose bioactivity was determined.

  6. Alanine racemase mutants of Mycobacterium tuberculosis require D-alanine for growth and are defective for survival in macrophages and mice.

    PubMed

    Awasthy, Disha; Bharath, Sowmya; Subbulakshmi, Venkita; Sharma, Umender

    2012-02-01

    Alanine racemase (Alr) is an essential enzyme in most bacteria; however, some species (e.g. Listeria monocytogenes) can utilize d-amino acid transaminase (Dat) to generate d-alanine, which renders Alr non-essential. In addition to the conflicting reports on gene knockout of alr in Mycobacterium smegmatis, a recent study concluded that depletion of Alr does not affect the growth of M. smegmatis. In order to get an unambiguous answer on the essentiality of Alr in Mycobacterium tuberculosis and validate it as a drug target in vitro and in vivo, we have inactivated the alr gene of M. tuberculosis and found that it was not possible to generate an alr knockout in the absence of a complementing gene copy or d-alanine in the growth medium. The growth kinetics of the alr mutant revealed that M. tuberculosis requires very low amounts of d-alanine (5-10 µg ml(-1)) for optimum growth. Survival kinetics of the mutant in the absence of d-alanine indicated that depletion of this amino acid results in rapid loss of viability. The alr mutant was found to be defective for growth in macrophages. Analysis of phenotype in mice suggested that non-availability of d-alanine in mice leads to clearance of bacteria followed by stabilization of bacterial number in lungs and spleen. Additionally, reversal of d-cycloserine inhibition in the presence of d-alanine in M. tuberculosis suggested that Alr is the primary target of d-cycloserine. Thus, Alr of M. tuberculosis is a valid drug target and inhibition of Alr alone should result in loss of viability in vitro and in vivo.

  7. Elevated creatine kinase and transaminases in asymptomatic SBMA.

    PubMed

    Sorenson, Eric J; Klein, Christopher J

    2007-02-01

    X-linked spinal and bulbar muscular atrophy (SBMA or Kennedy's disease) has a variable prognosis. Most male carriers are affected by their fourth or fifth decade of life, while some remain asymptomatic lifelong. Elevations of serum creatine kinase are well known to occur in clinically manifesting SBMA patients. Elevations prior to the onset of the clinical syndrome have not been reported. Here we report two cases of SBMA presenting with 'idiopathic' elevations of serum transaminases and creatine kinase a decade in advance of their symptomatic onset. These cases emphasize the need to consider SBMA and genetic testing for the androgen receptor trinucleotide CAG expansion in males otherwise healthy with 'idiopathic' elevated creatinine kinase.

  8. L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate beta-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?

    PubMed Central

    Cooper, Arthur J L; Krasnikov, Boris F; Okuno, Etsuo; Jeitner, Thomas M

    2003-01-01

    Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate beta-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate beta-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253-261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181-192] exhibit beta-lyase activity toward S -(1,2-dichlorovinyl)-L-cysteine (the cysteine S-conjugate of trichloroethylene) and S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial L-alanine-glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate beta-lyase reactions with S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(benzothiazolyl-L-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and S -(1,2-dichlorovinyl)-L-cysteine is toxic by acting as a thioacylating agent - particularly of lysine residues in nearby proteins. Toxicity, however, may also involve 'self-inactivation' of key enzymes. The present findings suggest that alanine-glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine-glyoxylate aminotransferase II is absent

  9. Studies on optical, mechanical and transport properties of NLO active L-alanine formate single crystal grown by modified Sankaranarayanan Ramasamy (SR) method

    NASA Astrophysics Data System (ADS)

    Justin Raj, C.; Dinakaran, S.; Krishnan, S.; Milton Boaz, B.; Robert, R.; Jerome Das, S.

    2008-04-01

    Bulk single crystals of L-alanine formate of 10 mm diameter and 50 mm length have been grown with an aid of modified Sankaranarayanan-Ramasamy (SR) uniaxial crystal growth method within a period of ten days. The optical properties of the grown crystal were calculated from UV transmission spectral analysis. The second harmonic generation efficiency of the grown crystal was confirmed by Kurtz powder test. In order to determine the mechanical strength of the crystal, Vicker's microhardness test was carried along the growth plane (0 0 1). Dielectric studies reveal that both dielectric constant and dielectric loss decreases with increase in frequency. Photoconductivity study confirms the negative photoconducting nature of the crystal.

  10. Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis.

    PubMed

    Genz, Maika; Vickers, Clare; van den Bergh, Tom; Joosten, Henk-Jan; Dörr, Mark; Höhne, Matthias; Bornscheuer, Uwe T

    2015-11-11

    To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5'-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions.

  11. Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis

    PubMed Central

    Genz, Maika; Vickers, Clare; van den Bergh, Tom; Joosten, Henk-Jan; Dörr, Mark; Höhne, Matthias; Bornscheuer, Uwe T.

    2015-01-01

    To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5′-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions. PMID:26569229

  12. Antifibrotic activity of galangin, a novel function evaluated in animal liver fibrosis model.

    PubMed

    Wang, Xinhui; Gong, Guoqing; Yang, Wenhui; Li, Yunzhan; Jiang, Meiling; Li, Linlin

    2013-09-01

    This study aimed to investigate the effects of galangin on liver fibrosis in rats induced by subcutaneous injection of carbon tetrachloride (CCl4). The administration of CCl4 to rats for 12 weeks caused significant increase of hyaluronic acid, laminin, alanine transaminase, aspartate transaminase and decrease of total protein, albumin in serum, while the influences could be reversed by galangin. Galangin markedly reduced hepatic malondialdehyde, hydroxyproline concentration, increased activities of liver superoxide dismutase, glutathione peroxidase compared with CCl4-treated rats. Histological results indicated that galangin alleviated liver damage. In addition, treatment with galangin significantly down-regulated expressions of α-smooth muscle actin and transforming growth factor β1. These results suggest galangin can inhibit liver fibrosis induced by CCl4 in rats, which was probably associated with its effect on removing oxygen free radicals, decreasing lipid peroxidation, as well as inhibiting hepatic stellate cells activation and proliferation.

  13. Peptide aromatic interactions modulated by fluorinated residues: Synthesis, structure and biological activity of Somatostatin analogs containing 3-(3′,5′difluorophenyl)-alanine

    PubMed Central

    Martín-Gago, Pablo; Rol, Álvaro; Todorovski, Toni; Aragón, Eric; Martin-Malpartida, Pau; Verdaguer, Xavier; Vallès Miret, Mariona; Fernández-Carneado, Jimena; Ponsati, Berta; Macias, Maria J.; Riera, Antoni

    2016-01-01

    Somatostatin is a 14-residue peptide hormone that regulates the endocrine system by binding to five G-protein-coupled receptors (SSTR1–5). We have designed six new Somatostatin analogs with L-3-(3′,5′-difluorophenyl)-alanine (Dfp) as a substitute of Phe and studied the effect of an electron-poor aromatic ring in the network of aromatic interactions present in Somatostatin. Replacement of each of the Phe residues (positions 6, 7 and 11) by Dfp and use of a D-Trp8 yielded peptides whose main conformations could be characterized in aqueous solution by NMR. Receptor binding studies revealed that the analog with Dfp at position 7 displayed a remarkable affinity to SSTR2 and SSTR3. Analogs with Dfp at positions 6 or 11 displayed a π-π interaction with the Phe present at 11 or 6, respectively. Interestingly, these analogs, particularly [D-Trp8,L-Dfp11]-SRIF, showed high selectivity towards SSTR2, with a higher value than that of Octreotide and a similar one to that of native Somatostatin. PMID:27271737

  14. EPR/alanine dosimetry for two therapeutic proton beams

    NASA Astrophysics Data System (ADS)

    Marrale, Maurizio; Carlino, Antonio; Gallo, Salvatore; Longo, Anna; Panzeca, Salvatore; Bolsi, Alessandra; Hrbacek, Jan; Lomax, Tony

    2016-02-01

    In this work the analysis of the electron paramagnetic resonance (EPR) response of alanine pellets exposed to two different clinical proton beams employed for radiotherapy is performed. One beam is characterized by a passive delivery technique and is dedicated to the eyes treatment (OPTIS2 beam line). Alanine pellets were irradiated with a 70 MeV proton beam corresponding to 35 mm range in eye tissue. We investigated how collimators with different sizes and shape used to conform the dose to the planned target volume influence the delivered dose. For this purpose we performed measurements with varying the collimator size (Output Factor) and the results were compared with those obtained with other dosimetric techniques (such as Markus chamber and diode detector). This analysis showed that the dosimeter response is independent of collimator diameter if this is larger than or equal to 10 mm. The other beam is characterized by an active spot-scanning technique, the Gantry1 beam line (maximum energy 230 MeV), and is used to treat deep-seated tumors. The dose linearity of alanine response in the clinical dose range was tested and the alanine dose response at selected locations in depth was measured and compared with the TPS planned dose in a quasi-clinical scenario. The alanine response was found to be linear in the dose in the clinical explored range (from 10 to 70 Gy). Furthermore, a depth dose profile in a quasi-clinical scenario was measured and compared to the dose computed by the Treatment Planning System PSIPLAN. The comparison of calibrated proton alanine measurements and TPS dose shows a difference under 1% in the SOBP and a "quenching" effect up to 4% in the distal part of SOBP. The positive dosimetric characteristics of the alanine pellets confirm the feasibility to use these detectors for "in vivo" dosimetry in clinical proton beams.

  15. Evaluation of hepatoprotective effect of Gentiana olivieri herbs on subacute administration and isolation of active principle.

    PubMed

    Orhan, Didem Deliorman; Aslan, Mustafa; Aktay, Göknur; Ergun, Ender; Yesilada, Erdem; Ergun, Fatma

    2003-04-04

    Hepatoprotective effect of Gentiana olivieri Griseb. (Gentianaceae) flowering herbs on subacute administration were studied using in vivo models in rats. For the activity assessment on carbon tetrachloride-induced hepatic damage following biochemical parameters were evaluated; plasma and hepatic tissue malondialdehyde formation, and liver tissue glutathione level, as well as plasma transaminase enzyme levels (aspartate transferase and alanine transferase). Results of biochemical tests were also confirmed by histopathological examination. Through bioassay-guided fractionation procedures isoorientin, a known C-glycosylflavone, was isolated from the ethyl acetate fraction as the active antihepatotoxic constituent by silica gel column chromatography. Isoorientin exhibited significant hepatoprotective effect at 15 mg/kg b.w. dose.

  16. Antihyperglycemic and antihyperlipidemic activities of 2-(4-[(2-hydroxybenzyl) amino]-phenyl amino-methyl)-phenol in STZ induced diabetic rats.

    PubMed

    Sirasanagandla, Swapna; Kasetti, Ramesh Babu; Shaik, Abdul Nabi; Natava, Rajesh; Surtineni, Venkata Prasad; Cirradur, Suresh Reddy; Chippada, Apparao

    2013-08-01

    Oral administration of 2-(4-[(2-hydroxybenzyl) amino]-phenyl amino-methyl)-phenol (HBPMP) (30 mg/kg) to Streptozotocin (STZ) rats produced significant antidiabetic activity after 6 h of HBPMP administration. Treatment of the STZ rats with HBPMP (30 mg/kg/day) for 30 days resulted in a significant decrease in their Fasting Blood Glucose (FBG), Serum Total Cholesterol (TC), Low Density Lipoprotein-Cholesterol (LDL-C), Very Low Density Lipoprotein-Cholesterol (VLDL-C) and triglycerides (TG) along with an increase in serum High Density Lipoprotein-Cholesterol (HDL-C) levels. Activities of Serum Aspartate transaminase (AST), Alanine transaminase (ALT) and Alkaline phosphatase (ALP) and levels of blood urea and creatinine were improved to near normal levels in the treated STZ rats indicating the protective role of the HBPMP against liver and kidney damage and its non-toxic property. In conclusion, HBPMP possesses antihyperglycemic and antihyperlipidemic activities.

  17. Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium.

    PubMed

    Venir, Elena; Del Torre, Manuela; Cunsolo, Vincenzo; Saletti, Rosaria; Musetti, Rita; Stecchini, Mara Lucia

    2014-02-01

    The L-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of D-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting L-alanine to the germination inhibitor D-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of L- to D-alanine. Unlike ATCC 14579 spores, L-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.

  18. Inhibitors of alanine racemase enzyme: a review.

    PubMed

    Azam, Mohammed Afzal; Jayaram, Unni

    2016-08-01

    Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalysing the conversion of l-alanine to d-alanine utilised by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The standard inhibitors of this enzyme include O-carbamyl-d-serine, d-cycloserine, chlorovinyl glycine, alaphosphin, etc. d-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase.

  19. [Raman scattering study of DL-alanine].

    PubMed

    Gong, Yan; Wang, Wen-qing

    2006-01-01

    Studies of Raman vibration spectra are useful to obtaining information on biomolecular crystals. The cell dimensions of the L- and DL-alanine crystals are nearly identical, and both structures belong to the orthorhombic system, but the space group is P2(1) 2(1) 2(1) for the L-isomer, and Pna2(1) for the racemate crystal. The Raman spectrum of L-alanine has been measured by many authors. The present work is focusing on the Raman scattering study of DL-alanine powder. Based on the analysis of the differences between DL-alanine and L-alanine Raman spectra, the authors obtained indispensable information on hydrogen bond and the motion of the molecular conformation in alanine crystals.

  20. Vibrational dynamics of crystalline L-alanine

    SciTech Connect

    Bordallo, H.N.; Eckert, J.; Barthes, M.

    1997-11-01

    The authors report a new, complete vibrational analysis of L-alanine and L-alanine-d{sub 4} which utilizes IINS intensities in addition to frequency information. The use of both isotopomers resulted in a self-consistent force field for and assignment of the molecular vibrations in L-alanine. Some details of the calculation as well as a comparison of calculated and observed IINS spectra are presented. The study clarifies a number of important issues on the vibrational dynamics of this molecule and presents a self-consistent force field for the molecular vibrations in crystalline L-alanine.

  1. Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine

    NASA Technical Reports Server (NTRS)

    Profy, A. T.; Usher, D. A.

    1984-01-01

    The aminoacylation of diinosine monophosphate was studied experimentally. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40 percent enantiomeric excess of the isomer was incorporated at the 2' site and the positions of equilibrium for the reversible 2'-3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups was found to decrease on the order 2'(3') less than internal 2' and less than 5', and the extent of the reaction was affected by the concentration of the imidazole buffer. Reaction of IpI with imidazolide of unprotected DL-alanine, by contrast, led to an excess of the D isomer at the internal 2' site. Finally, reaction with the N-carboxy anhydride of DL-alanine occurred without stereoselection. These results are found to be relevant to the study of the evolution of optical chemical activity and the origin of genetically directed protein synthesis.

  2. Catalytic Stereoinversion of L-Alanine to Deuterated D-Alanine.

    PubMed

    Moozeh, Kimia; So, Soon Mog; Chin, Jik

    2015-08-03

    A combination of an achiral pyridoxal analogue and a chiral base has been developed for catalytic deuteration of L-alanine with inversion of stereochemistry to give deuterated D-alanine under mild conditions (neutral pD and 25 °C) without the use of any protecting groups. This system can also be used for catalytic deuteration of D-alanine with retention of stereochemistry to give deuterated D-alanine. Thus a racemic mixture of alanine can be catalytically deuterated to give an enantiomeric excess of deuterated D-alanine. While catalytic deracemization of alanine is forbidden by the second law of thermodynamics, this system can be used for catalytic deracemization of alanine with deuteration. Such green and biomimetic approach to catalytic stereocontrol provides insights into efficient amino acid transformations.

  3. Identification of novel transaminases from a 12-aminododecanoic acid-metabolizing Pseudomonas strain.

    PubMed

    Wilding, Matthew; Walsh, Ellen F A; Dorrian, Susan J; Scott, Colin

    2015-07-01

    A Pseudomonas species [Pseudomonas sp. strain amino alkanoate catabolism (AAC)] was identified that has the capacity to use 12-aminododecanoic acid, the constituent building block of homo-nylon-12, as a sole nitrogen source. Growth of Pseudomonas sp. strain AAC could also be supported using a range of additional ω-amino alkanoates. This metabolic function was shown to be most probably dependent upon one or more transaminases (TAs). Fourteen genes encoding putative TAs were identified from the genome of Pseudomonas sp. AAC. Each of the 14 genes was cloned, 11 of which were successfully expressed in Escherichia coli and tested for activity against 12-aminododecanoic acid. In addition, physiological functions were proposed for 9 of the 14 TAs. Of the 14 proteins, activity was demonstrated in 9, and of note, 3 TAs were shown to be able to catalyse the transfer of the ω-amine from 12-aminododecanoic acid to pyruvate. Based on this study, three enzymes have been identified that are promising biocatalysts for the production of nylon and related polymers.

  4. Data in the activities of caspases and the levels of reactive oxygen species and cytochrome c in the •OH-induced fish erythrocytes treated with alanine, citrulline, proline and their combination

    PubMed Central

    Li, Huatao; Jiang, Weidan; Liu, Yang; Jiang, Jun; Zhang, Yongan; Wu, Pei; Zhao, Juan; Duan, Xudong; Zhou, Xiaoqiu; Feng, Lin

    2016-01-01

    The present study explored the effects of alanine (Ala), citrulline (Cit), proline (Pro) and their combination (Ala10Pro4Cit1) on the activities of caspases and levels of reactive oxygen species (ROS) and cytochrome c in hydroxyl radicals (•OH)-induced carp erythrocytes. The data displayed that •OH induced the increases in the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c in carp erythrocytes. However, Ala, Cit, Pro and Ala10Pro4Cit1 effectively suppressed the •OH-induced increases in the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c in carp erythrocytes. Furthermore, the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c were gradually decreased with increasing concentrations of Ala, Cit, Pro and Ala10Pro4Cit1 (0.175−1.400 mM) in the •OH-induced carp erythrocytes. These data demonstrated that the 50% inhibitory doses (ID50) of Ala10Pro4Cit1 on the activities of caspase−8, caspase−9 and caspase−3 and levels of ROS and cytochrome c were respectively estimated to be the minimum values among amino acids examined so far. The 5% inhibitory doses (ID5) of Ala, Cit, Pro and Ala10Pro4Cit1 on the activities of caspase−8, caspase−9 and caspase−3 and levels of ROS and cytochrome c were estimated to be at their physiological concentrations in mammalian. Our research article for further interpretation and discussion from these data in Li et al. (2016) [1]. PMID:26952131

  5. Basic aspects of GABA-transmission in alcoholism, with particular reference to GABA-transaminase.

    PubMed

    Sherif, F M; Tawati, A M; Ahmed, S S; Sharif, S I

    1997-02-01

    Neuronal dysfunction is the neurobiological basis for alcoholic behaviour, and ethanol craving seems related to hypofunction of the GABA-ergic activity. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). In several studies, GABA has been shown to be an important target of ethanol in the CNS, partly, as a consequence of damage to membrane-bound enzymes and receptors. GABA is involved in mediating pre- and post-synaptic inhibition of neuronal activity. It is speculated that the initial excitatory effects of ethanol may be due to inhibition of GABA-ergic activity whereas the sedative effects of the higher doses may be mediated by the activation of this inhibitory system. In the CNS, GABA is synthesised from glutamic acid by the enzyme glutamate decarboxylase (GAD) and catabolized into succinic semialdehyde by the enzyme GABA-transaminase (GABA-T), which are pyridoxal phosphate (PLP) dependent enzymes. Platelet GABA-T was characterized as being similar to central GABA-T. Inhibition of GABA-T with certain potent and selective compounds markedly increases the levels of brain GABA. Experimentally, acute ethanol treatment does not alter GABA-T activity whereas chronic treatment produces an increase in the activity, though, with some reservations since a bimodal effect has been found in chronically ethanol-treated rats. Thus, as it will be discussed below, it may be suggested that GABA-T inhibitors (e.g. vigabatrin) could have a potential role in the treatment of alcoholism and in some of the problems of ethanol withdrawal and of other drugs of abuse. Related studies on metabolism and concentrations of GABA are also promising and show a greater increase in our understanding of the aetiology and treatment of ethanol dependence and withdrawal. In general, this article also reviews both the animal and clinical observations in the field of alcoholism with regard to the GABA system.

  6. Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides.

    PubMed

    Doan, Thanh Thi Ngoc; Kim, Jin-Kwang; Ngo, Ho-Phuong-Thuy; Tran, Huyen-Thi; Cha, Sun-Shin; Min Chung, Kyung; Huynh, Kim-Hung; Ahn, Yeh-Jin; Kang, Lin-Woo

    2014-03-01

    D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of d-alanyl-d-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 Å. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with d-alanyl-d-alanine and ATP-bound TtDDL structure, the γ-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the ω-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped ω-loops. Results showed that the direct interactions between ω-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation.

  7. The 1.9 A Structure of the Branched-Chain Amino-Acid Transaminase (IlvE) from Mycobacterium tuberculosis

    SciTech Connect

    Tremblay, L.; Blanchard, J

    2009-01-01

    Unlike mammals, bacteria encode enzymes that synthesize branched-chain amino acids. The pyridoxal 5'-phosphate-dependent transaminase performs the final biosynthetic step in these pathways, converting keto acid precursors into {alpha}-amino acids. The branched-chain amino-acid transaminase from Mycobacterium tuberculosis (MtIlvE) has been crystallized and its structure has been solved at 1.9 {angstrom} resolution. The MtIlvE monomer is composed of two domains that interact to form the active site. The biologically active form of IlvE is a homodimer in which each monomer contributes a substrate-specificity loop to the partner molecule. Additional substrate selectivity may be imparted by a conserved N-terminal Phe30 residue, which has previously been observed to shield the active site in the type IV fold homodimer. The active site of MtIlvE contains density corresponding to bound PMP, which is likely to be a consequence of the presence of tryptone in the crystallization medium. Additionally, two cysteine residues are positioned at the dimer interface for disulfide-bond formation under oxidative conditions. It is unknown whether they are involved in any regulatory activities analogous to those of the human mitochondrial branched-chain amino-acid transaminase.

  8. The effects of alanine-substituted conantokin-G and ifenprodil on the human spermine-activated N-methyl-D-aspartate receptor.

    PubMed

    Tsai, V W-W; Dodd, P R; Lewis, R J

    2005-01-01

    We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [(3)H]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degrees C until assay. Both Con-G(A7) and ifenprodil inhibited [(3)H]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC(50) changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC(50) with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [(3)H]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC(50), while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-D-aspartate receptor.

  9. Alanine increases blood pressure during hypotension

    NASA Technical Reports Server (NTRS)

    Conlay, L. A.; Maher, T. J.; Wurtman, R. J.

    1990-01-01

    The effect of L-alanine administration on blood pressure (BP) during haemorrhagic shock was investigated using anesthetized rats whose left carotid arteries were cannulated for BP measurement, blood removal, and drug administration. It was found that L-alanine, in doses of 10, 25, 50, 100, and 200 mg/kg, increased the systolic BP of hypotensive rats by 38 to 80 percent (while 100 mg/kg pyruvate increased BP by only 9.4 mmhg, not significantly different from saline). The results suggest that L-alanine might influence cardiovascular function.

  10. Regulation of the ald gene encoding alanine dehydrogenase by AldR in Mycobacterium smegmatis.

    PubMed

    Jeong, Ji-A; Baek, Eun-Young; Kim, Si Wouk; Choi, Jong-Soon; Oh, Jeong-Il

    2013-08-01

    The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding L-alanine dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of L-alanine. The purified AldR protein exists as a homodimer in the absence of L-alanine, while it adopts the quaternary structure of a homohexamer in the presence of L-alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by L-alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N₂-ATC-N₂-TC and one putative AldR binding site with the sequence GA-N₂-GTT-N₂-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of L-alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

  11. Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics

    PubMed Central

    Mamidi, Ranganath; Gollapudi, Sampath K.; Mallampalli, Sri Lakshmi; Chandra, Murali

    2012-01-01

    Ala/Asp substitutions at Ser23/24 have been employed to investigate the functional impact of cardiac troponin I (cTnI) phosphorylation by protein kinase A (PKA). Some limitations of previous studies include the use of heterologous proteins and confounding effects arising from phosphorylation of cardiac myosin binding protein-C. Our goal was to probe the effects of cTnI phosphorylation using a homologous assay, so that altered function could be solely attributed to changes in cTnI. We reconstituted detergent-skinned rat cardiac papillary fibers with homologous rat cardiac troponin subunits to study the impact of Ala and Asp substitutions at Ser23/24 of rat cTnI (RcTnI S23A/24A and RcTnI S23D/24D). Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~36% decrease in Ca2+-activated maximal tension. Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~18% decrease in ATPase activity. Muscle fiber stiffness measurements suggested that the decrease in thin filament activation observed in RcTnI S23A/24A and RcTnI S23D/24D was due to a decrease in the number of strongly-bound crossbridges. Another major finding was that Ala and Asp substitutions in cTnI did not affect crossbridge detachment kinetics. PMID:22684024

  12. Catalytic Promiscuity of Transaminases: Preparation of Enantioenriched β-Fluoroamines by Formal Tandem Hydrodefluorination/Deamination.

    PubMed

    Cuetos, Aníbal; García-Ramos, Marina; Fischereder, Eva-Maria; Díaz-Rodríguez, Alba; Grogan, Gideon; Gotor, Vicente; Kroutil, Wolfgang; Lavandera, Iván

    2016-02-24

    Transaminases are valuable enzymes for industrial biocatalysis and enable the preparation of optically pure amines. For these transformations they require either an amine donor (amination of ketones) or an amine acceptor (deamination of racemic amines). Herein transaminases are shown to react with aromatic β-fluoroamines, thus leading to simultaneous enantioselective dehalogenation and deamination to form the corresponding acetophenone derivatives in the absence of an amine acceptor. A series of racemic β-fluoroamines was resolved in a kinetic resolution by tandem hydrodefluorination/deamination, thus giving the corresponding amines with up to greater than 99 % ee. This protocol is the first example of exploiting the catalytic promiscuity of transaminases as a tool for novel transformations.

  13. Biochemical changes of the synovial liquid of corpses with regard to the cause of death. 2: Alkaline phosphatase, lactic acid dehydrogenase (LDH), and glutamic oxalacetic transaminase (GOT).

    PubMed

    More, D S; Arroyo, M C

    1985-04-01

    We studied the activity of various enzymes in the synovial liquid of 100 corpses with regard to the cause of death finding that the alkaline phospatase and glutamic oxalacetic transaminase (GOT) are increased in cranioencephalic trauma, possibly as a result of the important cellular lysis which goes with them; and lactic acid dehydrogenase (LDH) is increased in the pulmonary processes, almost certainly with relation to the great quantity of this enzyme in the lung.

  14. Kinetic mechanism and inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by the antibiotic D-cycloserine.

    PubMed

    Prosser, Gareth A; de Carvalho, Luiz Pedro S

    2013-02-01

    D-cycloserine (DCS) is an antibiotic that is currently used in second-line treatment of tuberculosis. DCS is a structural analogue of D-alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over-expressed and purified recombinant Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl is activated by K(+), follows an ordered ter ter mechanism and displays distinct affinities for D-Ala at each D-Ala binding site (K(m,D-Ala1) = 0.075 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 μm, K(i,DCS2) = 25 μm); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K(i,DCS2) revealed a loss of DCS binding affinity at high pH (pK(a) = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel Ddl-specific inhibitors for use as anti-mycobacterial agents.

  15. Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: isolation and characterization of a phosphinothricin-specific transaminase from Escherichia coli.

    PubMed Central

    Schulz, A; Taggeselle, P; Tripier, D; Bartsch, K

    1990-01-01

    An aminotransferase capable of transaminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid to L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG), was purified to apparent homogeneity from Escherichia coli K-12. The enzyme catalyzes the transamination of L-phosphinothricin and various analogs with 2-ketoglutarate as the amino group acceptor. The transaminase has a molecular mass of 43 kilodaltons by sodium dodecyl sulfate-gel analysis and an isoelectric point of 4.35. The enzyme was most active in the high-pH region, with a maximum at pH 8.0 to 9.5, and had a temperature optimum of 55 degrees C. Heat stability was observed up to 70 degrees C. Substrate specificity studies suggested that the enzyme is identical with the 4-aminobutyrate:2-ketoglutarate transaminase (EC 2.6.1.19). The first 30 amino acids of the N terminus of the protein were determined by gas phase sequencing. The transaminase was immobilized by coupling to the epoxy-activated carrier VA-Biosynth (Riedel de Haen) and used in a column reactor for the continuous production of L-phosphinothricin. The enzyme reactor was operated for 7 weeks with only a slight loss of catalytic capacity. Production rates of more than 50 g of L-phosphinothricin per liter of column per h were obtained. Images PMID:2178550

  16. Production of Alanine by Fusarium moniliforme

    PubMed Central

    Carito, Sebastian L.; Pisano, Michael A.

    1966-01-01

    Fusarium moniliforme grown in a chemically defined medium in submerged culture accumulated amino acids extracellularly. Alanine and glutamic acid were present in greatest amounts, with traces of glycine, lysine, threonine, and valine detectable. Increasing the glucose and urea concentrations of the medium increased yields of alanine. Further increases in alanine production occurred with elevated levels of mineral salts in the medium, whereas the addition of a vitamin mixture proved to be inhibitory. Chemical changes resulting from the growth of F. moniliforme in the final fermentation medium disclosed maximal alanine production, mycelial weight, and glucose consumption after 72 hr of incubation at 28.5 C. Total soluble nitrogen, by contrast, was minimal at the same time period. The pH remained in the alkaline range throughout the fermentation. PMID:5914495

  17. Solved? The reductive radiation chemistry of alanine.

    PubMed

    Pauwels, Ewald; De Cooman, Hendrik; Waroquier, Michel; Hole, Eli O; Sagstuen, Einar

    2014-02-14

    The structural changes throughout the entire reductive radiation-induced pathway of l-α-alanine are solved on an atomistic level with the aid of periodic DFT and nudged elastic band (NEB) simulations. This yields unprecedented information on the conformational changes taking place, including the protonation state of the carboxyl group in the "unstable" and "stable" alanine radicals and the internal transformation converting these two radical variants at temperatures above 220 K. The structures of all stable radicals were verified by calculating EPR properties and comparing those with experimental data. The variation of the energy throughout the full radiochemical process provides crucial insight into the reason why these structural changes and rearrangements occur. Starting from electron capture, the excess electron quickly localizes on the carbon of a carboxyl group, which pyramidalizes and receives a proton from the amino group of a neighboring alanine molecule, forming a first stable radical species (up to 150 K). In the temperature interval 150-220 K, this radical deaminates and deprotonates at the carboxyl group, the detached amino group undergoes inversion and its methyl group sustains an internal rotation. This yields the so-called "unstable alanine radical". Above 220 K, triggered by the attachment of an additional proton on the detached amino group, the radical then undergoes an internal rotation in the reverse direction, giving rise to the "stable alanine radical", which is the final stage in the reductive radiation-induced decay of alanine.

  18. Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus.

    PubMed

    Okubo, Y; Yokoigawa, K; Esaki, N; Soda, K; Kawai, H

    1999-03-16

    A psychrophilic alanine racemase gene from Bacillus psychrosaccharolyticus was cloned and expressed in Escherichia coli SOLR with a plasmid pYOK3. The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T. The enzyme purified to homogeneity showed the high catalytic activity even at 0 degrees C and was extremely labile over 35 degrees C. The enzyme was found to have a markedly large Km value (5.0 microM) for the pyridoxal 5'-phosphate (PLP) cofactor in comparison with other reported alanine racemases, and was stabilized up to 50 degrees C in the presence of excess amounts of PLP. The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature. The enzyme has a distinguishing hydrophilic region around the residue no. 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of the region in the three dimensional structure of C atoms of the enzyme was predicted to be in a surface loop surrounding the active site. The region may interact with solvent and reduce the compactness of the active site.

  19. Inducible Glutamate Oxaloacetate Transaminase as a Therapeutic Target Against Ischemic Stroke

    PubMed Central

    Khanna, Savita; Briggs, Zachary

    2015-01-01

    Abstract Significance: Glutamate serves multi-faceted (patho)physiological functions in the central nervous system as the most abundant excitatory neurotransmitter and under pathological conditions as a potent neurotoxin. Regarding the latter, elevated extracellular glutamate is known to play a central role in ischemic stroke brain injury. Recent Advances: Glutamate oxaloacetate transaminase (GOT) has emerged as a new therapeutic target in protecting against ischemic stroke injury. Oxygen-sensitive induction of GOT expression and activity during ischemic stroke lowers glutamate levels at the stroke site while sustaining adenosine triphosphate levels in brain. The energy demands of the brain are among the highest of all organs underscoring the need to quickly mobilize alternative carbon skeletons for metabolism in the absence of glucose during ischemic stroke. Recent work builds on the important observation of Hans Krebs that GOT-mediated metabolism of glutamate generates tri-carboxylic acid (TCA) cycle intermediates in brain tissue. Taken together, outcomes suggest GOT may enable the transformative switch of otherwise excitotoxic glutamate into life-sustaining TCA cycle intermediates during ischemic stroke. Critical Issues: Neuroprotective strategies that focus solely on blocking mechanisms of glutamate-mediated excitotoxicity have historically failed in clinical trials. That GOT can enable glutamate to assume the role of a survival factor represents a paradigm shift necessary to develop the overall significance of glutamate in stroke biology. Future Directions: Ongoing efforts are focused to develop the therapeutic significance of GOT in stroke-affected brain. Small molecules that target induction of GOT expression and activity in the ischemic penumbra are the focus of ongoing studies. Antioxid. Redox Signal. 22, 175–186. PMID:25343301

  20. Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine, and its implications for the mechanism of catalysis and substrate activation.

    PubMed

    Andersen, Ole Andreas; Flatmark, Torgeir; Hough, Edward

    2002-07-26

    Phenylalanine hydroxylase catalyzes the stereospecific hydroxylation of L-phenylalanine, the committed step in the degradation of this amino acid. We have solved the crystal structure of the ternary complex (hPheOH-Fe(II).BH(4).THA) of the catalytically active Fe(II) form of a truncated form (DeltaN1-102/DeltaC428-452) of human phenylalanine hydroxylase (hPheOH), using the catalytically active reduced cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and 3-(2-thienyl)-L-alanine (THA) as a substrate analogue. The analogue is bound in the second coordination sphere of the catalytic iron atom with the thiophene ring stacking against the imidazole group of His285 (average interplanar distance 3.8A) and with a network of hydrogen bonds and hydrophobic contacts. Binding of the analogue to the binary complex hPheOH-Fe(II).BH(4) triggers structural changes throughout the entire molecule, which adopts a slightly more compact structure. The largest change occurs in the loop region comprising residues 131-155, where the maximum r.m.s. displacement (9.6A) is at Tyr138. This loop is refolded, bringing the hydroxyl oxygen atom of Tyr138 18.5A closer to the iron atom and into the active site. The iron geometry is highly distorted square pyramidal, and Glu330 adopts a conformation different from that observed in the hPheOH-Fe(II).BH(4) structure, with bidentate iron coordination. BH(4) binds in the second coordination sphere of the catalytic iron atom, and is displaced 2.6A in the direction of Glu286 and the iron atom, relative to the hPheOH-Fe(II).BH(4) structure, thus changing its hydrogen bonding network. The active-site structure of the ternary complex gives new insight into the substrate specificity of the enzyme, notably the low affinity for L-tyrosine. Furthermore, the structure has implications both for the catalytic mechanism and the molecular basis for the activation of the full-length tetrameric enzyme by its substrate. The large conformational change, moving

  1. Synthesis of pharmaceutically relevant 17-α-amino steroids using an ω-transaminase.

    PubMed

    Richter, Nina; Simon, Robert C; Kroutil, Wolfgang; Ward, John M; Hailes, Helen C

    2014-06-11

    An efficient and sustainable biocatalytic route for the synthesis of important 17-α-amino steroids has been developed using an ω-transaminase variant from Arthrobacter sp. Optimisation of the reaction conditions facilitated the synthesis of these valuable synthons on a preparative scale, affording excellent isolated yields and stereocontrol.

  2. ApoSOD1 lacking dismutase activity neuroprotects motor neurons exposed to beta-methylamino-L-alanine through the Ca2+/Akt/ERK1/2 prosurvival pathway

    PubMed Central

    Petrozziello, Tiziana; Secondo, Agnese; Tedeschi, Valentina; Esposito, Alba; Sisalli, MariaJosè; Scorziello, Antonella; Di Renzo, Gianfranco; Annunziato, Lucio

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe human adult-onset neurodegenerative disease affecting lower and upper motor neurons. In >20% of cases, the familial form of ALS is caused by mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1). Interestingly, administration of wild-type SOD1 to SOD1G93A transgenic rats ameliorates motor symptoms through an unknown mechanism. Here we investigated whether the neuroprotective effects of SOD1 are due to the Ca2+-dependent activation of such prosurvival signaling pathway and not to its catalytic activity. To this aim, we also examined the mechanism of neuroprotective action of ApoSOD1, the metal-depleted state of SOD1 that lacks dismutase activity, in differentiated motor neuron-like NSC-34 cells and in primary motor neurons exposed to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA). Preincubation of ApoSOD1 and SOD1, but not of human recombinant SOD1G93A, prevented cell death in motor neurons exposed to L-BMAA. Moreover, ApoSOD1 elicited ERK1/2 and Akt phosphorylation in motor neurons through an early increase of intracellular Ca2+ concentration ([Ca2+]i). Accordingly, inhibition of ERK1/2 by siMEK1 and PD98059 counteracted ApoSOD1- and SOD1-induced neuroprotection. Similarly, transfection of the dominant-negative form of Akt in NSC-34 motor neurons and treatment with the selective PI3K inhibitor LY294002 prevented ApoSOD1- and SOD1-mediated neuroprotective effects in L-BMAA-treated motor neurons. Furthermore, ApoSOD1 and SOD1 prevented the expression of the two markers of L-BMAA-induced ER stress GRP78 and caspase-12. Collectively, our data indicate that ApoSOD1, which is devoid of any catalytic dismutase activity, exerts a neuroprotective effect through an early activation of Ca2+/Akt/ERK1/2 pro-survival pathway that, in turn, prevents ER stress in a neurotoxic model of ALS. PMID:28085149

  3. Effects of high-salinity seawater acclimation on the levels of D-alanine in the muscle and hepatopancreas of kuruma prawn, Marsupenaeus japonicus.

    PubMed

    Yoshikawa, Naoko; Yokoyama, Masahumi

    2015-12-10

    Changes in D- and L-alanine contents were determined in the muscle and hepatopancreas of kuruma prawn Marsupenaeus japonicus, during acclimation from seawater containing 100% salinity to artificial seawater containing 150% salinity. In the hepatopancreas, contents of both amino acids increased by approximately threefold. The activity of alanine racemase, which catalyzes the interconversion of D- and L-alanine, also increased in the high-salinity seawater. In addition, the expression of the gene encoding alanine racemase increased in the hepatopancreas with an increase in the alanine racemase activity. These data indicate that the biosynthesis of D- and L-alanine is controlled by the gene expression level of alanine racemase, and D-alanine in the hepatopancreas functions as a major osmolyte for isosmotic regulation. In contrast, the content of D-alanine and alanine racemase activity did not change in the muscle during hyper-osmotic acclimation. Therefore, we suggest that D-alanine, which exists in the several tissues of M. japonicus, is considered to be utilized in some different physiological phenomena in different tissues.

  4. Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.

    PubMed

    Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2017-01-01

    The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.

  5. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  6. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  7. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  8. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  9. 21 CFR 582.5118 - Alanine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Alanine. 582.5118 Section 582.5118 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  10. [Alanine solution as enzyme reaction buffer used in A to O blood group conversion].

    PubMed

    Li, Su-Bo; Zhang, Xue; Zhang, Yin-Ze; Tan, Ying-Xia; Bao, Guo-Qiang; Wang, Ying-Li; Ji, Shou-Ping; Gong, Feng; Gao, Hong-Wei

    2014-06-01

    The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

  11. Repeated Supramaximal Exercise-Induced Oxidative Stress: Effect of β-Alanine Plus Creatine Supplementation

    PubMed Central

    Belviranli, Muaz; Okudan, Nilsel; Revan, Serkan; Balci, Serdar; Gokbel, Hakki

    2016-01-01

    Background: Carnosine is a dipeptide formed from the β-alanine and histidine amino acids and found in mainly in the brain and muscle, especially fast twitch muscle. Carnosine and creatine has an antioxidant effect and carnosine accounts for about 10% of the muscle's ability to buffer the H+ ions produced by exercise. Objectives: The aim of the study was to investigate the effects of beta alanine and/or creatine supplementation on oxidant and antioxidant status during repeated Wingate tests (WTs). Patients and Methods: Forty four sedentary males participated in the study. Participants performed three 30s WTs with 2 minutes rest between exercise bouts. After the first exercise session, the subjects were assigned to one of four groups: Placebo, Creatine, Beta-alanine and Beta-alanine plus creatine. Participants ingested twice per day for 22 consecutive days, then four times per day for the following 6 days. After the supplementation period the second exercise session was applied. Blood samples were taken before and immediately after the each exercise session for the analysis of oxidative stress and antioxidant markers. Results: Malondialdehyde levels and superoxide dismutase activities were affected by neither supplementation nor exercise. During the pre-supplementation session, protein carbonyl reduced and oxidized glutathione (GSH and GSSG) levels increased immediately after the exercise. However, during the post-supplementation session GSH and GSSG levels increased in beta-alanine and beta-alanine plus creatine groups immediately after the exercise compared to pre-exercise. In addition, during the post-supplementation session total antioxidant capacity increased in beta-alanine group immediately after the exercise. Conclusions: Beta-alanine supplementation has limited antioxidant effect during the repeated WTs. PMID:27217925

  12. Isolation and characterization of tryptophan transaminase and indolepyruvate C-methyltransferase. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus.

    PubMed

    Speedie, M K; Hornemann, U; Floss, H G

    1975-10-10

    Two enzymes, tryptophan transaminase and indolepyruvate C-methyltransferase, which are active in the initial steps of the biosynthetic pathway of the antibiotic indolmycin, have been detected and partially purified from cell-free extracts of Streptomyces griseus. The transaminase has been purified 3-fold by ammonium sulfate fractionation. At this stage of purification, it catalyzes the alpha-ketoglutarate and pyridoxal phosphate-dependent transamination of L-tryptophan, 3-methyltryptophan, L-pphenylalanine, and L-tyrosine. The C-methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine to position 3 of the aliphatic side chain of indolepyruvate. No cofactors are required. The C-methyltransferase has been purified 110-fold by ammonium sulfate fractionation, Sephadex G-150 gel filtration, DEAE-Sephadex column chromotography, and Bio-Gel A-5m gel filtration. The enzyme has a broad pH optimum of 7.5 to 8.5. A molecular weight of 55,000 +/- 5,000 has been determined by Sephadex G-200 gel filtration with reference proteins and a molecular weight of 58,000 +/- 8,000 has been determined by sucrose density gradient centrifugation. The enzyme is relatively stable at temperatures of 0-5 degrees but is destroyed by freezing or by heating. The C-methyltransferase is inhibited strongly by the thiol reagents p-chloromercuribenzoate and N-ethylmaleimide. The Zn2+ and Fe2+ chelators 1,10-phenanthroline and 2,2'-bipyridine also inhibit the enzyme activity but EDTA does not. Michaelis-Menten constants have been determined for the 110-fold purified enzyme as 1.2 X 10(-5) M for S-adenosylmethionine and 4.8 X 10(-6) M for indolepyruvate. The enzyme activity in the crude extract is inhibited competitively by indolmycin (Ki equals 2.3 mM) and L-tryptophan (Ki equals 0.17 mM), but these effects are not observed after the enzyme has been passed through the Sephades G-150 column during purification. The crude extract is capable of methylating phenylpyruvate and p

  13. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true DL-Alanine. 172.540 Section 172.540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents and Related Substances § 172.540 DL-Alanine. DL-Alanine (a racemic mixture of D- and...

  14. On the existence of ‘L-alanine cadmium bromide'

    NASA Astrophysics Data System (ADS)

    Srinivasan, Bikshandarkoil R.

    2013-12-01

    It is argued that the recently reported nonlinear optical crystal L-alanine cadmium bromide, grown by slow solvent evaporation method at room temperature [P. Ilayabarathi, J. Chandrasekaran, Spectrochim. Acta 96A (2012) 684-689] is the well-known L-alanine crystal. The isolation of L-alanine crystal is explained due to fractional crystallization.

  15. On the existence of 'L-alanine cadmium bromide'.

    PubMed

    Srinivasan, Bikshandarkoil R

    2013-12-01

    It is argued that the recently reported nonlinear optical crystal L-alanine cadmium bromide, grown by slow solvent evaporation method at room temperature [P. Ilayabarathi, J. Chandrasekaran, Spectrochim. Acta 96A (2012) 684-689] is the well-known L-alanine crystal. The isolation of L-alanine crystal is explained due to fractional crystallization.

  16. Knockout of the alanine racemase gene in Aeromonas hydrophila HBNUAh01 results in cell wall damage and enhanced membrane permeability.

    PubMed

    Liu, Dong; Zhang, Lu; Xue, Wen; Wang, Yaping; Ju, Jiansong; Zhao, Baohua

    2015-07-01

    This study focused on the alanine racemase gene (alr-2), which is involved in the synthesis of d-alanine that forms the backbone of the cell wall. A stable alr-2 knockout mutant of Aeromonas hydrophila HBNUAh01 was constructed. When the mutant was supplemented with d-alanine, growth was unaffected; deprivation of d-alanine caused the growth arrest of the starved mutant cells, but not cell lysis. No alanine racemase activity was detected in the culture of the mutant. Additionally, a membrane permeability assay showed increasing damage to the cell wall during d-alanine starvation. No such damage was observed in the wild type during culture. Scanning and transmission electron microscopy analyses revealed deficiencies of the cell envelope and perforation of the cell wall. Leakage of UV-absorbing substances from the mutants was also observed. Thus, the partial viability of the mutants and their independence of d-alanine for growth indicated that inactivation of alr-2 does not impose an auxotrophic requirement for d-alanine.

  17. Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence

    PubMed Central

    Giffin, Michelle M.; Shi, Lanbo; Gennaro, Maria L.; Sohaskey, Charles D.

    2016-01-01

    Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation. PMID:27203084

  18. Acute pancreatitis and elevated liver transaminases after rapid titration of oral levetiracetam.

    PubMed

    Azar, Nabil J; Aune, Patsy

    2014-06-01

    We report a 25-year-old woman with new onset convulsive episodes. The patient initially failed to respond to phenytoin and was switched to levetiracetam (LEV) which was rapidly titrated to 3000 mg daily over 1 week. At initiation of LEV therapy, she developed mild nausea and decrease in appetite. This was rapidly followed by severe digestive symptoms consistent with acute pancreatitis. She was also found to have elevated liver transaminases. An extensive work-up failed to reveal an organic cause for her symptoms, suggesting a direct relationship to LEV. Clinical symptoms and laboratory abnormalities normalized after LEV discontinuation, along with supportive therapy.

  19. Elevation of Alanine Aminotransferase Activity Occurs after Activation of the Cell-Death Signaling Initiated by Pattern-Recognition Receptors ‎but before Activation of Cytolytic Effectors in NK or CD8+ T Cells in the Liver During Acute HCV Infection

    PubMed Central

    Choi, Youkyung H.; Jin, Nancy; Kelly, Fiona; Sakthivel, SenthilKumar K.; Yu, Tianwei

    2016-01-01

    Pattern-recognition receptors (PRRs) promote host defenses against HCV infection by binding to their corresponding adapter molecules leading to the initiation of innate immune responses including cell death. We investigated the expression of PRR genes, biomarkers of liver cell-death, and T cell and NK cell activation/inhibition-related genes in liver and serum obtained from three experimentally infected chimpanzees with acute HCV infection, and analyzed the correlation between gene expression levels and clinical profiles. Our results showed that expression of hepatic RIG-I, TLR3, TLR7, 2OAS1, and CXCL10 mRNAs was upregulated as early as 7 days post-inoculation and peaked 12 to 83 days post-inoculation. All of the three HCV infected chimpanzees exhibited significant elevations of serum alanine aminotransferase (ALT) activity between 70 and 95 days after inoculation. Elevated levels of serum cytokeratin 18 (CK-18) and caspases 3 and 7 activity coincided closely with the rise of ALT activity, and were preceded by significant increases in levels of caspase 3 and caspase 7 mRNAs in the liver. Particularly we found that significant positive auto-correlations were observed between RIG-I, TLR3, CXCL10, 2OAS1, and PD-L1 mRNA and ALT activity at 3 to 12 days before the peak of ALT activity. However, we observed substantial negative auto-correlations between T cell and NK cell activation/inhibition-related genes and ALT activity at 5 to 32 days after the peak of ALT activity. Our results indicated cell death signaling is preceded by early induction of RIG-I, TLR3, 2OAS1, and CXCL10 mRNAs which leads to elevation of ALT activity and this signaling pathway occurs before the activation of NK and T cells during acute HCV infection. Our study suggests that PRRs and type I IFN response may play a critical role in development of liver cell injury related to viral clearance during acute HCV infection. PMID:27788241

  20. Characterization of the metabolic effect of β-alanine on markers of oxidative metabolism and mitochondrial biogenesis in skeletal muscle

    PubMed Central

    Sunderland, Kyle L.; Kuennen, Matthew R.; Vaughan, Roger A.

    2016-01-01

    [Purpose] β-alanine is a common component of numerous sports supplements purported to improve athletic performance through enhanced carnosine biosynthesis and related intracellular buffering. To date, the effects of β-alanine on oxidative metabolism remain largely unexplored. This work investigated the effects of β-alanine on the expression of proteins which regulate cellular energetics. [Methods] C2C12 myocytes were cultured and differentiated under standard conditions followed by treatment with either β-alanine or isonitrogenous non-metabolizable control D-alanine at 800μM for 24 hours. Metabolic gene and protein expression were quantified by qRT-PCR and immunoblotting, respectively. Glucose uptake and oxygen consumption were measured via fluorescence using commercially available kits. [Results] β-alanine-treated myotubes displayed significantly elevated markers of improved oxidative metabolism including elevated peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and mitochondrial transcription factor a (TFAM) which led to increased mitochondrial content (evidenced by concurrent increases in cytochrome c content). Additionally, β-alanine-treated cells exhibited significantly increased oxygen consumption compared to control in a PPARβ/δ-dependent manner. β-alanine significantly enhanced expression of myocyte enhancer factor 2 (MEF-2) leading to increased glucose transporter 4 (GLUT4) content. [Conclusion] β-alanine appears to increase cellular oxygen consumption as well as the expression of several cellular proteins associated with improved oxidative metabolism, suggesting β-alanine supplementation may provide additional metabolic benefit (although these observations require in vivo experimental verification). PMID:27508152

  1. Antihypernociceptive activity of anethole in experimental inflammatory pain.

    PubMed

    Ritter, Alessandra M V; Domiciano, Talita P; Verri, Waldiceu A; Zarpelon, Ana Carla; da Silva, Lorena G; Barbosa, Carmem P; Natali, Maria Raquel M; Cuman, Roberto K N; Bersani-Amado, Ciomar A

    2013-04-01

    Anethole has been reported to have antioxidant, antibacterial, antifungal, antiinflammatory, and anesthetic properties. In the present study, we evaluated the effects of anethole in two pain models of inflammatory origin: acute inflammation induced by carrageenan and persistent inflammation induced by Complete Freund's adjuvant. We evaluated the effects of anethole (125, 250, and 500 mg/kg) on the development of paw oedema and mechanical hypernociception. The liver was collected for histological analysis. Paw skin was collected to determine the levels of the cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-17 (IL-17), and myeloperoxidase activity. Blood was collected to assess alanine transaminase (ALT) and aspartate transaminase (AST). The chemical composition of star anise oil was determined by gas chromatography/mass spectrometry (GC/MS), showing a presence of anethole of 98.1%. Oral pretreatment with anethole in mice inhibited paw oedema, mechanical pernociception, myelopewroxidase activity, TNF-α, IL-1β and IL-17 levels in acute and persistent inflammation models. Additionally, anethole treatment did not alter prostaglandin E2-induced mechanical hypernociception. Possible side effects were also examined. Seven-day anethole treatment did not alter plasma AST and ALT levels, and the histological profile of liver tissue was normal. The present study provides evidence of the antiinflammatory and analgesic activities of anethole in acute and persistent inflammation models.

  2. Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase.

    PubMed

    Masola, B; Devlin, T M

    1995-12-01

    The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.

  3. Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics.

    PubMed

    Sharma, Vangmayee; Wang, Yane-Shih; Liu, Wenshe R

    2016-12-16

    Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine, can be genetically encoded using engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows a dramatic loss in the enzyme's catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme.

  4. NQR in Alanine and Lysine Iodates

    NASA Astrophysics Data System (ADS)

    Petrosyan, A. M.; Burbelo, V. M.; Tamazyan, R. A.; Karapetyan, H. A.; Sukiasyan, R. P.

    2000-02-01

    The structure o f iodates of α- and β-alanine ( Ala) (2(β-Ala • HIO3) • H2O , β-Ala-2HIO3 , D L-Ala• HIO3 • 2H2O, L-Ala • HIO3) and L-lysine (L-Lys) (L-Lys • HIO3, L-Lys • 2HIO3,L-Lys • 3HIO3, L-Lys • 6HIO3) have been investigated by means of iodine-127 NQR, IR spectroscopy and X-ray diffraction

  5. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    SciTech Connect

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. ); Wada, H.; Horio, Y. )

    1990-05-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP{sub PM} and mGOT are closely related.

  6. Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

    SciTech Connect

    Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen; Tang, De-Wei; Liu, Xiang-Yu; Zhao, Xiaojun; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous protein was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.

  7. Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

    SciTech Connect

    Faraci, W.S.; Walsh, C.T.

    1988-05-03

    Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L ..-->.. D and D..-->.. L directions for all three enzymes to assess the degree to which abstraction of the ..cap alpha..-proton or protonation of substrate PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of ..cap alpha..-/sup 3/H from substrate to product and solvent exchange/substrate conversion experiments in /sup 3/H/sub 2/O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.

  8. Evidence for disorder in L-alanine lattice detected by Pulsed-EPR spectroscopy at cryogenic temperatures.

    PubMed

    Maltar-Strmecki, N; Rakvin, B

    2006-03-13

    The unusual behavior of lattice dynamics of L-alanine has been assigned to intermolecular dynamics and localization of vibrational energy. Recent heat capacity and Pulsed-EPR measurements support presence of thermally activated dynamic orientational disorder in the L-alanine lattice below 20 K. In the present study, the additional evidence for possible thermally activated disordered behavior of L-alanine lattice have been obtained by investigating dependences of longitudinal relaxation time of first stable L-alanine radical, SAR1, on sample cooling rates for the same low temperature interval. The obtained relaxation time by Pulsed-EPR shows clear dependence on cooling rates and this behavior can be explained within two types of suggested spin-lattice relaxation mechanisms for the paramagnetic centers in the hydrogen-bonded organic crystal.

  9. International society of sports nutrition position stand: Beta-Alanine.

    PubMed

    Trexler, Eric T; Smith-Ryan, Abbie E; Stout, Jeffrey R; Hoffman, Jay R; Wilborn, Colin D; Sale, Craig; Kreider, Richard B; Jäger, Ralf; Earnest, Conrad P; Bannock, Laurent; Campbell, Bill; Kalman, Douglas; Ziegenfuss, Tim N; Antonio, Jose

    2015-01-01

    The International Society of Sports Nutrition (ISSN) provides an objective and critical review of the mechanisms and use of beta-alanine supplementation. Based on the current available literature, the conclusions of the ISSN are as follows: 1) Four weeks of beta-alanine supplementation (4-6 g daily) significantly augments muscle carnosine concentrations, thereby acting as an intracellular pH buffer; 2) Beta-alanine supplementation currently appears to be safe in healthy populations at recommended doses; 3) The only reported side effect is paraesthesia (tingling), but studies indicate this can be attenuated by using divided lower doses (1.6 g) or using a sustained-release formula; 4) Daily supplementation with 4 to 6 g of beta-alanine for at least 2 to 4 weeks has been shown to improve exercise performance, with more pronounced effects in open end-point tasks/time trials lasting 1 to 4 min in duration; 5) Beta-alanine attenuates neuromuscular fatigue, particularly in older subjects, and preliminary evidence indicates that beta-alanine may improve tactical performance; 6) Combining beta-alanine with other single or multi-ingredient supplements may be advantageous when supplementation of beta-alanine is high enough (4-6 g daily) and long enough (minimum 4 weeks); 7) More research is needed to determine the effects of beta-alanine on strength, endurance performance beyond 25 min in duration, and other health-related benefits associated with carnosine.

  10. Exchange of aspartate and alanine. Mechanism for development of a proton-motive force in bacteria.

    PubMed

    Abe, K; Hayashi, H; Maloney, P C; Malone, P C

    1996-02-09

    We examined the idea that aspartate metabolism by Lactobacillus subsp. M3 is organized as a proton-motive metabolic cycle by using reconstitution to monitor the activity of the carrier, termed AspT, expected to carry out the electrogenic exchange of precursor (aspartate) and product (alanine). Membranes of Lactobacillus subsp. M3 were extracted with 1.25% octyl glucoside in the presence of 0. 4% Escherichia coli phospholipid and 20% glycerol. The extracts were then used to prepare proteoliposomes loaded with either aspartate or alanine. Aspartate-loaded proteoliposomes accumulated external [3H]aspartate by exchange with internal substrate; this homologous self-exchange (Kt = 0.4 mm) was insensitive to potassium or proton ionophores and was unaffected by the presence or absence of Na+, K+, or Mg2+. Alanine-loaded proteoliposomes also took up [3H]aspartate in a heterologous antiport reaction that was stimulated or inhibited by an inside-positive or inside-negative membrane potential, respectively. Several lines of evidence suggest that these homologous and heterologous exchange reactions were catalyzed by the same functional unit. Thus, [3H]aspartate taken up by AspT during self-exchange was released by a delayed addition of alanine. In addition, the spontaneous loss of AspT activity that occurs when a detergent extract is held at 37 degrees C prior to reconstitution was prevented by the presence of either aspartate (KD(aspartate) = 0.3 mm) or alanine (KD(alanine) > or = 10 mm), indicating that both substrates interact directly with AspT. These findings are consistent with operation of a proton-motive metabolic cycle during aspartate metabolism by Lactobacillus subsp. M3.

  11. The Antitumor Activity of the Novel Compound Jesridonin on Human Esophageal Carcinoma Cells

    PubMed Central

    Wang, Saiqi; Shi, Hongge; Wang, Junwei; Wang, Ran; Li, Yongmei; Dou, Yinhui; Liu, Ying; Hou, Guiqin; Ke, Yu; Liu, Hongmin

    2015-01-01

    Jesridonin, a small molecule obtained through the structural modification of Oridonin, has extensive antitumor activity. In this study, we evaluated both its in vitro activity in the cancer cell line EC109 and its in vivo effect on tumor xenografts in nude mice. Apoptosis induced by Jesridonin was determined using an MTT assay, Annexin-V FITC assay and Hoechest 33258 staining. Apoptosis via mitochondrial and death receptor pathways were confirmed by detecting the regulation of MDM2, p53, and Bcl-2 family members and by activation of caspase-3/-8/-9. In addition, vena caudalis injection of Jesridonin showed significant inhibition of tumor growth in the xenograft model, and Jesridonin-induced cell apoptosis in tumor tissues was determined using TUNEL. Biochemical serum analysis of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT), total protein (TP) and albumin (ALB) indicated no obvious effects on liver function. Histopathological examination of the liver, kidney, lung, heart and spleen revealed no signs of JD-induced toxicity. Taken together, these results demonstrated that Jesridonin exhibits antitumor activity in human esophageal carcinomas EC109 cells both in vitro and in vivo and demonstrated no adverse effects on major organs in nude mice. These studies provide support for new drug development. PMID:26103161

  12. Incidence and risk factors for liver enzyme elevation during highly active antiretroviral therapy in HIV-HCV co-infected patients: results from the Italian EPOKA-MASTER Cohort

    PubMed Central

    Torti, Carlo; Lapadula, Giuseppe; Casari, Salvatore; Puoti, Massimo; Nelson, Mark; Quiros-Roldan, Eugenia; Bella, Daniele; Pastore, Giuseppe; Ladisa, Nicoletta; Minoli, Lorenzo; Sotgiu, Giovanni; Mazzotta, Francesco; Caputo, Sergio Lo; Di Perri, Giovanni; Filice, Gaetano; Tinelli, Carmine; Carosi, Giampiero

    2005-01-01

    Background The risk of hepatotoxicity associated with different highly active antiretroviral therapy (HAART) regimens (containing multiple-protease inhibitors, single-protease inhibitors or non nucleoside reverse transcriptase inhibitors) in HIV-HCV co-infected patients has not been fully assessed. Methods Retrospective analysis of a prospective cohort of 1,038 HIV-HCV co-infected patients who commenced a new HAART in the Italian MASTER database. Patients were stratified into naïve and experienced to antiretroviral therapy before starting the study regimens. Time to grade ≥III hepatotoxicity (as by ACTG classification) was the primary outcome. Secondary outcome was time to grade IV hepatotoxicity. Results Incidence of grade ≥III hepatotoxicity was 17.71 per 100 patient-years (p-yr) of follow up in naïve patient group and 8.22 per 100 p-yrs in experienced group (grade IV: 4.13 per 100 p-yrs and 1.08 per 100 p-yrs, respectively). In the latter group, the only independent factors associated with shorter time to the event at proportional hazards regression model were: previous liver transaminase elevations to grade ≥III, higher baseline alanine amino-transferase values, and use of a non nucleoside reverse transcriptase inhibitor based regimen. In the naive group, baseline aspartate transaminase level was associated with the primary outcome. Conclusion Use of a single or multiple protease inhibitor based regimen was not associated with risk of hepatotoxicity in either naïve or experienced patient groups to a statistically significant extent. A cautious approach with strict monitoring should be applied in HIV-HCV co-infected experienced patients with previous liver transaminase elevations, higher baseline alanine amino-transferase values and who receive regimens containing non nucleoside reverse transcriptase inhibitors. PMID:16018804

  13. Beta-alanine/alpha-ketoglutarate aminotransferase for 3-hydroxypropionic acid production

    DOEpatents

    Jessen, Holly Jean [Chanhassen, MN; Liao, Hans H [Eden Prairie, MN; Gort, Steven John [Apple Valley, MN; Selifonova, Olga V [Plymouth, MN

    2011-10-04

    The present disclosure provides novel beta-alanine/alpha ketoglutarate aminotransferase nucleic acid and protein sequences having increased biological activity. Also provided are cells containing such enzymes, as well as methods of their use, for example to produce malonyl semialdehyde and downstream products thereof, such as 3-hydroxypropionic acid and derivatives thereof.

  14. Beta-alanine/alpha-ketoglutarate aminotransferase for 3-hydroxypropionic acid production

    SciTech Connect

    Jessen, Holly Jean; Liao, Hans H; Gort, Steven John; Selifonova, Olga V

    2014-11-18

    The present disclosure provides novel beta-alanine/alpha ketoglutarate aminotransferase nucleic acid and protein sequences having increased biological activity. Also provided are cells containing such enzymes, as well as methods of their use, for example to produce malonyl semialdehyde and downstream products thereof, such as 3-hydroxypropionic acid and derivatives thereof.

  15. Levels of transaminases, alkaline phosphatase, and protein in tissues of Clarias gariepienus fingerlings exposed to sublethal concentrations of cadmium chloride.

    PubMed

    Velmurugan, Babu; Selvanayagam, Mariadoss; Cengiz, Elif I; Uysal, Ersin

    2008-12-01

    The freshwater fish, Clarias gariepienus fingerlings, were exposed to sublethal concentrations (1.7 and 3.4 mg/L) of cadmium chloride for 12 days. Aspartate aminotransferase (AAT), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total protein levels were assayed in the gill, brain, and muscle of the fish at regular intervals of 6 and 12 days. The activities of AAT, ALT, and ALP of the treated fishes increased significantly in all the tissues compared with the control fish. Protein level in all the tissues showed a significant decrease in comparison to unexposed controls throughout the experimental periods. These results revealed that cadmium chloride effects the intermediary metabolism of C. gariepienus fingerlings and that the assayed enzymes can work as good biomarkers of contamination.

  16. Alanine aminotransferase controls seed dormancy in barley

    PubMed Central

    Sato, Kazuhiro; Yamane, Miki; Yamaji, Nami; Kanamori, Hiroyuki; Tagiri, Akemi; Schwerdt, Julian G.; Fincher, Geoffrey B.; Matsumoto, Takashi; Takeda, Kazuyoshi; Komatsuda, Takao

    2016-01-01

    Dormancy allows wild barley grains to survive dry summers in the Near East. After domestication, barley was selected for shorter dormancy periods. Here we isolate the major seed dormancy gene qsd1 from wild barley, which encodes an alanine aminotransferase (AlaAT). The seed dormancy gene is expressed specifically in the embryo. The AlaAT isoenzymes encoded by the long and short dormancy alleles differ in a single amino acid residue. The reduced dormancy allele Qsd1 evolved from barleys that were first domesticated in the southern Levant and had the long dormancy qsd1 allele that can be traced back to wild barleys. The reduced dormancy mutation likely contributed to the enhanced performance of barley in industrial applications such as beer and whisky production, which involve controlled germination. In contrast, the long dormancy allele might be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley. PMID:27188711

  17. De novo alanine synthesis by bacteroids of Mesorhizobium loti is not required for nitrogen transfer in the determinate nodules of Lotus corniculatus.

    PubMed

    Kumar, Shalini; Bourdès, Alexandre; Poole, Philip

    2005-08-01

    Deletion of both alanine dehydrogenase genes (aldA) in Mesorhizobium loti resulted in the loss of AldA enzyme activity from cultured bacteria and bacteroids but had no effect on the symbiotic performance of Lotus corniculatus plants. Thus, neither indeterminate pea nodules nor determinate L. corniculatus nodules export alanine as the sole nitrogen secretion product.

  18. Crystal structure of the Apo form of D-Alanine:D-Alanine ligase (DDl) from Streptococcus mutans.

    PubMed

    Lu, Yongzhi; Xu, Hongyan; Zhao, Xiaojun

    2010-08-01

    D-Alanine:D-Alanine ligase (DDl) catalyzes the formation of D-Alanine:D-Alanine dipeptide and is an essential enzyme in bacterial cell wall biosynthesis.. This enzyme does not have a human ortholog, making it an attractive target for developing new antibiotic drugs. We determined the crystal structure at 2.23 A resolution of DDl from Streptococcus mutans (SmDDl), the principal aetiological agent of human dental caries. This structure reveals that SmDDl is a dimer and has a disordered omega-loop region.

  19. Radiolysis of alanine adsorbed in a clay mineral

    SciTech Connect

    Aguilar-Ovando, Ellen Y.; Negron-Mendoza, Alicia

    2013-07-03

    Optical activity in molecules is a chemical characteristic of living beings. In this work, we examine the hypothesis of the influence of different mineral surfaces on the development of a specific chirality in organic molecules when subjected to conditions simulating the primitive Earth during the period of chemical evolution. By using X-ray diffraction techniques and HPLC/ELSD to analyze aqueous suspensions of amino acids adsorbed on minerals irradiated in different doses with a cobalt-60 gamma source, the experiments attempt to prove the hypothesis that some solid surfaces (like clays and meteorite rocks) may have a concentration capacity and protective role against external sources of ionizing radiation (specifically {gamma}-ray) for some organic compounds (like some amino acids) adsorbed on them. Preliminary results show a slight difference in the adsorption and radiolysis of the D-and L-alanine.

  20. Human recombinant glutamate oxaloacetate transaminase 1 (GOT1) supplemented with oxaloacetate induces a protective effect after cerebral ischemia

    PubMed Central

    Pérez-Mato, M; Ramos-Cabrer, P; Sobrino, T; Blanco, M; Ruban, A; Mirelman, D; Menendez, P; Castillo, J; Campos, F

    2014-01-01

    Blood glutamate scavenging is a novel and attractive protecting strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. Glutamate oxaloacetate transaminase 1 (GOT1) activation by means of oxaloacetate administration has been used to reduce the glutamate concentration in the blood. However, the protective effect of the administration of the recombinant GOT1 (rGOT1) enzyme has not been yet addressed in cerebral ischemia. The aim of this study was to analyze the protective effect of an effective dose of oxaloacetate and the human rGOT1 alone and in combination with a non-effective dose of oxaloacetate in an animal model of ischemic stroke. Sixty rats were subjected to a transient middle cerebral artery occlusion (MCAO). Infarct volumes were assessed by magnetic resonance imaging (MRI) before treatment administration, and 24 h and 7 days after MCAO. Brain glutamate levels were determined by in vivo MR spectroscopy (MRS) during artery occlusion (80 min) and reperfusion (180 min). GOT activity and serum glutamate concentration were analyzed during the occlusion and reperfusion period. Somatosensory test was performed at baseline and 7 days after MCAO. The three treatments tested induced a reduction in serum and brain glutamate levels, resulting in a reduction in infarct volume and sensorimotor deficit. Protective effect of rGOT1 supplemented with oxaloacetate at 7 days persists even when treatment was delayed until at least 2 h after onset of ischemia. In conclusion, our findings indicate that the combination of human rGOT1 with low doses of oxaloacetate seems to be a successful approach for stroke treatment PMID:24407245

  1. Alanine with the Precipitate of Tomato Juice Administered to Rats Enhances the Reduction in Blood Ethanol Levels.

    PubMed

    Oshima, Shunji; Shiiya, Sachie; Tokumaru, Yoshimi; Kanda, Tomomasa

    2015-01-01

    Delay in gastric emptying (GE) lowers the blood ethanol concentration (BEC) after alcohol administration. We previously demonstrated that water-insoluble fractions, mainly comprising dietary fiber derived from many types of botanical foods, possessed the ability to absorb ethanol-containing aqueous solutions. Furthermore, there was a significant correlation between the absorption of ethanol and lowering of BEC because of delay in GE. Here we identified dietary nutrients that synergize with the water-insoluble fraction of tomatoes to lower BEC in rats. Consequently, unlike tomato juice without alanine, tomato juice with 5.0% alanine decreased BEC depending on the delay in GE and mediated the ethanol-induced decrease in the spontaneous motor activity (an indicator of drunkenness). Our findings indicate that the synergism between tomato juice and alanine to reduce the absorption of ethanol was attributable to the effect of alanine on precipitates such as the water-insoluble fraction of tomatoes.

  2. Alanine with the Precipitate of Tomato Juice Administered to Rats Enhances the Reduction in Blood Ethanol Levels

    PubMed Central

    Oshima, Shunji; Shiiya, Sachie; Tokumaru, Yoshimi; Kanda, Tomomasa

    2015-01-01

    Delay in gastric emptying (GE) lowers the blood ethanol concentration (BEC) after alcohol administration. We previously demonstrated that water-insoluble fractions, mainly comprising dietary fiber derived from many types of botanical foods, possessed the ability to absorb ethanol-containing aqueous solutions. Furthermore, there was a significant correlation between the absorption of ethanol and lowering of BEC because of delay in GE. Here we identified dietary nutrients that synergize with the water-insoluble fraction of tomatoes to lower BEC in rats. Consequently, unlike tomato juice without alanine, tomato juice with 5.0% alanine decreased BEC depending on the delay in GE and mediated the ethanol-induced decrease in the spontaneous motor activity (an indicator of drunkenness). Our findings indicate that the synergism between tomato juice and alanine to reduce the absorption of ethanol was attributable to the effect of alanine on precipitates such as the water-insoluble fraction of tomatoes. PMID:26713162

  3. Hypoglycemic activity and acute oral toxicity of chromium methionine complexes in mice.

    PubMed

    Tang, Hai-yan; Xiao, Qing-gui; Xu, Hong-bin; Zhang, Yi

    2015-01-01

    The hypoglycemic activity of chromium methionine (CrMet) in alloxan-induced diabetic (AID) mice was investigated and compared with those of chromium trichloride hexahydrate (CrCl3·6H2O) and chromium nicotinate (CrNic) through a 15-day feeding experiment. The acute oral toxicity of CrMet was also investigated in ICR (Institute for Cancer Research) mice by a single oral gavage. The anti-diabetic activity of CrMet was explored in detail from the aspects of body weight (BW), blood glucose, triglyceride, total cholesterol, liver glycogen levels, aspartate transaminase (AST) and alanine transaminase (ALT) levels. The obtained results showed that CrMet had beneficial effects on glucose and lipid metabolism, and might possess hepatoprotective efficacy for diabetes. Daily treatment with 500 and 1000μg Cr/kg BW of CrMet in AID mice for 15 days indicated that this low-molecular-weight organic chromium complex had better bioavailability and more beneficial effects on diabetics than CrCl3·6H2O. CrMet also had advantage over CrNic in the control of AST and ALT activities. Acute toxicity studies revealed that CrMet had low toxicity potential and relatively high safety margins in mice with the LD50 value higher than 10.0g/kg BW. These findings suggest that CrMet might be of potential value in the therapy and protection of diabetes.

  4. Production of D-Alanine by Corynebacterium fascians

    PubMed Central

    Yamada, Shigeki; Maeshima, Haruko; Wada, Mitsuru; Chibata, Ichiro

    1973-01-01

    A strain identified as Corynebacterium fascians was found to accumulate extracellular D-alanine from glycerol. Cultural conditions for the accumulation of D-alanine were investigated and, as a result, a yield of 7 g of D-alanine per liter was obtained after a 96-h incubation in a medium containing 5% glycerol, 4% (NH4)2HPO4, and 0.3% corn steep liquor. Optical purity of D-alanine was dependent upon the concentration of corn steep liquor. At the optimal condition, almost optically pure D-alanine was formed and readily isolated (5 g/liter) from the fermentation broth. The product was not contaminated with any detectable amount of other amino acids, except for glycine which was present at a concentration of less than 1 percent. PMID:4699220

  5. Oxygen-inducible glutamate oxaloacetate transaminase as protective switch transforming neurotoxic glutamate to metabolic fuel during acute ischemic stroke.

    PubMed

    Rink, Cameron; Gnyawali, Surya; Peterson, Laura; Khanna, Savita

    2011-05-15

    This work rests on our previous report (J Cereb Blood Flow Metab 30: 1275-1287, 2010) recognizing that glutamate (Glu) oxaloacetate transaminase (GOT) is induced when brain tissue hypoxia is corrected during acute ischemic stroke (AIS). GOT can metabolize Glu into tricarboxylic acid cycle intermediates and may therefore be useful to harness excess neurotoxic extracellular Glu during AIS as a metabolic substrate. We report that in cultured neural cells challenged with hypoglycemia, extracellular Glu can support cell survival as long as there is sufficient oxygenation. This effect is abrogated by GOT knockdown. In a rodent model of AIS, supplemental oxygen (100% O(2) inhaled) during ischemia significantly increased GOT expression and activity in the stroke-affected brain tissue and prevented loss of ATP. Biochemical analyses and in vivo magnetic resonance spectroscopy during stroke demonstrated that such elevated GOT decreased Glu levels at the stroke-affected site. In vivo lentiviral gene delivery of GOT minimized lesion volume, whereas GOT knockdown worsened stroke outcomes. Thus, brain tissue GOT emerges as a novel target in managing stroke outcomes. This work demonstrates that correction of hypoxia during AIS can help clear extracellular neurotoxic Glu by enabling utilization of this amino acid as a metabolic fuel to support survival of the hypoglycemic brain tissue. Strategies to mitigate extracellular Glu-mediated neurodegeneration via blocking receptor-mediated excitotoxicity have failed in clinical trials. We introduce the concept that under hypoglycemic conditions extracellular Glu can be transformed from a neurotoxin to a survival factor by GOT, provided there is sufficient oxygen to sustain cellular respiration.

  6. Comparison of EPR response of alanine and Gd₂O₃-alanine dosimeters exposed to TRIGA Mainz reactor.

    PubMed

    Marrale, M; Schmitz, T; Gallo, S; Hampel, G; Longo, A; Panzeca, S; Tranchina, L

    2015-12-01

    In this work we report some preliminary results regarding the analysis of electron paramagnetic resonance (EPR) response of alanine pellets and alanine pellets added with gadolinium used for dosimetry at the TRIGA research reactor in Mainz, Germany. Two set-ups were evaluated: irradiation inside PMMA phantom and irradiation inside boric acid phantom. We observed that the presence of Gd2O3 inside alanine pellets increases the EPR signal by a factor of 3.45 and 1.24 in case of PMMA and boric acid phantoms, respectively. We can conclude that in the case of neutron beam with a predominant thermal neutron component the addition of gadolinium oxide can significantly improve neutron sensitivity of alanine pellets. Monte Carlo (MC) simulations of both response of alanine and Gd-added alanine pellets with FLUKA code were performed and a good agreement was achieved for pure alanine dosimeters. For Gd2O3-alanine deviations between MC simulations and experimental data were observed and discussed.

  7. Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis

    PubMed Central

    Rodenburg, Anne; Khoury, Hania; de Chiara, Cesira; Howell, Steve; Snijders, Ambrosius P.

    2016-01-01

    The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis, where it has so far been underexploited. d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis. During our studies on alternative inhibitors of the d-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro–d-alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development. PMID:27480853

  8. Enhanced anti-diabetic activity of a combination of chromium(III) malate complex and propolis and its acute oral toxicity evaluation.

    PubMed

    Wu, Xiang-Yang; Li, Fang; Zhao, Ting; Mao, Guang-Hua; Li, Jing; Qu, Hong-Yuan; Ren, Yue-Na; Yang, Liu-Qing

    2012-07-01

    In order to obtain the additional benefit of anti-diabetic activity and protective effects of liver injury for diabetes, the anti-diabetic effect and acute oral toxicity of a combination of chromium(III) malate complex (Cr(2)(LMA)(3)) and propolis were assessed. The anti-diabetic activity of the combination of the Cr(2)LMA(3) and propolis was compared with Cr(2)(LMA)(3) and propolis alone in alloxan-induced diabetic mice by daily oral gavage for a period of 2 weeks. Acute oral toxicity of the combination of the Cr(2)LMA(3) and propolis was tested using ICR mice at the dose of 1.0-5.0 g/kg body mass by a single oral gavage and observed for a period of 2 weeks. The results of the anti-diabetic activity of the combination from the aspects of blood glucose level, liver glycogen level, and the activities of aspartate transaminase, alanine transaminase, and alkaline phosphatase indicated that the increased anti-diabetic activity and the protective efficacy of liver injury for diabetes were observed. In acute toxicity study, LD(50) (median lethal dose) value for the combination was greater than 5.0 g/kg body mass. The combination of Cr(2)LMA(3) and propolis might represent the nutritional supplement with potential therapeutic value to control blood glucose and exhibit protective efficacy of liver injury for diabetes and non-toxicity in acute toxicity.

  9. Characteristics of the transport of alanine, serine and glutamine across the plasma membrane of isolated rat liver cells.

    PubMed Central

    Joseph, S K; Bradford, N M; McGivan, J D

    1978-01-01

    1. Alanine, glutamine and serine were actively accumulated in liver cells isolated from starved rats. 2. This accumulation was inhibited when either Na+ or HCO3- ions were omitted from the incubation medium. In general the degree of dependence on Na+ was quantitatively similar to that on HCO3-. 3. The apparent Km values for the transport of all three amino acids were in the range 3--5mM with Vmax. values in the range 15--25nmol/min per mg of cell protein at 37 degrees C. 4. Alanine and serine transport were mutually competitive; glutamine inhibited the transport of alanine and serine non-competitively. 5. The initial rate of transport of these amino acids was inhibited when the intracellular content of ATP was decreased. 6. Ouabain inhibited the rate of alanine transport without inhibiting the rate of alanine metabolism. 7. It is concluded that a minimum of three transport systems must be postulated to exist in the liver cell plasma membrane to account for the transport of alanine, serine and glutamine. The rate of transport of these amino acids in isolated hepatocytes is unlikely to limit the rate at which they are metabolized. PMID:747655

  10. Transaminase abnormalities and adaptations of the liver lobule manifest at specific cut-offs of steatosis

    PubMed Central

    Hall, Andrew; Covelli, Claudia; Manuguerra, Roberta; Luong, Tu Vinh; Buzzetti, Elena; Tsochatzis, Emmanuel; Pinzani, Massimo; Dhillon, Amar Paul

    2017-01-01

    There is little documented evidence suggesting that liver fat is responsible for liver injury in the absence of other disease processes. We investigated the relationships between liver fat, aminotransferases and hepatic architecture in liver biopsies with simple steatosis. We identified 136 biopsies with simple steatosis from the Royal Free Hospital Archives with both clinical data and sufficient material. Digital image analysis was employed to measure fat proportionate area (mFPA). Hepatocyte area (HA) and lobule radius (LR) were also measured. There were significant increases in ALT (p < 0.001) and AST (p = 0.013) with increased fat content and evidence to suggest both 5% and 20% mFPA as a cut-off for raised ALT. In liver with increased fat content there were significant increases in HA (p < 0.001). LR also increased as mFPA increased to 10% (p < 0.001), at which point the lobule ceased to expand further and was counterbalanced with a decrease in the number of hepatocytes per lobule (p = 0.029). Consequently there are mechanisms of adaption in the liver architecture to accommodate the accumulation of fat and these are accompanied by significant increases in transaminases. These results support the generally accepted cut-off of 5% fat for steatosis and indicate 20% as a threshold of more severe liver injury. PMID:28106158

  11. A neuron-glia interaction involving GABA Transaminase contributes to sleep loss in sleepless mutants

    PubMed Central

    Chen, Wen-Feng; Maguire, Sarah; Sowcik, Mallory; Luo, Wenyu; Koh, Kyunghee; Sehgal, Amita

    2014-01-01

    Sleep is an essential process and yet mechanisms underlying it are not well understood. Loss of the Drosophila quiver/sleepless (qvr/sss) gene increases neuronal excitability and diminishes daily sleep, providing an excellent model for exploring the underpinnings of sleep regulation. Here, we used a proteomic approach to identify proteins altered in sss brains. We report that loss of sleepless post-transcriptionally elevates the CG7433 protein, a mitochondrial γ-aminobutyric acid transaminase (GABAT), and reduces GABA in fly brains. Loss of GABAT increases daily sleep and improves sleep consolidation, indicating that GABAT promotes wakefulness. Importantly, disruption of the GABAT gene completely suppresses the sleep phenotype of sss mutants, demonstrating that GABAT is required for loss of sleep in sss mutants. While SSS acts in distinct populations of neurons, GABAT acts in glia to reduce sleep in sss flies. Our results identify a novel mechanism of interaction between neurons and glia that is important for the regulation of sleep. PMID:24637426

  12. The crystal structure of the D-alanine-D-alanine ligase from Acinetobacter baumannii suggests a flexible conformational change in the central domain before nucleotide binding.

    PubMed

    Huynh, Kim-Hung; Hong, Myoung-ki; Lee, Clarice; Tran, Huyen-Thi; Lee, Sang Hee; Ahn, Yeh-Jin; Cha, Sun-Shin; Kang, Lin-Woo

    2015-11-01

    Acinetobacter baumannii, which is emerging as a multidrug-resistant nosocomial pathogen, causes a number of diseases, including pneumonia, bacteremia, meningitis, and skin infections. With ATP hydrolysis, the D-alanine-D-alanine ligase (DDL) catalyzes the synthesis of D-alanyl-D-alanine, which is an essential component of bacterial peptidoglycan. In this study, we determined the crystal structure of DDL from A. baumannii (AbDDL) at a resolution of 2.2 Å. The asymmetric unit contained six protomers of AbDDL. Five protomers had a closed conformation in the central domain, while one protomer had an open conformation in the central domain. The central domain with an open conformation did not interact with crystallographic symmetry-related protomers and the conformational change of the central domain was not due to crystal packing. The central domain of AbDDL can have an ensemble of the open and closed conformations before the binding of substrate ATP. The conformational change of the central domain is important for the catalytic activity and the detail information will be useful for the development of inhibitors against AbDDL and putative antibacterial agents against A. baumannii. The AbDDL structure was compared with that of other DDLs that were in complex with potent inhibitors and the catalytic activity of AbDDL was confirmed using enzyme kinetics assays.

  13. High-velocity intermittent running: effects of beta-alanine supplementation.

    PubMed

    Smith-Ryan, Abbie E; Fukuda, David H; Stout, Jeffrey R; Kendall, Kristina L

    2012-10-01

    The use of β-alanine in sport is widespread. However, the effects across all sport activities are inconclusive. The purpose of this study was to evaluate the effects of β-alanine supplementation on high-intensity running performance and critical velocity (CV) and anaerobic running capacity (ARC). Fifty recreationally trained men were randomly assigned, in a double-blind fashion, to a β-alanine group (BA, 2 × 800 mg tablets, 3 times daily; CarnoSyn; n = 26) or placebo group (PL, 2 × 800 mg maltodextrin tablets, 3 times daily; n = 24). A graded exercise test (GXT) was performed to establish peak velocity (PV). Three high-speed runs to exhaustion were performed at 110, 100, and 90% of PV, with 15 minutes of rest between bouts. The distances achieved were plotted over the time to exhaustion (TTE). Linear regression was used to determine the slope (CV) and y-intercept (ARC) of these relationships to assess aerobic and anaerobic performances, respectively. There were no significant treatment effects (p > 0.05) on CV or ARC for either men or women. Additionally, no TTE effects were evident for bouts at 90-110%PV lasting 1.95-5.06 minutes. There seems to be no ergogenic effect of β-alanine supplementation on CV, ARC, or high-intensity running lasting approximately 2-5 minutes in either men or women in the current study.

  14. Growth and characterization of pure and semiorganic nonlinear optical Lithium Sulphate admixtured l-alanine crystal

    NASA Astrophysics Data System (ADS)

    Vela, T.; Selvarajan, P.; Freeda, T. H.; Balasubramanian, K.

    2013-04-01

    Lithium sulphate admixtured l-alanine (LSLA) salt was synthesized and the solubility of the commercially available l-alanine and the synthesized LSLA sample was determined in de-ionized water at various temperatures. In accordance with the solubility data, the saturated aqueous solutions of l-alanine and lithium admixtured l-alanine were prepared separately and the single crystals of the samples were grown by the solution method with a slow evaporation technique. Studying single x-ray diffraction shows that pure and LSLA crystal belong to the orthorhombic system with a non-centrosymmetric space group P212121. Using the powder x-ray diffraction study, the crystallinity of the grown crystals is confirmed and the diffraction peaks are indexed. The various functional groups present in the pure and LSLA crystal are elucidated from Fourier transform infrared spectroscopy study. UV-visible transmittance is recorded to study the optical transmittance range for the grown crystals. The powder second harmonic generation test confirms the nonlinear optical property of the grown crystals. From the microhardness test, the hardness of the grown crystals is estimated. The dielectric behaviour, such as the dielectric constant and the loss of the sample, are measured as a function of temperature and frequency. The ac conductivity of the grown crystals is also studied and the activation energy is calculated.

  15. Structural and functional characterization of the alanine racemase from Streptomyces coelicolor A3(2).

    PubMed

    Tassoni, Raffaella; van der Aart, Lizah T; Ubbink, Marcellus; van Wezel, Gilles P; Pannu, Navraj S

    2017-01-29

    The conversion of l-alanine (L-Ala) into d-alanine (D-Ala) in bacteria is performed by pyridoxal phosphate-dependent enzymes called alanine racemases. D-Ala is an essential component of the bacterial peptidoglycan and hence required for survival. The Gram-positive bacterium Streptomyces coelicolor has at least one alanine racemase encoded by alr. Here, we describe an alr deletion mutant of S. coelicolor which depends on D-Ala for growth and shows increased sensitivity to the antibiotic d-cycloserine (DCS). The crystal structure of the alanine racemase (Alr) was solved with and without the inhibitors DCS or propionate, at 1.64 Å and 1.51 Å resolution, respectively. The crystal structures revealed that Alr is a homodimer with residues from both monomers contributing to the active site. The dimeric state of the enzyme in solution was confirmed by gel filtration chromatography, with and without L-Ala or d-cycloserine. The activity of the enzyme was 66 ± 3 U mg(-1) for the racemization of L- to D-Ala, and 104 ± 7 U mg(-1) for the opposite direction. Comparison of Alr from S. coelicolor with orthologous enzymes from other bacteria, including the closely related d-cycloserine-resistant Alr from S. lavendulae, strongly suggests that structural features such as the hinge angle or the surface area between the monomers do not contribute to d-cycloserine resistance, and the molecular basis for resistance therefore remains elusive.

  16. Structural features and kinetic characterization of alanine racemase from Staphylococcus aureus (Mu50).

    PubMed

    Scaletti, Emma R; Luckner, Sylvia R; Krause, Kurt L

    2012-01-01

    Staphylococcus aureus is an opportunistic Gram-positive bacterium which causes a wide variety of diseases ranging from minor skin infections to potentially fatal conditions such as pneumonia, meningitis and septicaemia. The pathogen is a leading cause of nosocomial acquired infections, a problem that is exacerbated by the existence of methicillin- and glycopeptide antibiotic-resistant strains which can be challenging to treat. Alanine racemase (Alr) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes reversible racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell-wall peptidoglycan, inhibition of Alr is lethal to prokaryotes. Additionally, while ubiquitous amongst bacteria, this enzyme is absent in humans and most eukaryotes, making it an excellent antibiotic drug target. The crystal structure of S. aureus alanine racemase (Alr(Sas)), the sequence of which corresponds to that from the highly antibiotic-resistant Mu50 strain, has been solved to 2.15 Å resolution. Comparison of the Alr(Sas) structure with those of various alanine racemases demonstrates a conserved overall fold, with the enzyme sharing most similarity to those from other Gram-positive bacteria. Structural examination indicates that the active-site binding pocket, dimer interface and active-site entryway of the enzyme are potential targets for structure-aided inhibitor design. Kinetic constants were calculated in this study and are reported here. The potential for a disulfide bond in this structure is noted. This structural and biochemical information provides a template for future structure-based drug-development efforts targeting Alr(Sas).

  17. Effect of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of certain plasma enzymes in CCl4-induced liver injury in low-protein fed rats.

    PubMed

    Nkosi, C Z; Opoku, A R; Terblanche, S E

    2005-04-01

    The effects of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of lactate dehydrogenase (LD), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) against carbon tetrachloride (CCl4)-induced acute liver injury in low-protein fed rats were investigated. A group of male Sprague-Dawley rats maintained on a low-protein diet for 5 days were divided into three subgroups. Two subgroups were injected with carbon tetrachloride and the other group with an equivalent amount of olive oil. Two hours after CCl4 intoxication one of the two subgroups was administered with pumpkin seed protein isolate. All three subgroups of rats were maintained on the low-protein diet for the duration of the investigation. Groups of rats from the different subgroups were killed at 24, 48 and 72 h after their respective treatments. After 5 days on the low-protein diet the activity levels of all four enzymes were significantly higher than their counterparts on a normal balanced diet. CCl4 intoxication resulted in significant increases in the activity levels of all four enzymes investigated. The administration of pumpkin seed protein isolate after CCl4 intoxication resulted in significantly reduced activity levels of all four enzymes. It is concluded that pumpkin seed protein isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition.

  18. Crystallization and preliminary X-ray diffraction analysis of ω-amino acid:pyruvate transaminase from Chromobacterium violaceum

    SciTech Connect

    Sayer, Christopher; Isupov, Michail N.; Littlechild, Jennifer A.

    2007-02-01

    An ω-amino acid:pyruvate transaminase from C. violaceum has been purified and crystallized in two crystal forms. The structure has been solved using molecular replacement. The enzyme ω-transaminase catalyses the conversion of chiral ω-amines to ketones. The recombinant enzyme from Chromobacterium violaceum has been purified to homogeneity. The enzyme was crystallized from PEG 4000 using the microbatch method. Data were collected to 1.7 Å resolution from a crystal belonging to the triclinic space group P1, with unit-cell parameters a = 58.9, b = 61.9, c = 63.9 Å, α = 71.9, β = 87.0, γ = 74.6°. Data were also collected to 1.95 Å from a second triclinic crystal form. The structure has been solved using the molecular-replacement method.

  19. Evaluation of hepatoprotective activity of Colocasia esculenta (L. Schott) leaves on thioacetamide-induced hepatotoxicity in rats.

    PubMed

    Chinonyelum, Azubike Nkiruka; Uwadiegwu, Achukwu Peter; Nwachukwu, Okwuosa Chukwugozie; Emmanuel, Oduah

    2015-11-01

    The hepatoprotective effect of orally administered leaf aqueous extract of Colocasia esculenta (CCLE) in thioacetamide-induced liver toxicity in rats was investigated in this study. Adult male Wistar rats (weight range: 120-150g) were divided into 5 groups (n=5) and received no treatment (normal control), distilled water (negative control), 50mg/kg silymarin (positive control) and CCLE (250 and 500mg/kg) respectively once daily for 3 consecutive days. Thioacetamide (TAA) (150mg/kg b.w.) was administered intraperitoneally on the 4th day to rats in all groups except the normal control. Evaluations were made for serum levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphate (ALP) and serum albumin. Histopathological examination was performed on the excised liver tissues. TAA-induced hepatotoxicity increased ALT, AST, ALP and decreased serum albumin. Histopathological results revealed extensive disruption of the liver histoarchitecture when compared to the normal control liver sections. Pre-treatment with CCLE showed protective effects by normalizing the liver enzymes markers. These results were supported by the histopathological observations. The activity of the CCLE was comparable to that of the standard hepatoprotective drug, silymarin (50mg/kg). Overall findings suggest that CCLE possesses in vivo hepatoprotective activity against thioacetamide in rats.

  20. Crystallization and preliminary X-ray diffraction studies of the (R)-selective amine transaminase from Aspergillus fumigatus.

    PubMed

    Thomsen, Maren; Skalden, Lilly; Palm, Gottfried J; Höhne, Matthias; Bornscheuer, Uwe T; Hinrichs, Winfried

    2013-12-01

    The (R)-selective amine transaminase from Aspergillus fumigatus was expressed in Escherichia coli and purified to homogeneity. Bright yellow crystals appeared while storing the concentrated solution in the refrigerator and belonged to space group C222(1). X-ray diffraction data were collected to 1.27 Å resolution, as well as an anomalous data set to 1.84 Å resolution that was suitable for S-SAD phasing.

  1. BarR, an Lrp-type transcription factor in Sulfolobus acidocaldarius, regulates an aminotransferase gene in a β-alanine responsive manner.

    PubMed

    Liu, Han; Orell, Alvaro; Maes, Dominique; van Wolferen, Marleen; Lindås, Ann-Christin; Bernander, Rolf; Albers, Sonja-Verena; Charlier, Daniel; Peeters, Eveline

    2014-05-01

    In archaea, nothing is known about the β-alanine degradation pathway or its regulation. In this work, we identify and characterize BarR, a novel Lrp-like transcription factor and the first one that has a non-proteinogenic amino acid ligand. BarR is conserved in Sulfolobus acidocaldarius and Sulfolobus tokodaii and is located in a divergent operon with a gene predicted to encode β-alanine aminotransferase. Deletion of barR resulted in a reduced exponential growth rate in the presence of β-alanine. Furthermore, qRT-PCR and promoter activity assays demonstrated that BarR activates the expression of the adjacent aminotransferase gene, but only upon β-alanine supplementation. In contrast, auto-activation proved to be β-alanine independent. Heterologously produced BarR is an octamer in solution and forms a single complex by interacting with multiple sites in the 170 bp long intergenic region separating the divergently transcribed genes. In vitro, DNA binding is specifically responsive to β-alanine and site-mutant analyses indicated that β-alanine directly interacts with the ligand-binding pocket. Altogether, this work contributes to the growing body of evidence that in archaea, Lrp-like transcription factors have physiological roles that go beyond the regulation of α-amino acid metabolism.

  2. Hepatoprotective, antinociceptive and antioxidant activities of cimetidine, ranitidine and famotidine as histamine H2 receptor antagonists.

    PubMed

    Ahmadi, Amirhossein; Ebrahimzadeh, Mohammad Ali; Ahmad-Ashrafi, Saeb; Karami, Mohammad; Mahdavi, Mohammad Reza; Saravi, Seyed Soheil Saeedi

    2011-02-01

    The antioxidant, antinociceptive and hepatoprotective effects of H(2) receptor blockers were examined with different experimental models. Antioxidant activities were determined by employing various in vitro assay systems such as 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical-scavenging activity assays, reducing power determination assays, nitric oxide-scavenging activity assays and hydrogen peroxide-scavenging activity assays. Antinociceptive effects were determined using the hot plate test in mice. The hepatoprotective effects of cimetidine, ranitidine and famotidine against hepatotoxicity induced by carbon tetrachloride (CCl(4) ) were determined by measuring the levels of serum enzymes alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities in mice. We found that the IC(50) values of cimetidine, ranitidine and famotidine on DPPH radical-scavenging activity were 671±28, 538±21 and 955±43 μg/mL, respectively. Famotidine showed very strong nitric oxide-scavenging activity. All three compounds showed very weak hydrogen peroxide-scavenging activity. Moreover, the compounds did not exhibit any reducing power activity until concentrations of 1.6 mg/mL. All compounds also showed a dose-dependent and marked analgesic activity in mice relative to controls. Pretreatment of mice with cimetidine, ranitidine or famotidine for three consecutive days reduced CCl(4)-induced hepatotoxicity in mice. Treatment with 200 mg/kg ranitidine reduced AST, AST and ALP serum levels, while 200 and 40 mg/kg of cimetidine and famotidine, respectively, reduced AST and ALP serum levels. H(2) blockers exhibited varying levels of antioxidant activities in various assays. Our results indicate that the antioxidant activities of H(2) blockers have an analgesic activity and protective effect on CCl(4)-induced hepatotoxicity in mice. These effects were greater with ranitidine than with the other compounds.

  3. D-Amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity.

    PubMed

    Sato, Masaru; Kirimura, Kohtaro; Kino, Kuniki

    2005-06-01

    D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.

  4. Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori.

    PubMed

    Wu, Dalei; Zhang, Liang; Kong, Yunhua; Du, Jiamu; Chen, Shuai; Chen, Jing; Ding, Jianping; Jiang, Hualiang; Shen, Xu

    2008-09-01

    D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SS1 (HpDdl) was kinetically and structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of the enzyme. Our present work hopefully provides useful information for understanding the D-Ala-D-Ala ligase of Helicobacter pylori.

  5. Noncovalent and covalent functionalization of a (5, 0) single-walled carbon nanotube with alanine and alanine radicals.

    PubMed

    Rajarajeswari, Muthusivarajan; Iyakutti, Kombiah; Kawazoe, Yoshiyuki

    2012-02-01

    We have systematically investigated the noncovalent and covalent adsorption of alanine and alanine radicals, respectively, onto a (5, 0) single-walled carbon nanotube using first-principles calculation. It was found that XH···π (X = N, O, C) interactions play a crucial role in the non-ovalent adsorption and that the functional group close to the carbon nanotube exhibits a significant influence on the binding strength. Noncovalent functionalization of the carbon nanotube with alanine enhances the conductivity of the metallic (5, 0) nanotube. In the covalent adsorption of each alanine radical onto a carbon nanotube, the binding energy depends on the adsorption site on CNT and the electronegative atom that binds with the CNT. The strongest complex is formed when the alanine radical interacts with a (5, 0) carbon nanotube through the amine group. In some cases, the covalent interaction of the alanine radical introduces a half-filled band at the Fermi level due to the local sp (3) hybridization, which modifies the conductivity of the tube.

  6. Structure of D-alanine-D-alanine ligase from Yersinia pestis: nucleotide phosphate recognition by the serine loop.

    PubMed

    Tran, Huyen Thi; Hong, Myoung Ki; Ngo, Ho Phuong Thuy; Huynh, Kim Hung; Ahn, Yeh Jin; Wang, Zhong; Kang, Lin Woo

    2016-01-01

    D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.5 Å resolution: apo, AMP-bound, ADP-bound, adenosine 5'-(β,γ-imido)triphosphate-bound, and D-alanyl-D-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the ω-loop) and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the ω-loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both the D-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops.

  7. Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis γ-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate

    PubMed Central

    Clark, Shawn M.; Di Leo, Rosa; Dhanoa, Preetinder K.; Van Cauwenberghe, Owen R.; Mullen, Robert T.; Shelp, Barry J.

    2009-01-01

    γ-Aminobutyrate transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, the previously identified Arabidopsis thaliana (L.) Heyhn GABA-T (AtGABA-T) was characterized in more detail. Full-length AtGABA-T contains an N-terminal 36 amino acid long targeting pre-sequence (36 amino acids) that is both sufficient and necessary for targeting the enzyme to mitochondria. Removal of the pre-sequence encoding this N-terminal targeting domain and co-expression of the resulting truncated AtGABA-T cDNA with the GroES/EL molecular chaperone complex in Escherichia coli yielded good recovery of the soluble recombinant proteins. Activity assays indicated that purified recombinant GABA-T has both pyruvate- and glyoxylate-dependent activities, but cannot utilize 2-oxoglutarate as amino acceptor. Kinetic parameters for glyoxylate- and pyruvate-dependent GABA-T activities were similar, with physiologically relevant affinities. Assays of GABA-T activity in cell-free leaf extracts from wild-type Arabidopsis and two knockout mutants in different genetic backgrounds confirmed that the native enzyme possesses both pyruvate- and glyoxylate-dependent activities. The GABA-T transcript was present throughout the plant, but its expression was highest in roots and increased as a function of leaf development. A GABA-T with dual functions suggests the potential for interaction between GABA metabolism and photorespiratory glyoxylate production. PMID:19264755

  8. Beta-alanine and taurine as endogenous agonists at glycine receptors in rat hippocampus in vitro.

    PubMed

    Mori, Masahiro; Gähwiler, Beat H; Gerber, Urs

    2002-02-15

    Electrophysiological and pharmacological properties of glycine receptors were characterized in hippocampal organotypic slice cultures. In the presence of ionotropic glutamate and GABA(B) receptor antagonists, pressure-application of glycine onto CA3 pyramidal cells induced a current associated with increased chloride conductance, which was inhibited by strychnine. Similar chloride currents could also be induced with beta-alanine or taurine. Whole-cell glycine responses were significantly greater in CA3 pyramidal cells than in CA1 pyramidal cells and dentate granule cells, while responses to GABA were similar among these three cell types. Although these results demonstrate the presence of functional glycine receptors in the hippocampus, no evidence for their activation during synaptic stimulation was found. Gabazine, a selective GABA(A) receptor antagonist, totally blocked evoked IPSCs in CA3 pyramidal cells. Glycine receptor activation is not dependent on transporter-controlled levels of extracellular glycine, as no chloride current was observed in response to sarcosine, an inhibitor of glycine transporters. In contrast, application of guanidinoethanesulfonic acid, an uptake inhibitor of beta-alanine and taurine, induced strychnine-sensitive chloride current in the presence of gabazine. These data indicate that modulation of transporters for the endogenous amino acids, beta-alanine and taurine, can regulate tonic activation of glycine receptors, which may function in maintenance of inhibitory tone in the hippocampus.

  9. The polyproline II conformation in short alanine peptides is noncooperative.

    PubMed

    Chen, Kang; Liu, Zhigang; Kallenbach, Neville R

    2004-10-26

    The finding that short alanine peptides possess a high fraction of polyproline II (PII) structure (Phi=-75 degrees, Psi=+145 degrees ) at low temperature has broad implications for unfolded states of proteins. An important question concerns whether or not this structure is locally determined or cooperative. We have monitored the conformation of alanine in a series of model peptides AcGGAnGGNH2 (n=1-3) over a temperature range from -10 degrees C to +80 degrees C. Use of 15N-labeled alanine substitutions makes it possible to measure 3JalphaN coupling constants accurately over the full temperature range. Based on a 1D next-neighbor model, the cooperative parameter sigma of PII nucleation is evaluated from the coupling constant data. The finding that sigma is close to unity (1 +/- 0.2) indicates a noncooperative role for alanine in PII structure formation, consistent with statistical surveys of the Protein Data Bank that suggest that most PII structure occurs in isolated residues. Lack of cooperativity in these models implies that hydration effects that influence PII conformation in water are highly localized. Using a nuclear Overhauser effect ratio strategy to define the alanine Psi angle, we estimate that, at 40 degrees C, the time-averaged alanine conformation (Phi=-80 degrees, Psi=+170 degrees ) deviates from canonical PII structure, indicating that PII melts at high temperature. Thus, the high-temperature state of short alanine peptides seems to be an unfolded ensemble with higher distribution in the extended beta structure basin, but not a coil.

  10. Does acute alcohol intoxication cause transaminase elevations in children and adolescents?

    PubMed

    Binder, Christoph; Knibbe, Karoline; Kreissl, Alexandra; Repa, Andreas; Thanhaeuser, Margarita; Greber-Platzer, Susanne; Berger, Angelika; Jilma, Bernd; Haiden, Nadja

    2016-03-01

    Several long-term effects of alcohol abuse in children and adolescents are well described. Alcohol abuse has severe effects on neurodevelopmental outcome, such as learning disabilities, memory deficits, and decreased cognitive performance. Additionally, chronic alcohol intake is associated with chronic liver disease. However, the effects of acute alcohol intoxication on liver function in children and adolescents are not well characterized. The aim of this study was to determine if a single event of acute alcohol intoxication has short-term effects on liver function and metabolism. All children and adolescents admitted to the Department of Pediatrics and Adolescent Medicine between 2004 and 2011 with the diagnosis "acute alcohol intoxication" were included in this retrospective analysis. Clinical records were evaluated for age, gender, alcohol consumption, blood alcohol concentration, symptoms, and therapy. Blood values of the liver parameters, CK, creatinine, LDH, AP, and the values of the blood gas analysis were analyzed. During the 8-year study period, 249 children and adolescents with the diagnosis "acute alcohol intoxication" were admitted, 132 (53%) girls and 117 (47%) boys. The mean age was 15.3 ± 1.2 years and the mean blood alcohol concentration was 0.201 ± 0.049%. Girls consumed significantly less alcohol than boys (64 g vs. 90 g), but reached the same blood alcohol concentration (girls: 0.199 ± 0.049%; boys: 0.204 ± 0.049%). The mean values of liver parameters were in normal ranges, but AST was increased in 9.1%, ALT in 3.9%, and γGT in 1.4%. In contrast, the mean value of AST/ALT ratio was increased and the ratio was elevated in 92.6% of all patients. Data of the present study showed significant differences in the AST/ALT ratio (p < 0.01) in comparison to a control group. Data of the present study indicate that there might be an effect of acute alcohol intoxication on transaminase levels. The AST/ALT ratio seems to reflect the damage in hepatocytes

  11. The Safety of Tenofovir–Emtricitabine for HIV Pre-Exposure Prophylaxis (PrEP) in Individuals With Active Hepatitis B

    PubMed Central

    Schechter, Mauro; Liu, Albert Y.; McManhan, Vanessa M.; Guanira, Juan V.; Hance, Robert J.; Chariyalertsak, Suwat; Mayer, Kenneth H.; Grant, Robert M.

    2016-01-01

    Background: Pre-exposure prophylaxis (PrEP) with daily oral emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) prevents HIV infection. The safety and feasibility of HIV PrEP in the setting of hepatitis B virus (HBV) infection were evaluated. Methods: The Iniciativa Profilaxis Pre-Exposición study randomized 2499 HIV-negative men and transgender women who have sex with men to once-daily oral FTC/TDF versus placebo. Hepatitis serologies and transaminases were obtained at screening and at the time PrEP was discontinued. HBV DNA was assessed by polymerase chain reaction, and drug resistance was assessed by population sequencing. Vaccination was offered to individuals susceptible to HBV infection. Results: Of the 2499 participants, 12 (0.5%; including 6 randomized to FTC/TDF) had chronic HBV infection. After stopping FTC/TDF, 5 of the 6 participants in the active arm had liver function tests performed at follow-up. Liver function tests remained within normal limits at post-stop visits except for a grade 1 elevation in 1 participant at post-stop week 12 (alanine aminotransferase = 90, aspartate aminotransferase = 61). There was no evidence of hepatic flares. Polymerase chain reaction of stored samples showed that 2 participants in the active arm had evidence of acute HBV infection at enrollment. Both had evidence of grade 4 transaminase elevations with subsequent resolution. Overall, there was no evidence of TDF or FTC resistance among tested genotypes. Of 1633 eligible for vaccination, 1587 (97.2%) received at least 1 vaccine; 1383 (84.7%) completed the series. Conclusions: PrEP can be safely provided to individuals with HBV infection if there is no evidence of cirrhosis or substantial transaminase elevation. HBV vaccination rates at screening were low globally, despite recommendations for its use, yet uptake and efficacy were high when offered. PMID:26413853

  12. Mutation of glycine receptor subunit creates beta-alanine receptor responsive to GABA.

    PubMed

    Schmieden, V; Kuhse, J; Betz, H

    1993-10-08

    The amino acid at position 160 of the ligand-binding subunit, alpha 1, is an important determinant of agonist and antagonist binding to the glycine receptor. Exchange of the neighboring residues, phenylalanine at position 159 and tyrosine at position 161, increased the efficacy of amino acid agonists. Whereas wild-type alpha 1 channels expressed in Xenopus oocytes required 0.7 millimolar beta-alanine for a half-maximal response, the doubly mutated (F159Y,Y161F) alpha 1 subunit had an affinity for beta-alanine (which was more potent than glycine) that was 110-fold that of the wild type. Also, gamma-aminobutyric acid and D-serine, amino acids that do not activate wild-type alpha 1 receptors, efficiently gated the mutant channel. Thus, aromatic hydroxyl groups are crucial for ligand discrimination at inhibitory amino acid receptors.

  13. Metabolic Engineering of Escherichia coli for the Production of 3-Hydroxypropionic Acid and Malonic Acid through β-Alanine Route.

    PubMed

    Song, Chan Woo; Kim, Je Woong; Cho, In Jin; Lee, Sang Yup

    2016-11-18

    Escherichia coli was metabolically engineered to produce industrially important platform chemicals, 3-hydroxypropionic acid (3-HP) and malonic acid (MA), through the β-alanine (BA) route. First, various combinations of downstream enzymes were screened and BA pyruvate transaminase (encoded by pa0132) from P. aeruginosa was selected to generate malonic semialdehyde (MSA) from BA. This platform strain was engineered by introducing E. coli MSA reductase (encoded by ydfG) to reduce MSA to 3-HP. Replacement of native promoter of the sdhC gene with the strong trc promoter in the genome increased 3-HP production to 3.69 g/L in flask culture. Introduction of E. coli semialdehyde dehydrogenase (encoded by yneI) into the platform strain resulted in the production of MA, and additional deletion of the ydfG gene increased MA production to 0.450 g/L in flask culture. Fed-batch cultures of final engineered strains resulted in the production of 31.1 g/L 3-HP or 3.60 g/L MA from glucose.

  14. Effects of α-methyl-p-tyrosine, p-chlorophenylalanine, L-β-(3,4-dihydroxyphenyl)alanine, 5-hydroxytryptophan and diethyldithiocarbamate on the analgesic activity of morphine and methylamphetamine in the mouse

    PubMed Central

    Major, C. T.; Pleuvry, Barbara J.

    1971-01-01

    1. The analgesic activity of sympathomimetic drugs does not appear to involve a peripheral component. 2. Drugs causing changes in morphine analgesia have similar effects on the analgesia produced by methylamphetamine. 3. The analgesia produced by morphine and methylamphetamine is increased by drugs which increase the ratio of brain 5-hydroxytryptamine (5-HT) to dopamine. 4. The analgesia is decreased by drugs causing a fall in brain 5-HT or a rise in dopamine relative to 5-HT. PMID:4256024

  15. On the existence of ``l-threonine formate'', ``l-alanine lithium chloride'' and ``bis l-alanine lithium chloride'' crystals

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

    We argue that the recently reported crystals "L-threonine formate" as well as "L-alanine lithium chloride" and "bis L-alanine lithium chloride" actually are the well-known crystals L-threonine and L-alanine, respectively.

  16. [Effects of ß-alanine supplementation on athletic performance].

    PubMed

    Domínguez, Raúl; Hernández Lougedo, Juan; Maté-Muñoz, José Luis; Garnacho-Castaño, Manuel Vicente

    2014-10-06

    Carnosine, dipeptide formed by amino acids ß-alanine and L-histidine, has important physiological functions among which its antioxidant and related memory and learning. However, in connection with the exercise, the most important functions would be associated with muscle contractility, improving calcium sensitivity in muscle fibers, and the regulatory function of pH. Thus, it is proposed that carnosine is the major intracellular buffer, but could contribute to 7-10% in buffer or buffer capacity. Since carnosine synthesis seems to be limited by the availability of ß-alanine supplementation with this compound has been gaining increasing popularity among the athlete population. Therefore, the objective of this study literature review was to examine all those research works have shown the effect of ß-alanine supplementation on athletic performance. Moreover, it also has attempted to establish a specific dosage that maximizing the potential benefits, minimize paresthesia, the main side effect presented in response to supplementation.

  17. First-principles studies of pure and fluorine substituted alanines

    NASA Astrophysics Data System (ADS)

    Ahmad, Sardar; Vaizie, Hamide; Rahnamaye Aliabad, H. A.; Ahmad, Rashid; Khan, Imad; Ali, Zahid; Jalali-Asadabadi, S.; Ahmad, Iftikhar; Khan, Amir Abdullah

    2016-05-01

    This paper communicates the structural, electronic and optical properties of L-alanine, monofluoro and difluoro substituted alanines using density functional calculations. These compounds exist in orthorhombic crystal structure and the calculated structural parameters such as lattice constants, bond angles and bond lengths are in agreement with the experimental results. L-alanine is an indirect band gap insulator, while its fluorine substituted compounds (monofluoroalanine and difluoroalanine) are direct band gap insulators. The substitution causes reduction in the band gap and hence these optically tailored direct wide band gap materials have enhanced optical properties in the ultraviolet (UV) region of electromagnetic spectrum. Therefore, optical properties like dielectric function, refractive index, reflectivity and energy loss function are also investigated. These compounds have almost isotropic nature in the lower frequency range while at higher energies, they have a significant anisotropic nature.

  18. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

    PubMed

    Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-06-01

    We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

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

    PubMed

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

    2017-04-01

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

  20. Relation between Liver Transaminases and Dyslipidaemia among 2-10 y.o. Northern Mexican Children

    PubMed Central

    Bibiloni, Maria del Mar; Salas, Rogelio; Nuñez, Georgina M.; Villarreal, Jesús Z.; Sureda, Antoni

    2016-01-01

    Background and Aims The increase in overweight and obese children may be linked to increased rates of liver damage and dyslipidaemia. This study aimed to explore the associations of liver biomarkers with overweight/obesity and dyslipidaemia in Mexican children. Methods The study was a population-based cross-sectional nutritional survey carried out in the State of Nuevo León, Mexico. The study included a 414 subjects aged between 2 and 10 years old (47.8% girls) who took part in the State Survey of Nutrition and Health–Nuevo León 2011/2012. Associations between alanine aminotransferase (ALT) and aspartate aminotransferase (AST), ALT/AST ratio, and major components of serum lipid profile were assessed. Results Children with high ALT (defined as ≥P75) showed higher prevalence of dyslipidaemia than their counterparts, with high prevalence of high TChol (P = 0.053), non-HDL-chol, TG, and low HDL-chol. Children with an AST/ALT ≥T3 ratio were 0.43-times (95% CI: 0.25–0.74) and 0.27-times (95% CI: 0.17–0.44) low likely to be overweight/obese and to have dyslipidaemia than those with an AST/ALT

  1. Atomic Layer Deposition of L-Alanine Polypeptide

    SciTech Connect

    Fu, Yaqin; Li, Binsong; Jiang, Ying-Bing; Dunphy, Darren R.; Tsai, Andy; Tam, Siu-Yue; Fan, Hongyou Y.; Zhang, Hongxia; Rogers, David; Rempe, Susan; Atanassov, Plamen; Cecchi, Joseph L.; Brinker, C. Jeffrey

    2014-10-30

    L-Alanine polypeptide thin films were synthesized via atomic layer deposition (ALD). Rather, instead of using an amino acid monomer as the precursor, an L-alanine amino acid derivatized with a protecting group was used to prevent self-polymerization, increase the vapor pressure, and allow linear cycle-by-cycle growth emblematic of ALD. Moreover, the successful deposition of a conformal polypeptide film has been confirmed by FTIR, TEM, and Mass Spectrometry, and the ALD process has been extended to polyvaline.

  2. Exogenous alanine and/or glucose plus kanamycin kills antibiotic-resistant bacteria.

    PubMed

    Peng, Bo; Su, Yu-Bin; Li, Hui; Han, Yi; Guo, Chang; Tian, Yao-Mei; Peng, Xuan-Xian

    2015-02-03

    Multidrug-resistant bacteria are an increasingly serious threat to human and animal health. However, novel drugs that can manage infections by multidrug-resistant bacteria have proved elusive. Here we show that glucose and alanine abundances are greatly suppressed in kanamycin-resistant Edwardsiella tarda by GC-MS-based metabolomics. Exogenous alanine or glucose restores susceptibility of multidrug-resistant E. tarda to killing by kanamycin, demonstrating an approach to killing multidrug-resistant bacteria. The mechanism underlying this approach is that exogenous glucose or alanine promotes the TCA cycle by substrate activation, which in turn increases production of NADH and proton motive force and stimulates uptake of antibiotic. Similar results are obtained with other Gram-negative bacteria (Vibrio parahaemolyticus, Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacterium (Staphylococcus aureus), and the results are also reproduced in a mouse model for urinary tract infection. This study establishes a functional metabolomics-based strategy to manage infection by antibiotic-resistant bacteria.

  3. Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma.

    PubMed

    Guo, Min; Chong, Yeeting E; Shapiro, Ryan; Beebe, Kirk; Yang, Xiang-Lei; Schimmel, Paul

    2009-12-10

    Mistranslation arising from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences. Throughout evolution, two editing checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly. One checkpoint is the AlaRS editing centre, and the other is from widely distributed AlaXps-free-standing, genome-encoded editing proteins that clear Ser-tRNA(Ala). The paradox of misincorporating both a smaller (glycine) and a larger (serine) amino acid suggests a deep conflict for nature-designed AlaRS. Here we show the chemical basis for this conflict. Nine crystal structures, together with kinetic and mutational analysis, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the alpha-amino group of the bound amino acid by using an acidic residue. This design, dating back more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently because no better architecture for the recognition of alanine could be found, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.

  4. Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.

    PubMed

    Hammer, Diana; Wild, Jens; Ludwig, Christine; Asbach, Benedikt; Notka, Frank; Wagner, Ralf

    2008-06-01

    Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

  5. Crystal Structure of a Thermostable Alanine Racemase from Thermoanaerobacter tengcongensis MB4 Reveals the Role of Gln360 in Substrate Selection.

    PubMed

    Sun, Xiaoliang; He, Guangzheng; Wang, Xiaoyan; Xu, Shujing; Ju, Jiansong; Xu, Xiaoling

    2015-01-01

    Pyridoxal 5'-phosphate (PLP) dependent alanine racemase catalyzes racemization of L-Ala to D-Ala, a key component of the peptidoglycan network in bacterial cell wall. It has been extensively studied as an important antimicrobial drug target due to its restriction in eukaryotes. However, many marketed alanine racemase inhibitors also act on eukaryotic PLP-dependent enzymes and cause side effects. A thermostable alanine racemase (AlrTt) from Thermoanaerobacter tengcongensis MB4 contains an evolutionarily non-conserved residue Gln360 in inner layer of the substrate entryway, which is supposed to be a key determinant in substrate specificity. Here we determined the crystal structure of AlrTt in complex with L-Ala at 2.7 Å resolution, and investigated the role of Gln360 by saturation mutagenesis and kinetic analysis. Compared to typical bacterial alanine racemase, presence of Gln360 and conformational changes of active site residues disrupted the hydrogen bonding interactions necessary for proper PLP immobilization, and decreased both the substrate affinity and turnover number of AlrTt. However, it could be complemented by introduction of hydrophobic amino acids at Gln360, through steric blocking and interactions with a hydrophobic patch near active site pocket. These observations explained the low racemase activity of AlrTt, revealed the essential role of Gln360 in substrate selection, and its preference for hydrophobic amino acids especially Tyr in bacterial alanine racemization. Our work will contribute new insights into the alanine racemization mechanism for antimicrobial drug development.

  6. Case-control study on prednisolone combined with ursodeoxycholic acid and azathioprine in pure primary biliary cirrhosis with high levels of immunoglobulin G and transaminases: efficacy and safety analysis.

    PubMed

    Fang, Yu-Qing; Lv, Dong-Xia; Jia, Wei; Li, Jun; Deng, Yong-Qiong; Wang, Yan; Yu, Min; Wang, Gui-Qiang

    2014-10-01

    To the best of our knowledge, this is the first study to address the use of glucocorticoids in the comparatively special population of pure primary biliary cirrhosis (PBC) patients who have high levels of immunoglobulin G (IgG) and transaminases but do not have PBC-autoimmune hepatitis overlap syndrome. Ursodeoxycholic acid (UDCA) is now assumed to be the standard therapy for PBC patients. However, patients treated with UDCA still have a risk of progression to cirrhosis and end-stage liver disease. The most recent European Association for the Study of the Liver guidelines of 2009 declared that further studies on glucocorticoid therapy in this disease should be a priority. Therefore, we designed this 3-year longitudinal retrospective study, which might provide deep insight into the treatment for PBC.The aim of this study was to assess whether the combination of prednisolone, UDCA, and azathioprine was superior to UDCA alone in these PBC patients.Sixty patients were enrolled in this study. Thirty-one patients underwent UDCA monotherapy, and 29 patients were treated with prednisolone, UDCA, and azathioprine. We analyzed their biochemistries, immune parameters, liver synthetic function, and noninvasive assessments of liver fibrosis, as well as treatment efficacy and adverse effects at baseline and at 1, 3, 6, 12, 24, and 36 months.Alkaline phosphatase (ALP), γ-glutamyl transpeptidase, alanine aminotransferase, and aspartate aminotransferase levels and the aspartate aminotransferase-to-platelet ratio index (APRI) and S-index improved dramatically in both groups, whereas IgG levels only decreased in the combination group (all P < 0.05). Albumin (ALB) levels decreased in the UDCA group but increased with the combination treatment at 36 months. Significant differences between the 2 groups were observed at 36 months in ALP (P = 0.005), IgG (P = 0.002), ALB (P = 0.002), APRI (P = 0.015), and S-index (P = 0.020). Prednisolone combined with UDCA and

  7. Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

    PubMed Central

    Abe, Keietsu; Ohnishi, Fumito; Yagi, Kyoko; Nakajima, Tasuku; Higuchi, Takeshi; Sano, Motoaki; Machida, Masayuki; Sarker, Rafiquel I.; Maloney, Peter C.

    2002-01-01

    Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high. PMID:12003930

  8. Plasmid-encoded asp operon confers a proton motive metabolic cycle catalyzed by an aspartate-alanine exchange reaction.

    PubMed

    Abe, Keietsu; Ohnishi, Fumito; Yagi, Kyoko; Nakajima, Tasuku; Higuchi, Takeshi; Sano, Motoaki; Machida, Masayuki; Sarker, Rafiquel I; Maloney, Peter C

    2002-06-01

    Tetragenococcus halophila D10 catalyzes the decarboxylation of L-aspartate with nearly stoichiometric release of L-alanine and CO(2). This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an L-aspartate-beta-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter --> aspD --> aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate-beta-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of L-aspartate-beta-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

  9. Physical activity as a protective factor for development of non-alcoholic fatty liver in men

    PubMed Central

    Pinto, Carla Giuliano de Sá; Marega, Marcio; de Carvalho, José Antonio Maluf; Carmona, Felipe Gambetta; Lopes, Carlos Eduardo Felix; Ceschini, Fabio Luis; Bocalini, Danilo Sales; Figueira, Aylton José

    2015-01-01

    Objective To determine the impact of physical activity on the prevalence of fatty liver, metabolic and cardiovascular disease in adult men. Methods This study evaluated 1,399 men (40.7±8.18 years) with body mass index of 26.7kg/m2 (±3.4) who participated in the Protocol of Preventive Health Check-up at Hospital Israelita Albert Einstein from January to October 2011. We conducted tests of serum blood glucose, total cholesterol, LDL, HDL, triglycerides, reactive c-protein, aspartate transaminase, alanine transaminase and gamma-glutamyl transpeptidase. The statistical analysis comprised in the comparison of mean and standard deviation. The analysis of variance was based in two paths of two way ANOVA, Student’s t-test, Mann Whitney U test, Wald test and χ2. We considered a significance level at p<0.05 and correlation of univariate Poison with 95% confidence interval. Results :Fatty liver was diagnosed in 37.0% of the sample. Triglyceride levels of active men with fatty liver were 148.2±77.6mg/dL while inactive men with fatty liver had 173.4±15.6mg/dL. The remaining serum levels were normal. Inactive individuals showed higher values than active. In addition, inactive individuals have 10.68 times higher risk of developing fatty liver compared with active. Conclusion Physical activity improves metabolic parameters such as triglycerides, weight control, HDL, which interfere in the development of fatty liver. Physically active individuals had lower fatty liver prevalence regardless of values of body composition and lipid profile, leading the conclusion that physical activity has a protective role against development of fatty liver. PMID:25993066

  10. Deletion of the Saccharomyces cerevisiae ARO8 gene, encoding an aromatic amino acid transaminase, enhances phenylethanol production from glucose.

    PubMed

    Romagnoli, Gabriele; Knijnenburg, Theo A; Liti, Gianni; Louis, Edward J; Pronk, Jack T; Daran, Jean-Marc

    2015-01-01

    Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products.

  11. Formation of {gamma}-alumina nanorods in presence of alanine

    SciTech Connect

    Dabbagh, Hossein A.; Rasti, Elham; Yalfani, Mohammad S.; Medina, Francesc

    2011-02-15

    Graphical abstract: Nanorod aluminas with a possible hexagonal symmetry, high surface area and relatively narrow pore size distribution were obtained. Research highlights: {yields} Research highlights {yields} Boehmite was prepared using a green sol-gel process in the presence of alanine. {yields} Nanorod aluminas with a high surface area were obtained. {yields} Addition of alanine would shape the size of the holes and crevices. {yields} The morphologies of the nanorods were revealed by transmission electron microscope. -- Abstract: Boehmite and alumina nanostructures were prepared using a simple green sol-gel process in the presence of alanine in water medium at room temperature. The uncalcined (dried at 200 {sup o}C) and the calcined materials (at 500, 600 and 700 {sup o}C for 4 h) were characterized using XRD, TEM, SEM, N{sub 2} physisorption and TGA. Nanorod aluminas with a possible hexagonal symmetry, high surface area and relatively narrow pore size distribution were obtained. The surface area was enhanced and crystallization was retarded as the alanine content increased. The morphologies of the nanoparticles and nanorods were revealed by a transmission electron microscope (TEM).

  12. A theoretical study of alanine dipeptide and analogs

    SciTech Connect

    Head-Gordon, T.; Head-Gordon, M.; Brooks, C. III; Pople, J. ); Frisch, M.J. )

    1989-01-01

    We Present a preliminary report on the conformational and energetic analysis of the molecule (S)-2-acetylamino-N-methylpropanamide (alanine dipeptide) and an analog molecule, (S)-{alpha}-formylaminopropanamide, using high-quality ab initio methods. Alanine dipeptide and its analogs are of interest since they incorporate many of the structural features found in proteins, such as intramolecular hydrogen bonds, conformational flexibility, and a variety of chemical functionality. One purpose of this study is to provide a useful benchmark calculation, MP2/6-31+G{sup **}//HF/6-31+G{sup *}, for a number of conformations of the alanine system. Based on the comparison of these benchmark calculations with lower-level basis sets, HF/3-21G was chosen to generate a fully relaxed {phi}, {psi} dihedral map. These calculations are the first of their kind on systems of this size. Features of the {phi},{psi} alanine dipeptide map that are discussed include the energetically accessible conformations and possible pathways for their interconversion. In addition, we illustrate the importance of fully optimized geometries and the proper evaluation of correlation energies,

  13. Spectrophotometric readout for an alanine dosimeter for food irradiation applications

    NASA Astrophysics Data System (ADS)

    Ebraheem, S.; Beshir, W. B.; Eid, S.; Sobhy, R.; Kovács, A.

    2003-06-01

    The alanine-electron spin resonance (EPR) readout system is well known as a reference and transfer dosimetry system for the evaluation of high doses in radiation processing. The high cost of an EPR/alanine dosimetry system is a serious handicap for large-scale routine application in irradiation facilities. In this study, the use of a complex produced by dissolving irradiated L-alanine in 1,4-phenyl diammonium dichloride solution was investigated for dosimetry purposes. This complex—having a purple colour—has an increasing absorbance with increasing dose in the range of 1-20 kGy. The applicability of spectrophotometric evaluation was studied by measuring the absorbance intensity of this complex at 360 and 505 nm, respectively. Fluorimetric evaluation was also investigated by measuring the emission of the complex at 435 nm as a function of dose. The present method is easy for routine application. The effect of the dye concentration as well as the suitable amount of irradiated alanine has been studied. With respect to routine application, the stability of the product complex after its formation was also investigated.

  14. Computational alanine scanning with linear scaling semiempirical quantum mechanical methods.

    PubMed

    Diller, David J; Humblet, Christine; Zhang, Xiaohua; Westerhoff, Lance M

    2010-08-01

    Alanine scanning is a powerful experimental tool for understanding the key interactions in protein-protein interfaces. Linear scaling semiempirical quantum mechanical calculations are now sufficiently fast and robust to allow meaningful calculations on large systems such as proteins, RNA and DNA. In particular, they have proven useful in understanding protein-ligand interactions. Here we ask the question: can these linear scaling quantum mechanical methods developed for protein-ligand scoring be useful for computational alanine scanning? To answer this question, we assembled 15 protein-protein complexes with available crystal structures and sufficient alanine scanning data. In all, the data set contains Delta Delta Gs for 400 single point alanine mutations of these 15 complexes. We show that with only one adjusted parameter the quantum mechanics-based methods outperform both buried accessible surface area and a potential of mean force and compare favorably to a variety of published empirical methods. Finally, we closely examined the outliers in the data set and discuss some of the challenges that arise from this examination.

  15. Liver Fibrosis Regression Measured by Transient Elastography in Human Immunodeficiency Virus (HIV)-Hepatitis B Virus (HBV)-Coinfected Individuals on Long-Term HBV-Active Combination Antiretroviral Therapy

    PubMed Central

    Audsley, Jennifer; Robson, Christopher; Aitchison, Stacey; Matthews, Gail V.; Iser, David; Sasadeusz, Joe; Lewin, Sharon R.

    2016-01-01

    Background. Advanced fibrosis occurs more commonly in human immunodeficiency virus (HIV)-hepatitis B virus (HBV) coinfected individuals; therefore, fibrosis monitoring is important in this population. However, transient elastography (TE) data in HIV-HBV coinfection are lacking. We aimed to assess liver fibrosis using TE in a cross-sectional study of HIV-HBV coinfected individuals receiving combination HBV-active (lamivudine and/or tenofovir/tenofovir-emtricitabine) antiretroviral therapy, identify factors associated with advanced fibrosis, and examine change in fibrosis in those with >1 TE assessment. Methods. We assessed liver fibrosis in 70 HIV-HBV coinfected individuals on HBV-active combination antiretroviral therapy (cART). Change in fibrosis over time was examined in a subset with more than 1 TE result (n = 49). Clinical and laboratory variables at the time of the first TE were collected, and associations with advanced fibrosis (≥F3, Metavir scoring system) and fibrosis regression (of least 1 stage) were examined. Results. The majority of the cohort (64%) had mild to moderate fibrosis at the time of the first TE, and we identified alanine transaminase, platelets, and detectable HIV ribonucleic acid as associated with advanced liver fibrosis. Alanine transaminase and platelets remained independently advanced in multivariate modeling. More than 28% of those with >1 TE subsequently showed liver fibrosis regression, and higher baseline HBV deoxyribonucleic acid was associated with regression. Prevalence of advanced fibrosis (≥F3) decreased 12.3% (32.7%–20.4%) over a median of 31 months. Conclusions. The observed fibrosis regression in this group supports the beneficial effects of cART on liver stiffness. It would be important to study a larger group of individuals with more advanced fibrosis to more definitively assess factors associated with liver fibrosis regression. PMID:27006960

  16. Liver Fibrosis Regression Measured by Transient Elastography in Human Immunodeficiency Virus (HIV)-Hepatitis B Virus (HBV)-Coinfected Individuals on Long-Term HBV-Active Combination Antiretroviral Therapy.

    PubMed

    Audsley, Jennifer; Robson, Christopher; Aitchison, Stacey; Matthews, Gail V; Iser, David; Sasadeusz, Joe; Lewin, Sharon R

    2016-01-01

    Background.  Advanced fibrosis occurs more commonly in human immunodeficiency virus (HIV)-hepatitis B virus (HBV) coinfected individuals; therefore, fibrosis monitoring is important in this population. However, transient elastography (TE) data in HIV-HBV coinfection are lacking. We aimed to assess liver fibrosis using TE in a cross-sectional study of HIV-HBV coinfected individuals receiving combination HBV-active (lamivudine and/or tenofovir/tenofovir-emtricitabine) antiretroviral therapy, identify factors associated with advanced fibrosis, and examine change in fibrosis in those with >1 TE assessment. Methods.  We assessed liver fibrosis in 70 HIV-HBV coinfected individuals on HBV-active combination antiretroviral therapy (cART). Change in fibrosis over time was examined in a subset with more than 1 TE result (n = 49). Clinical and laboratory variables at the time of the first TE were collected, and associations with advanced fibrosis (≥F3, Metavir scoring system) and fibrosis regression (of least 1 stage) were examined. Results.  The majority of the cohort (64%) had mild to moderate fibrosis at the time of the first TE, and we identified alanine transaminase, platelets, and detectable HIV ribonucleic acid as associated with advanced liver fibrosis. Alanine transaminase and platelets remained independently advanced in multivariate modeling. More than 28% of those with >1 TE subsequently showed liver fibrosis regression, and higher baseline HBV deoxyribonucleic acid was associated with regression. Prevalence of advanced fibrosis (≥F3) decreased 12.3% (32.7%-20.4%) over a median of 31 months. Conclusions.  The observed fibrosis regression in this group supports the beneficial effects of cART on liver stiffness. It would be important to study a larger group of individuals with more advanced fibrosis to more definitively assess factors associated with liver fibrosis regression.

  17. Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress.

    PubMed

    Limami, Anis M; Glévarec, Gaëlle; Ricoult, Claudie; Cliquet, Jean-Bernard; Planchet, Elisabeth

    2008-01-01

    The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.

  18. The unresolved puzzle why alanine extensions cause disease.

    PubMed

    Winter, Reno; Liebold, Jens; Schwarz, Elisabeth

    2013-08-01

    The prospective increase in life expectancy will be accompanied by a rise in the number of elderly people who suffer from ill health caused by old age. Many diseases caused by aging are protein misfolding diseases. The molecular mechanisms underlying these disorders receive constant scientific interest. In addition to old age, mutations also cause congenital protein misfolding disorders. Chorea Huntington, one of the most well-known examples, is caused by triplet extensions that can lead to more than 100 glutamines in the N-terminal region of huntingtin, accompanied by huntingtin aggregation. So far, nine disease-associated triplet extensions have also been described for alanine codons. The extensions lead primarily to skeletal malformations. Eight of these proteins represent transcription factors, while the nuclear poly-adenylate binding protein 1, PABPN1, is an RNA binding protein. Additional alanines in PABPN1 lead to the disease oculopharyngeal muscular dystrophy (OPMD). The alanine extension affects the N-terminal domain of the protein, which has been shown to lack tertiary contacts. Biochemical analyses of the N-terminal domain revealed an alanine-dependent fibril formation. However, fibril formation of full-length protein did not recapitulate the findings of the N-terminal domain. Fibril formation of intact PABPN1 was independent of the alanine segment, and the fibrils displayed biochemical properties that were completely different from those of the N-terminal domain. Although intranuclear inclusions have been shown to represent the histochemical hallmark of OPMD, their role in pathogenesis is currently unclear. Several cell culture and animal models have been generated to study the molecular processes involved in OPMD. These studies revealed a number of promising future therapeutic strategies that could one day improve the quality of life for the patients.

  19. Clinical Safety of a High Dose of Phycocyanin-Enriched Aqueous Extract from Arthrospira (Spirulina) platensis: Results from a Randomized, Double-Blind, Placebo-Controlled Study with a Focus on Anticoagulant Activity and Platelet Activation.

    PubMed

    Jensen, Gitte S; Drapeau, Cassandra; Lenninger, Miki; Benson, Kathleen F

    2016-07-01

    The goal for this study was to evaluate safety regarding anticoagulant activity and platelet activation during daily consumption of an aqueous cyanophyta extract (ACE), containing a high dose of phycocyanin. Using a randomized, double-blind, placebo-controlled study design, 24 men and women were enrolled after informed consent, and consumed either ACE (2.3 g/day) or placebo daily for 2 weeks. The ACE dose was equivalent to ∼1 g phycocyanin per day, chosen based on the highest dose Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration. Consuming ACE did not alter markers for platelet activation (P-selectin expression) or serum P-selectin levels. No changes were seen for activated partial thromboplastin time, thrombin clotting time, or fibrinogen activity. Serum levels of aspartate transaminase (AST) showed a significant reduction after 2 weeks of ACE consumption (P < .001), in contrast to placebo where no changes were seen; the difference in AST levels between the two groups was significant at 2 weeks (P < .02). Reduced levels of alanine transaminase (ALT) were also seen in the group consuming ACE (P < .08). Previous studies showed reduction of chronic pain when consuming 1 g ACE per day. The higher dose of 2.3 g/day in this study was associated with significant reduction of chronic pain at rest and when physically active (P < .05). Consumption of ACE showed safety regarding markers pertaining to anticoagulant activity and platelet activation status, in conjunction with rapid and robust relief of chronic pain. Reduction in AST and ALT suggested improvement in liver function and metabolism.

  20. Clinical Safety of a High Dose of Phycocyanin-Enriched Aqueous Extract from Arthrospira (Spirulina) platensis: Results from a Randomized, Double-Blind, Placebo-Controlled Study with a Focus on Anticoagulant Activity and Platelet Activation

    PubMed Central

    Drapeau, Cassandra; Lenninger, Miki; Benson, Kathleen F.

    2016-01-01

    Abstract The goal for this study was to evaluate safety regarding anticoagulant activity and platelet activation during daily consumption of an aqueous cyanophyta extract (ACE), containing a high dose of phycocyanin. Using a randomized, double-blind, placebo-controlled study design, 24 men and women were enrolled after informed consent, and consumed either ACE (2.3 g/day) or placebo daily for 2 weeks. The ACE dose was equivalent to ∼1 g phycocyanin per day, chosen based on the highest dose Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration. Consuming ACE did not alter markers for platelet activation (P-selectin expression) or serum P-selectin levels. No changes were seen for activated partial thromboplastin time, thrombin clotting time, or fibrinogen activity. Serum levels of aspartate transaminase (AST) showed a significant reduction after 2 weeks of ACE consumption (P < .001), in contrast to placebo where no changes were seen; the difference in AST levels between the two groups was significant at 2 weeks (P < .02). Reduced levels of alanine transaminase (ALT) were also seen in the group consuming ACE (P < .08). Previous studies showed reduction of chronic pain when consuming 1 g ACE per day. The higher dose of 2.3 g/day in this study was associated with significant reduction of chronic pain at rest and when physically active (P < .05). Consumption of ACE showed safety regarding markers pertaining to anticoagulant activity and platelet activation status, in conjunction with rapid and robust relief of chronic pain. Reduction in AST and ALT suggested improvement in liver function and metabolism. PMID:27362442

  1. β-alanine supplementation improves tactical performance but not cognitive function in combat soldiers

    PubMed Central

    2014-01-01

    Background There are no known studies that have examined β-alanine supplementation in military personnel. Considering the physiological and potential neurological effects that have been reported during sustained military operations, it appears that β-alanine supplementation may have a potential benefit in maintaining physical and cognitive performance during high-intensity military activity under stressful conditions. The purpose of this study was to examine the effect of 28 days of β-alanine ingestion in military personnel while fatigued on physical and cognitive performance. Methods Twenty soldiers (20.1 ± 0.9 years) from an elite combat unit were randomly assigned to either a β-alanine (BA) or placebo (PL) group. Soldiers were involved in advanced military training, including combat skill development, navigational training, self-defense/hand-to-hand combat and conditioning. All participants performed a 4-km run, 5-countermovement jumps using a linear position transducer, 120-m sprint, a 10-shot shooting protocol with assault rifle, including overcoming a misfire, and a 2-min serial subtraction test to assess cognitive function before (Pre) and after (Post) 28 days of supplementation. Results The training routine resulted in significant increases in 4-km run time for both groups, but no between group differences were seen (p = 0.597). Peak jump power at Post was greater for BA than PL (p = 0.034), while mean jump power for BA at Post was 10.2% greater (p = 0.139) than PL. BA had a significantly greater (p = 0.012) number of shots on target at Post (8.2 ± 1.0) than PL (6.5 ± 2.1), and their target engagement speed at Post was also significantly faster (p = 0.039). No difference in serial subtraction performance was seen between the groups (p = 0.844). Conclusion Results of this study indicate that 4-weeks of β-alanine ingestion in young, healthy soldiers did not impact cognitive performance, but did enhance power

  2. Synthesis of platinum(II) and palladium(II) complexes with 9,9-dihexyl-4,5-diazafluorene and their in vivo antitumour activity against Hep3B xenografted mice.

    PubMed

    Wang, Q-W; Lam, P-L; Wong, R S-M; Cheng, G Y-M; Lam, K-H; Bian, Z-X; Ho, C-L; Feng, Y-H; Gambari, R; Lo, Y-H; Wong, W-Y; Chui, C-H

    2016-11-29

    Two complexes dichloro(9,9-dihexyl-4,5-diazafluorene)platinum(II) (Pt-DHF) and dichloro(9,9-dihexyl-4,5-diazafluorene)palladium(II) (Pd-DHF) were synthesized and their in vivo antitumour activity was investigated using an athymic nude mice model xenografted with human Hep3B carcinoma cells. Pt-DHF- and Pd-DHF-treated groups showed significant tumour growth inhibition (with about 9-fold and 3-fold tumour growth retardation) when compared with the vehicle control group. The liver toxicology effects on the animals of the two compounds were investigated. Pt-DHF and Pd-DHF-treated groups had a lower alanine transaminase and aspartate transaminase values than those of the vehicle treated group as the animals from the vehicle control group had very heavy hepatoma burden. We assume that both complexes could be further investigated as effective antitumour agents and it is worthwhile to study their underlying working mechanism.

  3. Formation of simple biomolecules from alanine in ocean by impacts

    NASA Astrophysics Data System (ADS)

    Umeda, Y.; Sekine, T.; Furukawa, Y.; Kakegawa, T.; Kobayashi, T.

    2013-12-01

    The biomolecules on the Earth are thought either to have originated from the extraterrestrial parts carried with flying meteorites or to have been formed from the inorganic materials on the Earth through given energy. From the standpoint to address the importance of impact energy, it is required to simulate experimentally the chemical reactions during impacts, because violent impacts may have occurred 3.8-4.0 Gyr ago to create biomolecules initially. It has been demonstrated that shock reactions among ocean (H2O), atmospheric nitrogen, and meteoritic constitution (Fe) can induce locally reduction environment to form simple bioorganic molecules such as ammonia and amino acid (Nakazawa et al., 2005; Furukawa et al., 2009). We need to know possible processes for alanine how chemical reactions proceed during repeated impacts and how complicated biomolecules are formed. Alanine can be formed from glycine (Umeda et al., in preparation). In this study, we carried out shock recovery experiments at pressures of 4.4-5.7 GPa to investigate the chemical reactions of alanine. Experiments were carried out with a propellant gun. Stainless steel containers (30 mm in diameter, 30 mm long) with 13C-labeled alanine aqueous solution immersed in olivine or hematite powders were used as targets. Air gap was present in the sample room (18 mm in diameter, 2 mm thick) behind the sample. The powder, solution, and air represent meteorite, ocean, and atmosphere on early Earth, respectively. Two powders of olivine and hematite help to keep the oxygen fugacity low and high during experiments, respectively in order to investigate the effect of oxygen fugacity on chemical processes of alanine. The recovered containers, after cleaned completely, were immersed into liquid nitrogen to freeze sample solution and then we drilled on the impact surface to extract water-soluble run products using pure water. Thus obtained products were analyzed by LC/MS for four amino acids (glycine, alanine, valine, and

  4. Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine

    SciTech Connect

    Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia; Barletta, Raúl G.; Sacchettini, James C.

    2011-09-28

    D-Alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 {angstrom}. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC{sub 50}) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.

  5. Β-alanine and l-histidine transport across the inner blood-retinal barrier: potential involvement in L-carnosine supply.

    PubMed

    Usui, Takuya; Kubo, Yoshiyuki; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi

    2013-08-01

    The supply of L-carnosine, a bioactive dipeptide of β-alanine and l-histidine, to the retina across the blood-retinal barrier (BRB) was studied. The in vivo and in vitro studies revealed low uptake activities for [(3)H]Gly-Sar, a representative dipeptide, suggesting that l-carnosine transport plays only a minor role at the BRB. The in vivo study using rats showed approximately 18- and 23-fold greater retinal uptake indexes (RUI) for [(3)H]β-alanine and [(3)H]l-histidine compared with that of a paracellular marker, respectively. The RUI of [(3)H]β-alanine was taurine- and γ-aminobutyric acid-sensitive, and the in vitro uptake by TR-iBRB2 cells showed time- concentration- and temperature-dependent [(3)H]β-alanine uptake, suggesting that a carrier-mediated process was involved in β-alanine transport across the inner BRB. [(3)H]β-Alanine uptake was inhibited by taurine and β-guanidinopropionic acid, suggesting that taurine transporter (TAUT/SLC6A6) is responsible for the influx transport of β-alanine across the inner BRB. Regarding l-histidine, the l-leucine-sensitive RUI of [(3)H]l-histidine was identified, and the in vitro [(3)H]l-histidine uptake by TR-iBRB2 cells suggested that a carrier-mediated process was involved in l-histidine transport across the inner BRB. The inhibition profile suggested that L-type amino acid transporter (LAT1/SLC7A5) is responsible for the influx transport of l-histidine across the inner BRB. These results show that the influx transports of β-alanine and l-histidine across the inner BRB is carried out by TAUT and LAT1, respectively, suggesting that the retinal l-carnosine is supplied by enzymatic synthesis from two kinds of amino acids transported across the inner BRB.

  6. Characterization of the Genes Encoding d-Amino Acid Transaminase and Glutamate Racemase, Two d-Glutamate Biosynthetic Enzymes of Bacillus sphaericus ATCC 10208

    PubMed Central

    Fotheringham, Ian G.; Bledig, Stefan A.; Taylor, Paul P.

    1998-01-01

    In Bacillus sphaericus and other Bacillus spp., d-amino acid transaminase has been considered solely responsible for biosynthesis of d-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding d-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28.8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus, Pediococcus, and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the d-amino acid transaminase of the thermophilic Bacillus sp. strain YM1. PMID:9696787

  7. Structural and biochemical analyses of alanine racemase from the multidrug-resistant Clostridium difficile strain 630.

    PubMed

    Asojo, Oluwatoyin A; Nelson, Sarah K; Mootien, Sara; Lee, Yashang; Rezende, Wanderson C; Hyman, Daniel A; Matsumoto, Monica M; Reiling, Scott; Kelleher, Alan; Ledizet, Michel; Koski, Raymond A; Anthony, Karen G

    2014-07-01

    Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.

  8. Amino acid oxidation and alanine production in rat hemidiaphragm in vitro. Effects of dichloroacetate.

    PubMed Central

    Palmer, T N; Caldecourt, M A; Sugden, M C

    1984-01-01

    Dichloroacetate (an activator of pyruvate dehydrogenase) stimulates 14CO2 production from [U-14C]glucose, but not from [U-14C]glutamate, [U-14C]aspartate, [U-14C]- and [1-14C]-valine and [U-14C]- and [1-14C]-leucine. It is concluded (1) that pyruvate dehydrogenase is not rate-limiting in the oxidation to CO2 of amino acids that are metabolized to tricarboxylic acid-cycle intermediates, and (2) that carbohydrate (and not amino acids) is the main carbon precursor in alanine formation in muscle. PMID:6149743

  9. The GerW protein is essential for L-alanine-stimulated germination of Bacillus subtilis spores.

    PubMed

    Kuwana, Ritsuko; Takamatsu, Hiromu

    2013-11-01

    GerW (formerly called YtfJ) is a protein found in dormant spores of Bacillus subtilis. We have studied spore proteins in B. subtilis before, and here we report the characterization of GerW protein. Northern blot analysis revealed that gerW mRNA was transcribed by SigF-containing RNA polymerase beginning 1 h after the initiation of sporulation. Fluorescence was detected in forespores and dormant spores of B. subtilis recombinant strains expressing GerW-GFP. During germination in the presence of L-alanine or a mixture of L-asparagine, D-glucose, D-fructose and potassium ions (AGFK), normal spores of B. subtilis became darkened, stained positive with Hoechst 33342 and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), and released dipicolinic acid (DPA). In the case of gerW-deficient spores, AGFK triggered germination in a manner similar to that seen in the wild-type spores, whereas spores stimulated by L-alanine remained refractive under the phase contrast microscope, failed to stain positive with Hoechst 33342 or CFDA-SE, and released almost no DPA. These results indicate that GerW is essential for the L-alanine-induced breakdown of spore dormancy followed by core rehydration and the resumption of enzymatic activity, and suggest that GerW is involved in the early stages of germination in the presence of l-alanine.

  10. Degradation of glycine and alanine on irradiated quartz.

    PubMed

    Pawlikowski, Maciej; Benko, Aleksandra; Wróbel, Tomasz P

    2013-04-01

    Recent researches suggest participation of minerals in the formation of life under primordial conditions. Among all of the minerals, quartz seems to be one of the most probable to take part in such processes. However, an external source of energy is needed, e.g. electric discharge. A device simulating the proposed conditions was designed and was used to simulate prebiotic conditions. Investigation of processes occurring during the stimulation of quartz with electric discharge was studied by means of Ultraviolet-visible (UV-VIS) spectroscopy, in order to monitor the generation kinetics of free radicals. Additionally, infrared spectroscopy was applied to identify chemical reaction products created in a solution of alanine or glycine, in the presence of quartz treated with electric discharge. Formation of increased amounts of free radicals, compared to experiments performed without quartz and/or amino acid, is reported, along with identification of possible degradation products of alanine. No synthetic reactions were observed.

  11. Clinical applications of alanine/electron spin resonance dosimetry.

    PubMed

    Baffa, Oswaldo; Kinoshita, Angela

    2014-05-01

    This paper discusses the clinical applications of electron spin resonance (ESR) dosimetry focusing on the ESR/alanine system. A review of few past studies in this area is presented offering a critical overview of the challenges and opportunities for extending this system into clinical applications. Alanine/ESR dosimetry fulfills many of the required properties for several clinical applications such as water-equivalent composition, independence of the sensitivity for the energy range used in therapy and high precision. Improvements in sensitivity and the development of minidosimeters coupled with the use of a spectrometer of higher microwave frequency expanded the possibilities for clinical applications to the new modalities of radiotherapy (intensity-modulated radiation therapy and radiosurgery) and to the detection of low doses such as those present in some radiological image procedures.

  12. Physiological hypercortisolemia increases proteolysis, glutamine, and alanine production

    SciTech Connect

    Darmaun, D.; Matthews, D.E.; Bier, D.M. Cornell Univ. Medical College, New York, NY )

    1988-09-01

    Physiological elevations of plasma cortisol levels, as are encountered in stress and severe trauma, were produced in six normal subjects by infusing them with hydrocortisone for 64 h. Amino acid kinetics were measured in the postabsorptive state using three 4-h infusions of L-(1-{sup 13}C)leucine, L-phenyl({sup 2}H{sub 5})phenylalanine, L-(2-{sup 15}N)glutamine, and L-(1-{sup 13}C)alanine tracers (1) before, (2) at 12 h, and (3) at 60 h of cortisol infusion. Before and throughout the study, the subjects ate a normal diet of adequate protein and energy intake. The cortisol infusion raised plasma cortisol levels significantly from 10 {plus minus} 1 to 32 {plus minus} 4 {mu}g/dl, leucine flux from 83 {plus minus} 3 to 97 {plus minus} 3 {mu}mol{center dot}kg{sup {minus}1}{center dot}h{sup {minus}1}, and phenylalanine flux from 34 {plus minus} 1 to 39 {plus minus} 1 (SE) {mu}mol{center dot}kg{sup {minus}1}{center dot}h{sup {minus}1} after 12 h of cortisol infusion. These increases were maintained until the cortisol infusion was terminated. These nearly identical 15% increases in two different essential amino acid appearance rates are reflective of increased whole body protein breakdown. Glutamine flux rose by 12 h of cortisol infusion and remained elevated at the same level at 64 h. The increase in flux was primarily due to a 55% increase in glutamine de novo synthesis. Alanine flux increased with acute hypercortisolemia and increased further at 60 h of cortisol infusion, a result primarily of increased alanine de novo synthesis. Insulin, alanine, and lactate plasma levels responded similarly with significant rises between the acute and chronic periods of cortisol infusion. Thus hypercortisolemia increases both protein breakdown and the turnover of important nonessential amino acids for periods of up to 64 h.

  13. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion.

    PubMed

    Sousa, Cristovão M; Biancur, Douglas E; Wang, Xiaoxu; Halbrook, Christopher J; Sherman, Mara H; Zhang, Li; Kremer, Daniel; Hwang, Rosa F; Witkiewicz, Agnes K; Ying, Haoqiang; Asara, John M; Evans, Ronald M; Cantley, Lewis C; Lyssiotis, Costas A; Kimmelman, Alec C

    2016-08-25

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by an intense fibrotic stromal response and deregulated metabolism. The role of the stroma in PDAC biology is complex and it has been shown to play critical roles that differ depending on the biological context. The stromal reaction also impairs the vasculature, leading to a highly hypoxic, nutrient-poor environment. As such, these tumours must alter how they capture and use nutrients to support their metabolic needs. Here we show that stroma-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumour’s dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment. Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic interaction between PSCs and cancer cells, in which PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment.

  14. Pressure-induced phase transitions in L-alanine, revisited.

    PubMed

    Tumanov, N A; Boldyreva, E V; Kolesov, B A; Kurnosov, A V; Quesada Cabrera, R

    2010-08-01

    The effect of pressure on L-alanine has been studied by X-ray powder diffraction (up to 12.3 GPa), single-crystal X-ray diffraction, Raman spectroscopy and optical microscopy (up to approximately 6 GPa). No structural phase transitions have been observed. At approximately 2 GPa the cell parameters a and b become accidentally equal to each other, but without a change in space-group symmetry. Neither of two transitions reported by others (to a tetragonal phase at approximately 2 GPa and to a monoclinic phase at approximately 9 GPa) was observed. The changes in cell parameters were continuous up to the highest measured pressures and the cells remained orthorhombic. Some important changes in the intermolecular interactions occur, which also manifest themselves in the Raman spectra. Two new orthorhombic phases could be crystallized from a MeOH/EtOH/H(2)O pressure-transmitting mixture in the pressure range 0.8-4.7 GPa, but only if the sample was kept at these pressures for at least 1-2 d. The new phases converted back to L-alanine on decompression. Judging from the Raman spectra and cell parameters, the new phases are most probably not L-alanine but its solvates.

  15. Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase.

    PubMed

    Szymańska-Majchrzak, Jolanta; Pałka, Katarzyna; Kańska, Marianna

    2017-05-01

    Synthesis of 3-fluoro-[2-(2)H]-L-alanine (3-F-[(2)H]-L-Ala) in reductive amination of 3-fluoropyruvic acid catalysed by L-alanine dehydrogenase (AlaDH) was described. Fluorine derivative was used to study oxidative deamination catalysed by AlaDH applied kinetic (for 3-F-L-Ala in H2O - KIE's on Vmax: 1.1; on Vmax/KM: 1.2; for 3-F-L-Ala in (2)H2O - on Vmax: 1.4; on Vmax/KM: 2.1) and solvent isotope effect methods (for 3-F-L-Ala - SIE's on Vmax: 1.0; on Vmax/KM: 0.87; for 3-F-[2-(2)H]-L-Ala - on Vmax: 1.4; on Vmax/KM: 1.5). Studies explain some details of reaction mechanism.

  16. Suppression of γ-aminobutyric acid (GABA) transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato (Solanum lycopersicum L.).

    PubMed

    Koike, Satoshi; Matsukura, Chiaki; Takayama, Mariko; Asamizu, Erika; Ezura, Hiroshi

    2013-05-01

    Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1(RNAi) lines were 1.3-2.0 times and 6.8-9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits.

  17. Reconfiguration of N Metabolism upon Hypoxia Stress and Recovery: Roles of Alanine Aminotransferase (AlaAT) and Glutamate Dehydrogenase (GDH)

    PubMed Central

    Diab, Houssein; Limami, Anis M.

    2016-01-01

    In the context of climatic change, more heavy precipitation and more frequent flooding and waterlogging events threaten the productivity of arable farmland. Furthermore, crops were not selected to cope with flooding- and waterlogging-induced oxygen limitation. In general, low oxygen stress, unlike other abiotic stresses (e.g., cold, high temperature, drought and saline stress), received little interest from the scientific community and less financial support from stakeholders. Accordingly, breeding programs should be developed and agronomical practices should be adapted in order to save plants’ growth and yield—even under conditions of low oxygen availability (e.g., submergence and waterlogging). The prerequisite to the success of such breeding programs and changes in agronomical practices is a good knowledge of how plants adapt to low oxygen stress at the cellular and the whole plant level. In the present paper, we summarized the recent knowledge on metabolic adjustment in general under low oxygen stress and highlighted thereafter the major changes pertaining to the reconfiguration of amino acids syntheses. We propose a model showing (i) how pyruvate derived from active glycolysis upon hypoxia is competitively used by the alanine aminotransferase/glutamate synthase cycle, leading to alanine accumulation and NAD+ regeneration. Carbon is then saved in a nitrogen store instead of being lost through ethanol fermentative pathway. (ii) During the post-hypoxia recovery period, the alanine aminotransferase/glutamate dehydrogenase cycle mobilizes this carbon from alanine store. Pyruvate produced by the reverse reaction of alanine aminotransferase is funneled to the TCA cycle, while deaminating glutamate dehydrogenase regenerates, reducing equivalent (NADH) and 2-oxoglutarate to maintain the cycle function. PMID:27258319

  18. Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress.

    PubMed

    Rocha, Marcio; Sodek, Ladaslav; Licausi, Francesco; Hameed, Muhammad Waqar; Dornelas, Marcelo Carnier; van Dongen, Joost T

    2010-10-01

    Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again.

  19. Comparing anti-hyperglycemic activity and acute oral toxicity of three different trivalent chromium complexes in mice.

    PubMed

    Li, Fang; Wu, Xiangyang; Zou, Yanmin; Zhao, Ting; Zhang, Min; Feng, Weiwei; Yang, Liuqing

    2012-05-01

    Three different ligands (rutin, folate and stachyose) of chromium(III) complexes were compared to examine whether they have similar effect on anti-hyperglycemic activity as well as the acute toxicity status. Anti-hyperglycemic activities of chromium rutin complex (CrRC), chromium folate complex (CrFC) and chromium stachyose complex (CrSC) were examined in alloxan-induced diabetic mice with daily oral gavage for a period of 2 weeks at the dose of 0.5-3.0 mg Cr/kg. Acute toxicities of CrRC and CrFC were tested using ICR mice at the dose of 1.0-5.0 g/kg with a single oral gavage and observed for a period of 2 weeks. Biological activities results indicated that only CrRC and CrFC could decrease blood glucose level, reduce the activities of aspartate transaminase, alanine transaminase, alkaline phosphatase, and increase liver glycogen level. In acute toxicity study, LD(50) values for both CrRC and CrFC were above 5.0 g/kg. The minimum lethal dose for CrFC was above 5.0 g/kg, while that for CrRC was 1.0 g/kg. Anti-diabetic activity of those chromium complexes was not similar and their acute toxicities were also different. CrFC represent an optimal chromium supplement among those chromium complexes with potential therapeutic value to control blood glucose in diabetes and non-toxicity in acute toxicity.

  20. Enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant Escherichia coli

    PubMed Central

    Lacmata, Stephen Tamekou; Kuiate, Jules-Roger; Ding, Yamei; Xian, Mo; Liu, Huizhou; Boudjeko, Thaddée; Feng, Xinjun; Zhao, Guang

    2017-01-01

    Poly(3-hydroxypropionate) (P3HP) is a thermoplastic with great compostability and biocompatibility, and can be produced through several biosynthetic pathways, in which the glycerol pathway achieved the highest P3HP production. However, exogenous supply of vitamin B12 was required to maintain the activity of glycerol dehydratase, resulting in high production cost. To avoid the addition of VB12, we have previously constructed a P3HP biosynthetic route with β-alanine as intermediate, and the present study aimed to improve the P3HP production of this pathway. L-aspartate decarboxylase PanD was found to be the rate-limiting enzyme in the β-alanine pathway firstly. To improve the pathway efficiency, PanD was screened from four different sources (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Corynebacterium glutamicum). And PanD from C. glutamicum was found to have the highest activity, the P3HP production was improved in flask cultivation with this enzyme. To further improve the production, the host strain was screened and the culture condition was optimized. Under optimal conditions, production and content of P3HP reached to 10.2 g/L and 39.1% (wt/wt [cell dry weight]) in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without VB12. PMID:28253372

  1. R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport.

    PubMed

    Suzuki, Satomi; Nanatani, Kei; Abe, Keietsu

    2016-01-01

    The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)-a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport.

  2. A study on the involvement of GABA-transaminase in MCT induced pulmonary hypertension.

    PubMed

    Lingeshwar, Poorella; Kaur, Gurpreet; Singh, Neetu; Singh, Seema; Mishra, Akanksha; Shukla, Shubha; Ramakrishna, Rachumallu; Laxman, Tulsankar Sachin; Bhatta, Rabi Sankar; Siddiqui, Hefazat H; Hanif, Kashif

    2016-02-01

    Increased sympathetic nervous system (SNS) activity is associated with cardiovascular diseases but its role has not been completely explored in pulmonary hypertension (PH). Increased SNS activity is distinguished by elevated level of norepinephrine (NE) and activity of γ-Amino butyric acid Transminase (GABA-T) which degrades GABA, an inhibitory neurotransmitter within the central and peripheral nervous system. Therefore, we hypothesized that GABA-T may contribute in pathophysiology of PH by modulating level of GABA and NE. The effect of daily oral administration of GABA-T inhibitor, Vigabatrin (GVG, 50 and 75 mg/kg/day, 35 days) was studied following a single subcutaneous administration of monocrotaline (MCT, 60 mg/kg) in male SD rats. The pressure and hypertrophy of right ventricle (RV), oxidative stress, inflammation, pulmonary vascular remodelling were assessed after 35 days in MCT treated rats. The expression of GABA-T and HIF-1α was studied in lung tissue. The levels of plasma NE (by High performance liquid chromatography coupled with electrochemical detector; HPLC-ECD) and lung GABA (by liquid chromatography-mass spectrometry) were also estimated. GVG at both doses significantly attenuated increased in pressure (35.82 ± 4.80 mm Hg, p < 0.001; 28.37 ± 3.32 mm Hg, p < 0.001 respectively) and hypertrophy of RV, pulmonary vascular remodelling, oxidative stress and inflammation in lungs of MCT exposed rats. GVG also reduced the expression of GABA-T and HIF-1α in MCT treated rats. Increased NE level and decreased GABA level was also reversed by GVG in MCT exposed rats. GABA-T plays an important role in PH by modulating SNS activity and may be considered as a therapeutic target in PH.

  3. Structural insight into the inhibition of human kynurenine aminotransferase I/glutamine transaminase K.

    PubMed

    Han, Qian; Robinson, Howard; Cai, Tao; Tagle, Danilo A; Li, Jianyong

    2009-05-14

    Human kynurenine aminotransferase I (hKAT I) catalyzes the formation of kynurenic acid, a neuroactive compound. Here, we report three high-resolution crystal structures (1.50-1.55 A) of hKAT I that are in complex with glycerol and each of two inhibitors of hKAT I: indole-3-acetic acid (IAC) and Tris. Because Tris is able to occupy the substrate binding position, we speculate that this may be the basis for hKAT I inhibition. Furthermore, the hKAT/IAC complex structure reveals that the binding moieties of the inhibitor are its indole ring and a carboxyl group. Six chemicals with both binding moieties were tested for their ability to inhibit hKAT I activity; 3-indolepropionic acid and DL-indole-3-lactic acid demonstrated the highest level of inhibition, and as they cannot be considered as substrates of the enzyme, these two inhibitors are promising candidates for future study. Perhaps even more significantly, we report the discovery of two different ligands located simultaneously in the hKAT I active center for the first time.

  4. Structural Insight into the Inhibition of Human Kynurenine Aminotransferase I/Glutamine transaminase K∥

    PubMed Central

    Han, Qian; Robinson, Howard; Cai, Tao; Tagle, Danilo A.; Li, Jianyong

    2010-01-01

    Human kynurenine aminotransferase I (hKAT I) catalyzes the formation of kynurenic acid, a neuroactive compound. Here, we report three high-resolution crystal structures (1.50–1.55 Å) of hKAT I that are in complex with glycerol and each of two inhibitors of hKAT I: indole-3-acetic acid (IAC) and Tris. Because Tris is able to occupy the substrate binding position, we speculate that this may be the basis for hKAT I inhibition. Furthermore, the hKAT/IAC complex structure reveals that the binding moieties of the inhibitor are its indole ring and a carboxyl group. Six chemicals with both binding moieties were tested for their ability to inhibit hKAT I activity; 3-indolepropionic acid and DL-indole-3-lactic acid demonstrated the highest level of inhibition, and as they cannot be considered as substrates of the enzyme, these two inhibitors are promising candidates for future study. Perhaps even more significantly, we report the discovery of two different ligands located simultaneously in the hKAT I active center for the first time. PMID:19338303

  5. Oxidation of 3,4-dehydro-D-proline and other D-amino acid analogues by D-alanine dehydrogenase from Escherichia coli.

    PubMed

    Deutch, Charles E

    2004-09-15

    3,4-Dehydro-DL-proline is a toxic analogue of L-proline which has been useful in studying the uptake and metabolism of this key amino acid. When membrane fractions from Escherichia coli strain UMM5 (putA1::Tn5 proC24) lacking both L-proline dehydrogenase and L-Delta(1)-pyrroline-5-carboxylate reductase were incubated with 3,4-dehydro-DL-proline, pyrrole-2-carboxylate was formed. There was no enzyme activity with 3,4-dehydro-L-proline, but activity was restored after racemization of the substrate. Oxidation of 3,4-dehydro-DL-proline by membrane fractions from strain UMM5 was induced by growth in minimal medium containing D- or L-alanine, had a pH optimum of 9, and was competitively inhibited by D-alanine. An E. coli strain with no D-alanine dehydrogenase activity due to the dadA237 mutation was unable to oxidize either 3,4-dehydro-D-proline or D-alanine, as were spontaneous Dad(-) mutants of E. coli strain UMM5. Membrane fractions containing D-alanine dehydrogenase also catalyzed the oxidation of D-2-aminobutyrate, D-norvaline, D-norleucine, cis-4-hydroxy-D-proline, and DL-ethionine. These results indicate that d-alanine dehydrogenase is responsible for the residual 3,4-dehydro-DL-proline oxidation activity in putA proC mutants of E. coli and provide further evidence that this enzyme plays a general role in the metabolism of D-amino acids and their analogues.

  6. Glutamate dehydrogenase activator BCH stimulating reductive amination prevents high fat/high fructose diet-induced steatohepatitis and hyperglycemia in C57BL/6J mice

    PubMed Central

    Han, Seung Jin; Choi, Sung-E; Yi, Sang-A; Jung, Jong Gab; Jung, Ik-Rak; Shin, Maureen; Kang, Seok; Oh, Hyunhee; Kim, Hae Jin; Kim, Dae Jung; Kwon, Ji Eun; Choi, Cheol Soo; Lee, Kwan Woo; Kang, Yup

    2016-01-01

    Individuals with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) induced by high calorie western diet are characterized by enhanced lipogenesis and gluconeogenesis in the liver. Stimulation of reductive amination may shift tricarboxylic acid cycle metabolism for lipogenesis and gluconeogenesis toward glutamate synthesis with increase of NAD+/NADH ratio and thus, ameliorate high calorie diet-induced fatty liver and hyperglycemia. Stimulation of reductive amination through glutamate dehydrogenase (GDH) activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced both de novo lipogenesis and gluconeogenesis but increased the activities of sirtuins and AMP-activated kinase in primary hepatocytes. Long-term BCH treatment improved most metabolic alterations induced by high fat/high fructose (HF/HFr) diet in C57BL/6J mice. BCH prevented HF/HFr-induced fat accumulation and activation of stress/inflammation signals such as phospho-JNK, phospho-PERK, phospho-p38, and phospho-NFκB in liver tissues. Furthermore, BCH treatment reduced the expression levels of inflammatory cytokines such as TNF-α and IL-1β in HF/HFr-fed mouse liver. BCH also reduced liver collagen and plasma levels of alanine transaminase and aspartate transaminase. On the other hand, BCH significantly improved fasting hyperglycemia and glucose tolerance in HF/HFr-fed mice. In conclusion, stimulation of reductive amination through GDH activation can be used as a strategy to prevent high calorie western diet-induced NAFLD and T2D. PMID:27874078

  7. Oxidative stress induced by Cremophor EL is not accompanied by changes in NF-kappaB activation or iNOS expression.

    PubMed

    Gutiérrez, María Belén; Miguel, Beatriz San; Villares, Carmen; Gallego, Javier González; Tuñón, María Jesús

    2006-05-01

    The effects of polyoxyethylenglycerol triricinoleate 35 (Cremophor EL, CrEL) on markers of oxidative stress, nuclear factor kappa B (NF-kappaB) activation and inducible nitric oxide synthase (iNOS) expression were studied in the liver of male Wistar rats. Animals were randomly divided into three groups. Group Cr1 received, i.p., CrEL at 0.046ml/kg daily for 7 days, group Cr2 received CrEL at 0.33ml/kg and the controls were injected with CrEL vehicle (saline solution with 25% ethanol). Both alanine transaminase (ALT) and aspartate transaminase (AST) serum activities were significantly increased in the Cr2 group (+16% and +25%, respectively). AST activity was also higher in the Cr1 group when compared to control animals (+20%). The cytosolic concentration of thiobarbituric acid reactive substances (TBARS) increased in both groups of rats receiving CrEL (Cr1: +24%; Cr2: +33%). Reduced glutathione (GSH) concentration was not significantly modified at any of the CrEL doses, but both the hepatic concentration of oxidised glutathione (GSSG) (Cr1: +37%; Cr2: +84%) and the GSH/GSSG ratio (Cr1: -21%; Cr2: -45%) were significantly modified. CrEL induced no significant NF-kappaB activation, changes in p50 and p65 NF-kappaB subunits or induction of iNOS protein. Data obtained indicate that although high doses of CrEL cause oxidative stress, this is not enough to induce changes in NF-kappaB activation or iNOS expression.

  8. Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

    PubMed

    Börner, Tim; Grey, Carl; Adlercreutz, Patrick

    2016-08-01

    Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.

  9. Thermodynamics of Deca-alanine Folding in Water.

    PubMed

    Hazel, Anthony; Chipot, Christophe; Gumbart, James C

    2014-07-08

    The determination of the folding dynamics of polypeptides and proteins is critical in characterizing their functions in biological systems. Numerous computational models and methods have been developed for studying structure formation at the atomic level. Due to its small size and simple structure, deca-alanine is used as a model system in molecular dynamics (MD) simulations. The free energy of unfolding in vacuum has been studied extensively using the end-to-end distance of the peptide as the reaction coordinate. However, few studies have been conducted in the presence of explicit solvent. Previous results show a significant decrease in the free energy of extended conformations in water, but the α-helical state is still notably favored over the extended state. Although sufficient in vacuum, we show that end-to-end distance is incapable of capturing the full complexity of deca-alanine folding in water. Using α-helical content as a second reaction coordinate, we deduce a more descriptive free-energy landscape, one which reveals a second energy minimum in the extended conformations that is of comparable free energy to the α-helical state. Equilibrium simulations demonstrate the relative stability of the extended and α-helical states in water as well as the transition between the two states. This work reveals both the necessity and challenge of determining a proper reaction coordinate to fully characterize a given process.

  10. The effect of immunonutrition (glutamine, alanine) on fracture healing

    PubMed Central

    Küçükalp, Abdullah; Durak, Kemal; Bayyurt, Sarp; Sönmez, Gürsel; Bilgen, Muhammed S.

    2014-01-01

    Background There have been various studies related to fracture healing. Glutamine is an amino acid with an important role in many cell and organ functions. This study aimed to make a clinical, radiological, and histopathological evaluation of the effects of glutamine on fracture healing. Methods Twenty rabbits were randomly allocated into two groups of control and immunonutrition. A fracture of the fibula was made to the right hind leg. All rabbits received standard food and water. From post-operative first day for 30 days, the study group received an additional 2 ml/kg/day 20% L-alanine L-glutamine solution via a gastric catheter, and the control group received 2 ml/kg/day isotonic via gastric catheter. At the end of 30 days, the rabbits were sacrificed and the fractures were examined clinically, radiologically, and histopathologically in respect to the degree of union. Results Radiological evaluation of the control group determined a mean score of 2.5 according to the orthopaedists and 2.65 according to the radiologists. In the clinical evaluation, the mean score was 1.875 for the control group and 2.0 for the study group. Histopathological evaluation determined a mean score of 8.5 for the control group and 9.0 for the study group. Conclusion One month after orally administered glutamine–alanine, positive effects were observed on fracture healing radiologically, clinically, and histopathologically, although no statistically significant difference was determined.

  11. Formation of chloroform during chlorination of alanine in drinking water.

    PubMed

    Chu, Wen-Hai; Gao, Nai-Yun; Deng, Yang; Dong, Bing-Zhi

    2009-11-01

    Currently, dissolved nitrogenous organic matters in water, important precursors of disinfection by-products (DBPs), are of significant concern. This study was to explore the formation of chloroform (CF) during chlorination of alanine (Ala), an important nitrogenous organic compound commonly present in water sources. Our results indicated that the CF yield reached a maximum value of 0.143% at the molar ratio of chlorine atom to nitrogen atom (Cl/N)=1.0 over a Cl/N range of 0.2-5.0 (pH=7.0, reaction time=5d, and initial Ala=0.1mM). At an acidic-neutral condition (pH 4-7), the formation of CF was suppressed. However, the highest CF yield (0.227%) occurred at weakly alkaline condition (pH 8.0) (initial Ala=0.1mM, and Cl/N=1.0). The increase of Br(-) in water can increase total trihalomethanes (THMs) and bromo-THMs. However, the bromo-THMs level reached a plateau at Br(-)/Cl>0.04. Finally, based on the computation of frontier electron density and identification and measurement of key intermediates during Ala chlorination, we proposed a formation pathway of CF from Ala chlorination: Ala-->monochloro-N-alanine (MC-N-Ala)-->acetaldehyde (AAld)-->monochloroacetaldehyde acetaldehyde (MCAld)-->dichloroacetaldehyde (DCAld)-->trichloroacetaldehyde (TCAld)-->CF.

  12. Toxicity and biodistribution of activated and non-activated intravenous iron oxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Tate, J. A.; Ogden, J. A.; Strawbridge, R. R.; Pierce, Z. E.; Hoopes, P. J.

    2009-02-01

    The use of nanoparticles in medical treatment has prompted the question of their safety. In this study, the pathophysiology and biodistribution of three different concentrations of intravenously-delivered dextran-coated Fe3O4 iron oxide nanoparticles (IONP) were evaluated in mice. Some groups of mice were exposed to an AC magnetic field (AMF) at levels comparable with those proposed for cancer treatments. Iron biodistribution analysis for both AMF and non-AMF treated mice was performed for all three concentrations used (.6 mg Fe/mouse, 1.8 mg Fe/mouse, and 5.6 mg Fe/mouse). Blood urea nitrogen, alanine transaminase, alkaline phosphatase, total serum protein, and creatinine were also assessed at 4 hours, 7 days, and 14 days post-injection. Histological analysis of lung, spleen, heart, liver, and kidney tissue was conducted at 7 and 14 days post-injection. Prussian blue and H&E stains were used to histomorphometrically assess iron content in the tissues studied. Preliminary results demonstrate small temporary elevation in liver enzymes and hepatocyte vacuolization at all iron concentrations studied. Liver and spleen were the primary sites of IONP deposition. None of the animals demonstrated systemic or local toxicity or illness, with or without AMF activation.

  13. An archaeal glutamate decarboxylase homolog functions as an aspartate decarboxylase and is involved in β-alanine and coenzyme A biosynthesis.

    PubMed

    Tomita, Hiroya; Yokooji, Yuusuke; Ishibashi, Takuya; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-03-01

    β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5'-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4'-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.

  14. An Archaeal Glutamate Decarboxylase Homolog Functions as an Aspartate Decarboxylase and Is Involved in β-Alanine and Coenzyme A Biosynthesis

    PubMed Central

    Tomita, Hiroya; Yokooji, Yuusuke; Ishibashi, Takuya; Imanaka, Tadayuki

    2014-01-01

    β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5′-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4′-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms. PMID:24415726

  15. Recurrent truncating mutations in alanine-glyoxylate aminotransferase gene in two South Indian families with primary hyperoxaluria type 1 causing later onset end-stage kidney disease

    PubMed Central

    Dutta, A. K.; Paulose, B. K.; Danda, S.; Alexander, S.; Tamilarasi, V.; Omprakash, S.

    2016-01-01

    Primary hyperoxaluria type 1 is an autosomal recessive inborn error of metabolism due to liver-specific peroxisomal enzyme alanine-glyoxylate transaminase deficiency. Here, we describe two unrelated patients who were diagnosed to have primary hyperoxaluria. Homozygous c.445_452delGTGCTGCT (p.L151Nfs*14) (Transcript ID: ENST00000307503; human genome assembly GRCh38.p2) (HGMD ID CD073567) mutation was detected in both the patients and the parents were found to be heterozygous carriers. Our patients developed end-stage renal disease at 23 years and 35 years of age. However, in the largest series published from OxalEurope cohort, the median age of end-stage renal disease for null mutations carriers was 9.9 years, which is much earlier than our cases. Our patients had slower progressions as compared to three unrelated patients from North India and Pakistan, who had homozygous c.302T>C (p.L101P) (HGMD ID CM093792) mutation in exon 2. Further, patients need to be studied to find out if c.445_452delGTGCTGCT mutation represents a founder mutation in Southern India. PMID:27512303

  16. Controlled radical polymerization of an acrylamide containing L-alanine moiety via ATRP.

    PubMed

    Rafiee, Zahra

    2016-02-01

    Homopolymerization of an optically active acrylamide having an amino acid moiety in the side chain, N-acryloyl-L-alanine (AAla) was carried out via atom transfer radical polymerization (ATRP) at room temperature using 2-hydroxyethyl-2'-methyl-2'-bromopropionate (HMB) or sodium-4-(bromomethyl)benzoate (SBB) as initiator in pure water, methanol/water mixture and pure methanol solvents. The polymerization reaction resulted in the optically active biocompatible amino acid-based homopolymer in good yield with narrow molecular weight distribution. The number average molecular weight increased with conversion and polydispersity was low. The structure and molecular weight of synthesized polymer were characterized by (1)H NMR, FT-IR spectroscopic techniques and size-exclusion chromatography.

  17. The Helical Alanine Controversy: An (Ala)6 Insertion Dramatically Increases Helicity

    PubMed Central

    Lin, Jasper C.; Barua, Bipasha

    2013-01-01

    Employing chemical shift melts and hydrogen/deuterium exchange NMR techniques, we have determined the stabilization of the Trp-cage miniprotein due to multiple alanine insertions within the N-terminal α-helix. Alanine is shown to be uniquely helix-stabilizing and this stabilization is reflected in the global fold stability of the Trp-cage. The associated free energy change per alanine can be utilized to calculate the alanine propagation value. From the Lifson–Roig formulation, the calculated value (wAla = 1.6) is comparable to those obtained for short, solubilized, alanine-rich helices and is much larger than the values obtained by prior host–guest techniques or in N-terminally templated helices and peptides bearing long contiguous strings of alanines with no capping or solubilizing units present. PMID:15493925

  18. Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo0352, D-alanine-D-alanine ligase A, from Xanthomonas oryzae pv. oryzae.

    PubMed

    Doan, Thanh Thi Ngoc; Kim, Jin-Kwang; Kim, Hyesoon; Ahn, Yeh-Jin; Kim, Jeong-Gu; Lee, Byoung-Moo; Kang, Lin-Woo

    2008-12-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. D-Alanine-D-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in D-alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of D-alanyl-D-alanine from two D-alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 A resolution and belonged to the primitive tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 83.0, c = 97.6 A. There is one molecule in the asymmetric unit, with a corresponding V(M) of 1.88 A(3) Da(-1) and a solvent content of 34.6%. The initial structure was determined by molecular replacement using D-alanine-D-alanine ligase from Staphylococcus aureus (PDB code 2i87) as a template model.

  19. Precision and sensitivity of the measurement of 15N enrichment in D-alanine from bacterial cell walls using positive/negative ion mass spectrometry

    NASA Technical Reports Server (NTRS)

    Tunlid, A.; Odham, G.; Findlay, R. H.; White, D. C.

    1985-01-01

    Sensitive detection of cellular components from specific groups of microbes can be utilized as 'signatures' in the examination of microbial consortia from soils, sediments or biofilms. Utilizing capillary gas chromatography/mass spectrometry and stereospecific derivatizing agents, D-alanine, a component localized in the prokaryotic (bacterial) cell wall, can be detected reproducibly. Enrichments of D-[15N]alanine determined in E. coli grown with [15N]ammonia can be determined with precision at 1.0 atom%. Chemical ionization with methane gas and the detection of negative ions (M - HF)- and (M - F or M + H - HF)- formed from the heptafluorobutyryl D-2 butanol ester of D-alanine allowed as little as 8 pg (90 fmol) to be detected reproducibly. This method can be utilized to define the metabolic activity in terms of 15N incorporation at the level of 10(3)-10(4) cells, as a function of the 15N-14N ratio.

  20. (2R,1'S,2'R)- and (2S,1'S,2'R)-3-[2-Mono(di,tri)fluoromethylcyclopropyl]alanines and their incorporation into hormaomycin analogues

    PubMed Central

    Kozhushkov, Sergei I; Yufit, Dmitrii S; Grosse, Christian; Kaiser, Marcel

    2014-01-01

    Summary Efficient and scalable syntheses of enantiomerically pure (2R,1'S,2'R)- and (2S,1'S,2'R)-3-[2-mono(di,tri)fluoromethylcyclopropyl]alanines 9a–c, as well as allo-D-threonine (4) and (2S,3R)-β-methylphenylalanine (3), using the Belokon' approach with (S)- and (R)-2-[(N-benzylprolyl)amino]benzophenone [(S)- and (R)-10] as reusable chiral auxiliaries have been developed. Three new fluoromethyl analogues of the naturally occurring octadepsipeptide hormaomycin (1) with (fluoromethylcyclopropyl)alanine moieties have been synthesized and subjected to preliminary tests of their antibiotic activity. PMID:25550751

  1. Role of the "helix clamp" in HIV-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis.

    PubMed

    Beard, W A; Minnick, D T; Wade, C L; Prasad, R; Won, R L; Kumar, A; Kunkel, T A; Wilson, S H

    1996-05-24

    Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the "helix clamp" (Hermann, T., Meier, T., Gotte, M., and Heumann, H. (1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two alpha-helices (alpha H and alpha I) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in alpha H make important interactions with nucleic acid and influence frameshift fidelity. To define the role of alpha I (residues 278-286) during catalytic cycling, we performed systematic site-directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine. Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized. In contrast to alanine mutants of alpha H, alanine substitution in alpha I did not have a significant effect on template.primer (T.P) binding as revealed by a lack of an effect on Km, T.P, Ki for 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, koff, T.P and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of alpha I lowered the apparent activity of every mutant relative to wild-type enzyme. Titration of two mutants exhibiting the lowest activity with T.P (L282A and R284A) demonstrated that these mutant enzymes could bind T.P stoichiometrically and tightly. In contrast, active site concentrations determined from "burst" experiments suggest that the lower activity is due to a smaller populations of enzyme bound productively to T.P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent. In contrast, interactions between several residues of alpha H and the DNA minor groove, 3-5 nucleotides from the 3

  2. Neonatal taurine and alanine modulate anxiety-like behavior and decelerate cortical spreading depression in rats previously suckled under different litter sizes.

    PubMed

    Francisco, Elian da Silva; Guedes, Rubem Carlos Araújo

    2015-11-01

    The amino acids taurine and alanine play a role in several physiological processes, including behavior and the electrical activity of the brain. In this study, we investigated the effect of treatment with taurine or alanine on anxiety-like behavior and the excitability-dependent phenomenon known as cortical spreading depression (CSD), using rats suckled in litters with 9 and 15 pups (groups L9 and L15). From postnatal days 7 to 27, the animals received per gavage 300 mg/kg/day of taurine or alanine or both. At 28 days, we tested the animals in the elevated plus maze, and at 33-35 days, we recorded CSD and analyzed its velocity of propagation, amplitude, and duration. Compared with water-treated controls, the L9 groups treated with taurine or alanine displayed anxiolytic behavior (higher number of entries in the open arms; p < 0.05), and reduced CSD velocity (p < 0.001). The effect of both amino acids on CSD was also found in the L15 groups and in five additional L9 groups (naïve, water, taurine, alanine, or both) treated at adulthood (90-110 days). The L15 condition resulted in smaller durations and higher CSD velocities compared with the L9 condition. Besides reinforcing previous evidence of behavioral modulation by taurine and alanine, our data are the first confirmation that treatment with these amino acids decelerates CSD regardless of lactation conditions (normal versus unfavorable lactation) or age at amino acid administration (young versus adult). The results suggest a modulating role for both amino acids on anxiety behavior and neuronal electrical activity.

  3. Asymmetric synthesis of aromatic β-amino acids using ω-transaminase: Optimizing the lipase concentration to obtain thermodynamically unstable β-keto acids.

    PubMed

    Mathew, Sam; Jeong, Seong-Su; Chung, Taeowan; Lee, Sang-Hyeup; Yun, Hyungdon

    2016-01-01

    Synthesized aromatic β-amino acids have recently attracted considerable attention for their application as precursors in many pharmacologically relevant compounds. Previous studies on asymmetric synthesis of aromatic β-amino acids using ω-transaminases could not be done efficiently due to the instability of β-keto acids. In this study, a strategy to circumvent the instability problem of β-keto acids was utilized to generate β-amino acids efficiently via asymmetric synthesis. In this work, thermodynamically stable β-ketoesters were initially converted to β-keto acids using lipase, and the β-keto acids were subsequently aminated using ω-transaminase. By optimizing the lipase concentration, we successfully overcame the instability problem of β-keto acids and enhanced the production of β-amino acids. This strategy can be used as a general approach to efficiently generate β-amino acids from β-ketoesters.

  4. Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4'-substituted acetophenones, and follows Swain-Lupton parameterisation.

    PubMed

    Cassimjee, Karim Engelmark; Humble, Maria Svedendahl; Land, Henrik; Abedi, Vahak; Berglund, Per

    2012-07-28

    For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

  5. Free Radical Scavenging Activity of Calotropis gigantea on Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Rathod, N. R.; Raghuveer, I.; Chitme, H. R.; Chandra, R.

    2009-01-01

    Swarnabhasma, an Ayurvedic preparation containing Calotropis gigantea R. Br. (Asclepiadaceae) is extensively used by Ayurvedic physicians for treatment of diabetes mellitus, bronchial asthma, rheumatoid arthritis and nervous disorders. In the present study, we report the effect of chloroform extracts of Calotropis gigantea leaf and flower on free radical scavenging activity, and lipid profile in streptozotozin-induced diabetic rats. The lipid peroxidation, superoxide dismutase, and catalase were measured in liver homogenate and serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, alkaline phosphatase, lipid profile were measured in blood serum. Administration of single dose of streptozotozin (55 mg/kg, i.p.) caused significant increases in lipid peroxidation, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, alkaline phosphatase, cholesterol and triglyceride levels, while superoxide dismutase and catalase levels were significantly decreased. Further, administration of chloroform extracts of Calotropis gigantea leaf and flower to streptozotocin-induced diabetes rats at a dose of 10, 20 and 50 mg/kg orally for 27 d lead to a significant decrease in lipid peroxidation, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, alkaline phosphatase, cholesterol and triglyceride levels. Consequently, superoxide dismutase and catalase levels were significantly increased. Glibenclamide was used as a positive control (10 mg/kg). It was observed that the effect of chloroform extracts of Calotropis gigantea on alkaline phosphatase, cholesterol, superoxide dismutase, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, levels are comparable to that of those produced by the positive control. PMID:20376213

  6. Prolonged continuous intravenous infusion of the dipeptide L-alanine- L-glutamine significantly increases plasma glutamine and alanine without elevating brain glutamate in patients with severe traumatic brain injury

    PubMed Central

    2014-01-01

    Introduction Low plasma glutamine levels are associated with worse clinical outcome. Intravenous glutamine infusion dose- dependently increases plasma glutamine levels, thereby correcting hypoglutaminemia. Glutamine may be transformed to glutamate which might limit its application at a higher dose in patients with severe traumatic brain injury (TBI). To date, the optimal glutamine dose required to normalize plasma glutamine levels without increasing plasma and cerebral glutamate has not yet been defined. Methods Changes in plasma and cerebral glutamine, alanine, and glutamate as well as indirect signs of metabolic impairment reflected by increased intracranial pressure (ICP), lactate, lactate-to-pyruvate ratio, electroencephalogram (EEG) activity were determined before, during, and after continuous intravenous infusion of 0.75 g L-alanine-L-glutamine which was given either for 24 hours (group 1, n = 6) or 5 days (group 2, n = 6) in addition to regular enteral nutrition. Lab values including nitrogen balance, urea and ammonia were determined daily. Results Continuous L-alanine-L-glutamine infusion significantly increased plasma and cerebral glutamine as well as alanine levels, being mostly sustained during the 5 day infusion phase (plasma glutamine: from 295 ± 62 to 500 ± 145 μmol/ l; brain glutamine: from 183 ± 188 to 549 ± 120 μmol/ l; plasma alanine: from 327 ± 91 to 622 ± 182 μmol/ l; brain alanine: from 48 ± 55 to 89 ± 129 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test). Plasma glutamate remained unchanged and cerebral glutamate was decreased without any signs of cerebral impairment. Urea and ammonia were significantly increased within normal limits without signs of organ dysfunction (urea: from 2.7 ± 1.6 to 5.5 ± 1.5 mmol/ l; ammonia: from 12 ± 6.3 to 26 ± 8.3 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test). Conclusions High dose L-alanine-L-glutamine infusion (0

  7. First-principles study of fluorination of L-Alanine

    NASA Astrophysics Data System (ADS)

    Sreepad, H. R.; Ravi, H. R.; Ahmed, Khaleel; Dayananda, H. M.; Umakanth, K.; Manohara, B. M.

    2013-02-01

    First-principles calculations based on Density Functional Theory have been done on effect of fluorination of an important amino acid - L-Alanine. Its structure has been simulated. The unit cell is orthorhombic with lattice parameters a=5.90Å, b=13.85Å and c=5.75Å with volume 470 (Å)3. Bond lengths and bond angles have been estimated. Electronic Density of States calculations show that the material has a band gap of 4.47eV. Electronic band structure indicates that the material can be effectively used for NLO applications. The electronic contribution to the dielectric constant has been calculated and its average value comes out to be 2.165.

  8. Alanine Aminotransferase Variants Conferring Diverse NUE Phenotypes in Arabidopsis thaliana

    PubMed Central

    McAllister, Chandra H.; Good, Allen G.

    2015-01-01

    Alanine aminotransferase (AlaAT, E.C. 2.6.1.2), is a pyridoxal-5’-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT, in contrast to other AlaAT enzyme homologues in creating this phenotype has not yet been explored. To address the role of AlaAT in NUE, AlaAT variants from diverse sources and different subcellular locations, were expressed in the wild-type Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background in various N environments. The analysis and comparison of both the physical and physiological properties of AlaAT over-expressing transgenic plants demonstrated significant differences between plants expressing the different AlaAT enzymes under different external conditions. This analysis indicates that the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed. PMID:25830496

  9. Alanine aminotransferase variants conferring diverse NUE phenotypes in Arabidopsis thaliana.

    PubMed

    McAllister, Chandra H; Good, Allen G

    2015-01-01

    Alanine aminotransferase (AlaAT, E.C. 2.6.1.2), is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT, in contrast to other AlaAT enzyme homologues in creating this phenotype has not yet been explored. To address the role of AlaAT in NUE, AlaAT variants from diverse sources and different subcellular locations, were expressed in the wild-type Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background in various N environments. The analysis and comparison of both the physical and physiological properties of AlaAT over-expressing transgenic plants demonstrated significant differences between plants expressing the different AlaAT enzymes under different external conditions. This analysis indicates that the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed.

  10. [The prevalence of hepatitis C antibodies among volunteer blood donors with elevated blood transaminase and antibodies to the B virus core antigen].

    PubMed

    Gavilán Carrasco, J C; González Santos, P; Rosario Díaz, E

    1996-05-01

    The use of non-specific markers before 1989 (increased serum transaminase values and antibodies to hepatitis B core antigen) as a screening method for blood donors in an attempt to decrease the incidence of post-transfusional non-A non-B hepatitis (currently hepatitis C virus) was a matter of controversy. To determine the impact of the use of these markers on the detection of blood donors infected with hepatitis C virus, a prospective study was undertaken in Málaga (1988-1989) with 5,003 volunteer donors with two objectives: a) to know the prevalence of these non-specific markers (anti-HBc and increased serum transaminase) and antibodies to HCV (anti-C100) in our blood donor population; b) to determine whether the presence of some of these non specific markers in blood donors was associated with a higher rate of virus C infection. The prevalence of antibodies to HCV in blood donors with increased serum transaminase and/or anti-HBc was significantly higher than the prevalence found among the general blood donor population.

  11. Ste20-related proline/alanine-rich kinase: A novel regulator of intestinal inflammation

    PubMed Central

    Yan, Yutao; Merlin, Didier

    2008-01-01

    Recently, inflammatory bowel disease (IBD) has been the subject of considerable research, with increasing attention being paid to the loss of intestinal epithelial cell barrier function as a mechanism of pathogenesis. Ste20-related proline/alanine-rich kinase (SPAK) is involved in regulating barrier function. SPAK is known to interact with inflammation-related kinases (such as p38, JNK, NKCC1, PKCtheta;, WNK and MLCK), and with transcription factor AP-1, resulting in diverse biological phenomena, including cell differentiation, cell transformation and proliferation, cytoskeleton rearrangement, and regulation of chloride transport. This review examines the involvement of Ste20-like kinases and downstream mitogen-activated protein kinases (MAPKs) pathways in the pathogenesis and control of intestinal inflammation. The primary focus will be on the molecular features of intestinal inflammation, with an emphasis on the interaction between SPAK and other molecules, and the effect of these interactions on homeostatic maintenance, cell volume regulation and increased cell permeability in intestinal inflammation. PMID:18985800

  12. Gliotoxicity of the cyanotoxin, β-methyl-amino-L-alanine (BMAA).

    PubMed

    Chiu, Alexander S; Gehringer, Michelle M; Braidy, Nady; Guillemin, Gilles J; Welch, Jeffrey H; Neilan, Brett A

    2013-01-01

    The amino acid variant β-methyl-amino-L-alanine (BMAA) has long been associated with the increased incidence and progression of the amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). Previous studies have indicated that BMAA damages neurons via excitotoxic mechanisms. We have challenged rat olfactory ensheathing cells (OECs) with exogenous BMAA and found it to be cytotoxic. BMAA also induces a significant increase in Ca2+ influx, enhanced production of reactive oxygen species (ROS), and disrupts mitochondrial activity in OECs. This is the first study investigating BMAA toxicity using pure glial cells. These findings align BMAA with the three proposed mechanisms of degeneration in ALS, those being non-cell autonomous death, excitotoxicity and mitochondrial dysfunction.

  13. Experimental and DFT computational studies of L-alanine cadmium chloride crystals

    NASA Astrophysics Data System (ADS)

    Ignatius, I. Cicili; Dheivamalar, S.; Kirubavathi, K.; Selvaraju, K.

    2016-05-01

    In this work, we report the combined experimental and theoretical study on molecular structure and vibrational spectra of nonlinear optical crystal L-alanine cadmium chloride (LACC). The single X-ray diffraction studies have revealed that the compound crystallizes in monoclinic system C2 space group with cell parameters a = 16.270, b = 7.358, c = 7.887 and Z = 4. FTIR and Raman spectra of the nonlinear optical materials LACC have been recorded and analyzed. The optimized geometric bond length and bond angles are obtained with the help of density functional theory (DFT) (B3LYP) calculation. The optimized geometric bond lengths and bond angles obtained by using DFT show good agreement with the experimental data. Using the natural bond orbital analysis the electronic effect and hydrogen bonding were confirmed. The HOMO-LUMO energy gap and the first order hyperpolarizability were calculated and it supports the nonlinear optical activity of LACC crystal.

  14. Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli.

    PubMed

    Zhou, Li; Deng, Can; Cui, Wen-Jing; Liu, Zhong-Mei; Zhou, Zhe-Min

    2016-01-01

    L-Alanine has important applications in food, pharmaceutical and veterinary and is used as a substrate for production of engineered thermoplastics. Microbial fermentation could reduce the production cost and promote the application of L-alanine. However, the presence of L-alanine significantly inhibit cell growth rate and cause a decrease in the ultimate L-alanine productivity. For efficient L-alanine production, a thermo-regulated genetic switch was designed to dynamically control the expression of L-alanine dehydrogenase (alaD) from Geobacillus stearothermophilus on the Escherichia coli B0016-060BC chromosome. The optimal cultivation conditions for the genetically switched alanine production using B0016-060BC were the following: an aerobic growth phase at 33 °C with a 1-h thermo-induction at 42 °C followed by an oxygen-limited phase at 42 °C. In a bioreactor experiment using the scaled-up conditions optimized in a shake flask, B0016-060BC accumulated 50.3 g biomass/100 g glucose during the aerobic growth phase and 96 g alanine/100 g glucose during the oxygen-limited phase, respectively. The L-alanine titer reached 120.8 g/l with higher overall and oxygen-limited volumetric productivities of 3.09 and 4.18 g/l h, respectively, using glucose as the sole carbon source. Efficient cell growth and L-alanine production were reached separately, by switching cultivation temperature. The results revealed the application of a thermo-regulated strategy for heterologous metabolic production and pointed to strategies for improving L-alanine production.

  15. Effect of adrenergic agonists and antagonists on alanine amino transferase, fructose-1:6-bisphosphatase and glucose production in hepatocytes.

    PubMed

    Begum, N A; Datta, A G

    1992-08-18

    Using rat hepatocytes we confirmed our previous results that glucagon and beta-adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (alpha-agonist and alpha-antagonist respectively) also alpha-antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistent in hepatocyte system. Fructose-1:6-bisphosphatase (Fru-P2-ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other beta-adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P2-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P2-ase by glucagon is purely a b-receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known alpha-agonist and antagonists are behaving as beta-agonists. Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol. The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in

  16. Hepatoprotective activity of Moringa oleifera against cadmium toxicity in rats

    PubMed Central

    Toppo, Reetu; Roy, Birendra Kumar; Gora, Ravuri Halley; Baxla, Sushma Lalita; Kumar, Prabhat

    2015-01-01

    Aim: The present investigation has been conducted to evaluate the hepatoprotective activity of Moringa oleifera against cadmium-induced toxicity in rats. Materials and Methods: For this study, 18 Wistar albino rats were taken. Control group, Group I rats were given cadmium chloride @ 200 ppm per kg and Group II rats were treated with M. oleifera extract @ 500 mg/kg along with cadmium chloride @ 200 ppm per kg (daily oral for 28 days). On 29th day, animals were slaughtered and various parameters were determined. Serum biomarkers, oxidative stress parameters, histomorphological examination were carried out with estimation of cadmium concentration in liver tissues. Results: Oral administration of cadmium chloride @ 200 ppm/kg for 28 days resulted in a significant increase in aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), significant (p≤0.01) increase of lipid peroxidation (LPO) and decrease in superoxide dismutase (SOD), and increase in cadmium accumulation in liver. Treatment with M. oleifera @ 500 mg/kg significantly (p<0.01) decreased the elevated ALP, AST, ALT, LPO levels and increase in SOD levels, and as compared to cadmium chloride treated group. However, there was no significant difference in cadmium concentration in liver when compared with cadmium chloride treated group. Conclusion: The study conclude that supplementation of M. oleifera (500 mg/kg), daily oral for 28 days has shown protection against cadmium-induced hepatotoxicity. PMID:27047130

  17. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  18. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  19. Polymerization of alanine in the presence of a non-swelling montmorillonite

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.; Lahav, N.

    1977-01-01

    Alanine, starting from alanine-adenylate, has been polymerized in the presence of non-swelling Al-montmorillonite. The yield of polymerization is much lower than that obtained in the presence of swelling Na-montmorillonite. The possibility that the changing interlayer spacing in Na-montmorillonite might be responsible for its catalytic properties, is discussed.

  20. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  1. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  2. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-, salt. 721.520 Section 721.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for...

  3. Glutathione peroxidase 1 deficiency attenuates concanavalin A-induced hepatic injury by modulation of T-cell activation

    PubMed Central

    Lee, D H; Son, D J; Park, M H; Yoon, D Y; Han, S B; Hong, J T

    2016-01-01

    Concanavalin A (Con A)-induced hepatitis model is well-established experimental T cell-mediated liver disease. Reactive oxygen species (ROS) is associated with T-cell activation and proliferation, but continued ROS exposure induces T-cell hyporesponsiveness. Because glutathione peroxidase 1 (Gpx1) is an antioxidant enzyme and is involved in T-cell development, we investigated the role of Gpx1 during Con A-induced liver injury in Gpx1 knockout (KO) mice. Male wild-type (WT) mice and Gpx1 KO mice were intravenously injected with Con A (10 mg/kg), and then killed after 8 h after Con A injection. Serum levels of aspartate transaminase and alanine transaminase were measured to assess hepatic injury. To identify that Gpx1 affects T cell-mediated inflammation, we pretreated Gpx1 inhibitor to Human Jurkat T cells then treated Con A. Con A-induced massive liver damage in WT mice but its damage was attenuated in Gpx1 KO mice. Con A-induced Th1 cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin (IL)-2 were also decreased in the liver and spleen of Gpx1 KO mice compared with WT mice. In Jurkat T cells, Con A-induced mRNA levels of IL-2, IFN-γ and TNF-α were downregulated by pretreatment of Gpx inhibitor, mercaptosuccinic acid. We also observed that Gpx1 KO mice showed increasing oxidative stress in the liver and spleen compared with WT mice. These results suggest that Gpx1 deficiency attenuates Con A-induced liver injury by induction of T-cell hyporesponsiveness through chronic ROS exposure. PMID:27124582

  4. How similar is the electronic structures of β-lactam and alanine?

    NASA Astrophysics Data System (ADS)

    Chatterjee, Subhojyoti; Ahmed, Marawan; Wang, Feng

    2016-02-01

    The C1s spectra of β-lactam i.e. 2-azetidinone (C3H5NO), a drug and L-alanine (C3H7NO2), an amino acid, exhibit striking similarities, which may be responsible for the competition between 2-azetidinone and the alanyl-alanine moiety in biochemistry. The present study is to reveal the degree of similarities and differences between their electronic structures of the two model molecular pairs. It is found that the similarities in C1s and inner valence binding energy spectra are due to their bonding connections but other properties such as ring structure (in 2-azetidinone) and chiral carbon (alanine) can be very different. Further, the inner valence region of ionization potential greater than 18 eV for 2-azetidinone and alanine is also significantly similar. Finally the strained lactam ring exhibits more chemical reactivity measured at all non-hydrogen atoms by Fukui functions with respect to alanine.

  5. Evaluation of the effects of hydroalcoholic extract of Berberis vulgaris root on the activity of liver enzymes in male hypercholesterolemic rats

    PubMed Central

    Taheri, Soheila; Zarei, Ali; Changizi Ashtiyani, Saeed; Rezaei, Azam; Zaheiri, Saeed

    2012-01-01

    Objectives: Hyperlipidemia can cause a variety of diseases such as atherosclerosis, diabetes, and fatty liver which is followed by increased liver enzymes. Since Berberis vulgaris (B. vulgaris) root possesses antioxidant properties, the present study was conducted to investigate the effect of its extract on the activity of liver enzymes in rats. Materials and Methods: In this experimental study, sixty Wistar rats were selected and allocated to six groups of ten each. The control group received a normal diet and the sham group received a fatty diet while the other groups including experimental groups received a fatty diet and the alcoholic extract of B. vulgaris at minimum (75 mg/kg), moderate (150 mg/kg), and maximum (300 mg/kg) doses by intraperitoneal injection (i.p.) or oral atorvastatin (10 mg /kg) with a fatty diet. At the end of this 21-day period, blood samples were drawn and the levels of the intended factors were measured. Data were analyzed using SPSS software version 11.5. Results: The comparison of the obtained results showed that the levels of alanine transaminase (ALT) and alkaline phosphatase (ALP) enzymes in the sham group that only received fatty food increased (p≤0.05), whereas in the treatment groups receiving B. vulgaris extract as well as in the group receiving Atorvastatin, these enzymes significantly decreased; however, no significant changes were observed in aspartate transaminase (AST) levels. Conclusion: Noticing the antioxidant properties of B. vulgaris root extract and its effects on reducing the activity of liver enzymes, the extract of this plant can be a good choice for improving the function of liver. PMID:25050245

  6. Alanine mutagenesis of the primary antigenic escape residue cluster, c1, of apical membrane antigen 1.

    PubMed

    Dutta, Sheetij; Dlugosz, Lisa S; Clayton, Joshua W; Pool, Christopher D; Haynes, J David; Gasser, Robert A; Batchelor, Adrian H

    2010-02-01

    Antibodies against apical membrane antigen 1 (AMA1) inhibit invasion of Plasmodium merozoites into red cells, and a large number of single nucleotide polymorphisms on AMA1 allow the parasite to escape inhibitory antibodies. The availability of a crystal structure makes it possible to test protein engineering strategies to develop a monovalent broadly reactive vaccine. Previously, we showed that a linear stretch of polymorphic residues (amino acids 187 to 207), localized within the C1 cluster on domain 1, conferred the highest level of escape from inhibitory antibodies, and these were termed antigenic escape residues (AER). Here we test the hypothesis that immunodampening the C1 AER will divert the immune system toward more conserved regions. We substituted seven C1 AER of the FVO strain Plasmodium falciparum AMA1 with alanine residues (ALA). The resulting ALA protein was less immunogenic than the native protein in rabbits. Anti-ALA antibodies contained a higher proportion of cross-reactive domain 2 and domain 3 antibodies and had higher avidity than anti-FVO. No overall enhancement of cross-reactive inhibitory activity was observed when anti-FVO and anti-ALA sera were compared for their ability to inhibit invasion. Alanine mutations at the C1 AER had shifted the immune response toward cross-strain-reactive epitopes that were noninhibitory, refuting the hypothesis but confirming the importance of the C1 cluster as an inhibitory epitope. We further demonstrate that naturally occurring polymorphisms that fall within the C1 cluster can predict escape from cross-strain invasion inhibition, reinforcing the importance of the C1 cluster genotype for antigenic categorization and allelic shift analyses in future phase 2b trials.

  7. Essential oil of Artemisia vestita exhibits potent in vitro and in vivo antibacterial activity: Investigation of the effect of oil on biofilm formation, leakage of potassium ions and survival curve measurement.

    PubMed

    Yang, Chang; Hu, Dong-Hui; Feng, Yan

    2015-10-01

    The aim of the present study was to investigate the chemical composition of the essential oil of Artemisia vestita and to determine the antibacterial activity of the essential oil and its two major components, grandisol and 1,8‑cineole, against certain respiratory infection‑causing bacterial strains, in vitro and in vivo. The chemical composition of the essential oil was analyzed using gas chromatography‑mass spectrometry. A micro‑well dilution method was used to determine the minimum inhibition concentration (MIC) values of the essential oil and its major constituents. A model of Streptococcus pyogenes infection in mice was used to determine its in vivo activities. Lung and blood samples were obtained to assess bacterial cell counts. Toxicity evaluation of the essential oil and its components was completed by performing biochemical analysis of the serum, particularly monitoring aspartate transaminase, alanine transaminase, urea and creatinine. The essential oil exhibited potent antibacterial activity, whereas the two major constituents were less potent. The essential oil exhibited MIC values between 20 and 80 µg/ml, while the values of the two constituents were between 130 and 200 µg/ml. Scanning electron microscopy results demonstrated that the essential oil inhibited biofilm formation and altered its architecture. Survival curves indicated that the essential oil led to a reduction in the viability of different bacteria. The essential oil also induced significant leakage of potassium ions from S. pyogenes. The essential oil (100 µg/mouse) and grandisol (135 µg/mouse) significantly reduced the number of viable bacterial cells in the lungs (P<0.01). However, intake of 100 µg/mouse of essential oil or grandisol 135 µg/mouse once or twice each day for 9 days did not produce any toxic effects in the mice. In conclusion, the in vitro and in vivo results suggested that the essential oil of A. vestita and one of its major constituents, grandisol, can

  8. Alanine synthesis from glyceraldehyde and ammonium ion in aqueous solution

    NASA Technical Reports Server (NTRS)

    Weber, A. L.

    1985-01-01

    The formation of alanine (ala) form C(14)-glyceraldehyde and ammonium phosphate in the presence or absence of a thiol is reported. At ambient temperature, ala synthesis was six times more rapid in the presence of 3-mercaptopropionic acid than in its absence (0.6 and 0.1 percent, respectively, after 60 days). Similarly, the presence of another thiol, N-acetylcysteinate, increased the production of ala, as well as of lactate. The reaction pathway of thiol-catalyzed synthesis of ala, with the lactic acid formed in a bypath, is suggested. In this, dehydration of glyceraldehyde is followed by the formation of hemithioacetal. In the presence of ammonia, an imine is formed, which eventually yields ala. This pathway is consistent with the observation that the rate ratio of ala/lactate remains constant throughout the process. The fact that the reaction takes place under anaerobic conditions in the presence of H2O and with the low concentrations of simple substrates and catalysts makes it an attractive model prebiotic reaction in the process of molecular evolution.

  9. Effects of lixisenatide on elevated liver transaminases: systematic review with individual patient data meta-analysis of randomised controlled trials on patients with type 2 diabetes

    PubMed Central

    Gluud, Lise L; Knop, Filip K; Vilsbøll, Tina

    2014-01-01

    Objective To evaluate the effects of the glucagon-like peptide-1 receptor agonist lixisenatide on elevated liver blood tests in patients with type 2 diabetes. Design Systematic review. Data sources Electronic and manual searches were combined. Study selection Randomised controlled trials (RCTs) on lixisenatide versus placebo or active comparators for type 2 diabetes were included. Participants Individual patient data were retrieved to calculate outcomes for patients with elevated liver blood tests. Main outcome measures Normalisation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Data synthesis The results of included trials were combined in meta-analyses. Sequential, subgroup and regression analyses were performed to evaluate heterogeneity and bias. Results We included 12 RCTs on lixisenatide versus placebo and 3 RCTs with the active comparators liraglutide, exenatide or sitagliptin. The mean treatment duration was 29 weeks. Lixisenatide increased the proportion of patients with normalisation of ALT (risk difference: 0.07; 95% CI 0.01 to 0.14; number needed to treat: 14 patients, p=0.042). The effect was not confirmed in sequential analysis. No effects of lixisenatide were identified on AST, alkaline phosphatase or bilirubin. No evidence of bias was identified. Mixed effect multilevel meta-regression analyses suggest that the benefit of lixisenatide on ALT was limited to patients who were overweight or obese. Conclusions This review suggests that lixisenatide increases the proportion of obese or overweight patients with type 2 diabetes who achieve normalisation of ALT. Additional research is needed to determine if the findings translate to clinical outcome measures. Trial registration number PROSPERO; CRD42013005779. PMID:25526792

  10. Taiwanofungus camphoratus activates peroxisome proliferator-activated receptors and induces hypotriglyceride in hypercholesterolemic rats.

    PubMed

    Suk, Fat-Moon; Lin, Shyr-Yi; Chen, Chien-Ho; Yen, Shish-Jung; Su, Ching-Hua; Liu, Der-Zen; Hou, Wen-Chi; Hung, Ling-Fang; Lin, Pei-Jung; Liang, Yu-Chih

    2008-07-01

    Taiwanofungus camphoratus (T. camphoratus), a fungus and a Taiwan-specific, well-known traditional Chinese medicine, has long been used to treat diarrhea, hypertension, itchy skin, and liver cancer. To gain a large amount of T. camphoratus, several culture techniques have been developed, including solid-state culture and liquid-state fermentation. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been described as a hypoglycemic agent that increases insulin sensitivity in peripheral tissues and results in reduced blood glucose, insulin, and triglyceride levels in insulin-resistant animals and in type-2 (non-insulin-dependent) diabetic patients. In this study, we investigate the possibility that T. camphoratus might activate PPARgamma in vitro and hypolipidemic activity in vivo. The results show that an aqueous extract of the wild fruiting bodies of T. camphoratus was able to increase the PPARgamma activity in cells transfected with the PPARgamma expression plasmid and the AOx-TK reporter plasmid. Based on the cell experiment, we examined the hypolipidemic effect of wild fruiting bodies (WFT) and a solid-state culture (SST) of T. camphoratus on SD rats fed on a high-cholesterol (HC) diet. The results show that WFT significantly decreased the serum triglyceride level, but could not affect the cholesterol level. SST only slightly decreased the serum triglyceride level. In addition, both WFT and SST significantly decreased the serum alanine transaminase (ALT) level and protected against the liver damage induced by the HC diet from the results of a histological examination. These results suggest that T. camphoratus might contain PPARgamma ligands and result in a hypotriglyceridemic effect, and that it also exhibits a liver protective activity.

  11. Characterization of a Thermostable d-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

    PubMed Central

    Baek, Dae Heoun; Kwon, Seok-Joon; Hong, Seung-Pyo; Kwak, Mi-Sun; Lee, Mi-Hwa; Song, Jae Jun; Lee, Seung-Goo; Yoon, Ki-Hong; Sung, Moon-Hee

    2003-01-01

    A gene encoding a new thermostable d-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l-amino acid amides, d-amino acid-containing peptides, and NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+. The kcat/Km for d-alaninamide was measured as 544.4 ± 5.5 mM−1 min−1 at 50°C with 1 mM Co2+. PMID:12571020

  12. Effects of Monovalent Cations on the Sodium-Alanine Interaction in Rabbit Ileum

    PubMed Central

    Frizzell, Raymond A.; Schultz, Stanley G.

    1970-01-01

    H, K, Rb, and Li inhibit Na-dependent alanine influx across the brush border of rabbit ileum. Kinetic analysis indicates that H and K behave as competitive inhibitors of influx so that increasing the concentration of H or K in the mucosal solution is kinetically indistinguishable from decreasing the Na concentration. In addition the coupling between alanine and Na influxes is markedly reduced at pH 2.5. With the exception of H and Li, none of these monovalent cations significantly affects carrier-mediated alanine influx in the absence of Na indicating that their inhibitory effects are largely restricted to the Na-dependent fraction of influx. Increasing H concentration from 0.03 to 3 mM does not affect influx in the absence of Na but markedly inhibits influx in the presence of Na. Li significantly enhances alanine influx in the absence of Na. Ag, UO2, and La also inhibit the Na-dependent fraction of alanine influx. These findings suggest that anionic groups having a pKa of approximately 4 are involved in the interaction between Na and the alanine-carrier complex; present evidence implicates carboxylate groups however, phosphoryl residues cannot be ruled out. The previously proposed kinetic model for the Na-alanine interaction has been extended to accommodate these effects of H and other monovalent cations. The mechanistic and physiological implications of these findings are discussed. PMID:5507092

  13. Revised mechanism of D-alanine incorporation into cell wall polymers in Gram-positive bacteria.

    PubMed

    Reichmann, Nathalie T; Cassona, Carolina Picarra; Gründling, Angelika

    2013-09-01

    Teichoic acids (TAs) are important for growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. The chemical structures of the TAs vary between bacteria, though they typically consist of zwitterionic polymers that are anchored to either the peptidoglycan layer as in the case of wall teichoic acid (WTA) or the cell membrane and named lipoteichoic acid (LTA). The polymers are modified with D-alanines and a lack of this decoration leads to increased susceptibility to cationic antimicrobial peptides. Four proteins, DltA-D, are essential for the incorporation of d-alanines into cell wall polymers and it has been established that DltA transfers D-alanines in the cytoplasm of the cell onto the carrier protein DltC. However, two conflicting models have been proposed for the remainder of the mechanism. Using a cellular protein localization and membrane topology analysis, we show here that DltC does not traverse the membrane and that DltD is anchored to the outside of the cell. These data are in agreement with the originally proposed model for D-alanine incorporation through a process that has been proposed to proceed via a D-alanine undecaprenyl phosphate membrane intermediate. Furthermore, we found that WTA isolated from a Staphylococcus aureus strain lacking LTA contains only a small amount of D-alanine, indicating that LTA has a role, either direct or indirect, in the efficient D-alanine incorporation into WTA in living cells.

  14. Alanine-EPR as a transfer standard dosimetry system for low energy X radiation

    NASA Astrophysics Data System (ADS)

    Khoury, H. J.; da Silva, E. J.; Mehta, K.; de Barros, V. S.; Asfora, V. K.; Guzzo, P. L.; Parker, A. G.

    2015-11-01

    The purpose of this paper is to evaluate the use of alanine-EPR as a transfer standard dosimetry system for low energy X radiation, such as that in RS-2400, which operates in the range from 25 to 150 kV and 2 to 45 mA. Two types of alanine dosimeters were investigated. One is a commercial alanine pellets from Aérial-Centre de Ressources Technologiques, France and one was prepared in our laboratory (LMRI-DEN/UFPE). The EPR spectra of the irradiated dosimeters were recorded in the Nuclear Energy Department of UFPE, using a Bruker EMX10 EPR spectrometer operating in the X-band. The alanine-EPR dosimetry system was calibrated in the range of 20-220 Gy in this X-ray field, against an ionization chamber calibrated at the relevant X-ray energy with traceability to PTB. The results showed that both alanine dosimeters presented a linear dose response the same sensitivity, when the EPR signal was normalized to alanine mass. The total uncertainty in the measured dose was estimated to be about 3%. The results indicate that it is possible to use the alanine-EPR dosimetry system for validation of a low-energy X ray irradiator, such as RS-2400.

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

    PubMed

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

    2009-10-01

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

  16. Kinetic study of interaction between BRL 42715, beta-lactamases, and D-alanyl-D-alanine peptidases.

    PubMed Central

    Matagne, A; Ledent, P; Monnaie, D; Felici, A; Jamin, M; Raquet, X; Galleni, M; Klein, D; François, I; Frère, J M

    1995-01-01

    A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4) is presented. The compound was a very efficient inactivator of all active-site serine beta-lactamases but was hydrolyzed by the class B, Zn(2+)-containing enzymes, with very different kcat values. Inactivation of the Streptomyces sp. strain R61 extracellular DD-peptidase was not observed, and the Actinomadura sp. strain R39 DD-peptidase exhibited a low level of sensitivity to the compound. PMID:7695311

  17. Crystal structures of yeast beta-alanine synthase complexes reveal the mode of substrate binding and large scale domain closure movements.

    PubMed

    Lundgren, Stina; Andersen, Birgit; Piskur, Jure; Dobritzsch, Doreen

    2007-12-07

    Beta-alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu(159) and Arg(322) as crucial for catalysis and His(262) and His(397) as functionally important but not essential. We determined the crystal structures of wild-type beta-alanine synthase in complex with the reaction product beta-alanine, and of the mutant E159A with the substrate N-carbamyl-beta-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a approximately 30 degrees rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

  18. Crystal structure of an extensively simplified variant of bovine pancreatic trypsin inhibitor in which over one-third of the residues are alanines.

    PubMed

    Islam, Mohammad Monirul; Sohya, Shihori; Noguchi, Keiichi; Yohda, Masafumi; Kuroda, Yutaka

    2008-10-07

    We report the high-resolution crystal structures of an extensively simplified variant of bovine pancreatic trypsin inhibitor containing 20 alanines (BPTI-20st) and a reference single-disulfide-bonded variant (BPTI-[5,55]st) at, respectively, 1.39 and 1.09 A resolutions. The sequence was simplified based on the results of an alanine scanning experiment, as reported previously. The effects of the multiple alanine substitutions on the overall backbone structure were surprisingly small (C(alpha) atom RMSD of 0.53 A) being limited to small local structural perturbations. Both BPTI variants retained a wild-type level of trypsin inhibitory activity. The side-chain configurations of residues buried in the hydrophobic cores (<30% accessible surface area) were almost perfectly retained in both BPTI-20st and BPTI-[5,55]st, indicating that neither multiple alanine replacements nor the removal of the disulfide bonds affected their precise placements. However, the side chains of three partially buried residues (Q31, R20, and to some extent Y21) and several unburied residues rearranged into alternative dense-packing structures, suggesting some plasticity in their shape complementarity. These results indicate that a protein sequence simplified over its entire length can retain its densely packed, native side-chain structure, and suggest that both the design and fold recognition of natively folded proteins may be easier than previously thought.

  19. Importance of intrahepatic mechanisms to gluconeogenesis from alanine during exercise and recovery

    SciTech Connect

    Wasserman, D.H.; Williams, P.E.; Lacy, D.B.; Green, D.R.; Cherrington, A.D.

    1988-04-01

    These studies were performed to assess the importance of intrahepatic mechanisms to gluconeogenesis in the dog during 150 min of treadmill exercise and 90 min of recovery. Sampling catheters were implanted in an artery and portal and hepatic veins 16 days before experimentation. Infusions of (U-/sup 14/C)alanine, (3-/sup 3/H)glucose, and indocyanine green were used to assess gluconeogenesis. During exercise, a decline in arterial and portal vein plasma alanine and in hepatic blood flow led to a decrease in hepatic alanine delivery. During recovery, hepatic blood flow was restored to basal, causing an increase in hepatic alanine delivery beyond exercise rates but still below resting rates. Hepatic fractional alanine extraction increased from 0.26 +/- 0.02 at rest to 0.64 +/- 0.03 during exercise and remained elevated during recovery. Net hepatic alanine uptake was 2.5 +/- 0.2 mumol.kg-1.min-1 at rest and remained unchanged during exercise but was increased during recovery. The conversion rate of (/sup 14/C)alanine to glucose had increased by 248 +/- 38% by 150 min of exercise and had increased further during recovery. The efficiency with which alanine was channeled into glucose in the liver was accelerated to a rate of 338 +/- 55% above basal by 150 min of exercise but declined slightly during recovery. In conclusion, 1) gluconeogenesis from alanine is accelerated during exercise, due to an increase in the hepatic fractional extraction of the amino acid and through intrahepatic mechanisms that more efficiently channel it into glucose.

  20. Determination of D- and L-alanine concentrations using a pyruvic acid sensor.

    PubMed

    Inaba, Yohei; Hamada-Sato, Naoko; Kobayashi, Takeshi; Imada, Chiaki; Watanabe, Etsuo

    2003-08-01

    The concentrations of D- and L-alanine in bivalves are useful as indicators of environmental pollution. Amino acid oxidase with a low substrate specificity catalyzes the oxidation of various amino acids. Among the various amino acids, pyruvic acid can be generated from alanine only by the catalytic oxidative reaction of this oxidase. Therefore, in this study, the concentrations of D- and L-alanine were determined from the concentration of pyruvic acid, which was determined from the consumption of oxygen based on the oxidative reaction of pyruvate oxidase. From this point of view, there is a very strong possibility that biosensors utilizing enzymes with a low substrate specificity can be developed. The results obtained were as follows. (1) The optimum conditions for the use of pyruvic acid sensor were as follows: temperature of 25 degrees C, pH of 6.8, flow rate of 0.1 ml/min, thiamin diphosphate concentration of 1.5 mM, and injection volume of 50 microl. (2) D-Alanine and L-alanine optimally reacted with D- and L-amino acid oxidase at 30 degrees C, pH 8.2, for 30 min and at 37 degrees C, pH 7.8, for 90 min, respectively. (3) The linear relationships between the concentrations of D- and L-alanine and the output of the sensor were obtained at 3.56-106.8 microg of D-alanine and 5.34-71.3 microg of L-alanine. (4) The concentrations of D- and L-alanine in Meretrix iusoria, Patinopecten yessonsi, and Corbicula leana obtained by the proposed assay were in good agreement with those determined by a conventional method.

  1. Serum Alanine Aminotransferase Levels, Hematocrit Rate and Body Weight Correlations Before and After Hemodialysis Session

    PubMed Central

    Lopes, Edmundo Pessoa; Sette, Luis Henrique B. C.; Sette, Jorge Bezerra C.; Luna, Carlos F.; Andrade, Amaro M.; Moraes, Maviael; Sette, Paulo C. A.; Menezes, Roberto; Cavalcanti, Rui L.; Conceição, Sergio C.

    2009-01-01

    PURPOSE To evaluate alanine aminotransferase levels before and after a hemodialysis session and to correlate these values with the hematocrit rate and weight loss during hemodialysis. PATIENTS AND METHODS The serum alanine aminotransferase levels, hematocrit rate and body weight were measured and correlated before and after a single hemodialysis session for 146 patients with chronic renal failure. An receiver operating characteristic (ROC) curve for the serum alanine aminotransferase levels collected before and after hemodialysis was plotted to identify hepatitis C virus-infected patients. RESULTS The mean weight loss of the 146 patients during hemodialysis was 5.3% (p < 0.001). The mean alanine aminotransferase levels before and after hemodialysis were 18.8 and 23.9 IU/, respectively, denoting a significant 28.1% increase. An equally significant increase of 16.4% in the hematocrit rate also occurred after hemodialysis. The weight loss was inversely correlated with the rise in both the alanine aminotransferase level (r = 0.3; p < 0.001) and hematocrit rate (r = 0.5; p < 0.001). A direct correlation was found between the rise in alanine aminotransferase levels and the hematocrit during the hemodialysis session (r = 0.4; p < 0.001). Based on the ROC curve, the upper limit of the normal alanine aminotransferase level should be reduced by 40% relative to the upper limit of normal if the blood samples are collected before the hemodialysis session or by 60% if blood samples are collected after the session. CONCLUSION In the present study, significant elevations in the serum alanine aminotransferase levels and hematocrit rates occurred in parallel to a reduction in body weight after the hemodialysis session. These findings suggest that one of the factors for low alanine aminotransferase levels prior to hemodialysis could be hemodilution in patients with chronic renal failure. PMID:19841699

  2. Boron Induces Early Matrix Mineralization via Calcium Deposition and Elevation of Alkaline Phosphatase Activity in Differentiated Rat Bone Marrow Mesenchymal Stem Cells

    PubMed Central

    Movahedi Najafabadi, Bent-al-hoda; Abnosi, Mohammad Hussein

    2016-01-01

    Objective Boron (B) is essential for plant development and might be an essential micronutrient for animals and humans. This study was conducted to characterize the impact of boric acid (BA) on the cellular and molecular nature of differentiated rat bone marrow mesenchymal stem cells (BMSCs). Materials and Methods In this experimental study, BMSCs were extracted and expanded to the 3rdpassage, then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) complemented with osteogenic media as well as 6 ng/ml and 6 µg/ml of BA. After 5, 10, 15 and 21 days the viability and the level of mineralization was determined using MTT assay and alizarin red respectively. In addition, the morphology, nuclear diameter and cytoplasmic area of the cells were studied with the help of fluorescent dye. The concentration of calcium, activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) as well as sodium and potassium levels were also evaluated using commercial kits and a flame photometer respectively. Results Although 6 µg/ml of BA was found to be toxic, a concentration of 6 ng/ml increased the osteogenic ability of the cell significantly throughout the treatment. In addition it was observed that B treatment caused the early induction of matrix mineralization compared to controls. Conclusion Although more investigation is required, we suggest the prescription of a very low concentration of B in the form of BA or foods containing BA, in groups at high risk of osteoporosis or in the case of bone fracture. PMID:27054120

  3. Thymoquinone, an active constituent of Nigella sativa seeds, binds with bilirubin and protects mice from hyperbilirubinemia and cyclophosphamide-induced hepatotoxicity.

    PubMed

    Laskar, Amaj A; Khan, Masood A; Rahmani, Arshad H; Fatima, Sana; Younus, Hina

    2016-08-01

    Some reports indicate that thymoquinone (TQ), the main constituent of Nigella sativa seeds, is hepatoprotective. The aim of this study was to determine whether TQ is able to bind directly to bilirubin, and whether TQ or liposomal formulation of TQ (Lip-TQ) can reduce cyclophosphamide (CYP)-induced liver toxicity, serum bilirubin level in mice. The binding of TQ with bilirubin was studied by UV-VIS, fluorescence and Near-UV CD spectroscopy. Inhibition of binding of bilirubin to erythrocytes by TQ was also examined. To increase the in vivo efficacy, Lip-TQ was prepared and used against CYP-induced toxicity. The protective role of TQ or Lip-TQ against CYP-induced toxicity was assessed by determining the liver function parameters, the levels of superoxide dismutase (SOD) and catalase (CAT), and histological studies. It was found that TQ binds to bilirubin and significantly inhibits the binding of bilirubin to erythrocytes. Lip-TQ (10 mg/kg) significantly reduced the levels of aspartate transaminase (AST) from 254 ± 48 to 66 ± 18 IU/L (P < 0.001), alanine transaminase (ALT) from 142 ± 28 to 47.8 ± 16 IU/L (P < 0.05) and serum bilirubin from 2.8 ± 0.50 to 1.24 ± 0.30 mg/dl (P < 0.05). Treatment with Lip-TQ reduced the CYP-induced inflammation and hemorrhage in liver tissues. Moreover, treatment with free or Lip-TQ protected the activity of SOD and CAT in CYP-injected mice. Therefore, TQ can reduce the level of bilirubin in systemic circulation in disease conditions that lead to hyperbilirubinemia and liver toxicity and hence may be used as a supplement in the treatment of liver ailments.

  4. The effect of platelet activating factor antagonist BN 52021 on bacterial translocation and ICAM-I expression in experimental obstructive jaundice.

    PubMed

    Akyürek, Nusret; Salman, Bülent; Irkörücü, Oktay; Tezcaner, Tugan; Azili, Cem; Erdem, Ozlem; Akca, Gülçin; Akin, Okan; Tatlicioglu, Ertan

    2005-01-01

    Expression of intracellular adhesion molecule-1 (ICAM-1) in an obstructive jaundice model and the potential protective role of platelet activating factor antagonist over small intestine and liver together with its effects on bacterial translocation are examined in this study. Forty-eight male Wistar albino rats were assigned into four equal groups of 12. In groups I and II, animals were sham operated. In groups III and IV, common bile duct ligation and division were performed. In group I and group III, 0.5 ml/day normal saline was applied intraperitoneally daily from day 2 to 6 of the study; in group II and group IV, 1 mg/kg/day BN 52021 was applied intraperitoneally daily from day 2 to 6 of the study. All animals were sacrificed on postoperative day 7. ICAM-1 expression (CD54 positivity) was analyzed in the liver and ileum tissue by immunohistochemical method. Samples from blood, liver mesenteric lymph nodes, and spleen were cultured under aerobic conditions. It is revealed that ICAM-1 expression was statistically higher in group III, with highest bacterial translocation and liver and spleen injury when compared to other groups. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (GGT), bilirubin, tumor necrosis factor alpha (TNFalpha), and interleukin 1beta(IL-1beta) values were at the highest level in group III, and there was a statistical decrease in group IV compared to group III. The administration of BN52021 in experimental obstructive jaundice is a useful way to reduce liver and intestinal mucosal villi damage by inhibiting bacterial translocation and systemic inflammatory response.

  5. Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

    PubMed

    Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Hori, Hatsuhiro; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2015-08-01

    We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [(3) H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [(3) H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [(3) H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.

  6. Progress towards an alanine/ESR therapy level reference dosimetry service at NPL.

    PubMed

    Sharpe, P H; Rajendran, K; Sephton, J P

    1996-01-01

    This paper describes work being carried out at the National Physical Laboratory towards the establishment of an alanine reference dosimetry service for radiotherapy applications. A precision fused quartz holder has been constructed to allow precise positioning of alanine dosimeters in the ESR cavity. A novel method of signal analysis based on spectrum fitting has been developed to minimize the effect of baseline distortions. Data are also presented on the relative response of alanine to 60Co gamma rays and high energy photons (4-12 MeV).

  7. Applicability of EPR/alanine dosimetry for quality assurance in proton eye radiotherapy.

    PubMed

    Michalec, B; Mierzwinska, G; Ptaszkiewicz, M; Sowa, U; Stolarczyk, L; Weber, A

    2014-06-01

    A new quality assurance and quality control method for proton eye radiotherapy based on electron paramagnetic resonance (EPR)/alanine dosimetry has been developed. It is based on Spread-Out Bragg Peak entrance dose measurement with alanine detectors. The entrance dose is well correlated with the dose at the facility isocenter, where, during the therapeutic irradiation, the tumour is placed. The unique alanine detector features namely keeping the dose record in a form of stable radiation-induced free radicals trapped in the material structure, and the non-destructive read-out makes this type of detector a good candidate for additional documentation of the patient's exposure over the therapy course.

  8. Interactions of L-alanine with alumina as studied by vibrational spectroscopy.

    PubMed

    Garcia, Ana R; de Barros, Ricardo Brito; Fidalgo, Alexandra; Ilharco, Laura M

    2007-09-25

    The interactions of L-alanine with gamma- and alpha-alumina have been investigated by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). L-alanine/alumina samples were dried from aqueous suspensions, at 36.5 degrees C, with two amino acid concentrations (0.4 and 0.8 mmol g-1) and at different pH values (1, 6, and 13). The vibrational spectra proved that the nature of L-alanine interactions with both aluminas is the same (hydrogen bonding), although the groups involved depend on the L-alanine form and on alumina surface groups, both controlled by the pH. For samples prepared at pH 1, cationic L-alanine [CH3CH(NH3+)COOH] displaces physisorbed water from alumina, and strong hydrogen bonds are established between the carbonyl groups of alanine, as electron donors, and the surface Al-OH2+ groups of alumina. This occurs at the expense of alanine dimer dissociation and breaking of intramolecular bonds. When samples are prepared at pH 6, the interacting groups are Al-OH2+ and the carboxylate groups of zwitterionic L-alanine [CH3CH(NH3+)COO-]. The affinity of L-alanine toward alumina decreases, as the strong NH3+...-OOC intermolecular hydrogen bonds prevail over the interactions with alumina. Thus, for a load of 0.8 mmol g-1, phase segregation is observed. On alpha-alumina, crystal deposition is even observed for a load of 0.4 mmol g-1. At pH 13, the carboxylate groups of anionic L-alanine [CH3CH(NH2)COO-] are not affected by alumina. Instead, hydrogen bond interactions occur between NH2 and the Al-OH surface groups of the substrate. Complementary N2 adsorption-desorption isotherms showed that adsorption of L-alanine occurs onto the alumina pore network for samples prepared at pH 1 and 13, whereas at pH 6 the amino acid/alumina interactions are not strong enough to promote adsorption. The mesoporous structure and the high specific surface area of gamma-alumina make it a more efficient substrate for adsorption of L-alanine. For each alumina, however, it is

  9. Temperature dependences of piezoelectric, elastic and dielectric constants of L-alanine crystal

    NASA Astrophysics Data System (ADS)

    Tylczyński, Z.; Sterczyńska, A.; Wiesner, M.

    2011-09-01

    Temperature changes in the components of piezoelectric, elastic and dielectric tensors were studied in L-alanine crystals in the range 100-300 K. A jumpwise increase in the c55 component of the elastic stiffness accompanied by maxima in damping of all face-shear modes observed at 199 K in L-alanine crystal were interpreted as a result of changes in the NH3+ vibrations occurring through electron-phonon coupling. All components of the piezoelectric tensor show small anomalies in this temperature range. The components of the electromechanical coupling coefficient determined indicate that L-alanine is a weak piezoelectric.

  10. Optical and Spectral Studies on β Alanine Metal Halide Hybrid Crystals

    NASA Astrophysics Data System (ADS)

    Sweetlin, M. Daniel; Selvarajan, P.; Perumal, S.; Ramalingom, S.

    2011-10-01

    We have synthesized and grown β alanine metal halide hybrid crystals viz. β alanine cadmium chloride (BACC), an amino acid transition metal halide complex crystal and β alanine potassium chloride (BAPC), an amino acid alkali metal halide complex crystal by slow evaporation method. The grown crystals were found to be transparent and have well defined morphology. The optical characteristics of the grown crystals were carried out with the help of UV-Vis Spectroscopy. The optical transmittances of the spectrums show that BAPC is more transparent than BACC. The Photoluminescence of the materials were determined by the Photoluminescent Spectroscopy

  11. Temperature dependences of piezoelectric, elastic and dielectric constants of L-alanine crystal.

    PubMed

    Tylczyński, Z; Sterczyńska, A; Wiesner, M

    2011-09-07

    Temperature changes in the components of piezoelectric, elastic and dielectric tensors were studied in L-alanine crystals in the range 100-300 K. A jumpwise increase in the c(55) component of the elastic stiffness accompanied by maxima in damping of all face-shear modes observed at 199 K in L-alanine crystal were interpreted as a result of changes in the NH(3)(+) vibrations occurring through electron-phonon coupling. All components of the piezoelectric tensor show small anomalies in this temperature range. The components of the electromechanical coupling coefficient determined indicate that L-alanine is a weak piezoelectric.

  12. Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae.

    PubMed

    De Benedetti, Stefania; Bühl, Henrike; Gaballah, Ahmed; Klöckner, Anna; Otten, Christian; Schneider, Tanja; Sahl, Hans-Georg; Henrichfreise, Beate

    2014-01-01

    For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.

  13. PPAR{alpha} regulates the hepatotoxic biomarker alanine aminotransferase (ALT1) gene expression in human hepatocytes

    SciTech Connect

    Thulin, Petra; Rafter, Ingalill; Stockling, Kenneth; Tomkiewicz, Celine; Norjavaara, Ensio; Aggerbeck, Martine; Hellmold, Heike; Ehrenborg, Ewa; Andersson, Ulf; Cotgreave, Ian; Glinghammar, Bjoern

    2008-08-15

    In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) {alpha} agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPAR{alpha} agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at - 574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum.

  14. Effects of Acute Beta-Alanine Supplementation on Anaerobic Performance in Trained Female Cyclists.

    PubMed

    Glenn, Jordan M; Smith, Keyona; Moyen, Nicole E; Binns, Ashley; Gray, Michelle

    2015-01-01

    Longitudinal beta-alanine (BA) supplementation can improve exercise performance in males through increases in carnosine; however, females experience greater relative increases in carnosine compared to males. This potentially allows females to benefit from acute BA doses; however, effects of an acute BA dose on performance in females remain unknown. The purpose of this investigation was to evaluate how an acute dose of 1.6 g BA affects anaerobic performance in female cyclists. Twelve females (age=26.6±1.3 y) volunteered to participate in this randomized, double-blind study. All participants completed two supplement trials: 1) Placebo=34 g dextrose and 2) BA=1.6 g BA + 34 g dextrose. Thirty-minutes after supplementation, participants performed three repeated Wingate cycling tests with 2 min of active rest after each. Fatigue index, mean power, and peak power were measured during each Wingate. Lactate, heart rate, and rating of perceived exertion (RPE) were measured at rest, immediately after each Wingate, and after each active rest period. RPE significantly decreased (p<0.001) immediately following Wingates 1 and 2 and after each 2-min rest period for the BA trials; however, no differences were observed immediately after Wingate 3 (p>0.05). No significant supplementation effect was observed for any performance or physiological variable (p>0.05 for all variables). Findings suggest that an acute dose of BA (1.6 g) decreases RPE during anaerobic power activities in trained female cyclists.

  15. Solvation Free Energies of Alanine Peptides: The Effect of Flexibility

    SciTech Connect

    Kokubo, Hironori; Harris, Robert C.; Asthagiri, Dilip; Pettitt, Bernard M.

    2013-12-03

    The electrostatic (?Gel), cavity-formation (?Gvdw), and total (?G) solvation free energies for 10 alanine peptides ranging in length (n) from 1 to 10 monomers were calculated. The free energies were computed both with xed, extended conformations of the peptides and again for some of the peptides without constraints. The solvation free energies, ?Gel, ?Gvdw, and ?G, were found to be linear in n, with the slopes of the best-fit lines being gamma_el, gamma_vdw, and gamma, respectively. Both gamma_el and gamma were negative for fixed and flexible peptides, and gamma_vdw was negative for fixed peptides. That gamma_vdw was negative was surprising, as experimental data on alkanes, theoretical models, and MD computations on small molecules and model systems generally suggest that gamma_vdw should be positive. A negative gamma_vdw seemingly contradicts the notion that ?Gvdw drives the initial collapse of the protein when it folds by favoring conformations with small surface areas, but when we computed ?Gvdw for the flexible peptides, thereby allowing the peptides to assume natural ensembles of more compact conformations, gamma-vdw was positive. Because most proteins do not assume extended conformations, a ?Gvdw that increases with increasing surface area may be typical for globular proteins. An alternative hypothesis is that the collapse is driven by intramolecular interactions. We show that the intramolecular van der Waal's interaction energy is more favorable for the flexible than for the extended peptides, seemingly favoring this hypothesis, but the large fluctuations in this energy may make attributing the collapse of the peptide to this intramolecular energy difficult.

  16. Rapid Ti(III) reduction of perchlorate in the presence of beta-alanine: kinetics, pH effect, complex formation, and beta-alanine effect.

    PubMed

    Wang, Chao; Huang, Zhengdao; Lippincott, Lee; Meng, Xiaoguang

    2010-03-15

    Ti(III) reduction of perchlorate might be a useful method for the treatment of highly perchlorate-contaminated water. Though the reaction rate was usually low, we observed that beta-alanine (HOOCCH(2)CH(2)NH(2)) could significantly promote the reaction. A complete (>99.9%) perchlorate removal was obtained in a solution containing [ClO(4)(-)]=1.0mM, [Ti(III)]=40 mM, and [beta-alanine]=120 mM after 2.5h of reaction under 50 degrees C. The effects of both pH and complex formation on the reaction were then studied. The results showed that without beta-alanine the optimal pH was 2.3. When pH increased from 1.6 to 2.3, the reduction rate increased remarkably. In the pH range >2.3, however, the reduction was significantly inhibited, attributed to the formation of Ti(III) precipitate. The presence of beta-alanine at a molar ratio of [beta-alanine]:[Ti(III)]=3:1 significantly increased the reduction rate of perchlorate even at near neutral pH. This is because beta-alanine formed complexes with Ti(III), which greatly improved the total soluble [Ti(III)] in the pH range between 3.5 and 6. The findings may lead to the development of rapid treatment methods for intermittent and small stream of highly perchlorate-contaminated water, which are resulted from the manufacturing, storage, handling, use and/or disposal of large quantities of perchlorate salts.

  17. Assessing microbial utilization of free versus sorbed Alanine by using position-specific 13C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Herschbach, Jennifer; Apostel, Carolin; Spielvogel, Sandra; Kuzyakov, Yakov; Dippold, Michaela

    2016-04-01

    Microbial utilization is a key transformation process of soil organic matter (SOM). Sorption of low molecular weight organic substances (LMWOS) to soil mineral surfaces blocks or delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil science, combined with 13C-phospholipid fatty acid (PLFA) analysis, to assess microbial utilization of sorbed and non-sorbed Alanine in soil. Alanine has various functional groups enabling different sorption mechanisms via its positive charge (e.g. to clay minerals by cation exchange), as well as via its negative charge (e.g. to iron oxides by ligand exchange). To assess changes in the transformation pathways caused by sorption, we added uniformly and position-specifically 13C and 14C labeled Alanine to the Ap of a loamy Luvisol in a short-term (10 days) incubation experiment. To allow for sorption of the tracer solution to an aliquot of this soil, microbial activity was minimized in this subsample by sterilizing the soil by γ-radiation. After shaking, the remaining solutions were filtered and the non-sorbed Alanine was removed with Millipore water and then added to non-sterilized soil. For the free Alanine treatment, solutions with Alanine of similar amount and isotopic composition were prepared, added to the soil and incubated as well. The respired CO2 was trapped in NaOH and its 14C-activity was determined at increasing times intervals. Microbial utilization of Alanine's individual C positions was evaluated in distinct microbial groups classified by 13C-PLFA analysis. Sorption to soil minerals delayed respiration to CO2 and reduced initial respiration rate by 80%. Irrespective of sorption, the highest amount was respired from the carboxylic position (C-1), whereas the amino-bound (C-2) and the methylic position (C-3) were preferentially incorporated into PLFA of microorganisms due to the

  18. Inducible l-Alanine Exporter Encoded by the Novel Gene ygaW (alaE) in Escherichia coli ▿

    PubMed Central

    Hori, Hatsuhiro; Yoneyama, Hiroshi; Tobe, Ryuta; Ando, Tasuke; Isogai, Emiko; Katsumata, Ryoichi

    2011-01-01

    We previously isolated a mutant hypersensitive to l-alanyl-l-alanine from a non-l-alanine-metabolizing Escherichia coli strain and found that it lacked an inducible l-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular l-alanine and a reduction in the l-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes, ygaW and ytfF, and two characterized genes, yddG and yeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellular l-alanine level and enhancement of the l-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of l-alanine. Since ygaW exhibited the most striking impact on both the intra- and the extracellular l-alanine levels among the four genes identified, we disrupted the ygaW gene in the non-l-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular l-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the ygaW gene conferred on the cells the ability to excrete alanine. In addition, expression of the ygaW gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for l-alanine in E. coli and proposed that the gene be redesignated alaE for alanine export. PMID:21531828

  19. A comparative study on the growth and characterization of nonlinear optical amino acid crystals: L-alanine (LA) and L-alanine alaninium nitrate (LAAN).

    PubMed

    Aravindan, A; Srinivasan, P; Vijayan, N; Gopalakrishnan, R; Ramasamy, P

    2008-11-15

    A comparative study on the properties of L-alanine and LAAN crystals has been made and discussed. It may be concluded that the protonation of the amino group in the L-alanine molecule is the key factor in increasing the relative SHG efficiency of LAAN. The protonation is justified by the crystal structure analysis, FTIR and photoluminescence studies. The factor group vibrations are compared and found that there is an increase in vibrational modes of LA when reacted with nitric acid forming LAAN.

  20. Determination of the carbon, hydrogen and nitrogen contents of alanine and their uncertainties using the certified reference material L-alanine (NMIJ CRM 6011-a).

    PubMed

    Itoh, Nobuyasu; Sato, Ayako; Yamazaki, Taichi; Numata, Masahiko; Takatsu, Akiko

    2013-01-01

    The carbon, hydrogen, and nitrogen (CHN) contents of alanine and their uncertainties were estimated using a CHN analyzer and the certified reference material (CRM) L-alanine. The CHN contents and their uncertainties, as measured using the single-point calibration method, were 40.36 ± 0.20% for C, 7.86 ± 0.13% for H, and 15.66 ± 0.09% for N; the results obtained using the bracket calibration method were also comparable. The method described in this study is reasonable, convenient, and meets the general requirement of having uncertainties ≤ 0.4%.

  1. Titration of Alanine Monitored by NMR Spectroscopy: A Biochemistry Laboratory Experiment

    ERIC Educational Resources Information Center

    Waller, Francis J.; And Others

    1977-01-01

    The experiment described here involves simultaneous monitoring of pH and NMR chemical shifts during an aqueous titration of alpha- and beta-alanine. This experiment is designed for use in an undergraduate biochemistry course. (MR)

  2. Second harmonic generation studies in L-alanine single crystals grown from solution

    NASA Astrophysics Data System (ADS)

    Boomadevi, Shanmugam; Pandiyan, Krishnamoorthy

    2014-01-01

    Single crystals of L-alanine of dimensions 2×1.1×0.5 cm3 were grown by evaporation method using deionised water as a solvent. The morphology of the grown crystals had (1 2 0) and (0 1 1) as their prominent faces. UV-vis-near IR spectrum shows the transparency range of L-alanine crystal available for frequency doubling from 250 to 1400 nm. Phase-matched second harmonic generation was observed in L-alanine sample by using 7 ns Q-switched Nd:YAG laser with OPO set up. In the present work, phase matching was achieved by angle and wavelength tuning. The angular and spectral phase-matching bandwidths were determined experimentally for a 1.5 mm thick L-alanine crystal and the results have been compared with their theoretical results. Further the possible reasons for the broadening of SHG spectrum have been discussed.

  3. Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis

    SciTech Connect

    Feild, M.J.

    1988-01-01

    The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase of Salmonella typhimurium was determined by automated Edman degradation of peptide fragments generated by chemical and enzymatic digestion of S-carboxymethylated and S-pyridylethylated transaminase B. Peptide fragments of transaminase B were generated by treatment of the enzyme with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. Protocols were developed for separation of the peptide fragments by reverse-phase high performance liquid chromatography (HPLC), ion-exchange HPLC, and SDS-urea gel electrophoresis. The enzyme subunit contains 308 amino acid residues and has a molecular weight of 33,920 daltons. The coenzyme-binding site was determined by treatment of the enzyme, containing bound pyridoxal 5-phosphate, with tritiated sodium borohydride prior to trypsin digestion. Monitoring radioactivity incorporation and peptide map comparisons with an apoenzyme tryptic digest, allowed identification of the pyridoxylated-peptide which was isolated by reverse-phase HPLC and sequenced. The coenzyme-binding site is a lysyl residue at position 159. Some peptides were further characterized by fast atom bombardment mass spectrometry.

  4. SU-E-T-643: Pure Alanine Dosimeter for Verification Dosimetry in IMRT

    SciTech Connect

    Al-Karmi, Anan M.; Zraiqat, Fadi

    2015-06-15

    Purpose: The objective of this study was evaluation of accuracy of pure alanine dosimeters measuring intensity-modulated radiation therapy (IMRT) dose distributions in a thorax phantom. Methods: Alanine dosimeters were prepared in the form of 110 mg pure L-α-alanine powder filled into clear tissue-equivalent polymethylmethacrylate (PMMA) plastic tubes with the dimensions 25 mm length, 3 mm inner diameter, and 1 mm wall thickness. A dose-response calibration curve was established for the alanine by placing the dosimeters at 1.5 cm depth in a 30×30×30 cm{sup 3} solid water phantom and then irradiating on a linac with 6 MV photon beam at 10×10 cm{sup 2} field size to doses ranging from 1 to 5 Gy. Electron paramagnetic resonance (EPR) spectroscopy was used to determine the absorbed dose in alanine. An IMRT treatment plan was designed for a commercial heterogeneous CIRS thorax phantom and the dose values were calculated at three different points located in tissue, lung, and bone equivalent materials. A set of dose measurements was carried out to compare measured and calculated dose values by placing the alanine dosimeters at those selected locations inside the thorax phantom and delivering the IMRT to the phantom. Results: The alanine dose measurements and the IMRT plan dose calculations were found to be in agreement within ±2%. Specifically, the deviations were −0.5%, 1.3%, and −1.7% for tissue, lung, and bone; respectively. The slightly large deviations observed for lung and bone may be attributed to tissue inhomogeneity, steep dose gradients in these regions, and uncontrollable changes in spectrometer conditions. Conclusion: The results described herein confirmed that pure alanine dosimeter was suitable for in-phantom dosimetry of IMRT beams because of its high sensitivity and acceptable accuracy. This makes the dosimeter a promising option for quality control of the therapeutic beams, complementing the commonly used ionization chambers, TLDs, and films.

  5. Oligomerization of Glycine and Alanine Catalyzed by Iron Oxides: Implications for Prebiotic Chemistry

    NASA Astrophysics Data System (ADS)

    Shanker, Uma; Bhushan, Brij; Bhattacharjee, G.; Kamaluddin

    2012-02-01

    Iron oxide minerals are probable constituents of the sediments present in geothermal regions of the primitive earth. They might have adsorbed different organic monomers (amino acids, nucleotides etc.) and catalyzed polymerization processes leading to the formation of the first living cell. In the present work we tested the catalytic activity of three forms of iron oxides (Goethite, Akaganeite and Hematite) in the intermolecular condensation of each of the amino acids glycine and L-alanine. The effect of zinc oxide and titanium dioxide on the oligomerization has also been studied. Oligomerization studies were performed for 35 days at three different temperatures 50, 90 and 120°C without applying drying/wetting cycling. The products formed were characterized by HPLC and ESI-MS techniques. All three forms of iron oxides catalyzed peptide bond formation (23.2% of gly2 and 10.65% of ala2). The reaction was monitored every 7 days. Formation of peptides was observed to start after 7 days at 50°C. Maximum yield of peptides was found after 35 days at 90°C. Reaction at 120°C favors formation of diketopiperazine derivatives. It is also important to note that after 35 days of reaction, goethite produced dimer and trimer with the highest yield among the oxides tested. We suggest that the activity of goethite could probably be due to its high surface area and surface acidity.

  6. Computational analysis of binding affinity and neural response at the l-alanine receptor

    NASA Astrophysics Data System (ADS)

    Venanzi, Thomas J.; Bryant, Bruce P.; Venanzi, Carol A.

    1995-10-01

    A model of analogue-receptor binding is developed for the l-alanine receptor in the channel catfish using the AM1-SM2 and ab initio SCRF computational methods. Besides interactions involving the zwitterionic moiety of the amino acid analogue and complementary subsites on the receptor, the model suggests the presence of a hydrophobic pocket with dispersion interactions between the receptor and the residue on the amino acid analogue. Conformational analysis suggests not only a small compact active site on the receptor, but also that the analogues with the highest affinity occupy nearly identical regions of space. Although the binding interaction is dominated by the ionic terms, AM1-SM2 calculations indicate that free energy terms associated with cavity formation, solvent reorganization, and dispersion interactions can be correlated to activation and neural response. From a consideration of this model, molecular features of the analogues that are important for binding and neural response were deduced and other analogues or ligands were developed and tested.

  7. Assessing functional diversity in the soybean β-substituted alanine synthase enzyme family.

    PubMed

    Yi, Hankuil; Jez, Joseph M

    2012-11-01

    In plants, proteins of the β-substituted alanine synthase (BSAS) enzyme family perform a diverse range of reactions, including formation of cysteine from O-acetylserine and sulfide, detoxification of cyanide by its addition to cysteine, the breakdown of cysteine into pyruvate, ammonia, and sulfide, and the synthesis of S-sulfocysteine. With the completed genome sequence of soybean (Glycine max (L.) Merr. cv. Williams 82), the functional diversity of the BSAS in this highly duplicated plant species was examined to determine whether soybean BSAS enzymes catalyze the various reactions connected to cysteine metabolism. The 16 soybean BSAS can be grouped into clades that are similar to those observed in Arabidopsis. Biochemical analysis of soybean BSAS proteins demonstrate that enzymes of clades I and III function as O-acetylserine sulfhydrylases for cysteine synthesis, clade II encodes cysteine desulfhydrase activity, and that clade V proteins function as β-cyanoalanine synthase for cyanide detoxification. Although clade IV is similar to Arabidopsis S-sulfocysteine synthase, this activity was not detected in the soybean homolog. Overall, our results show that bioinformatics approach provides a useful method to assess the biochemical properties of BSAS enzymes in plant species.

  8. Alanine scanning mutagenesis of anti-TRAP (AT) reveals residues involved in binding to TRAP.

    PubMed

    Chen, Yanling; Gollnick, Paul

    2008-04-11

    The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic (trp) genes in response to changes in intracellular levels of free l-tryptophan in many Gram-positive bacteria. When activated by binding tryptophan, TRAP binds to the mRNAs of several genes involved in tryptophan metabolism, and down-regulates transcription or translation of these genes. Anti-TRAP (AT) is an antagonist of TRAP that binds to tryptophan-activated TRAP and prevents it from binding to its RNA targets, and thereby up-regulates trp gene expression. The crystal structure shows that AT is a cone-shaped trimer (AT(3)) with the N-terminal residues of the three subunits assembled at the apex of the cone and that these trimers can further assemble into a dodecameric (AT(12)) structure. Using alanine-scanning mutagenesis we found four residues, all located on the "top" region of AT(3), that are essential for binding to TRAP. Fluorescent labeling experiments further suggest that the top region of AT is in close juxtaposition to TRAP in the AT-TRAP complex. In vivo studies confirmed the importance of these residues on the top of AT in regulating TRAP mediated gene regulation.

  9. Effect of abomasal glucose infusion on alanine metabolism and urea production in sheep.

    PubMed

    Obitsu, T; Bremner, D; Milne, E; Lobley, G E

    2000-08-01

    The effect of abomasal infusion of glucose (120 kJ/d per kg body weight (BW)0.75, 758 mmol/d) on urea production, plasma alanine-N flux rate and the conversion of alanine-N to urea was studied in sheep offered a low-N diet at limited energy intake (500 kJ/d per kg BW0.75), based on hay and grass pellets. Glucose provision reduced urinary N (P = 0.040) and urea (P = 0.009) elimination but this was offset by poorer N digestibility. Urea-N production was significantly reduced (822 v. 619 mmol/d, P = 0.024) by glucose while plasma alanine-N flux rate was elevated (295 v. 342 mmol/d, P = 0.011). The quantity of urea-N derived from alanine tended to be decreased by glucose (127 v. 95 mmol/d) but the fraction of urea production from alanine was unaltered (15%). Plasma urea and alanine concentrations (plus those of the branched chain amino acids) decreased in response to exogenous glucose, an effect probably related to enhanced anabolic usage of amino acids and lowered urea production.

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

    PubMed Central

    2014-01-01

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

  11. Expression, crystallization and preliminary X-ray crystallographic analysis of D-alanine-D-alanine ligase from OXA-23-producing Acinetobacter baumannii K0420859.

    PubMed

    Huynh, Kim-Hung; Tran, Huyen-Thi; Pham, Tan-Viet; Ngo, Ho-Phuong-Thuy; Cha, Sun-Shin; Chung, Kyung Min; Lee, Sang Hee; Kang, Lin-Woo

    2014-04-01

    Acinetobacter baumannii causes bacteraemia, pneumonia, other respiratory-tract and urinary-tract infections in humans. OXA-23 carbapenemase-producing A. baumannii K0420859 (A. baumannii OXA-23) is resistant to carbapenem, a common antibacterial drug. To develop an efficient and novel antibacterial drug against A. baumannii OXA-23, D-alanine-D-alanine ligase, which is essential in bacterial cell-wall synthesis, is of interest. Here, the D-alanine-D-alanine ligase (AbDdl) gene from A. baumannii OXA-23 was cloned and expressed, and the AbDdl protein was purified and crystallized; this enzyme can be used as a novel target for an antibacterial drug against A. baumannii OXA-23. The AbDdl crystal diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 113.4, b = 116.7, c = 176.5 Å, a corresponding VM of 2.8 Å(3) Da(-1) and a solvent content of 56.3%, and six protomers in the asymmetric unit.

  12. Alanine scanning analyses of the three major loops in domain II of Bacillus thuringiensis mosquitocidal toxin Cry4Aa.

    PubMed

    Howlader, Mohammad Tofazzal Hossain; Kagawa, Yasuhiro; Miyakawa, Ai; Yamamoto, Ayaka; Taniguchi, Tetsuya; Hayakawa, Tohru; Sakai, Hiroshi

    2010-02-01

    Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.

  13. Cycas micronesica (Cycadales) plants devoid of endophytic cyanobacteria increase in beta-methylamino-L-alanine.

    PubMed

    Marler, Thomas E; Snyder, Laura R; Shaw, Christopher A

    2010-09-15

    Cycads are among the most ancient of extant Spermatophytes, and are known for their pharmacologically active compounds. beta-methylamino-l-alanine (BMAA) is one metabolite that been implicated as causal of human neurodegenerative diseases in Guam. We grew Cycas micronesica seedlings without endophytic cyanobacteria symbiosis, and quantified initial and ending BMAA in various plant tissues. BMAA increased 79% during nine months of seedling growth, and root tissue contained 75% of the ultimate BMAA pool. Endophytic cyanobacteria symbionts were not the source of BMAA increase in these seedlings, which contradicts previously reported claims that biosynthesis of this toxin by cyanobacteria initiates its accumulation in the Guam environment. The preferential loading of root tissue with BMAA does not support earlier reports that this toxin serves a defensive role against herbivory of leaf or seed tissues. The long history of conflicting results in Guam's cycad toxin research continues, and recent developments underscore the sense of urgency in continued research as this endangered cycad population approaches extirpation from the island.

  14. Cyclic side-chain-linked opioid analogs utilizing cis- and trans-4-aminocyclohexyl-D-alanine.

    PubMed

    Piekielna, Justyna; Gentilucci, Luca; De Marco, Rossella; Perlikowska, Renata; Adamska, Anna; Olczak, Jacek; Mazur, Marzena; Artali, Roberto; Modranka, Jakub; Janecki, Tomasz; Tömböly, Csaba; Janecka, Anna

    2014-12-01

    Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generating analogs with improved bioactivities. Cyclization can be achieved through various bridging bonds between peptide ends or side-chains. In our earlier paper we have reported the synthesis and biological activity of a cyclic peptide, Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (1), which can be viewed as an analog of endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2). Cyclization was achieved through an amide bond between side-chains of D-Lys and Asp residues. Here, to increase rigidity of the cyclic structure, we replaced d-Lys with cis- or trans-4-aminocyclohexyl-D-alanine (D-ACAla). Two sets of analogs incorporating either Tyr or Dmt (2',6'-dimethyltyrosine) residues in position 1 were synthesized. In the binding studies the analog incorporating Dmt and trans-D-ACAla showed high affinity for both, μ- and δ-opioid receptors (MOR and DOR, respectively) and moderate affinity for the κ-opioid receptor (KOR), while analog with Dmt and cis-D-ACAla was exceptionally MOR-selective. Conformational analyses by NMR and molecular docking studies have been performed to investigate the molecular structural features responsible for the noteworthy MOR selectivity.

  15. Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis

    PubMed Central

    Ma, Li-Juan; Ohmert, Iris; Vardanyan, Vitya

    2011-01-01

    Controlled opening and closing of an ion-selective pathway in response to changes of membrane potential is a fundamental feature of voltage-gated ion channels. In recent decades, various details of this process have been revealed with unprecedented precision based on studies of prototypic potassium channels. Though current scientific efforts are focused more on a thorough description of voltage-sensor movement, much less is known about the similarities and differences of the gating mechanisms among potassium channels. Here, we describe the peculiarities of the KCNQ1 gating process in parallel comparison to Shaker. We applied alanine scanning mutagenesis to the S4-S5 linker and pore region and followed the regularities of gating perturbations in KCNQ1. We found a fractional constitutive conductance for wild-type KCNQ1. This component increased significantly in mutants with considerably leftward-shifted steady-state activation curves. In contrast to Shaker, no correlation between V1/2 and Z parameters was observed for the voltage-dependent fraction of KCNQ1. Our experimental findings are explained by a simple allosteric gating scheme with voltage-driven and voltage-independent transitions. Allosteric features are discussed in the context of extreme gating adaptability of KCNQ1 upon interaction with KCNE β-subunits. PMID:21320432

  16. X MARCKS the spot: myristoylated alanine-rich C kinase substrate in neuronal function and disease

    PubMed Central

    Brudvig, Jon J.; Weimer, Jill M.

    2015-01-01

    Intracellular protein-protein interactions are dynamic events requiring tightly regulated spatial and temporal checkpoints. But how are these spatial and temporal cues integrated to produce highly specific molecular response patterns? A helpful analogy to this process is that of a cellular map, one based on the fleeting localization and activity of various coordinating proteins that direct a wide array of interactions between key molecules. One such protein, myristoylated alanine-rich C-kinase substrate (MARCKS) has recently emerged as an important component of this cellular map, governing a wide variety of protein interactions in every cell type within the brain. In addition to its well-documented interactions with the actin cytoskeleton, MARCKS has been found to interact with a number of other proteins involved in processes ranging from intracellular signaling to process outgrowth. Here, we will explore these diverse interactions and their role in an array of brain-specific functions that have important implications for many neurological conditions. PMID:26528135

  17. A Micro-Platinum Wire Biosensor for Fast and Selective Detection of Alanine Aminotransferase

    PubMed Central

    Thuy, Tran Nguyen Thanh; Tseng, Tina T.-C.

    2016-01-01

    In this study, a miniaturized biosensor based on permselective polymer layers (overoxidized polypyrrole (Ppy) and Nafion®) modified and enzyme (glutamate oxidase (GlutOx)) immobilized micro-platinum wire electrode for the detection of alanine aminotransferase (ALT) was fabricated. The proposed ALT biosensor was measured electrochemically by constant potential amperometry at +0.7 V vs. Ag/AgCl. The ALT biosensor provides fast response time (~5 s) and superior selectivity towards ALT against both negatively and positively charged species (e.g., ascorbic acid (AA) and dopamine (DA), respectively). The detection range of the ALT biosensor is found to be 10–900 U/L which covers the range of normal ALT levels presented in the serum and the detection limit and sensitivity are found to be 8.48 U/L and 0.059 nA/(U/L·mm2) (N = 10), respectively. We also found that one-day storage of the ALT biosensor at −20 °C right after the sensor being fabricated can enhance the sensor sensitivity (1.74 times higher than that of the sensor stored at 4 °C). The ALT biosensor is stable after eight weeks of storage at −20 °C. The sensor was tested in spiked ALT samples (ALT activities: 20, 200, 400, and 900 U/L) and reasonable recoveries (70%~107%) were obtained. PMID:27240366

  18. Sequential micellar electrokinetic chromatography analysis of racemization reaction of alanine enantiomers.

    PubMed

    Fu, Rao; Liu, Lina; Guo, Yingna; Guo, Liping; Yang, Li

    2014-02-28

    A novel method for online monitoring racemization reaction of alanine (Ala) enantiomers was developed, by combining sequential sample injection and micellar electrokinetic chromatography (MEKC) technique. Various conditions were investigated to optimize the sequential injection, Ala derivatization and MEKC chiral separation of d-/l-Ala. High reproducibility of the sequential MEKC analysis was demonstrated by analyzing the standard Ala samples, with relative standard deviation values (n=20) of 1.35%, 1.98%, and 1.09% for peak height, peak area and migration time, respectively. Ala racemization was automatically monitored every 40s from the beginning to the end of the reaction, by simultaneous detection of the consumption of the substrate enantiomer and the formation of the product enantiomer. The Michaelis constants of the racemization reaction were obtained by the sequential MEKC method, and were in good agreement with those obtained by traditional off-line enzyme assay. Our study indicated that the present sequential MEKC method can perform fast, efficient, accurate and reproducible analysis of racemization reaction of amino acids, which is of great importance for the determination of the activity of racemase and thus understanding its metabolic functions.

  19. Predicting three-dimensional conformations of peptides constructed of only glycine, alanine, aspartic acid, and valine.

    PubMed

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  20. Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

    NASA Astrophysics Data System (ADS)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  1. L-Alanine augments rhizobacteria-induced systemic resistance in cucumber.

    PubMed

    Park, K S; Paul, D; Kim, J S; Park, J W

    2009-01-01

    Bacillus vallismortis strain EXTN-1 is a proven biotic elicitor of systemic resistance in many crops against various pathogens. L: -Alanine (Ala) was tested in cucumber as a chemical elicitor of induced systemic resistance (ISR) against Colletotrichum orbiculare. In the greenhouse, both Ala and EXTN-1 induced significant levels of disease suppression in cucumber against anthracnose. When cucumber plants were treated with EXTN-1 and Ala together, augmentative disease suppression was observed. Experiments with transgenic tobacco plants carrying pathogenesis-related genes fused with the beta-glucuronidase (GUS) reported gene (PR-1a::GUS & PDF 1.2::GUS) showed an enhanced activation of both PR-1a and PDF 1.2 genes upon combined treatment with Ala and EXTN-1. RT-PCR analysis with transgenic (PR-1a or PDF 1.2 over expressing) Arabidopsis plant showed more enhanced expression of resistance genes PR-1a and PDF 1.2 upon combined treatment with Ala and EXTN-1 than either alone. An augmentative ISR effect, when the bacterial elicitor and chemical elicitor were combined together, was confirmed.

  2. Histological and Clinical Characteristics of Patients with Chronic Hepatitis C and Persistently Normal Alanine Aminotransferase Levels

    PubMed Central

    Guzman, Grace

    2014-01-01

    Patients with chronic hepatitis C virus (HCV) infection and persistently normal alanine aminotransferase (PNALT) are generally described to have mild liver disease. The aim of this study was to compare clinical and histological features in HCV-infected patients with PNALT and elevated ALT. Patients presenting to the University of Illinois Medical Center, Chicago, who had biopsy proven HCV, an ALT measurement at the time of liver biopsy, at least one additional ALT measurement over the next 12 months, and liver biopsy slides available for review were identified. PNALT was defined as ALT ≤ 30 on at least 2 different occasions over 12 months. Of 1200 patients with HCV, 243 met the study criteria. 13% (32/243) of patients had PNALT while 87% (211/243) had elevated ALT. Significantly more patients with PNALT had advanced fibrosis (F3 and F4) compared to those with elevated ALT (P = 0.007). There was no significant difference in the histology activity index score as well as mean inflammatory score between the two groups. In conclusion, in a well-characterized cohort of patients at a tertiary medical center, PNALT did not distinguish patients with mild liver disease. PMID:24891947

  3. Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference.

    PubMed

    Mahan, Sheri D; Ireton, Greg C; Stoddard, Barry L; Black, Margaret E

    2004-07-20

    Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.

  4. FTIR spectra and conformational structure of deutero-β-alanine isolated in argon matrices

    NASA Astrophysics Data System (ADS)

    Stepanian, Stepan G.; Ivanov, Alexander Yu; Adamowicz, Ludwik

    2016-02-01

    Low temperature FTIR spectra of β-alanine-d3 isolated in argon matrices are used to determine the conformational composition of this compound. UV irradiation of the matrix samples is found to change the relative populations of the β-alanine-d3 conformers. The populations of conformers I and II with an Nsbnd D⋯O intramolecular H-bond decrease after the UV irradiation while the populations of conformer V with an N⋯Dsbnd O H-bond and conformer IV which has no intramolecular H-bonds increase. This behavior of the β-alanine-d3 conformers are used to separate the bands of the different conformers. The analysis of the experimental FTIR spectra is based on the calculated harmonic B3LYP/6-311++G(df,pd) frequencies and on the MP2/aug-cc-pVDZ frequencies calculated with a method that includes anharmonic effects. Polynomial scaling of the calculated frequencies is used to achieve better agreement with the experimental data. The observation of the wide band of the OD stretching vibration at 2201 cm-1 is a direct evidence of the presence of the β-alanine-d3 conformer V in the Ar matrix. In total ten bands of conformer V are detected. The influence of the matrix environment on the structures and the IR spectra of the β-alanine and β-alanine-d3 conformers is investigated. This involves performing calculations of the β-alanine conformers embedded in argon clusters containing from 163 to 166 argon atoms using the M06-2X and B3LYP(GD3BJ) density-functional methods. Good agreement between the calculated and the experimental matrix splitting is demonstrated.

  5. Glucose and Alanine Metabolism in Children with Maple Syrup Urine Disease

    PubMed Central

    Haymond, Morey W.; Ben-Galim, Ehud; Strobel, Karen E.

    1978-01-01

    In vitro studies have suggested that catabolism of branched chain amino acids is linked with alanine and glutamine formed in, and released from, muscle. To explore this possibility in vivo, static and kinetic studies were performed in three patients with classical, and one patient with partial, branched chain α-ketoacid decarboxylase deficiency (maple syrup urine disease, MSUD) and compared to similar studies in eight age-matched controls. The subjects underwent a 24-30-h fast, and a glucose-alanine flux study using stable isotopes. Basal plasma leucine concentrations were elevated (P <0.001) in patients with MSUD (1,140±125 μM vs. 155±18 μM in controls); and in contrast to the controls, branched chain amino acid concentrations in plasma increased during the fast in the MSUD patients. Basal plasma alanine concentrations were lower (P <0.01) in patients with classical MSUD (153±8 μM vs. 495±27 μM in controls). This discrepancy was maintained throughout the fast despite a decrease in alanine concentrations in both groups. Plasma alanine and leucine concentrations in the patient with partial MSUD were intermediate between those of the controls and the subjects with the classical form of the disease. Circulating ketone bodies and glucoregulatory hormones concentrations were similar in the MSUD and normal subjects during the fast. Alanine flux rates in two patients with classical MSUD (3.76 and 4.00 μmol/Kg per min) and the patient with partial MSUD (5.76 μmol/Kg per min) were clearly lower than those of the controls (11.72±2.53 [SD] μmol/Kg per min). After short-term starvation, glucose flux and fasting concentrations were similar in the MSUD patients and normal subjects. These data indicate that branched chain amino acid catabolism is an important rate limiting event for alanine production in vivo. PMID:670400

  6. Mycobacterium abscessus ᴅ-alanyl-ᴅ-alanine dipeptidase induces the maturation of dendritic cells and promotes Th1-biased immunity

    PubMed Central

    Lee, Seung Jun; Jang, Jong-Hwa; Yoon, Gun Young; Kang, Da Rae; Park, Hee Jo; Shin, Sung Jae; Han, Hee Dong; Kang, Tae Heung; Park, Won Sun; Yoon, Young Kyung; Soh, Byoung Yul; Jung, In Duk; Park, Yeong-Min

    2016-01-01

    Mycobacterium abscessus, a member of the group of non-tuberculous mycobacteria, has been identified as an emerging pulmonary pathogen in humans. However, little is known about the protective immune response of antigen-presenting cells, such as dendritic cells (DCs), which guard against M. abscessus infection. The M. abscessus gene MAB1843 encodes ᴅ-alanyl-ᴅ-alanine dipeptidase, which catalyzes the hydrolysis of ᴅ-alanyl-ᴅ-alanine dipeptide. We investigated whether MAB1843 is able to interact with DCs to enhance the effectiveness of the host’s immune response. MAB1843 was found to induce DC maturation via toll-like receptor 4 and its downstream signaling pathways, such as the mitogen-activated protein kinase and nuclear factor kappa B pathways. In addition, MAB1843-treated DCs stimulated the proliferation of T cells and promoted Th1 polarization. Our results indicate that MAB1843 could potentially regulate the immune response to M. abscessus, making it important in the development of an effective vaccine against this mycobacterium. [BMB Reports 2016; 49(10): 554-559] PMID:27439605

  7. ABS-Scan: In silico alanine scanning mutagenesis for binding site residues in protein-ligand complex.

    PubMed

    Anand, Praveen; Nagarajan, Deepesh; Mukherjee, Sumanta; Chandra, Nagasuma

    2014-01-01

    Most physiological processes in living systems are fundamentally regulated by protein-ligand interactions. Understanding the process of ligand recognition by proteins is a vital activity in molecular biology and biochemistry. It is well known that the residues present at the binding site of the protein form pockets that provide a conducive environment for recognition of specific ligands. In many cases, the boundaries of these sites are not well defined. Here, we provide a web-server to systematically evaluate important residues in the binding site of the protein that contribute towards the ligand recognition through in silico alanine-scanning mutagenesis experiments. Each of the residues present at the binding site is computationally mutated to alanine. The ligand interaction energy is computed for each mutant and the corresponding ΔΔG values are calculated by comparing it to the wild type protein, thus evaluating individual residue contributions towards ligand interaction. The server will thus provide a ranked list of residues to the user in order to obtain loss-of-function mutations. This web-tool can be freely accessed through the following address: http://proline.biochem.iisc.ernet.in/abscan/.

  8. Molecular dynamics simulations of mutated Mycobacterium tuberculosis L-alanine dehydrogenase to illuminate the role of key residues.

    PubMed

    Ling, Baoping; Bi, Siwei; Sun, Min; Jing, Zhihong; Li, Xiaoping; Zhang, Rui

    2014-05-01

    L-Alanine dehydrogenase from Mycobacterium tuberculosis (L-MtAlaDH) catalyzes the NADH-dependent interconversion of l-alanine and pyruvate, and it is considered to be a potential target for the treatment of tuberculosis. The experiment has verified that amino acid replacement of the conserved active-site residues which have strong stability and no great changes in biological evolutionary process, such as His96 and Asp270, could lead to inactive mutants [Ågren et al., J. Mol. Biol. 377 (2008) 1161-1173]. However, the role of these conserved residues in catalytic reaction still remains unclear. Based on the crystal structures, a series of mutant structures were constructed to investigate the role of the conserved residues in enzymatic reaction by using molecular dynamics simulations. The results show that whatever the conserved residues were mutated, the protein can still convert its conformation from open state to closed state as long as NADH is present in active site. Asp270 maintains the stability of nicotinamide ring and ribose of NADH through hydrogen bond interactions, and His96 is helpful to convert the protein conformation by interactions with Gln271, whereas, they would lead to the structural rearrangement in active site and lose the catalytic activity when they were mutated. Additionally, we deduce that Met301 plays a major role in catalytic reaction due to fixing the nicotinamide ring of NADH to prevent its rotation, and we propose that Met301 would be mutated to the hydrophobic residue with large steric hindrance in side chain to test the activity of the protein in future experiment.

  9. Microbial transformations of free versus sorbed alanine analyzed by position-specific 13C and 14C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Apostel, Carolin; Dippold, Michaela; Bore, Ezekiel; Kuzyakov, Yakov

    2015-04-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model representative for the pool of LMWOS. To assess transformations of sorbed alanine, we combined position-specifically and uniformly 13C and 14C labeled alanine tracer solution with a loamy haplic luvisol that had previously been sterilized by γ-radiation. After shaking the mixtures, the supernatant was removed, as was all non-sorbed alanine by repeated shaking with millipore water. The labeled soil was added to non-sterilized soil from the same site. To compare the effect of sorption, soil labeled with the same position-specifically labeled tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time steps. The incorporation of 14C into microbial biomass was determined by CFE, and utilization of individual C positions by distinct microbial groups was evaluated by 13C-PLFA analysis. A dual peak in the respired CO2 revealed the influence of two sorption mechanisms. Microbial uptake and transformation of the sorbed alanine was 3 times slower compared to non-sorbed alanine. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we introduced the divergence index (DI). The DI reveals the convergent or divergent behaviour of C from individual molecule positions during microbial utilization. The DI revealed, that alanines C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFAs. This indicates

  10. Treatment of Huntington disease with gamma-acetylenic GABA an irreversible inhibitor of GABA-transaminase: increased CSF GABA and homocarnosine without clinical amelioration.

    PubMed

    Tell, G; Böhlen, P; Schechter, P J; Koch-Weser, J; Agid, Y; Bonnet, A M; Coquillat, G; Chazot, G; Fischer, C

    1981-02-01

    gamma-Acetylenic GABA (GAG, RMI 71.645), a potent irreversible inhibitor of gamma-aminobutyric acid transaminase, was given orally in various dosage schedules to 14 patients with Huntington disease. The biochemical effects of the drug on cerebrospinal fluid (CSF) concentrations of gamma-aminobutyric acid (GABA) and the GABA-containing dipeptide, homocarnosine, were measured in 10 of 14 patients. Treatment with GAG increased CSF concentrations of GABA and homocarnosine as compared to pretreatment values, suggesting that the drug increased brain GABA concentration. Despite this neurochemical effect, the clinical state was not improved. Except for single seizure episodes in five patients, GAG therapy was well tolerated. These results do not exclude the possibility that agents that augment CNS GABAergic function may prove useful in therapy of Huntington disease.

  11. Perturbation correction for alanine dosimeters in different phantom materials in high-energy photon beams.

    PubMed

    von Voigts-Rhetz, P; Anton, M; Vorwerk, H; Zink, K

    2016-02-07

    In modern radiotherapy the verification of complex treatments plans is often performed in inhomogeneous or even anthropomorphic phantoms. For dose verification small detectors are necessary and therefore alanine detectors are most suitable. Though the response of alanine for a wide range of clinical photon energies in water is well know, the knowledge about the influence of the surrounding phantom material on the response of alanine is sparse. Therefore we investigated the influence of twenty different surrounding/phantom materials for alanine dosimeters in clinical photon fields via Monte Carlo simulations. The relative electron density of the used materials was in the range [Formula: see text] up to 1.69, covering almost all materials appearing in inhomogeneous or anthropomorphic phantoms used in radiotherapy. The investigations were performed for three different clinical photon spectra ranging from 6 to 25 MV-X and Co-60 and as a result a perturbation correction [Formula: see text] depending on the environmental material was established. The Monte Carlo simulation show, that there is only a small dependence of [Formula: see text] on the phantom material and the photon energy, which is below  ±0.6%. The results confirm the good suitability of alanine detectors for in-vivo dosimetry.

  12. Theoretical and experimental study of valence photoelectron spectrum of D,L-alanine amino acid.

    PubMed

    Farrokhpour, H; Fathi, F; De Brito, A Naves

    2012-07-05

    In this work, the He-I (21.218 eV) photoelectron spectrum of D,L-alanine in the gas phase is revisited experimentally and theoretically. To support the experiment, the high level ab initio calculations were used to calculate and assign the photoelectron spectra of the four most stable conformers of gaseous alanine, carefully. The symmetry adapted cluster/configuration interaction (SAC-CI) method based on single and double excitation operators (SD-R) and its more accurate version, termed general-R, was used to separately calculate the energies and intensities of the ionization bands of the L- and D-alanine conformers. The intensities of ionization bands were calculated based on the monopole approximation. Also, natural bonding orbital (NBO) calculations were employed for better spectral band assignment. The relative electronic energy, Gibbs free energy, and Boltzmann population ratio of the conformers were calculated at the experimental temperature (403 K) using several theoretical methods. The theoretical photoelectron spectrum of alanine was calculated by summing over the spectra of individual D and L conformers weighted by different population ratios. Finally, the population ratio of the four most stable conformers of alanine was estimated from the experimental photoelectron spectrum using theoretical calculations for the first time.

  13. Perturbation correction for alanine dosimeters in different phantom materials in high-energy photon beams

    NASA Astrophysics Data System (ADS)

    von Voigts-Rhetz, P.; Anton, M.; Vorwerk, H.; Zink, K.

    2016-02-01

    In modern radiotherapy the verification of complex treatments plans is often performed in inhomogeneous or even anthropomorphic phantoms. For dose verification small detectors are necessary and therefore alanine detectors are most suitable. Though the response of alanine for a wide range of clinical photon energies in water is well know, the knowledge about the influence of the surrounding phantom material on the response of alanine is sparse. Therefore we investigated the influence of twenty different surrounding/phantom materials for alanine dosimeters in clinical photon fields via Monte Carlo simulations. The relative electron density of the used materials was in the range {{n}e}/{{n}e,\\text{w}}=0.20 up to 1.69, covering almost all materials appearing in inhomogeneous or anthropomorphic phantoms used in radiotherapy. The investigations were performed for three different clinical photon spectra ranging from 6 to 25 MV-X and Co-60 and as a result a perturbation correction {{k}\\text{env}} depending on the environmental material was established. The Monte Carlo simulation show, that there is only a small dependence of {{k}\\text{env}} on the phantom material and the photon energy, which is below  ±0.6%. The results confirm the good suitability of alanine detectors for in-vivo dosimetry.

  14. Absorbed dose determination in kilovoltage X-ray synchrotron radiation using alanine dosimeters.

    PubMed

    Butler, D J; Lye, J E; Wright, T E; Crossley, D; Sharpe, P H G; Stevenson, A W; Livingstone, J; Crosbie, J C

    2016-12-01

    Alanine dosimeters from the National Physical Laboratory (NPL) in the UK were irradiated using kilovoltage synchrotron radiation at the imaging and medical beam line (IMBL) at the Australian Synchrotron. A 20 × 20 mm(2) area was irradiated by scanning the phantom containing the alanine through the 1 mm × 20 mm beam at a constant velocity. The polychromatic beam had an average energy of 95 keV and nominal absorbed dose to water rate of 250 Gy/s. The absorbed dose to water in the solid water phantom was first determined using a PTW Model 31014 PinPoint ionization chamber traceable to a graphite calorimeter. The alanine was read out at NPL using correction factors determined for (60)Co, traceable to NPL standards, and a published energy correction was applied to correct for the effect of the synchrotron beam quality. The ratio of the doses determined by alanine at NPL and those determined at the synchrotron was 0.975 (standard uncertainty 0.042) when alanine energy correction factors published by Waldeland et al. (Waldeland E, Hole E O, Sagstuen E and Malinen E, Med. Phys. 2010, 37, 3569) were used, and 0.996 (standard uncertainty 0.031) when factors by Anton et al. (Anton M, Büermann L., Phys Med Biol. 2015 60 6113-29) were used. The results provide additional verification of the IMBL dosimetry.

  15. Effect of 10 week beta-alanine supplementation on competition and training performance in elite swimmers.

    PubMed

    Chung, Weiliang; Shaw, Greg; Anderson, Megan E; Pyne, David B; Saunders, Philo U; Bishop, David J; Burke, Louise M

    2012-10-09

    Although some laboratory-based studies show an ergogenic effect with beta-alanine supplementation, there is a lack of field-based research in training and competition settings. Elite/Sub-elite swimmers (n = 23 males and 18 females, age = 21.7 ± 2.8 years; mean ± SD) were supplemented with either beta-alanine (4 weeks loading phase of 4.8 g/day and 3.2 g/day thereafter) or placebo for 10 weeks. Competition performance times were log-transformed, then evaluated before (National Championships) and after (international or national selection meet) supplementation. Swimmers also completed three standardized training sets at baseline, 4 and 10 weeks of supplementation. Capillary blood was analyzed for pH, bicarbonate and lactate concentration in both competition and training. There was an unclear effect (0.4%; ± 0.8%, mean, ± 90% confidence limits) of beta-alanine on competition performance compared to placebo with no meaningful changes in blood chemistry. While there was a transient improvement on training performance after 4 weeks with beta-alanine (-1.3%; ± 1.0%), there was an unclear effect at ten weeks (-0.2%; ± 1.5%) and no meaningful changes in blood chemistry. Beta-alanine supplementation appears to have minimal effect on swimming performance in non-laboratory controlled real-world training and competition settings.

  16. Specific proteolysis of native alanine racemases from Salmonella typhimurium: identification of the cleavage site and characterization of the clipped two-domain proteins

    SciTech Connect

    Galakatos, N.G.; Walsh, C.T.

    1987-12-15

    Native DadB and Alr alanine racemases (M/sub r/ 39,000) from Salmonella typhimurium are proteolyzed at homologous positions by ..cap alpha..-chymotrypsin, trypsin, and subtilisin to generate in all cases two nonoverlapping polypeptides of M/sub r/ 28,000 and 11,000. Under nondenaturing conditions, chymotryptic digest results in an associated form of the two fragments which possesses 3% of the original catalytic activity, incorporates 0.76 equiv of the mechanism-based inactivator ..beta..-chloro-(/sup 14/C)-D-alanine, and exhibits a UV circular dichroism profile identical with that of native enzyme. Protein sequence analysis of the denatured chymotryptic fragments indicates the presence of a tetrapeptide interdomain hinge (DadB, residues 254-257; Alr, residues 256-259) that is attacked at both ends during proteolysis. Under the previously employed digest conditions, NaB/sup 3/H/sub 4/-reduced DadB holoenzyme is resistant to ..cap alpha..-chymotrypsin and trypsin and is labile only toward subtilisin. These data suggest that the hinge structure is essential for a catalytically efficient enzyme species and is sensitive to active site geometry. The sequence at the hinge region is also conserved in alanine racemases from Gram-positive bacteria.

  17. Using Position-Specific 13C and 14C Labeling and 13C-PLFA Analysis to Assess Microbial Transformations of Free Versus Sorbed Alanine

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Herschbach, J.; Bore, E. K.; Kuzyakov, Y.; Dippold, M. A.

    2015-12-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model substance for the pool of LMWOS. To assess transformations of sorbed alanine, we added position-specific and uniformly 13C and 14C labeled alanine tracer to soil that had previously been sterilized by γ-radiation. The labeled soil was added to non-sterilized soil from the same site and incubated. Soil labeled with the same tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time intervals. The incorporation of 14C into microbial biomass was determined by chloroform fumigation extraction (CFE), and utilization of individual C positions by distinct microbial groups was evaluated by 13C-phospholipid fatty acid analysis (PLFA). A dual peak in the respired CO2 revealed two sorption mechanisms. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we applied the divergence index (DI). The DI reveals the convergent or divergent behavior of C from individual molecule positions during microbial utilization. Alanine C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFA. This indicates that sorption by the COOH group does not protect this group from preferential oxidation. Microbial metabolism was determinative for the preferential oxidation of individual molecule positions. The use of position-specific labeling revealed mechanisms and kinetics of microbial utilization of sorbed and non

  18. β-Alanine as an Ethylene Precursor. Investigations Towards Preparation, and Properties, of a Soluble Enzyme System From a Subcellular Particulate Fraction of Bean Cotyledons 1

    PubMed Central

    Stinson, Robert A.; Spencer, Mary

    1969-01-01

    A method is described for the preparation, from a subcellular particulate fraction of wax bean cotyledons, of a soluble enzyme system that is capable of converting β-alanine to ethylene. In the presence of ATP, CoA, thiamine pyrophosphate, MgSO4, and pyridoxal phosphate, ethylene production is maximum at a 0.5 mm concentration of β-alanine. The system exhibits a pH optimum at 7.0 but when the pH is raised above 8, evolution of the volatile again increases and continues to do so up to pH 12. The enzyme system is stimulated by either NADPH or NADH; the concentration of NADPH necessary to obtain maximum activity is twice that of NADH. The requirement for a reducing agent is in agreement with the proposal that malonate semialdehyde, formed by an aminotransferase reaction from β-alanine, is reduced to β-hydroxypropionate. Both malonate semialdehyde and β-hydroxypropionate are better stimulators of production of the volatile in the soluble system than is β-alanine, and β-hydroxypropionate is a better stimulator than malonate semialdehyde. This system is also able to incorporate tritium from tritiated water into ethylene; this supports the proposal that ethylene is formed by the decarboxylation of acrylate, the latter being formed from β-hydroxypropionate. Experiments with both cold and labeled malonate suggest that this compound stimulates ethylene production by acting as an end product inhibitor that prevents the loss of malonate semialdehyde from the pathway. Malonate does not appear to serve as a precursor. Addition of cytoplasmic enzymes to the `soluble system' (prepared from particulate enzymes) results in a considerable boost in ethylene production, but the specific activity (mμ1 / mg protein) is lowered from that of the particulate enzymes alone. PMID:16657194

  19. Hepatoprotective activity of Moringa oleifera on antitubercular drug-induced liver damage in rats.

    PubMed

    Pari, L; Kumar, N Ashok

    2002-01-01

    Moringa oleifera Lam (Moringaceae), commonly known as "Drumstick," is used in Indian folk medicine for the treatment of various illness. We have evaluated the hepatoprotective effect of an ethanolic extract of M. oleifera leaves on liver damage induced by antitubercular drugs such as isoniazid (INH), rifampicin (RMP), and pyrazinamide (PZA) in rats. Oral administration of the extract showed a significant protective action made evident by its effect on the levels of glutamic oxaloacetic transaminase (aspartate aminotransferase), glutamic pyruvic transaminase (alanine aminotransferase), alkaline phosphatase, and bilirubin in the serum; lipids, and lipid peroxidation levels in liver. This observation was supplemented by histopathological examination of liver sections. The results of this study showed that treatment with M. oleifera extracts or silymarin (as a reference) appears to enhance the recovery from hepatic damage induced by antitubercular drugs.

  20. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes*

    PubMed Central

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-01-01

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of the kinetic studies. We purified His6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (Km = 0.35 ± 0.03 mm for l-aspartate, Km = 0.098 ± 0 mm for d-aspartate, Km = 26 ± 2 mm for l-alanine, Km = 3.3 ± 0.2 mm for d-alanine). Competitive inhibition by various amino acids of l-aspartate or l-alanine in self-exchange reactions revealed that l-cysteine selectively inhibited l-aspartate self-exchange but only weakly inhibited l-alanine self-exchange. Additionally, l-serine selectively inhibited l-alanine self-exchange but barely inhibited l-aspartate self-exchange. The aspartate analogs l-cysteine sulfinic acid, l-cysteic acid, and d-cysteic acid competitively and strongly inhibited l-aspartate self-exchange compared with l-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of l-aspartate and l-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  1. Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

    PubMed

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-08-19

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.

  2. Sensitivity of alanine dosimeters with gadolinium exposed to 6 MV photons at clinical doses.

    PubMed

    Marrale, M; Longo, A; Spanò, M; Bartolotta, A; D'Oca, M C; Brai, M

    2011-12-01

    In this study we analyzed the ESR signal of alanine dosimeters with gadolinium exposed to 6 MV linear accelerator photons. We observed that the addition of gadolinium brings about an improvement in the sensitivity to photons because of its high atomic number. The experimental data indicated that the addition of gadolinium increases the sensitivity of the alanine to 6 MV photons. This enhancement was better observed at high gadolinium concentrations for which the tissue equivalence is heavily reduced. However, information about the irradiation setup and of the radiation beam features allows one to correct for this difference. Monte Carlo simulations were carried out to obtain information on the expected effect of the addition of gadolinium on the dose absorbed by the alanine molecules inside the pellets. These results are compared with the experimental values, and the agreement is discussed.

  3. On the roles of the alanine and serine in the β-sheet structure of fibroin.

    PubMed

    Carrascoza Mayen, Juan Francisco; Lupan, Alexandru; Cosar, Ciprian; Kun, Attila-Zsolt; Silaghi-Dumitrescu, Radu

    2015-02-01

    In its silk II form, fibroin is almost exclusively formed from layers of β-sheets, rich in glycine, alanine and serine. Reported here are computational results on fibroin models at semi-empirical, DFT levels of theory and molecular dynamics (MD) for (Gly)10, (Gly-Ala)5 and (Gly-Ser)5 decapeptides. While alanine and serine introduce steric repulsions, the alanine side-chain adds to the rigidity of the sheet, allowing it to maintain a properly pleated structure even in a single β-sheet, and thus avoiding two alternative conformations which would interfere with the formation of the multi-layer pleated-sheet structure. The role of the serine is proposed to involve modulation of the hydrophobicity in order to construct the supramolecular assembly as opposed to random precipitation due to hydrophobicity.

  4. A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine.

    PubMed

    Fonseca, Andrea S C; Gonçalves, M Sameiro T; Costa, Susana P G

    2012-12-01

    A novel fluorescent amino acid, L-4-chloromethylcoumarin-6-yl-alanine, was obtained from tyrosine by a Pechmann reaction. The assembly of the heterocyclic ring at the tyrosine side chain could be achieved before or after incorporation of tyrosine into a dipeptide, and amino acid and dipeptide ester conjugates were obtained by coupling to a model N-protected alanine. The behaviour of one of the fluorescent conjugates towards irradiation was studied in a photochemical reactor at different wavelengths (254, 300, 350 and 419 nm). The photoreaction course in methanol/HEPES buffer solution (80:20) was followed by HPLC/UV monitoring. It was found that the novel unnatural amino acid could act as a fluorescent label, due to its fluorescence properties, and, more importantly, as a photoactivable unit, due to the short irradiation times necessary to cleave the ester bond between the model amino acid and the coumarin-6-yl-alanine.

  5. Nucleation kinetics, growth and studies of β-alanine single crystals

    NASA Astrophysics Data System (ADS)

    Shanthi, D.; Selvarajan, P.; HemaDurga, K. K.; Lincy Mary Ponmani, S.

    2013-06-01

    Solubility and metastable zone width for the re-crystallized salt of β-alanine was determined. Induction period measurement for the selected supersaturation ratios at room temperature (31 °C) was carried out for supersaturated aqueous solutions of β-alanine and it is noticed that induction period decreases with increase of supersaturation ratio. The nucleation parameters such as Gibbs free energy change, radius and number of molecules of the critical nucleus, interfacial tension and the nucleation rate have been evaluated by classical nucleation theory. Single crystals of β-alanine were grown using the optimized nucleation parameters by solution method and grown crystals have been subjected to various studies like XRD studies, FTIR, optical, thermal and SHG studies.

  6. Relative response of the alanine dosimeter to medium energy x-rays.

    PubMed

    Anton, M; Büermann, L

    2015-08-07

    The response of the alanine dosimeter to kilovoltage x-rays with respect to the dose to water was measured, relative to the response to Co-60 radiation.Two series of x-ray qualities were investigated, one ranging from 30 kV to 100 kV tube voltage (TW series), the other one ranging from 70 kV to 280 kV (TH series). Due to the use of the water calorimeter as a primary standard, the uncertainty of the delivered dose is significantly lower than for other published data. The alanine response was measured as described in a previous publication (Anton et al 2013 Phys. Med. Biol. 58 3259-82). The uncertainty component due to the alanine measurement and analysis is ⩽0.4%, the major part of the combined uncertainty of the relative response originates from the uncertainty of the delivered dose. The relative uncertainties of the relative response vary from ⩽2% for the TW series to ⩽1.1% for the TH series.Different from the behaviour of the alanine dosimeter for megavoltage x-rays or electrons, the relative response drops significantly from unity for Co-60 radiation to less than 64% for the TW quality with a tube voltage of 30 kV. In order to reproduce this behaviour through Monte Carlo simulations, not only the ratio of the absorbed dose to alanine to the absorbed dose to water has to be known, but also the intrinsic efficiency, i.e. the dependence of the number of free radicals generated per unit of absorbed dose on the photon energy. This quantity is not yet accessible for the TW series.For a possible use of the alanine dosimeter for kilovoltage x-rays, for example in electronic brachytherapy, users should rely on the measured data for the relative response which have become available with this publication.

  7. Fragmentation of α- and β-alanine molecules by ions at Bragg-peak energies

    NASA Astrophysics Data System (ADS)

    Bari, S.; Sobocinski, P.; Postma, J.; Alvarado, F.; Hoekstra, R.; Bernigaud, V.; Manil, B.; Rangama, J.; Huber, B.; Schlathölter, T.

    2008-02-01

    The interaction of keV He+, He2+, and O5+ ions with isolated α and β isomers of the amino acid alanine was studied by means of high resolution coincidence time-of-flight mass spectrometry. We observed a strong isomer dependence of characteristic fragmentation channels which manifests in strongly altered branching ratios. Despite the ultrashort initial perturbation by the incoming ion, evidence for molecular rearrangement leading to the formation of H3+ was found. The measured kinetic energies of ionic alanine fragments can be sufficient to induce secondary damage to DNA in a biological environment.

  8. On the fragmentation of biomolecules: Fragmentation of alanine dipeptide along the polypeptide chain

    SciTech Connect

    Solov'yov, I. A. Yakubovich, A. V.; Solov'yov, A. V.; Greiner, W.

    2006-09-15

    The interaction potential between amino acids in alanine dipeptide has been studied for the first time taking into account exact molecular geometry. Ab initio calculation has been performed in the framework of density functional theory taking into account all electrons in the system. The fragmentation of dipeptide along the polypeptide chain, as well as the interaction between alanines, has been considered. The energy of the system has been analyzed as a function of the distance between fragments for all possible dipeptide fragmentation channels. Analysis of the energy barriers makes it possible to estimate the characteristic fragmentation times and to determine the degree of applicability of classical electrodynamics for describing the system energy.

  9. Neurotoxic Non-proteinogenic Amino Acid β-N-Methylamino-L-alanine and Its Role in Biological Systems.

    PubMed

    Popova, A A; Koksharova, O A

    2016-08-01

    Secondary metabolites of photoautotrophic organisms have attracted considerable interest in recent years. In particular, molecules of non-proteinogenic amino acids participating in various physiological processes and capable of producing adverse ecological effects have been actively investigated. For example, the non-proteinogenic amino acid β-N-methylamino-L-alanine (BMAA) is neurotoxic to animals including humans. It is known that BMAA accumulation via the food chain can lead to development of neurodegenerative diseases in humans such as Alzheimer's and Parkinson's diseases as well as amyotrophic lateral sclerosis. Moreover, BMAA can be mistakenly incorporated into a protein molecule instead of serine. Natural sources of BMAA and methods for its detection are discussed in this review, as well as the role of BMAA in metabolism of its producers and possible mechanisms of toxicity of this amino acid in different living organisms.

  10. Antibody and markers of T-cell activation illuminate the pathogenesis of HCV immune restoration disease in HIV/HCV co-infected patients commencing ART.

    PubMed

    Yunihastuti, Evy; Lee, Silvia; Gani, Rino A; Saraswati, Henny; Sundaru, Heru; Lesmana, L A; Sukmana, Nanang; Price, Patricia

    2011-04-01

    Some HIV/hepatitis C virus co-infected patients beginning ART experience Immune Restoration Disease (IRD) manifested as a rise in serum alanine transaminase. This was investigated in HIV/HCV co-infected individuals (n=50) commencing ART in Jakarta (Indonesia). Samples were collected at weeks 0, 4, 8, 12, 24 and at HCV IRD. Nine patients experienced HCV IRD (incidence=9.2 per 1000 person-weeks). These resolved without changing treatment. Markers of T-cell activation (sCD26, sCD30) and immune recruitment (CXCL10) increased in many HCV IRD cases, so T-cells may mediate HCV IRD. Total anti-HCV antibody (core, NS3, NS4) remained lower in HCV IRD cases, but levels of antibody to core were not lower in HCV IRD cases. Rises in HCV RNA on ART were independent of HCV IRD, but there was a negative correlation between baseline HCV RNA and total anti-HCV antibody. High levels of antibody may protect against HCV IRD, via lower HCV antigen loads.

  11. Effect of Beta alanine and sodium bicarbonate supplementation on repeated-sprint performance.

    PubMed

    Ducker, Kagan J; Dawson, Brian; Wallman, Karen E

    2013-12-01

    This study aimed to investigate if combining beta alanine (BA) and sodium bicarbonate (NaHCO3) supplementation could lead to enhanced repeated-sprint performance in team-sport athletes, beyond what is possible with either supplement alone. Participants (n = 24) completed duplicate trials of a repeated-sprint test (3 sets; 6 × 20 m departing every 25 seconds, 4 minutes active recovery between sets) and were then allocated into 4 groups as follows: BA only (n = 6; 28 days BA, acute sodium chloride placebo); NaHCO3 only (n = 6; 28 days glucose placebo, acute NaHCO3); BA/NaHCO3 (n = 6; 28 days BA, acute NaHCO3); placebo only (n = 6; 28 days glucose placebo, acute sodium chloride placebo), then completed duplicate trials postsupplementation. Sodium bicarbonate alone resulted in moderate effect size (d = 0.40-0.71) and "likely" and "very likely" benefit for overall total sprint times (TST) and for each individual set and for first sprint (sets 2 and 3) and best sprint time (sets 2 and 3). Combining BA and NaHCO3 resulted in "possible" to "likely" benefits for overall TST and for sets 2 and 3. First sprint (set 3) and best sprint time (sets 2 and 3) also showed "likely" benefit after this trial. The BA and placebo groups showed no differences in performance after supplementation. In conclusion, these results indicate that supplementation with acute NaHCO3 improved repeated-sprint performance more than either a combination of NaHCO3 and BA or BA alone.

  12. Alanine Expansions Associated with Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-mediated Dimerization and Nuclear Import*

    PubMed Central

    Di Lascio, Simona; Belperio, Debora

    2016-01-01

    Heterozygous mutations of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to congenital central hypoventilation syndrome (CCHS), a neurodevelopmental disorder characterized by a failure in the autonomic control of breathing. Polyalanine expansions in the 20-residues region of the C terminus of PHOX2B are the major mutations responsible for CCHS. Elongation of the alanine stretch in PHOX2B leads to a protein with altered DNA binding, transcriptional activity, and nuclear localization and the possible formation of cytoplasmic aggregates; furthermore, the findings of various studies support the idea that CCHS is not due to a pure loss of function mechanism but also involves a dominant negative effect and/or toxic gain of function for PHOX2B mutations. Because PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant negative effects of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A using a co-immunoprecipitation assay and the mammalian two-hybrid system. Our findings show that PHOX2B forms homodimers and heterodimerizes weakly with mutated proteins, exclude the direct involvement of the polyalanine tract in dimer formation, and indicate that mutated proteins retain partial ability to form heterodimers with PHOX2A. Moreover, in this study, we investigated the effects of the longest polyalanine expansions on the homeodomain-mediated nuclear import, and our data clearly show that the expanded C terminus interferes with this process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity. PMID:27129232

  13. Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice

    NASA Astrophysics Data System (ADS)

    de Souza, Ludmilla Regina; Alexandre Muehlmann, Luis; Carneiro Matos, Lívia; Simón-Vázquez, Rosana; Guerreiro Marques Lacava, Zulmira; Maurício Batista De-Paula, Alfredo; Mosiniewicz-Szablewska, Ewa; Suchocki, Piotr; César Morais, Paulo; González-Fernández, África; Nair Báo, Sônia; Bentes Azevedo, Ricardo

    2015-12-01

    Selol is a semi-synthetic compound containing selenite that is effective against cancerous cells and safer for clinical applications in comparison with other inorganic forms of selenite. Recently, we have developed a formulation of poly(methyl vinyl ether-co-maleic anhydride)-shelled selol nanocapsules (SPN), which reduced the proliferative activity of lung adenocarcinoma cells and presented little deleterious effects on normal cells in in vitro studies. In this study, we report on the antitumor activity and systemic effects induced by this formulation in chemically induced lung adenocarcinoma-bearing mice. The in vivo antitumor activity of the SPN was verified by macroscopic quantification, immunohistochemistry and morphological analyses. Toxicity analyses were performed by evaluations of the kidney, liver, and spleen; analyses of hemogram and plasma levels of alanine aminotransferase, aspartate transaminase, urea, and creatinine; and DNA fragmentation and cell cycle activity of the bone marrow cells. Furthermore, we investigated the potential of the SPN formulation to cause hemolysis, activate the complement system, provoke an inflammatory response and change the conformation of the plasma proteins. Our results showed that the SPN reduced the area of the surface tumor nodules but not the total number of tumor nodules. The biochemical and hematological findings were suggestive of the low systemic toxicity of the SPN formulation. The surface properties of the selol nanocapsules point to characteristics that are consistent with the treatment of the tumors in vivo: low hemolytic activity, weak inflammatory reaction with no activation of the complement system, and mild or absent conformational changes of the plasma proteins. In conclusion, this report suggests that the SPN formulation investigated herein exhibits anti-tumoral effects against lung adenocarcinoma in vivo and is associated with low systemic toxicity and high biocompatibility.

  14. Effects of glycine, beta-alanine and diazepam upon morphine-tolerant-dependent mice.

    PubMed

    Contreras, E; Tamayo, L

    1980-05-01

    The effects in mice of glycine, beta-alanine and diazepam on the analgesic response to morphine, on the intensity of tolerance and on the physical dependence on the analgesic have been examined. The two amino acids increased the analgesic response to morphine in a dose-related manner. However, both compounds were ineffective in the analgesic test (hot plate) when administered without morphine. Diazepam was ineffective in the analgesic test and it did not alter morphine analgesia, except when administered in a high dose which decreased and analgesic response. Glycine, either in single or repeated doses, did not modify tolerance to morphine, whereas beta-alanine induced a dose-related partial antagonism, which promptly reached a plateau. Diazepam induced a small decrease in the intensity of tolerance to the analgesic. The abstinence syndrome to morphine, induced by naloxone administration to primed mice, was reduced by single doses of glycine or beta-alanine. Diazepam behaved as a weak inhibitor of the abstinence syndrome when administered at a high dose. The potentiation of morphine analgesia and the antagonism of the abstinence syndrome induced by the amino acids may be related to their hyperpolarizing action in the c.n. system. The effects of beta-alanine on morphine tolerance cannot be explained by the same mechanism.

  15. Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I

    PubMed Central

    Pey, Angel L.; Albert, Armando; Salido, Eduardo

    2013-01-01

    Alanine-glyoxylate aminotransferase catalyzes the transamination between L-alanine and glyoxylate to produce pyruvate and glycine using pyridoxal 5′-phosphate (PLP) as cofactor. Human alanine-glyoxylate aminotransferase is a peroxisomal enzyme expressed in the hepatocytes, the main site of glyoxylate detoxification. Its deficit causes primary hyperoxaluria type I, a rare but severe inborn error of metabolism. Single amino acid changes are the main type of mutation causing this disease, and considerable effort has been dedicated to the understanding of the molecular consequences of such missense mutations. In this review, we summarize the role of protein homeostasis in the basic mechanisms of primary hyperoxaluria. Intrinsic physicochemical properties of polypeptide chains such as thermodynamic stability, folding, unfolding, and misfolding rates as well as the interaction of different folding states with protein homeostasis networks are essential to understand this disease. The view presented has important implications for the development of new therapeutic strategies based on targeting specific elements of alanine-glyoxylate aminotransferase homeostasis. PMID:23956997

  16. Influence of beta-alanine supplementation on skeletal muscle carnosine concentrations and high intensity cycling capacity.

    PubMed

    Hill, C A; Harris, R C; Kim, H J; Harris, B D; Sale, C; Boobis, L H; Kim, C K; Wise, J A

    2007-02-01

    Muscle carnosine synthesis is limited by the availability of beta-alanine. Thirteen male subjects were supplemented with beta-alanine (CarnoSyn) for 4 wks, 8 of these for 10 wks. A biopsy of the vastus lateralis was obtained from 6 of the 8 at 0, 4 and 10 wks. Subjects undertook a cycle capacity test to determine total work done (TWD) at 110% (CCT(110%)) of their maximum power (Wmax). Twelve matched subjects received a placebo. Eleven of these completed the CCT(110%) at 0 and 4 wks, and 8, 10 wks. Muscle biopsies were obtained from 5 of the 8 and one additional subject. Muscle carnosine was significantly increased by +58.8% and +80.1% after 4 and 10 wks beta-alanine supplementation. Carnosine, initially 1.71 times higher in type IIa fibres, increased equally in both type I and IIa fibres. No increase was seen in control subjects. Taurine was unchanged by 10 wks of supplementation. 4 wks beta-alanine supplementation resulted in a significant increase in TWD (+13.0%); with a further +3.2% increase at 10 wks. TWD was unchanged at 4 and 10 wks in the control subjects. The increase in TWD with supplementation followed the increase in muscle carnosine.

  17. Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography-tandem mass spectrometry.

    PubMed

    Burton, Casey; Gamagedara, Sanjeewa; Ma, Yinfa

    2013-04-01

    Since sarcosine and D,L-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine-alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R(2) = 0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of D,L-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb-1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from D,L-alanine leading to underivatized quantification of sarcosine.

  18. Synthesis, characterization, and biocompatible properties of alanine-grafted chitosan copolymers.

    PubMed

    Park, Gyu Han; Kang, Min-Sil; Knowles, Jonathan C; Gong, Myoung-Seon

    2016-04-01

    In order to overcome major problems regarding the lack of affinity to solvents and limited reactivity of the free amines of chitosan, introduction of appropriate spacer arms having terminal amine function is considered of interest. L-Alanine-N-carboxyanhydride was grafted onto chitosan via anionic ring-opening polymerization. The chemical and structural characterizations of L-alanine-grafted chitosan (Ala-g-Cts) were confirmed through Fourier transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy ((1)H NMR). In addition, the viscoelastic properties of Ala-g-Cts were examined by means of a rotational viscometer, and thermal analysis was carried out with a thermogravimetric analyzer and differential scanning calorimetry. Morphological changes in the chitosan L-alanine moiety were determined by x-ray diffraction. To determine the feasibility of using these films as biomedical materials, we investigated the effects of their L-alanine content on physical and mechanical properties. The biodegradation results of crosslinked Ala-g-Cts films were evaluated in phosphate-buffered solution containing lysozyme at 37℃. Proliferation of MC3T3-E1 cells on crosslinked Ala-g-Cts films was also investigated with use of the CCK-8 assay.

  19. Effect of alpha interferon on glucose and alanine transport by rat renal brush border membrane vesicles

    SciTech Connect

    Batuman, V.; Chadha, I. New Jersey Medical School, Newark )

    1990-01-01

    To investigate the pathogenetic mechanisms of interferon nephrotoxicity, we studied the effect of recombinant interferon alfa-2b on the uptake of {sup 14}C-D-glucose and {sup 14}C-L-alanine by rat renal brush-border-membrane vesicles. Interferon significantly inhibited 20 sec. sodium-dependent and 5 and 10 min. equilibrium uptake of both glucose and alanine. The inhibitory effect was dose dependent with maximum effect achieved at interferon concentration of 5 {times} 10{sup {minus}8}M in the uptake media. The half-maximal inhibitory concentrations, IC{sub 50}, of interferon on glucose uptake was 1.8 {times} 10{sup {minus}8}M, and 5.4 {times} 10{sup {minus}9}M on alanine uptake. Dixon plot analysis of uptake data was consistent with pure non-competitive inhibition. The inhibition constants, K{sub i}, 1.5 {times} 10{sup {minus}8}M for glucose uptake, and 7.3 {times} 10{sup {minus}9}M for alanine uptake, derived from Dixon plots were in close agreement with the IC{sub 50}s calculated from the semilog dose response curves. These observations reveal that direct interactions at the proximal tubule cell membrane are involved in the pathogenesis of interferon nephrotoxicity, and that its mechanism of nephrotoxicity is similar to that of other low molecular weight proteins.

  20. Investigation on physical properties of L-alanine: An effect of Methylene blue dye

    NASA Astrophysics Data System (ADS)

    Shkir, Mohd.; Yahia, I. S.; Al-Qahtani, A. M. A.; Ganesh, V.; AlFaify, S.

    2017-03-01

    In the present investigation, a bulk size (35 mm × 25 mm × 15 mm) single crystal of 0.1 wt% Methylene blue dye (MLB) added L-alanine is grown at room temperature using solution technique for the first time. The L-alanine crystals with higher concentrations of dye (0.5 and 1 wt%) were also grown. Solubility study was performed at different temperatures. Structural, vibrational and good quality was inveterate by powder XRD, FT-Raman and SEM analyses. High transmittance in dyed crystals was confirmed. The presence of MLB dye was confirmed by an absorption band centered at 650 nm. Optical band gap was calculated for pure and dyed L-alanine crystals and found to be 5.45 and 4.49 eV respectively. Photoluminescence intensity of UV-A emission band centered at 332 nm was found to be enhanced due to the presence of dye. The dielectric measurement was done in the wide frequency range. Furthermore, the third order nonlinear optical parameters are enhanced in dyed L-alanine crystals determined by Z-scan technique.

  1. Positron and electron scattering by glycine and alanine: Shape resonances and methylation effect

    NASA Astrophysics Data System (ADS)

    Nunes, Fernanda B.; Bettega, Márcio H. F.; Sanchez, Sergio d'Almeida

    2016-12-01

    We report integral cross sections (ICSs) for both positron and electron scattering by glycine and alanine amino acids. These molecules differ only by a methyl group. We computed the scattering cross sections using the Schwinger multichannel method for both glycine and alanine in different levels of approximation for both projectiles. The alanine ICSs are greater in magnitude than the glycine ICSs for both positron and electron scattering, probably due to the larger size of the molecule. In electron scattering calculations, we found two resonances for each molecule. Glycine presents one at 1.8 eV, and another centered at around 8.5 eV, in the static-exchange plus polarization (SEP) approximation. The ICS for alanine shows one resonance at 2.5 eV and another at around 9.5 eV, also in SEP approximation. The results are in good agreement with most of the data present in the literature. The comparison of the electron scattering ICSs for both molecules indicates that the methylation of glycine destabilizes the resonances, shifting them to higher energies.

  2. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  3. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  4. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  5. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  6. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  7. Synthesis of silver nanoparticles using DL-alanine for ESR dosimetry applications

    NASA Astrophysics Data System (ADS)

    Guidelli, Eder José; Ramos, Ana Paula; Zaniquelli, Maria Elisabete D.; Nicolucci, Patricia; Baffa, Oswaldo

    2012-03-01

    The potential use of alanine for the production of nanoparticles is presented here for the first time. Silver nanoparticles were synthesized using a simple green method, namely the thermal treatment of silver nitrate aqueous solutions with DL-alanine. The latter compound was employed both as a reducing and a capping agent. Particles with average size equal to 7.5 nm, face-centered cubic crystalline structure, narrow size distribution, and spherical shape were obtained. Interaction between the silver ions present on the surface of the nanoparticles and the amine group of the DL-alanine molecule seems to be responsible for reduction of the silver ions and for the stability of the colloid. The bio-hybrid nano-composite was used as an ESR dosimeter. The amount of silver nanoparticles in the nanocomposite was not sufficient to cause considerable loss of tissue equivalency. Moreover, the samples containing nanoparticles presented increased sensitivity and reduced energetic dependence as compared with pure DL-alanine, contributing to the construction of small-sized dosimeters.

  8. Probing the interaction of the amino acid alanine with the surface of ZnO(1010).

    PubMed

    Gao, Y K; Traeger, F; Shekhah, O; Idriss, H; Wöll, C

    2009-10-01

    The adsorption modes and stability of the amino acid alanine (NH(2)-CH(CH(3))-COOH) have been studied on the nonpolar single crystal surface of zinc oxide, ZnO(1010), experimentally by X-ray photoelectron spectroscopy (XPS) and computationally using density functional theory (DFT). Deposition at 200 K was found to lead to the formation of multilayers identified by an XPS N1s peak at 401.7 eV assigned to the NH(3)(+) group, a fingerprint of the zwitterionic structure of alanine in the solid state. Heating to 300 K resulted in the removal of most of the multilayers with the remaining surface coverage estimated to 0.4 with respect to Zn cations. At this temperature most of the alanine molecules are found to be deprotonated (dissociated), yielding a carboxylate species (NH(2)-CH(CH(3))-COO(-) (a) + OH (s); where O is surface oxygen, (a) for adsorbed and (s) for surface species). Further heating of the surface resulted in a gradual decrease of the surface coverage and by 500 K a large fraction of adsorbed alanine molecules have desorbed from the surface. Total energy DFT computations of different adsorbate species identified two stable dissociative adsorption modes: bidentate and monodentate. The bidentate species with adsorption energy of 1.75 eV was found to be more stable than the monodentate species by about 0.7 eV.

  9. Spectral characterization of a non-centrosymmetric organic compound: D-(-)-alanine

    NASA Astrophysics Data System (ADS)

    Moovendaran, K.; Martin Britto Dhas, S. A.; Natarajan, S.

    2013-08-01

    The crystal growth of D-(-)-alanine (1), a non-centrosymmetric solid is reported. It was characterized by NMR, infrared, Raman, UV-Vis-NIR and CD spectra. Experimental vibrational frequencies are compared with theoretically calculated values. Second harmonic generation (SHG) and first hyperpolarizability measurements are reported.

  10. Spectral characterization of a non-centrosymmetric organic compound: D-(-)-alanine.

    PubMed

    Moovendaran, K; Martin Britto Dhas, S A; Natarajan, S

    2013-08-01

    The crystal growth of D-(-)-alanine (1), a non-centrosymmetric solid is reported. It was characterized by NMR, infrared, Raman, UV-Vis-NIR and CD spectra. Experimental vibrational frequencies are compared with theoretically calculated values. Second harmonic generation (SHG) and first hyperpolarizability measurements are reported.

  11. Effects of endogenous D-alanine synthesis and autoinhibition of Bacillus anthracis germination on in vitro and in vivo infections.

    PubMed

    McKevitt, Matthew T; Bryant, Katie M; Shakir, Salika M; Larabee, Jason L; Blanke, Steven R; Lovchik, Julie; Lyons, C Rick; Ballard, Jimmy D

    2007-12-01

    Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of D-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.

  12. Rational design of mirror-like peptides with alanine regulation.

    PubMed

    Li, Weizhong; Tan, Tingting; Xu, Wei; Xu, Lin; Dong, Na; Ma, Deying; Shan, Anshan

    2016-02-01

    To generate effective antimicrobial peptides (AMPs) with good antimicrobial activities and cell selectivity, many synthetic strategies have been implemented to facilitate the development of AMPs. However, these synthetic strategies represent only a small proportion of the methods used for the development of AMPs and are not optimal with the requirements needed for the design of AMPs. In this investigation, we designed a mirror-like structure with a lower charge and a higher number of hydrophobic amino acids. The amino acid sequence of the designed mirror-like peptides was XXYXXXYXXXYXX [X represents L (Leu) and/or A (Ala); Y represents K (Lys)]. These mirror-like peptides displayed antimicrobial activity against both Gram-positive and Gram-negative bacteria. Hemolysis activity and cytotoxicity, detected by using human red blood cells (hRBCs) and human embryonic kidney cells (HEK293), respectively, demonstrated that the frequency of Ala residues in this structure had a regulatory effect on the high hydrophobic region. In particular, KL4A6 showed a greater antimicrobial potency than the other three mirror-like peptides, folded into an α-helical structure, and displayed the highest therapeutic index, suggesting its good cell selectivity. Observations from fluorescence spectroscopy, flow cytometry, and electron microscopy experiments indicated that KL4A6 exhibited good membrane penetration potential by inducing membrane blebbing, disruption and lysis. Therefore, generating mirror-like peptides is a promising strategy for designing effective AMPs with regions of high hydrophobicity.

  13. Alanine racemase is essential for the growth and interspecies competitiveness of Streptococcus mutans

    PubMed Central

    Wei, Yuan; Qiu, Wei; Zhou, Xue-Dong; Zheng, Xin; Zhang, Ke-Ke; Wang, Shi-Da; Li, Yu-Qing; Cheng, Lei; Li, Ji-Yao; Xu, Xin; Li, Ming-Yun

    2016-01-01

    D-alanine (D-Ala) is an essential amino acid that has a key role in bacterial cell wall synthesis. Alanine racemase (Alr) is a unique enzyme that interconverts L-alanine and D-alanine in most bacteria, making this enzyme a potential target for antimicrobial drug development. Streptococcus mutans is a major causative factor of dental caries. The factors involved in the survival, virulence and interspecies interactions of S. mutans could be exploited as potential targets for caries control. The current study aimed to investigate the physiological role of Alr in S. mutans. We constructed alr mutant strain of S. mutans and evaluated its phenotypic traits and interspecies competitiveness compared with the wild-type strain. We found that alr deletion was lethal to S. mutans. A minimal supplement of D-Ala (150 μg·mL−1) was required for the optimal growth of the alr mutant. The depletion of D-alanine in the growth medium resulted in cell wall perforation and cell lysis in the alr mutant strain. We also determined the compromised competitiveness of the alr mutant strain relative to the wild-type S. mutans against other oral streptococci (S. sanguinis or S. gordonii), demonstrated using either conditioned medium assays or dual-species fluorescent in situ hybridization analysis. Given the importance and necessity of alr to the growth and competitiveness of S. mutans, Alr may represent a promising target to modulate the cariogenicity of oral biofilms and to benefit the management of dental caries. PMID:27740612

  14. A preliminary optimization of alanine blends for ESR dosimetry in a mixed n-γ field: Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Hoseininaveh, M.; Ranjbar, A. H.

    2016-04-01

    In this study, a preliminary work on the enhancement of ESR response of several arrangements of alanine and boron compounds, exposed to a thermal neutron beam, is presented using FLUKA code. A multi-layer dosimeter consist of consecutive layers of alanine and boron compounds showed that the amount of energy deposited in the alanine layers is maximized when their thickness is 5 μm and the thickness of boron compound layers are between 2 and 3 μm. Furthermore, the optimum number of 10B layers in the dosimeter was found to be 35 layers. Moreover, the alanine samples consisting of small spherical grains of boron compounds, arranged regularly in the middle plane of the dosimeters, exposed to a thermal neutron beam, were modeled. The dependence of energy deposition in the alanine material on the size of grains, and on their composition were also studied, as well.

  15. Mechanism-based Inactivation by Aromatization of the Transaminase BioA Involved in Biotin Biosynthesis in Mycobaterium tuberculosis

    SciTech Connect

    Shi, Ce; Geders, Todd W.; Park, Sae Woong; Wilson, Daniel J.; Boshoff, Helena I.; Abayomi, Orishadipe; Barry, III, Clifton E.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2011-11-16

    BioA catalyzes the second step of biotin biosynthesis, and this enzyme represents a potential target to develop new antitubercular agents. Herein we report the design, synthesis, and biochemical characterization of a mechanism-based inhibitor (1) featuring a 3,6-dihydropyrid-2-one heterocycle that covalently modifies the pyridoxal 5'-phosphate (PLP) cofactor of BioA through aromatization. The structure of the PLP adduct was confirmed by MS/MS and X-ray crystallography at 1.94 {angstrom} resolution. Inactivation of BioA by 1 was time- and concentration-dependent and protected by substrate. We used a conditional knock-down mutant of M. tuberculosis to demonstrate the antitubercular activity of 1 correlated with BioA expression, and these results provide support for the designed mechanism of action.

  16. Complement activation and liver impairment in trichloroethylene-sensitized BALB/c mice.

    PubMed

    Zhang, Jiaxiang; Zha, Wansheng; Wang, Feng; Jiang, Tao; Xu, Shuhai; Yu, Junfeng; Zhou, Chengfan; Shen, Tong; Wu, Changhao; Zhu, Qixing

    2013-01-01

    Our recent studies have shown that trichloroethylene (TCE) was able to induce multisystem injuries in the form of occupational medicamentosa-like dermatitis, including skin, kidney, and liver damages. However, the role of complement activation in the immune-mediated liver injury is not known. This study examined the role of complement activation in the liver injury in a mouse model of TCE-induced sensitization. Treatment of female BALB/c mice with TCE under specific dosing protocols resulted in skin inflammation and sensitization. Skin edema and erythema occurred in TCE-sensitized groups. Trichloroethylene sensitization produced liver histopathological lesions, increased serum alanine aminotransferase, aspartate transaminase activities, and the relative liver weight. The concentrations of serum complement components C3a-desArg, C5a-desArg, and C5b-9 were significantly increased in 24-hour, 48-hour, and 72-hour sensitization-positive groups treated with TCE and peaked in the 72-hour sensitization-positive group. Depositions of C3a, C5a, and C5b-9 into the liver tissue were also revealed by immunohistochemistry. Immunofluorescence further verified high C5b-9 expression in 24-hour, 48-hour, and 72-hour sensitization-positive groups in response to TCE treatment. Reverse transcription-polymerase chain reaction detected C3 messenger RNA expression in the liver, and this was significantly increased in 24-hour and 48-hour sensitization-positive groups with a transient reduction at 72 hours. These results provide the first experimental evidence that complement activation may play a key role in the generation and progression of immune-mediated hepatic injury by exposure to TCE.

  17. D-Alanine-Controlled Transient Intestinal Mono-Colonization with Non-Laboratory-Adapted Commensal E. coli Strain HS.

    PubMed

    Cuenca, Miguelangel; Pfister, Simona P; Buschor, Stefanie; Bayramova, Firuza; Hernandez, Sara B; Cava, Felipe; Kuru, Erkin; Van Nieuwenhze, Michael S; Brun, Yves V; Coelho, Fernanda M; Hapfelmeier, Siegfried

    2016-01-01

    Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig)A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization.

  18. Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis.

    PubMed Central

    Cáceres, N E; Harris, N B; Wellehan, J F; Feng, Z; Kapur, V; Barletta, R G

    1997-01-01

    D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation. PMID:9260945

  19. D-Alanine-Controlled Transient Intestinal Mono-Colonization with Non-Laboratory-Adapted Commensal E. coli Strain HS

    PubMed Central

    Buschor, Stefanie; Bayramova, Firuza; Hernandez, Sara B.; Cava, Felipe; Kuru, Erkin; Van Nieuwenhze, Michael S.; Brun, Yves V.; Coelho, Fernanda M.; Hapfelmeier, Siegfried

    2016-01-01

    Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig)A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization. PMID:27002976

  20. Anthelmintic activity of Indigofera tinctoria against gastrointestinal nematodes of sheep

    PubMed Central

    Meenakshisundaram, Ambalathaduvar; Harikrishnan, Tirunelveli Jayagopal; Anna, Thavasi

    2016-01-01

    Aim: Gastrointestinal (GI) nematodes are considered as a major constraint for successful sheep production. Control of these parasites heavily relies on the use of chemical anthelmintics. Over the past decades, the development of anthelmintic resistance to various groups of anthelmintics and problem of drug residues in animal products has awakened interest in medicinal plants as an alternative source of anthelmintics. Hence, this study was undertaken to evaluate the anthelmintic efficacy of Indigofera tinctoria by scientifically validated in vitro and in vivo tests approved by the World Association for the Advancement of Veterinary Parasitology. Materials and Methods: In vitro assays such as egg hatch assay for ovicidal and larval migration inhibition and larval development assay for larvicidal properties were used to investigate in vitro effect of extracts on strongyle egg and larvae, respectively. Fecal egg count reduction test was conducted in vivo to evaluate the therapeutic efficacy of the extracts administered orally at dose rates of 125, 250, 500 mg/kg to sheep naturally infected with mixed GI nematodes. Results: Ethanolic extract of I. tinctoria demonstrated significant (p<0.01) inhibition on egg hatching at concentrations of 40 mg/ml and 80 mg/ml. In in vivo assay, the ethanolic extract of I. tinctoria reduced the fecal egg count ranging between 30.82% and 47.78% at various doses (125, 250 and 500 mg/kg). Although there was a slight variation, all the hematological parameters were within the normal range reported for sheep. Except for alanine transaminase, the overall mean of all the serum biochemical profile was within the normal range for sheep. Conclusion: Based on the results obtained by in vitro and in vivo assay, the ethanolic extract of I. tinctoria possesses anthelmintic activity and could replace the chemical anthelmintics used presently. PMID:27051192

  1. Effects of a combination of puerarin, baicalin and berberine on the expression of proliferator-activated receptor-γ and insulin receptor in a rat model of nonalcoholic fatty liver disease

    PubMed Central

    ZHAO, WEIHAN; LIU, LIJUAN; WANG, YUNLIANG; MAO, TANGYOU; LI, JUNXIANG

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a prevalent disease, with a clinical spectrum ranging from simple fatty liver disease to nonalcoholic steatohepatitis and cirrhosis. Puerarin, baicalin and berberine are herbal products widely used in Asia, which are believed to possess therapeutic benefits for alleviating the symptoms of NAFLD. In the present study, a rat model of NAFLD, induced by a high-fat diet, was established and orthographical experimentation was used to investigate the effects of various combinations of puerarin, baicalin and berberine on the hepatic expression of proliferator-activated receptor (PPAR)-γ and insulin receptor (IR). The present study demonstrated that serum levels of total cholesterol, alanine transaminase and low-density lipoproteins were significantly decreased in the puerarin-dominated groups (P<0.05 vs. the model group), whereas the concentrations of tumor necrosis factor-α and interleukin-6 were significantly improved in the baicalin- and berberine-dominated groups (P<0.05 vs. the model group). Furthermore, as compared with the control group, the levels of PPAR-γ/IR mRNA and protein expression were significantly decreased in the model group (P<0.01), and significantly increased in the rosiglitazone group and some of the orthogonal experiment groups (P<0.01). In conclusion, a combination of puerarin, baicalin and berberine induced favorable effects on NAFLD by upregulating hepatic PPAR-γ and IR expression levels, and different proportions of monomer compositions exerted variable positive effects on various stages of NAFLD. PMID:26889237

  2. Isotope labeling studies on the formation of multiple addition products of alanine in the pyrolysis residue of glucose/alanine mixtures by high-resolution ESI-TOF-MS.

    PubMed

    Chu, Fong Lam; Sleno, Lekha; Yaylayan, Varoujan A

    2011-11-09

    Pyrolysis was used as a microscale sample preparation tool to generate glucose/alanine reaction products to minimize the use of expensive labeled precursors in isotope labeling studies. The residue remaining after the pyrolysis at 250 °C was analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). It was observed that a peak at m/z 199.1445 in the ESI-TOF-MS spectrum appeared only when the model system contained at least 2-fold excess alanine. The accurate mass determination indeed indicated the presence of two nitrogen atoms in the molecular formula (C(10)H(18)N(2)O(2)). To verify the origin of the carbon atoms in this unknown compound, model studies with [(13)U(6)]glucose, [(13)C-1]alanine, [(13)C-2]alanine, [(13)C-3]alanine, and [(15)N]alanine were also performed. Glucose furnished six carbon atoms, and alanine provides four carbon (2 × C-2 and 2 × C-3) and two nitrogen atoms. When commercially available fructosylalanine (N-attached to C-1) was reacted with only 1 mol of alanine, a peak at m/z 199.1445 was once again observed. In addition, when 3-deoxyglucosone (3-DG) was reacted with a 2-fold excess of alanine, a peak at m/z 199.1433 was also generated, confirming the points of attachment of the two amino acids at C-1 and C-2 atoms of 3-DG. These studies have indicated that amino acids can undergo multiple addition reactions with 1,2-dicarbonyl compounds such as 3-deoxyglucosone and eventually form a tetrahydropyrazine moiety.

  3. Synthesis and evaluation of 18F labeled alanine derivatives as potential tumor imaging agents

    PubMed Central

    Wang, Limin; Zha, Zhihao; Qu, Wenchao; Qiao, Hongwen; Lieberman, Brian P.; Plössl, Karl; Kung, Hank F.

    2012-01-01

    Introduction This paper reports the synthesis and labeling of 18F alanine derivatives. We also investigate their biological characteristics as potential tumor imaging agents mediated by alanine-serine-cysteine preferring (ASC) transporter system. Methods Three new 18F alanine derivatives were prepared from corresponding tosylate-precursors through a two-step labelling reaction. In vitro uptake studies to evaluate and to compare these three analogs were carried out in 9L glioma and PC-3 prostate cancer cell lines. Potential transport mechanisms, protein incorporation and stability of 3-(1-[18F]fluoromethyl)-L-alanine (L[18F]FMA) were investigated in 9L glioma cells. Its biodistribution was determined in a rat-bearing 9L tumor model. PET imaging studies were performed on rat bearing 9L glioma tumors and transgenic mouse carrying spontaneous generated M/tomND tumor (mammary gland adenocarcinoma). Results New 18F alanine derivatives were prepared with 7–34% uncorrected radiochemical yields, excellent enantiomeric purity (>99%) and good radiochemical purity (>99%). In vitro uptake of the L-[18F]FMA in 9L glioma and PC-3 prostate cancer cells was higher than those observed for other two alanine derivatives and [18F]FDG in first 1 h. Inhibition of cell uptake studies suggested that L-[18F]FMA uptake in 9L glioma was predominantly via transport system ASC. After entering into cells, L-[18F]FMA remained stable and was not incorporated into protein within 2 h. In vivo biodistribution studies demonstrated that L-[18F]FMA had relatively high uptake in liver and kidney. Tumor uptake was fast, reaching a maximum within 30 min. The tumor-to-muscle, tumor-to-blood and tumor-to-brain ratios at 60 min post injection were 2.2, 1.9 and 3.0, respectively. In PET imaging studies, tumors were visualized with L-[18F]FMA in both 9L rat and transgenic mouse. Conclusion L-[18F]FMA showed promising properties as a PET imaging agent for up-regulated ASC transporter associated with tumor

  4. Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis.

    PubMed

    Wu, Dalei; Hu, Tiancen; Zhang, Liang; Chen, Jing; Du, Jiamu; Ding, Jianping; Jiang, Hualiang; Shen, Xu

    2008-06-01

    Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.

  5. β-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling.

    PubMed

    Engskog, Mikael K R; Ersson, Lisa; Haglöf, Jakob; Arvidsson, Torbjörn; Pettersson, Curt; Brittebo, Eva

    2017-02-04

    β-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC-MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

  6. Effect of glucose, independent of changes in insulin and glucagon secretion, on alanine metabolism in the conscious dog.

    PubMed Central

    Shulman, G I; Lacy, W W; Liljenquist, J E; Keller, U; Williams, P E; Cherrington, A D

    1980-01-01

    To study the effects of hyperglycemia on the metabolism of alanine and lactate independent of changes in plasma insulin and glucagon, glucose was infused into five 36-h-fasted dogs along with somatostatin and constant replacement amounts of both insulin and glucagon. Hepatic uptakes of alanine and lactate were calculated using the arteriovenous difference technique. [14C]Alanine was infused to measure the conversion of alanine and lactate into glucose. Hyperglycemia (delta 115 mg/dl) of 2 h duration caused the plasma alanine level to increase by over 50%. This change was caused by an increase in the inflow of alanine into plasma since the net hepatic uptake of the amino acid did not change. Taken together, the above findings indicate that glucose per se can significantly impair the fractional extraction of alanine by the liver. Hepatic extraction of lactate was also affected by hyperglycemia and had fallen to zero within 90 min of starting the glucose infusion. This fall was associated with a doubling of arterial lactate level. Conversion of [14C]-alanine and [14C]lactate into [14C]glucose was suppressed by 60 +/- 11% after 2 h of hyperglycemia, and because this fall could not be entirely accounted for by decreased lactate extraction an inhibitory effect of glucose on gluconeogenesis within the liver is suggested. These studies indicate that the plasma glucose level per se can be an important determinant of the level of alanine and lactate in plasma as well as the rate at which they are converted to glucose. PMID:7356691

  7. [Kinetics and equilibrium of reactions between nucleotides and methylol derivatives of beta-alanine].

    PubMed

    Khulordava, K G; Kosaganov, Iu N; Lazurkin, Iu S

    1978-01-01

    The rate constants of forward and reverse reactions between methylol derivatives of beta-alanine and deoxycytidine 5'-phosphate, deoxyadenosine 5'phosphate and deoxyguanosine 5'phosphate and the equilibrium constants of these reactions were determined by the spectrophotometric method at 39,5 degrees C and pH 6,95. Besides, the equilibrium constant of the reaction between beta-alanine and formaldehyde was determined. Unlike deoxycytidine and deoxyadenosine 5'-phosphates, interaction of deoxyguanosine 5'phosphate with methylol derivatives is more complicated. A model proposed for the interaction of deoxyguanosine 5'phosphate with methylol derivatives explains the behavior of this nucleotide in the reaction. The kinetic and equilibrium constants of the interaction of methylol derivatives with nucleotides investigated exceed by two or three orders of magnitude the corresponding constants of the interaction of formaldehyde with these nucleotides.

  8. Crystallization and preliminary X-ray analysis of an alanine dehydrogenase from Bacillus megaterium WSH-002.

    PubMed

    Lu, Xiaoyun; Yi, Qiufen; Zhang, Guofang; Zhu, Xianming; Zhou, Honggang; Dong, Hui

    2013-08-01

    Alanine dehydrogenase (L-AlaDH) from Bacillus megaterium WSH-002 catalyses the NAD⁺-dependent interconversion of L-alanine and pyruvate. The enzyme was expressed in Escherichia coli BL21 (DE3) cells and purified with a His6 tag by Ni²⁺-chelating affinity chromatography for X-ray crystallographic analysis. Crystals were grown in a solution consisting of 0.1 M HEPES pH 8.0, 12%(w/v) polyethylene glycol 8000, 8%(v/v) ethylene glycol at a concentration of 15 mg ml⁻¹ purified protein. The crystal diffracted to 2.35 Å resolution and belonged to the trigonal space group R32, with unit-cell parameters a = b = 125.918, c = 144.698 Å.

  9. Adsorption of di-l-alanine on Cu(110) investigated with scanning tunneling microscopy [rapid communication

    NASA Astrophysics Data System (ADS)

    Stensgaard, I.

    2003-11-01

    Sub-monolayer growth of a small chiral peptide, di- L-alanine, on Cu(1 1 0) was investigated by variable temperature scanning tunneling microscopy (STM). At low coverage and for temperatures above ≈-220 K the molecules nucleate along the [ 3¯ 3 2] direction to form short, mainly one-dimensional islands. An increase in coverage leads to the formation of [ 3¯ 3 2]-directed, elongated islands. Images with sub-molecular resolution reveal that the orientation of the molecules within one particular island depends on the deposition temperature. At higher coverage, up to one monolayer, the islands coalesce, giving rise to phase boundaries between domains of opposite orientation. An atomic-scale model for di- L-alanine on Cu(1 1 0) is presented.

  10. Unusual hydroxyl migration in the fragmentation of β-alanine dication in the gas phase.

    PubMed

    Piekarski, Dariusz Grzegorz; Delaunay, Rudy; Maclot, Sylvain; Adoui, Lamri; Martín, Fernando; Alcamí, Manuel; Huber, Bernd A; Rousseau, Patrick; Domaracka, Alicja; Díaz-Tendero, Sergio

    2015-07-14

    We present a combined experimental and theoretical study of the fragmentation of doubly positively charged β-alanine molecules in the gas phase. The dissociation of the produced dicationic molecules, induced by low-energy ion collisions, is analysed by coincidence mass spectrometric techniques; the coupling with ab initio molecular dynamics simulations allows rationalisation of the experimental observations. The present strategy gives deeper insights into the chemical mechanisms of multiply charged amino acids in the gas phase. In the case of the β-alanine dication, in addition to the expected Coulomb explosion and hydrogen migration processes, we have found evidence of hydroxyl-group migration, which leads to unusual fragmentation products, such as hydroxymethyl cation, and is necessary to explain some of the observed dominant channels.

  11. Formation of homochiral glycine/Cu(111) quantum corral array realized using alanine nuclei

    NASA Astrophysics Data System (ADS)

    Nakamura, Miki; Huang, Hui; Kanazawa, Ken; Taninaka, Atsushi; Yoshida, Shoji; Takeuchi, Osamu; Shigekawa, Hidemi

    2015-08-01

    Glycine has enantiomeric isomers on a Cu(111) surface through the dissociation of hydrogen from the carboxyl group and forms an array of quantum corrals of ∼1.3 nm diameter. Stable homo-chiral glycinate trimers are formed in the first step, which subsequently form a network with a hexagonal arrangement. However, domains with R- or S-chirality coexist with the same probability. On the other hand, α-alanine has D- and L-chirality in nature and forms a similar quantum corral array on Cu(111) with R- and S-chirality, respectively. Here, by using α-alanine molecules as nuclei, the chirality of glycine molecules was controlled and a homochiral quantum corral array was successfully formed, which indicates the possibility that the optical isomers can be separated through a method such as preferential crystallization.

  12. Chiral effects on helicity studied via the energy landscape of short (D, L)-alanine peptides.

    PubMed

    Neelamraju, Sridhar; Oakley, Mark T; Johnston, Roy L

    2015-10-28

    The homochirality of natural amino acids facilitates the formation of regular secondary structures such as α-helices and β-sheets. Here, we study the relationship between chirality and backbone structure for the example of hexa-alanine. The most stable stereoisomers are identified through global optimisation. Further, the energy landscape, a database of connected low-energy local minima and transition points, is constructed for various neutral and zwitterionic stereoisomers of hexa-alanine. Three order parameters for partial helicity are applied and metric disconnectivity graphs are presented with partial helicity as a metric. We also apply the Zimm-Bragg model to derive average partial helicities for Ace-(L-Ala)6-NHMe, Ace-(D-Ala-L-Ala)3-NHMe, and Ace-(L-Ala)3-(D-Ala)3-NHMe from the database of local minima and compare with previous studies.

  13. Membrane topology of the electrogenic aspartate-alanine antiporter AspT of Tetragenococcus halophilus.

    PubMed

    Nanatani, Kei; Ohonishi, Fumito; Yoneyama, Hiroshi; Nakajima, Tasuku; Abe, Keietsu

    2005-03-04

    AspT is an electrogenic aspartate:alanine exchange protein that represents the vectorial component of a proton-motive metabolic cycle found in some strains of Tetragenococcus halophilus. AspT is the sole member of a new family, the Aspartate: Alanine Exchanger (AAE) family, in secondary transporters, according to the computational classification proposed by Saier et al. (http://www.biology.ucsd.edu/~msaier/transport/). We analyzed the topology of AspT biochemically, by using fusion methods in combination with alkaline phosphatase or beta-lactamase. These results suggested that AspT has a unique topology; 8 TMS, a large cytoplasmic loop (183 amino acids) between TMS5 and TMS6, and N- and C-termini that both face the periplasm. These results demonstrated a unique 2D-structure of AspT as the novel AAE family.

  14. The potential of mean force surface for the alanine dipeptide in aqueous solution: a theoretical approach

    NASA Astrophysics Data System (ADS)

    Montgomery Pettitt, B.; Karplus, Martin

    1985-11-01

    Results of an application of integral equation theory to the determination of the intramolecular potential of mean force for the alanine dipeptide. N-methyl alanine acetamide, in aqueous solution are presented. The calculations are based on Ornstein—Zernike-like equations for polar systems with an intramolecular superposition approximation. The solvated free energy surface for the dipeptide as a function of the dihedral angles φ and ψ (Ramachandran plot) is determined and compared with the vaccum surface calculations. Conformations that are essentially forbidden in vaccum are found to be significant in aqueous solution. The solvent contributions to the free energy surface are decomposed into enthalpic and entropic terms. Possible applications and extensions of the method are outlined.

  15. Equine endurance exercise alters serum branched-chain amino acid and alanine concentrations.

    PubMed

    Trottier, N L; Nielsen, B D; Lang, K J; Ku, P K; Schott, H C

    2002-09-01

    Six 2-year-old Arabian horses were used to determine whether 60 km prolonged endurance exercise (approximately 4 h) alters amino acid concentrations in serum and muscle, and the time required for serum amino acid concentrations to return to basal resting values. Blood and muscle samples were collected throughout exercise and during a 3 day recovery period. Isoleucine concentration in muscle tended to increase and leucine and valine did not change due to exercise. Serum alanine concentrations did not increase immediately after exercise, but increased at 24, 48 and 72 h postexercise. Serum isoleucine, leucine, and valine concentrations decreased after exercise and time required to reach pre-exercising concentrations was 48 h. In conclusion, endurance exercise in the horse decreases serum isoleucine, leucine, and valine concentrations, and increases serum alanine concentration. The decrease in serum branched-chain amino acid concentrations did not correspond to a measurable increase in total muscle branched-chain amino acid concentrations.

  16. Crystallization and preliminary X-ray data analysis of β-alanine synthase from Drosophila melanogaster

    SciTech Connect

    Lundgren, Stina; Andersen, Birgit; Piškur, Jure; Dobritzsch, Doreen

    2007-10-01

    β-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine. Crystals of the recombinant enzyme from D. melanogaster belong to space group C2. Diffraction data to 3.3 Å resolution were collected and analyzed. β-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 Å at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 Å, β = 125.8°) and contain 8–10 molecules per asymmetric unit.

  17. Response of the alanine/ESR dosimeter to radiation from an Ir-192 HDR brachytherapy source.

    PubMed

    Anton, M; Hackel, T; Zink, K; von Voigts-Rhetz, P; Selbach, H-J

    2015-01-07

    The response of the alanine dosimeter to radiation from an Ir-192 source with respect to the absorbed dose to water, relative to Co-60 radiation, was determined experimentally as well as by Monte Carlo simulations. The experimental and Monte Carlo results for the response agree well within the limits of uncertainty. The relative response decreases with an increasing distance between the measurement volume and the source from approximately 98% at a 1 cm distance to 96% at 5 cm. The present data are more accurate, but agree well with data published by Schaeken et al (2011 Phys. Med. Biol. 56 6625-34). The decrease of the relative response with an increasing distance that had already been observed by these authors is confirmed. In the appendix, the properties of the alanine dosimeter with respect to volume and sensitivity corrections are investigated. The inhomogeneous distribution of the detection probability that was taken into account for the analysis was determined experimentally.

  18. Structures of a γ-aminobutyrate (GABA) transaminase from the s-triazine-degrading organism Arthrobacter aurescens TC1 in complex with PLP and with its external aldimine PLP–GABA adduct

    PubMed Central

    Bruce, Heather; Nguyen Tuan, Anh; Mangas Sánchez, Juan; Leese, Charlotte; Hopwood, Jennifer; Hyde, Ralph; Hart, Sam; Turkenburg, Johan P.; Grogan, Gideon

    2012-01-01

    Two complex structures of the γ-aminobutyrate (GABA) transaminase A1R958 from Arthrobacter aurescens TC1 are presented. The first, determined to a resolution of 2.80 Å, features the internal aldimine formed by reaction between the ∊-amino group of Lys295 and the cofactor pyridoxal phosphate (PLP); the second, determined to a resolution of 2.75 Å, features the external aldimine adduct formed between PLP and GABA in the first half-reaction. This is the first structure of a microbial GABA transaminase in complex with its natural external aldimine and reveals the molecular determinants of GABA binding in this enzyme. PMID:23027742

  19. Influence of lysine content and pH on the stability of alanine-based copolypeptides.

    PubMed

    Vila, J A; Ripoll, D R; Scheraga, H A

    2001-03-01

    To account for the relative contributions of lysine and alanine residues to the stability of alpha-helices of copolymers of these two residues, conformational energy calculations were carried out for several hexadecapeptides at several pHs. All the calculations considered explicitly the coupling between the conformation of the molecule and the ionization equilibria as a function of pH. The total free energy function used in these calculations included terms that account for the solvation free energy and free energy of ionization. These terms were evaluated by means of a fast multigrid boundary element method. Reasonable agreement with experimental values was obtained for the helix contents and vicinal coupling constants ((3)J(HNalpha)). The helix contents were found to depend strongly on the lysine content, in agreement with recent experimental results of Williams et al. (Journal of the American Chemical Society, 1998, Vol. 120, pp. 11033-11043) In the lowest energy conformation computed for a hexadecapeptide containing 3 lysine residues at pH 6, the lysine side chains are preferentially hydrated; this decreases the hydration of the backbone CO and NH groups, thereby forcing the latter to form hydrogen bonds with each other in the helical conformation. The lowest energy conformation computed for a hexadecapeptide containing 6 lysine residues at pH 6 shows a close proximity between the NH3(+) groups of the lysine side chains, a feature that was previously observed in calculations of short alanine-based oligopeptides. The calculation on a blocked 16-mer of alanine shows a 7% helix content based on the Boltzmann averaged vicinal coupling constants computed from the dihedral angles phi, consistent with previous experimental evidence on triblock copolymers containing a central block of alanines, and with earlier theoretical calculations.

  20. Photochemical redox reactions of copper(II)-alanine complexes in aqueous solutions.

    PubMed

    Lin, Chen-Jui; Hsu, Chao-Sheng; Wang, Po-Yen; Lin, Yi-Liang; Lo, Yu-Shiu; Wu, Chien-Hou

    2014-05-19

    The photochemical redox reactions of Cu(II)/alanine complexes have been studied in deaerated solutions over an extensive range of pH, Cu(II) concentration, and alanine concentration. Under irradiation, the ligand-to-metal charge transfer results in the reduction of Cu(II) to Cu(I) and the concomitant oxidation of alanine, which produces ammonia and acetaldehyde. Molar absorptivities and quantum yields of photoproducts for Cu(II)/alanine complexes at 313 nm are characterized mainly with the equilibrium Cu(II) speciation where the presence of simultaneously existing Cu(II) species is taken into account. By applying regression analysis, individual Cu(I) quantum yields are determined to be 0.094 ± 0.014 for the 1:1 complex (CuL) and 0.064 ± 0.012 for the 1:2 complex (CuL2). Individual quantum yields of ammonia are 0.055 ± 0.007 for CuL and 0.036 ± 0.005 for CuL2. Individual quantum yields of acetaldehyde are 0.030 ± 0.007 for CuL and 0.024 ± 0.007 for CuL2. CuL always has larger quantum yields than CuL2, which can be attributed to the Cu(II) stabilizing effect of the second ligand. For both CuL and CuL2, the individual quantum yields of Cu(I), ammonia, and acetaldehyde are in the ratio of 1.8:1:0.7. A reaction mechanism for the formation of the observed photoproducts is proposed.

  1. Weak BMAA toxicity compares with that of the dietary supplement β-alanine.

    PubMed

    Lee, Moonhee; McGeer, Patrick L

    2012-07-01

    β-N-methylamino-L-alanine (BMAA) is routinely described in the literature as a potent neurotoxin and as a possible cause of neurodegenerative disorders of aging such as Alzheimer's disease, amyotrophic lateral sclerosis, and the amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC) syndrome of Guam. To test for the toxicity of BMAA against human neurons, we chose 3 standard human neuronal cell lines for examination and compared the toxicity with the muscle-building nutritional supplement β-alanine, glutamic acid, and the established excitotoxins kainic acid, quisqualic acid, ibotenic acid, domoic acid, and quinolinic acid. Neurotoxicity was measured by the standard lactic dehydrogenase release assay after 5-day incubation of NT-2, SK-N-MC, and SH-SY5Y cells with BMAA and the comparative substances. The ED(50) of BMAA, corresponding to 50% death of neurons, varied from 1430 to 1604 μM while that of the nutritional supplement β-alanine was almost as low, varying from 1945 to 2134 μM. The ED(50) for glutamic acid and the 5 established excitotoxins was 200- to 360-fold lower, varying from 44 to 70 μM. These in vitro data are in accord with previously published in vivo data on BMAA toxicity in which mice showed no pathological effects from oral consumption of 500 mg/kg/day for more than 10 weeks. Because there are no known natural sources of BMAA that would make consumption of such amounts possible, and because the toxicity observed was in the same range as the nutritional supplement β-alanine, the hypothesis that BMAA is an environmental hazard and a contributor to degenerative neurological diseases becomes untenable.

  2. β-alanine supplementation improves isometric endurance of the knee extensor muscles

    PubMed Central

    2012-01-01

    Background We examined the effect of four weeks of β-alanine supplementation on isometric endurance of the knee extensors at 45% maximal voluntary isometric contraction (MVIC). Methods Thirteen males (age 23 ± 6 y; height 1.80 ± 0.05 m; body mass 81.0 ± 10.5 kg), matched for pre-supplementation isometric endurance, were allocated to either a placebo (n = 6) or β-alanine (n = 7; 6.4 g·d-1 over 4 weeks) supplementation group. Participants completed an isometric knee extension test (IKET) to fatigue, at an intensity of 45% MVIC, before and after supplementation. In addition, two habituation tests were completed in the week prior to the pre-supplementation test and a further practice test was completed in the week prior to the post-supplementation test. MVIC force, IKET hold-time, and impulse generated were recorded. Results IKET hold-time increased by 9.7 ± 9.4 s (13.2%) and impulse by 3.7 ± 1.3 kN·s-1 (13.9%) following β-alanine supplementation. These changes were significantly greater than those in the placebo group (IKET: t(11) = 2.9, p ≤0.05; impulse: t(11) = 3.1, p ≤ 0.05). There were no significant changes in MVIC force in either group. Conclusion Four weeks of β-alanine supplementation at 6.4 g·d-1 improved endurance capacity of the knee extensors at 45% MVIC, which most likely results from improved pH regulation within the muscle cell as a result of elevated muscle carnosine levels. PMID:22697405

  3. Stabilization of helices in glycine and alanine dipeptides in a reaction field model of solvent

    SciTech Connect

    Shang, H.S. Lawrence Berkeley Lab., CA ); Head-Gordon, T. )

    1994-02-23

    We present molecular orbital calculations of the full conformational space of blocked glycine and alanine dipeptide in the presence of a reaction field representation of water. Secondary structures of right- and left-handed helices are found, in contrast to recent gas-phase results, indicating that the origin of helical stabilization in dipeptides is strictly due to environment. Limitations of the reaction field model and the various implications of stabilization due to environment are discussed. 43 refs., 2 figs., 3 tabs.

  4. Dosimétrie RPE alanine, étude de faisabilité et applications possibles

    NASA Astrophysics Data System (ADS)

    Kuntz, F.; Chabanais, B.; Karamanoukian, D.; Delpech, J. P.; Marchioni, E.

    1998-04-01

    Alanine ESR dosimetry presents a great interest for quality controls in radiotherapy. This new developped water equivalent alamine dosimeter allows a reproducible dose measurement, by a non-destructif readout technique in a large dose range. In this paper the stability of the dosimeter response has been shown but also its independance with the energy or the dose rate of the absorbed radiation. Through this different studies, one can broaden the application field of alanine/ESR dosimetry especially for in-vivo dosimetry. The results of the experiments and the intra operative treatment, indicate that this kind of dosimetry seems to be a promising technique for in-vivo quality controls in electron beam, γ ray or X-ray radiotherapy. Le dosimètre à l'alanine dépouillé par Résonance Paramagnétique Électronique (EPE), est pratiquement équivalent tissu et présente plusieurs caractéristiques intéressantes : la reproductibilité de sa mesure, son dépouillement non destructif, son faible fading, sa large gamme de mesure de dose (0,5 à 100 kGy). La réponse de ce dosimètre est, de plus, indépendante du débit de dose du rayonnement qu'il a absorbé ainsi que de son énergie. À travers plusieurs études, et un essai in-viro, nous ouvrons un important champ d'applications, faisant de la dosimétie RPE/Alanine un Outil prometteur pour le contrôle de la qualité des traitements radiothérapeutiques par faisceaux d'électrons, rayonnement X et photons γ.

  5. Hepatic serine and alanine metabolism during endotoxin-induced fever in sheep.

    PubMed Central

    Southorn, B G; Thompson, J R

    1987-01-01

    Time course changes in plasma amino acid concentrations and the hepatic metabolism of serine and alanine were measured in six mature wethers during endotoxin-induced fever. In separate trials, the animals' responses to injections of saline and endotoxin were measured. The endotoxin was from Escherichia coli serotype 055:B5 and was injected intravenously (4 micrograms/kg body weight). Liver biopsies were obtained from the sheep at 6 h postinjection during both endotoxin and saline injection trials. Rectal temperature in the endotoxin treated animals was increased (P less than 0.05, above that in control animals from 4.25 h to 9 h postinjection, with a maximum rise of 2.43 degrees C at 5.5 h postinjection. Glucose concentration in jugular plasma decreased (P less than 0.05) by 3 h postinjection and remained depressed throughout the 24 h postinjection sampling period. Plasma serine concentration was decreased (P less than 0.05) by 3 h postinjection. Plasma alanine concentration was decreased significantly (P less than 0.05) only at 24 h postinjection. Endotoxin injection increased (P less than 0.05) hepatic oxidation of 14C-serine (162%) and the net incorporation of 14C-serine carbon into hepatic protein (173%) and glycogen (275%). The net incorporation of 14C-alanine carbon into hepatic protein (172%) and glycogen (323%) were increased (P less than 0.05) by endotoxin injection, while alanine oxidation was not affected by endotoxin treatment (P greater than 0.05). The increased hepatic use of serine may explain, in part, the dramatic decrease in plasma concentrations of this amino acid following endotoxin injection into sheep. PMID:3115552

  6. Structure and vibrational spectra of L-alanine L-alaninium picrate monohydrate

    NASA Astrophysics Data System (ADS)

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

    2012-05-01

    Preparation, crystal and molecular structure as well as vibrational spectra of the crystal L-alanine L-alaninium picrate monohydrate are described. The title crystal is monoclinic, space group P21. The asymmetric unit contains one dimeric (L-Ala⋯L-Ala+) cation, one picrate anion and a water molecule. The O⋯O distance in the dimeric cation is equal to 2.553(2) Å. The IR and Raman spectra are interpreted based on the structure.

  7. Determination of the anti-inflammatory and cytoprotective effects of l-glutamine and l-alanine, or dipeptide, supplementation in rats submitted to resistance exercise.

    PubMed

    Raizel, Raquel; Leite, Jaqueline Santos Moreira; Hypólito, Thaís Menezes; Coqueiro, Audrey Yule; Newsholme, Philip; Cruzat, Vinicius Fernandes; Tirapegui, Julio

    2016-08-01

    We evaluated the effects of chronic oral supplementation with l-glutamine and l-alanine in their free form or as the dipeptide l-alanyl-l-glutamine (DIP) on muscle damage, inflammation and cytoprotection, in rats submitted to progressive resistance exercise (RE). Wistar rats (n 8/group) were submitted to 8-week RE, which consisted of climbing a ladder with progressive loads. In the final 21 d before euthanasia, supplements were delivered in a 4 % solution in drinking water. Glutamine, creatine kinase (CK), lactate dehydrogenase (LDH), TNF-α, specific IL (IL-1β, IL-6 and IL-10) and monocyte chemoattractant protein-1 (MCP-1) levels were evaluated in plasma. The concentrations of glutamine, TNF-α, IL-6 and IL-10, as well as NF-κB activation, were determined in extensor digitorum longus (EDL) skeletal muscle. HSP70 level was assayed in EDL and peripheral blood mononuclear cells (PBMC). RE reduced glutamine concentration in plasma and EDL (P<0·05 v. sedentary group). However, l-glutamine supplements (l-alanine plus l-glutamine (GLN+ALA) and DIP groups) restored glutamine levels in plasma (by 40 and 58 %, respectively) and muscle (by 93 and 105 %, respectively). GLN+ALA and DIP groups also exhibited increased level of HSP70 in EDL and PBMC, consistent with the reduction of NF-κB p65 activation and cytokines in EDL. Muscle protection was also indicated by attenuation in plasma levels of CK, LDH, TNF-α and IL-1β, as well as an increase in IL-6, IL-10 and MCP-1. Our study demonstrates that chronic oral l-glutamine treatment (given with l-alanine or as dipeptide) following progressive RE induces cyprotective effects mediated by HSP70-associated responses to muscle damage and inflammation.

  8. Identification and Partial Characterization of a Novel UDP-N-Acetylenolpyruvoylglucosamine Reductase/UDP-N-Acetylmuramate:l-Alanine Ligase Fusion Enzyme from Verrucomicrobium spinosum DSM 4136T

    PubMed Central

    Naqvi, Kubra F.; Patin, Delphine; Wheatley, Matthew S.; Savka, Michael A.; Dobson, Renwick C. J.; Gan, Han Ming; Barreteau, Hélène; Blanot, Didier; Mengin-Lecreulx, Dominique; Hudson, André O.

    2016-01-01

    The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/CVs) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44–46°C. Its apparent Km values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum. PMID:27047475

  9. Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1)

    PubMed Central

    Chen, Qiushi; Müller, Juliane S.; Pang, Poh-Choo; Laval, Steve H.; Haslam, Stuart M.; Lochmüller, Hanns; Dell, Anne

    2015-01-01

    Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells. PMID:26501342

  10. Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

    PubMed

    Chen, Qiushi; Müller, Juliane S; Pang, Poh-Choo; Laval, Steve H; Haslam, Stuart M; Lochmüller, Hanns; Dell, Anne

    2015-10-16

    Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed "limb-girdle CMS with tubular aggregates". CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.

  11. Identification and expression analyses of the alanine aminotransferase (AlaAT) gene family in poplar seedlings.

    PubMed

    Xu, Zhiru; Ma, Jing; Qu, Chunpu; Hu, Yanbo; Hao, Bingqing; Sun, Yan; Liu, Zhongye; Yang, Han; Yang, Chengjun; Wang, Hongwei; Li, Ying; Liu, Guanjun

    2017-04-05

    Alanine aminotransferase (AlaAT, E.C.2.6.1.2) catalyzes the reversible conversion of pyruvate and glutamate to alanine and α-oxoglutarate. The AlaAT gene family has been well studied in some herbaceous plants, but has not been well characterized in woody plants. In this study, we identified four alanine aminotransferase homologues in Populus trichocarpa, which could be classified into two subgroups, A and B. AlaAT3 and AlaAT4 in subgroup A encode AlaAT, while AlaAT1 and AlaAT2 in subgroup B encode glutamate:glyoxylate aminotransferase (GGAT), which catalyzes the reaction of glutamate and glyoxylate to α-oxoglutarate and glycine. Four AlaAT genes were cloned from P. simonii × P. nigra. PnAlaAT1 and PnAlaAT2 were expressed predominantly in leaves and induced by exogenous nitrogen and exhibited a diurnal fluctuation in leaves, but was inhibited in roots. PnAlaAT3 and PnAlaAT4 were mainly expressed in roots, stems and leaves, and was induced by exogenous nitrogen. The expression of PnAlaAT3 gene could be regulated by glutamine or its related metabolites in roots. Our results suggest that PnAlaAT3 gene may play an important role in nitrogen metabolism and is regulated by glutamine or its related metabolites in the roots of P. simonii × P. nigra.

  12. beta-Alanine elevates dopamine levels in the rat nucleus accumbens: antagonism by strychnine.

    PubMed

    Ericson, Mia; Clarke, Rhona B C; Chau, PeiPei; Adermark, Louise; Söderpalm, Bo

    2010-04-01

    Glycine receptors (GlyRs) in the nucleus accumbens (nAc) have recently been suggested to be involved in the reinforcing and dopamine-elevating properties of ethanol via a neuronal circuitry involving the VTA. Apart from ethanol, both glycine and taurine have the ability to modulate dopamine output via GlyRs in the same brain region. In the present study, we wanted to explore whether yet another endogenous ligand for the GlyR, beta-alanine, had similar effects. To this end, we monitored dopamine in the nAc by means of in vivo microdialysis and found that local perfusion of beta-alanine increased dopamine output. In line with previous observations investigating ethanol, glycine and taurine, the competitive GlyR antagonist strychnine completely blocked the dopamine elevation. The present results suggest that beta-alanine has the ability to modulate dopamine levels in the nAc via strychnine-sensitive GlyRs, and are consistent with previous studies suggesting the importance of this receptor for modulating dopamine output.

  13. VUV photodynamics and chiral asymmetry in the photoionization of gas phase alanine enantiomers.

    PubMed

    Tia, Maurice; Cunha de Miranda, Barbara; Daly, Steven; Gaie-Levrel, François; Garcia, Gustavo A; Nahon, Laurent; Powis, Ivan

    2014-04-17

    The valence shell photoionization of the simplest proteinaceous chiral amino acid, alanine, is investigated over the vacuum ultraviolet region from its ionization threshold up to 18 eV. Tunable and variable polarization synchrotron radiation was coupled to a double imaging photoelectron/photoion coincidence (i(2)PEPICO) spectrometer to produce mass-selected threshold photoelectron spectra and derive the state-selected fragmentation channels. The photoelectron circular dichroism (PECD), an orbital-sensitive, conformer-dependent chiroptical effect, was also recorded at various photon energies and compared to continuum multiple scattering calculations. Two complementary vaporization methods-aerosol thermodesorption and a resistively heated sample oven coupled to an adiabatic expansion-were applied to promote pure enantiomers of alanine into the gas phase, yielding neutral alanine with different internal energy distributions. A comparison of the photoelectron spectroscopy, fragmentation, and dichroism measured for each of the vaporization methods was rationalized in terms of internal energy and conformer populations and supported by theoretical calculations. The analytical potential of the so-called PECD-PICO detection technique-where the electron spectroscopy and circular dichroism can be obtained as a function of mass and ion translational energy-is underlined and applied to characterize the origin of the various species found in the experimental mass spectra. Finally, the PECD findings are discussed within an astrochemical context, and possible implications regarding the origin of biomolecular asymmetry are identified.

  14. Survivability and reactivity of glycine and alanine in early oceans: effects of meteorite impacts.

    PubMed

    Umeda, Yuhei; Fukunaga, Nao; Sekine, Toshimori; Furukawa, Yoshihiro; Kakegawa, Takeshi; Kobayashi, Takamichi; Nakazawa, Hiromoto

    2016-01-01

    Prebiotic oceans might have contained abundant amino acids, and were subjected to meteorite impacts, especially during the late heavy bombardment. It is so far unknown how meteorite impacts affected amino acids in the early oceans. Impact experiments were performed under the conditions where glycine was synthesized from carbon, ammonia, and water, using aqueous solutions containing (13)C-labeled glycine and alanine. Selected amino acids and amines in samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). In particular, the (13)C-labeled reaction products were analyzed to distinguish between run products and contaminants. The results revealed that both amino acids survived partially in the early ocean through meteorite impacts, that part of glycine changed into alanine, and that large amounts of methylamine and ethylamine were formed. Fast decarboxylation was confirmed to occur during such impact processes. Furthermore, the formation of n-butylamine, detected only in the samples recovered from the solutions with additional nitrogen and carbon sources of ammonia and benzene, suggests that chemical reactions to form new biomolecules can proceed through marine impacts. Methylamine and ethylamine from glycine and alanine increased considerably in the presence of hematite rather than olivine under similar impact conditions. These results also suggest that amino acids present in early oceans can contribute further to impact-induced reactions, implying that impact energy plays a potential role in the prebiotic formation of various biomolecules, although the reactions are complicated and depend upon the chemical environments as well.

  15. Identification and expression analyses of the alanine aminotransferase (AlaAT) gene family in poplar seedlings

    PubMed Central

    Xu, Zhiru; Ma, Jing; Qu, Chunpu; Hu, Yanbo; Hao, Bingqing; Sun, Yan; Liu, Zhongye; Yang, Han; Yang, Chengjun; Wang, Hongwei; Li, Ying; Liu, Guanjun

    2017-01-01

    Alanine aminotransferase (AlaAT, E.C.2.6.1.2) catalyzes the reversible conversion of pyruvate and glutamate to alanine and α-oxoglutarate. The AlaAT gene family has been well studied in some herbaceous plants, but has not been well characterized in woody plants. In this study, we identified four alanine aminotransferase homologues in Populus trichocarpa, which could be classified into two subgroups, A and B. AlaAT3 and AlaAT4 in subgroup A encode AlaAT, while AlaAT1 and AlaAT2 in subgroup B encode glutamate:glyoxylate aminotransferase (GGAT), which catalyzes the reaction of glutamate and glyoxylate to α-oxoglutarate and glycine. Four AlaAT genes were cloned from P. simonii × P. nigra. PnAlaAT1 and PnAlaAT2 were expressed predominantly in leaves and induced by exogenous nitrogen and exhibited a diurnal fluctuation in leaves, but was inhibited in roots. PnAlaAT3 and PnAlaAT4 were mainly expressed in roots, stems and leaves, and was induced by exogenous nitrogen. The expression of PnAlaAT3 gene could be regulated by glutamine or its related metabolites in roots. Our results suggest that PnAlaAT3 gene may play an important role in nitrogen metabolism and is regulated by glutamine or its related metabolites in the roots of P. simonii × P. nigra. PMID:28378825

  16. Surface chemistry of alanine on Cu{111}: Adsorption geometry and temperature dependence

    NASA Astrophysics Data System (ADS)

    Baldanza, Silvia; Cornish, Alix; Nicklin, Richard E. J.; Zheleva, Zhasmina V.; Held, Georg

    2014-11-01

    Adsorption of L-alanine on the Cu{111} single crystal surface was investigated as a model system for interactions between small chiral modifier molecules and close-packed metal surfaces. Synchrotron-based X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy are used to determine the chemical state, bond coordination and out-of-plane orientation of the molecule on the surface. Alanine adsorbs in its anionic form at room temperature, whilst at low temperature the overlayer consists of anionic and zwitterionic molecules. NEXAFS spectra exhibit a strong angular dependence of the π* resonance associated with the carboxylate group, which allows determining the tilt angle of this group with respect to the surface plane (48° ± 2°) at room temperature. Low-energy electron diffraction (LEED) shows a p(2√{ 13} × 2√{ 13}) R 13 ° superstructure with only one domain, which breaks the mirror symmetry of the substrate and, thus, induces global chirality to the surface. Temperature-programmed XPS (TP-XPS) and temperature-programmed desorption (TPD) experiments indicate that the zwitterionic form converts into the anionic species (alaninate) at 293 K. The latter desorbs/decomposes between 435 K and 445 K.

  17. Qualitative analysis of collective mode frequency shifts in L-alanine using terahertz spectroscopy.

    PubMed

    Taulbee, Anita R; Heuser, Justin A; Spendel, Wolfgang U; Pacey, Gilbert E

    2009-04-01

    We have observed collective mode frequency shifts in deuterium-substituted L-alanine, three of which have previously only been calculated. Terahertz (THz) absorbance spectra were acquired at room temperature in the spectral range of 66-90 cm(-1), or 2.0-2.7 THz, for L-alanine (L-Ala) and four L-Ala compounds in which hydrogen atoms (atomic mass = 1 amu) were substituted with deuterium atoms (atomic mass = 2 amu): L-Ala-2-d, L-Ala-3,3,3-d(3), L-Ala-2,3,3,3-d(4), and L-Ala-d(7). The absorbance maxima of two L-Ala collective modes in this spectral range were recorded for multiple spectral measurements of each compound, and the magnitude of each collective mode frequency shift due to increased mass of these specific atoms was evaluated for statistical significance. Calculations were performed which predict the THz absorbance frequencies based on the estimated reduced mass of the modes. The shifts in absorbance maxima were correlated with the location(s) of the substituted deuterium atom(s) in the L-alanine molecule, and the atoms contributing to the absorbing delocalized mode in the crystal structure were deduced using statistics described herein. The statistical analyses presented also indicate that the precision of the method allows reproducible frequency shifts as small as 1 cm(-1) or 0.03 THz to be observed and that these shifts are not random error in the measurement.

  18. Effect of vitamins A, E and C on liver enzyme activity in rats exposed to organophosphate pesticide diazinon.

    PubMed

    Shokrzadeh, Mohammad; Shobi, Sepideh; Attar, Hossein; Shayegan, Sahel; Payam, Sakineh Sadat Hosseini; Ghorbani, Faezeh

    2012-10-01

    Diazinon, a commonly used organophosphorus pesticide, has been widely used throughout the world in agriculture and horticulture to control insects that feed on crops, ornamentals, lawns, fruits, vegetables and other food products. The toxicity of the DZN causes adverse effects on many organs. The purpose of this study was to examine the protective effect of vitamins A, E and C on liver enzymes alanine transaminase (ALT), Aspartate aminotransferase (AST) and Lactate Dehydrogenase (LDH) in rats exposed to diazinon. In this study, male wistar rats were randomly divided into 10 different groups. The groups were administered normal saline, soybean oil (as the solvent for diazinon and fat-soluble vitamins), diazinon, (30 mg kg(-1), vitamins E, C and A (100, 500 mg kg(-1) and 400 IU kg(-1), respectively) and a combination of diazinon with the same dose of each vitamin intraperitoneally i.p.daily for 14 days. Seven days after the final injection, the animals were anesthetized and blood samples were taken. The photometric method was used to measure the activity of the enzymes. The activities of ALT and AST in the diazinon group were significantly higher than that observed in the control group; however, the diazinon/vitamin E, A, C group displayed significant reduction in ALT and AST activities compared to the diazinon group. The lowest level of LDH enzyme activity was observed in the dazinon/vitamin C group and this was statistically lower than the diazinon group. The results of this study revealed that vitamin E, A and C have a potent protective effect against diazinon-induced hepatotoxicity in rats, which may be due to the scavenging of free radicals and increased antioxidant status.

  19. Unique food-entrained circadian rhythm in cysteine414-alanine mutant mCRY1 transgenic mice.

    PubMed

    Okano, Satoshi; Yasui, Akira; Hayasaka, Kiyoshi; Nakajima, Osamu

    Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Daily scheduled restricted feeding (RF), in which the food available time is restricted for several hours each day, elicits anticipatory activity. This food-anticipatory activity (FAA) is controlled by a food-entrainable oscillator (FEO) that is distinct from the suprachiasmatic nucleus (SCN), the master pacemaker in mammals. In an earlier report, we described generation of transgenic (Tg) mice ubiquitously overexpressing cysteine414-alanine mutant mCRY1. The Tg mice displayed long locomotor free-running periods (approximately 28 h) with rhythm splitting. Furthermore, their locomotor activity immediately re-adjusted to the advance of light-dark cycles (LD), suggesting some disorder in the coupling of SCN neurons. The present study examined the restricted feeding cycle (RF)-induced entrainment of locomotor activity in Tg mice in various light conditions. In LD, wild-type controls showed both FAA and LD-entrained activities. In Tg mice, almost all activity was eventually consolidated to a single bout before the feeding time. The result suggests a possibility that in Tg mice the feeding cycle dominates the LD cycle as an entrainment agent. In constant darkness (DD), wild-type mice exhibited robust free-run activity and FAA during RF. For Tg mice, only the rhythm entrained to RF was observed in DD. Furthermore, after returning to free feeding, the free-run started from the RF-entrained phase. These results suggest that the SCN of Tg mice is entrainable to RF and that the mCRY1 mutation alters the sensitivity of SCN to the cycle of nonphotic zeitgebers.

  20. Heterogeneity of L-alanine transport systems in brush-border membrane vesicles from rat placenta during late gestation.

    PubMed Central

    Alonso-Torre, S R; Serrano, M A; Medina, J M; Alvarado, F

    1992-01-01

    The placental uptake of L-alanine was studied by using purified brush-border membrane vesicles from rat trophoblasts. Saturation curves were carried out at 37 degrees C in buffers containing 100 mM (zero-trans)-NaSCN, -NaCl, -KSCN, -KCl, or -N-methyl-D-glucamine gluconate. The uncorrected uptake results were fitted by non-linear regression analysis to an equation involving one diffusional component either one or two saturable Michaelian transport terms. In the presence of NaCl, two distinct L-alanine transport systems were distinguished, named respectively System 1 (S-1; Vm1 about 760 pmol/s per mg of protein; KT1 = 0.5 mM) and System 2 (S-2; Vm2 about 1700 pmol/s per mg; KT2 = 9 mM). In contrast, in the presence of K+ (KCl = KSCN) or in the absence of any alkali-metal ions (N-methyl-D-glucamine gluconate), only one saturable system was apparent, which we identify as S-2. When Na+ is present, S-1, but not S-2, appears to be rheogenic, since its maximal transport capacity significantly increases in the presence of an inside-negative membrane potential, created either by replacing Cl- with the permeant anion thiocyanate (NaSCN > NaCl) or by applying an appropriate K+ gradient and valinomycin. alpha-(Methylamino)isobutyrate (methyl-AIB) appears to be a substrate of S-1, but not of S-2. For reasons that remain to be explained, however, methyl-AIB inhibits S-2. We conclude that S-1 represents a truly Na(+)-dependent mechanism, where Na+ behaves as an obligatory activator, whereas S-2 cannot discriminate between Na+ and K+, although its activity is higher in the presence of alkali-metal ions than in their absence (Na+ = K+ > N-methyl-D-glucammonium ion). S-2 appears to be fully developed 2 days before birth, whereas S-1 undergoes a capacity-type activation between days 19.5 and 21.5 of gestation, i.e. its apparent Vmax. nearly doubles, whereas its KT remains constant. PMID:1445280

  1. Probing the energy landscape of alanine dipeptide and decalanine using temperature as a tunable parameter in molecular dynamics

    NASA Astrophysics Data System (ADS)

    Chatterjee, A.; Bhattacharya, S.

    2016-10-01

    We perform several molecular dynamics (MD) calculations of solvated alanine dipeptide and decalanine in vacuum with temperature as a tunable parameter and in the process, generate Markov state models (MSMs) at each temperature. An interesting observation that the kinetic rates appear to obey the Arrhenius rate law allows us to predict the dynamics of alanine dipeptide at 300 K at the microsecond timescales using the nanoseconds long high temperature calculations without actually performing MD simulations at 300 K. We conclude that the energy landscape of alanine dipeptide contains superbasins deeper than kBT and determine the energy barriers associated with the moves from the Arrhenius rate expression. Similar insights regarding the energy landscape associated with folding/unfolding pathways of a deca-alanine molecule are obtained using kinetic rates calculated at different temperatures.

  2. Beta-alanine-hydrochloride (2:1) crystal: structure, 13C NMR and vibrational properties, protonation character.

    PubMed

    Godzisz, D; Ilczyszyn, M; Ciunik, Z

    2003-01-15

    The crystal structure of beta-alanine-hydrochloride (2:1) complex (2A-HCl) has been determined by X-ray diffraction method at 298 and 100 K as monoclinic, space group C2/c, Z=4. The crystal comprises chloride anions and protonated beta-alanine dimers: two beta-alanine zwitterions are joined by strong, symmetric (Ci) hydrogen bond with the O...O distance of 2.473 A at room temperature. Powder FT-IR and FT-Raman as well as solid state 13C NMR spectra provide insights into the solid structure of this complex, character of its hydrogen bonds and the beta-alanine protonation.

  3. Feasibility on using composite gel-alanine dosimetry on the validation of a multiple brain metastasis radiosurgery VMAT technique

    NASA Astrophysics Data System (ADS)

    Pavoni, J. F.; Neves-Junior, W. F. P.; Silveira, M. A.; Ramos, P. A. M. M.; Haddad, C. M. K.; Baffa, O.

    2015-01-01

    This work presents an end-to-end test using a composite Gel-Alanine phantom, in order to validate 3-dimensionally the dose distribution delivered by a single isocenter VMAT technique on the simultaneous treatment of multiple brain metastases. The results obtained with the gel and alanine dosimeters are consistent with the expected by the treatment planning system, showing the potential of this multidosimetric approach and validating dosimetrically the multiple brain metastases treatment using VMAT.

  4. Barrier-Free Intermolecular Proton Transfer Induced by Excess Electron Attachment to the Complex of Alanine with Uracil

    SciTech Connect

    Dabkowska, Iwona; Rak, Janusz; Gutowski, Maciej S.; Nilles, J.M.; Stokes, Sarah; Bowen, Kit H.

    2004-04-01

    The photoelectron spectrum of the uracil-alanine anionic complex (UA)- has been recorded with 2.540 eV photons. This spectrum reveals a broad feature with a maximum between 1.6-2.1 eV. The vertical electron detachment energy is too large to be attributed to an (UA)- anionic complex in which an intact uracil anion is solvated by alanine, or vice versa. The neutral and anionic complexes of uracil and alanine were studied at the B3LYP and second order Moeller-Plesset level of theory with 6-31++G** basis sets. The neutral complexes form cyclic hydrogen bonds and the three most stable neutral complexes are bound by 0.72, 0.61 and 0.57 eV. The electron hole in complexes of uracil with alaninie is localized on uracil, but the formation of a complex with alanine strongly modulates the vertical ionization energy of uracil. The theoretical results indicate that the excess electron in (UA)- occupies a p* orbital localized on uracil. The excess electron attachment to the complex can induce a barrier-free proton transfer (BFPT) from the carboxylic group of alanine to the O8 atom of uracil. As a result, the four most stable structures of the uracil-alanine anionic complex can be characterized as the neutral radical of hydrogenated uracil solvated by the anion of deprotonated alanine. Our current results for the anionic complex of uracil with alanine are similar to our previous results for the anion of uracil with glycine [Eur. Phys. J. D 20, 431 (2002)], and together they indicate that the BFPT process is not very sensitive to the nature of the amino acid's hydrophobic residual group. The BFPT to the O8 atom of uracil may be relevant to the damage suffered by nucleic acid bases due to exposure to low energy electrons.

  5. Stimulation of [3H] GABA and beta-[3H] alanine release from rat brain slices by cis-4-aminocrotonic acid.

    PubMed

    Chebib, M; Johnston, G A

    1997-02-01

    cis-4-Aminocrotonic acid (CACA; 100 microM), an analogue of GABA in a folded conformation, stimulated the passive release of [3H] GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of beta-[3H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 microM) did not stimulate the passive release of [3H]taurine from slices of cerebellum and spinal cord or of D-[3H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [3H]-taurine from the cerebellum and spinal cord and D-[3H]-aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and beta-alanine release are due to CACA acting as a substrate for a beta-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of beta-[3H]alanine into slices of rat cerebellum and cerebral cortex. The observed Ki for CACA against beta-[3H]alanine uptake in the cerebellum was 750 +/- 60 microM. CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABAc receptor. The effects of CACA on GABA and beta-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, beta-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, beta-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.

  6. The effect of beta-alanine supplementation on isokinetic force and cycling performance in highly trained cyclists.

    PubMed

    Howe, Samuel T; Bellinger, Phillip M; Driller, Matthew W; Shing, Cecilia M; Fell, James W

    2013-12-01

    Beta-alanine may benefit short-duration, high-intensity exercise performance. The aim of this randomized double-blind placebo-controlled study was to examine the effects of beta-alanine supplementation on aspects of muscular performance in highly trained cyclists. Sixteen highly trained cyclists (mean ± SD; age = 24 ± 7 yr; mass = 70 ± 7 kg; VO2max = 67 ± 4 ml · kg(-1) · min(-1)) supplemented with either beta-alanine (n = 8, 65 mg · kg - 1BM) or a placebo (n = 8; dextrose monohydrate) over 4 weeks. Pre- and postsupplementation cyclists performed a 4-minute maximal cycling test to measure average power and 30 reciprocal maximal isokinetic knee contractions at a fixed angular velocity of 180° · sec(-1) to measure average power/repetition, total work done (TWD), and fatigue index (%). Blood pH, lactate (La-) and bicarbonate (HCO3-) concentrations were measured pre- and postisokinetic testing at baseline and following the supplementation period. Beta-alanine supplementation was 44% likely to increase average power output during the 4-minute cycling time trial when compared with the placebo, although this was not statistically significant (p = .25). Isokinetic average power/repetition was significantly increased post beta-alanine supplementation compared with placebo (beta-alanine: 6.8 ± 9.9 W, placebo: -4.3 ± 9.5 W, p = .04, 85% likely benefit), while fatigue index was significantly reduced (p = .03, 95% likely benefit). TWD was 89% likely to be improved following beta-alanine supplementation; however, this was not statistically significant (p = .09). There were no significant differences in blood pH, lactate, and HCO3- between groups (p > .05). Four weeks of beta-alanine supplementation resulted in worthwhile changes in time-trial performance and short-duration muscular force production in highly trained cyclists.

  7. GABA, β-alanine and glycine in the digestive juice of privet-specialist insects: convergent adaptive traits against plant iridoids.

    PubMed

    Konno, Kotaro; Hirayama, Chikara; Yasui, Hiroe; Okada, Sachiko; Sugimura, Masahiro; Yukuhiro, Fumiko; Tamura, Yasumori; Hattori, Makoto; Shinbo, Hiroshi; Nakamura, Masatoshi

    2010-09-01

    The privet tree, Ligustrum obtusifolium (Oleaceae), defends its leaves against insects with a strong lysine-decreasing activity that make proteins non-nutritive. This is caused by oleuropein, an iridoid glycoside. We previously found that some privet-specialist caterpillars adapt by secreting glycine in the digestive juice as a neutralizer that prevents the loss of lysine. Here, we extended the survey into 42 lepidopteran and hymenopteran species. The average concentration of glycine in digestive juice for 11 privet-feeding species (40.396 mM) was higher than that for 32 non-privet-feeding species (2.198 mM). The glycine concentrations exceeded 10 mM in 7 out of 11 privet-feeding species. In Macrophya timida (Hymenoptera), it reached 164.8 mM. Three out of the four remaining privet-feeding species had other amino acids instead. Larvae of a privet-specialist butterfly, Artopoetes pryeri (Lycaenidae), had a high concentration (60.812 mM) of GABA. In two other specialists, β-alanine was found. GABA, β-alanine, and glycine as well as alanine, amines, and ammonium ion inhibited the lysine decrease, indicating that amino residues are responsible for the inhibition. However, the three amino acids found in the specialists were far more effective (20 mM showed 80% inhibition) than the rest (>140 mM was required for 80% inhibition). Our results show a clear and rare case of the apparent convergent evolution of herbivores' molecular adaptations of feeding on a plant with a chemical defense in a manner that minimizes the cost of adaptation. The novel role of GABA in plant-herbivore interactions shown here is probably the first reported non-neuronal role of animal-derived GABA.

  8. Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

    PubMed

    Pitluk, Z W; McDonough, M; Sangan, P; Gonda, D K

    1995-03-01

    CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.

  9. In Silico Screening, Alanine Mutation, and DFT Approaches for Identification of NS2B/NS3 Protease Inhibitors

    PubMed Central

    Balajee, R.; Srinivasadesikan, V.; Sakthivadivel, M.; Gunasekaran, P.

    2016-01-01

    To identify the ligand that binds to a target protein with high affinity is a nontrivial task in computer-assisted approaches. Antiviral drugs have been identified for NS2B/NS3 protease enzyme on the mechanism to cleave the viral protein using the computational tools. The consequence of the molecular docking, free energy calculations, and simulation protocols explores the better ligand. It provides in-depth structural insights with the catalytic triad of His51, Asp75, Ser135, and Gly133. The MD simulation was employed here to predict the stability of the complex. The alanine mutation has been performed and its stability was monitored by using the molecular dynamics simulation. The minimal RMSD value suggests that the derived complexes are close to equilibrium. The DFT outcome reveals that the HOMO-LUMO gap of Ligand19 is 2.86 kcal/mol. Among the considered ligands, Ligand19 shows the lowest gap and it is suggested that the HOMO of Ligand19 may transfer the electrons to the LUMO in the active regions. The calculated binding energy of Ligand19 using the DFT method is in good agreement with the docking studies. The pharmacological activity of ligand was performed and satisfies Lipinski rule of 5. Moreover, the computational results are compared with the available IC50 values of experimental results. PMID:27057355

  10. The use of L-serine to prevent β-methylamino-L-alanine (BMAA)-induced proteotoxic stress in vitro.

    PubMed

    Main, Brendan J; Dunlop, Rachael A; Rodgers, Kenneth J

    2016-01-01

    β-methylamino-L-alanine (BMAA), a non-protein amino acid synthesised by cyanobacteria, has been linked to a complex neurological disorder on Guam and more recently to other cases of sporadic ALS (sALS), however the mechanisms of BMAA toxicity are not completely understood. We have previously demonstrated that BMAA is misincorporated into newly synthesised proteins by human neuroblastoma cells and fibroblasts, resulting in the formation of autofluorescent material and the induction of apoptotic cell death. In the present study we show that BMAA at low levels does not cause an acute toxicity in neuroblastoma cells but increases the expression of the ER stress marker, C/EBP homologous protein (CHOP) and increases the activity of the pro-apoptotic enzyme caspase-3. We also observed an increase in the activity of the lysosomal cysteine proteases cathepsin B and L, characteristic of the accumulation of proteins in the lysosomal system. We were able to prevent these proteotoxic effects in neuroblastoma cells through co-treatment with l-serine suggesting that they resulted from incorporation of BMAA into proteins. Misincorporation provides a possible mechanism whereby BMAA could initiate misfolding, and the accumulation of aggregate-prone proteins in neurons. This build-up of misfolded proteins could explain the long latency period of the disease previously reported on Guam.

  11. The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode beta-cyano-L-alanine hydratase/nitrilase.

    PubMed

    Piotrowski, M; Schönfelder, S; Weiler, E W

    2001-01-26

    Nitrilases (nitrile aminohydrolases, EC ) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases of Arabidopsis thaliana, the isoform NIT4 is the most divergent one, and homologs of NIT4 are also known from species not belonging to the Brassicaceae like Nicotiana tabacum and Oryza sativa. We expressed A. thaliana NIT4 as hexahistidine tag fusion protein in Escherichia coli. The purified enzyme showed a strong substrate specificity for beta-cyano-l-alanine (Ala(CN)), an intermediate product of cyanide detoxification in higher plants. Interestingly, not only aspartic acid but also asparagine were identified as products of NIT4-catalyzed Ala(CN) hydrolysis. Asn itself was no substrate for NIT4, indicating that it is not an intermediate but one of two reaction products. NIT4 therefore has both nitrilase and nitrile hydratase activity. Several lines of evidence indicate that the catalytic center for both reactions is the same. The NIT4 homologs of N. tabacum were found to catalyze the same reactions and protein extracts of A. thaliana, N. tabacum and Lupinus angustifolius also converted Ala(CN) to Asp and Asn in vitro. NIT4 may play a role in cyanide detoxification during ethylene biosynthesis because extracts from senescent leaves of A. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue.

  12. Nepenthes insignis uses a C2-portion of the carbon skeleton of L-alanine acquired via its carnivorous organs, to build up the allelochemical plumbagin.

    PubMed

    Rischer, Heiko; Hamm, Andreas; Bringmann, Gerhard

    2002-03-01

    Tropical pitcher plants (Nepenthes) catch animals in their specialized cup-shaped leaves, digest the prey by secreting enzymes, and actively take up the resulting compounds. The benefit of this behaviour is the ability to grow and compete in nutrient-poor habitats. Our present in vitro study shows that not only the nitrogen of alanine fed to the carnivorous organs is used by the plant but that in addition intact C2-units derived from C-2 and C-3 of stable isotope labelled L-alanine serve as building blocks, here exemplarily for the synthesis of the secondary metabolite plumbagin, a potent allelochemical. This result adds a new facet to the benefit of carnivory for plants. The availability of plumbagin by a de novo synthesis probably enhances the plants' fitness in their defence against phytophagous and pathogenic organisms. A missing specific uptake or CoA activation mechanism might be the reason that acetate fed to the pitchers was not incorporated into the naphthoquinone plumbagin. The dihydronaphthoquinone glucosides rossoliside and plumbaside A, here isolated for the first time from Nepenthes, by contrast, showed no incorporation after feeding of any of the two precursors, suggesting these compounds to be storage forms with probably very low turnover rates.

  13. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

    PubMed

    Leke, Renata; Bak, Lasse K; Anker, Malene; Melø, Torun M; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Portela, Luis V; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

    2011-04-01

    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

  14. Analysis of Plasma Protein Concentrations and Enzyme Activities in Cattle within the Ex-Evacuation Zone of the Fukushima Daiichi Nuclear Plant Accident

    PubMed Central

    Endo, Satoru; Tanaka, Kenichi; Hirakawa, Yasuko; Hayashi, Gohei; Sekine, Tsutomu; Kino, Yasushi; Kuwahara, Yoshikazu; Suzuki, Masatoshi; Fukumoto, Motoi; Yamashiro, Hideaki; Abe, Yasuyuki; Fukuda, Tomokazu; Shinoda, Hisashi; Isogai, Emiko; Arai, Toshiro; Fukumoto, Manabu

    2016-01-01

    The effect of the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on humans and the environment is a global concern. We performed biochemical analyses of plasma from 49 Japanese Black cattle that were euthanized in the ex-evacuation zone set within a 20-km radius of FNPP. Among radionuclides attributable to the FNPP accident, germanium gamma-ray spectrometry detected photopeaks only from 134Cs and 137Cs (radiocesium) commonly in the organs and in soil examined. Radioactivity concentration of radiocesium was the highest in skeletal muscles. Assuming that the animal body was composed of only skeletal muscles, the median of internal dose rate from radiocesium was 12.5 μGy/day (ranging from 1.6 to 33.9 μGy/day). The median of external dose rate calculating from the place the cattle were caught was 18.8 μGy/day (6.0–133.4 μGy/day). The median of internal and external (total) dose rate of the individual cattle was 26.9 μGy/day (9.1–155.1 μGy/day). Plasma levels of malondialdehyde and superoxide dismutase activity were positively and glutathione peroxidase activity was negatively correlated with internal dose rate. Plasma alanine transaminase activity and percent activity of lactate dehydrogenase (LDH)-2, LDH-3 and LDH-4 were positively and LDH-1 was negatively correlated with both internal and total dose rate. These suggest that chronic exposure to low-dose rate of ionizing radiation induces slight stress resulting in modified plasma protein and enzyme levels. PMID:27159386

  15. Preparation of alanine and tyrosine functionalized graphene oxide nanoflakes and their modified carbon paste electrodes for the determination of dopamine

    NASA Astrophysics Data System (ADS)

    Kumar, Mohan; Swamy, B. E. Kumara; Asif, M. H. Mohammed; Viswanath, C. C.

    2017-03-01

    Herein, established the synthesis of graphene oxide (GO) by Hummers Method with addition of KMnO4 followed by thermal heating at 80 °C. The obtained GO was further functionalized by alanine and tyrosine. The prepared GO, alanine functionalized GO nanoflakes (AGONF) and tyrosine functionalized GO nanoflakes (TGONF) were characterized by spectroscopic technique using energy-dispersive spectroscopy (EDS), quantitatively by scanning electron microscopy (SEM) and structural studies along with interlayer distance verified through X-ray diffraction technique. Afterwards, the prepared AGONF and TGONF were used as the modifier for the carbon paste electrode (CPE). The electrochemical behavior of the AGONF and TGONF modified carbon paste electrodes (MCPEs) towards dopamine (DA) in phosphate buffer solution (PBS) were examined by cyclic voltammetric (CV) technique and the obtained consequences showed good electrocatalytic activity of MCPEs by increasing the redox peak current with a lower potential difference compared to the bare CPE (BCPE). The AGONF and TGONF MCPEs were further used for the optimization studies. From the pH studies, it was found that the equal number of proton and electron transfer reaction involved in both the modified electrodes. The scan rate studies demonstrate the adsorption controlled electrode process at AGONF MCPE and diffusion controlled at TGONF MCPE. The oxidation peak current increased linearly with two concentration interval of DA at a range of 2-7 μM and 10-30 μM in presence of PBS (pH 7.4) at MCPEs and the limit of detection (LOD) were found to be 0.84 μM and 0.96 μM for first interval DA concentration range (2-7 μM) at AGONF and TGONF MCPE. The stability, repeatability and reproducibility of functionalized GO nanoflakes MCPEs at DA were studied and established excellent characteristics. The newly developed functionalized GO nanoflake electrodes were successfully tested in DA injection sample. Furthermore the functionalized GO and

  16. Free Energy Landscapes of Alanine Oligopeptides in Rigid-Body and Hybrid Water Models.

    PubMed

    Nayar, Divya; Chakravarty, Charusita

    2015-08-27

    Replica exchange molecular dynamics is used to study the effect of different rigid-body (mTIP3P, TIP4P, SPC/E) and hybrid (H1.56, H3.00) water models on the conformational free energy landscape of the alanine oligopeptides (acAnme and acA5nme), in conjunction with the CHARMM22 force field. The free energy landscape is mapped out as a function of the Ramachandran angles. In addition, various secondary structure metrics, solvation shell properties, and the number of peptide-solvent hydrogen bonds are monitored. Alanine dipeptide is found to have similar free energy landscapes in different solvent models, an insensitivity which may be due to the absence of possibilities for forming i-(i + 4) or i-(i + 3) intrapeptide hydrogen bonds. The pentapeptide, acA5nme, where there are three intrapeptide backbone hydrogen bonds, shows a conformational free energy landscape with a much greater degree of sensitivity to the choice of solvent model, though the three rigid-body water models differ only quantitatively. The pentapeptide prefers nonhelical, non-native PPII and β-sheet populations as the solvent is changed from SPC/E to the less tetrahedral liquid (H1.56) to an LJ-like liquid (H3.00). The pentapeptide conformational order metrics indicate a preference for open, solvent-exposed, non-native structures in hybrid solvent models at all temperatures of study. The possible correlations between the properties of solvent models and secondary structure preferences of alanine oligopeptides are discussed, and the competition between intrapeptide, peptide-solvent, and solvent-solvent hydrogen bonding is shown to be crucial in the relative free energies of different conformers.

  17. Relative response of alanine dosemeters for high-energy electrons determined using a Fricke primary standard.

    PubMed

    Vörös, Sándor; Anton, Mathias; Boillat, Bénédicte

    2012-03-07

    A significant proportion of cancer patients is treated using MeV electron radiation. One of the measurement methods which is likely to furnish reliable dose values also under non-reference conditions is the dosimetry using alanine and read-out via electron spin resonance (ESR). The system has already proven to be suitable for QA purposes for modern radiotherapy involving megavoltage x-rays. In order to render the secondary standard measurement system of the Physikalisch-Technische Bundesanstalt based on alanine/ESR useable for dosimetry in radiotherapy, the dose-to-water (D(W)) response of the dosemeter needs to be known for relevant radiation qualities. For MeV electrons, the D(W) response was determined using the Fricke primary standard of the Swiss Federal Office of Metrology. Since there were no citable detailed publications on the Swiss primary standard available, this measurement system is described in some detail. The experimental results for the D(W) response are compared to results of Monte Carlo simulations which model in detail the beams furnished by the electron accelerator as well as the geometry of the detectors. The agreement between experiment and simulation is very good, as well as the agreement with results published by the National Research Council of Canada which are based on a different primary standard. No significant dependence of the D(W) response was found in the range between 6 and 20 MeV. It is therefore suggested to use a unique correction factor k(E) for alanine for all MeV qualities of k(E) = 1.012 ± 0.010.

  18. Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function.

    PubMed

    Lee, Shirley Y; Pullen, Lester; Virgil, Daniel J; Castañeda, Carlos A; Abeykoon, Dulith; Bolon, Daniel N A; Fushman, David

    2014-04-03

    Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.

  19. A new method for ligand docking to flexible receptors by dual alanine scanning and refinement (SCARE)

    NASA Astrophysics Data System (ADS)

    Bottegoni, Giovanni; Kufareva, Irina; Totrov, Maxim; Abagyan, Ruben

    2008-05-01

    Protein binding sites undergo ligand specific conformational changes upon ligand binding. However, most docking protocols rely on a fixed conformation of the receptor, or on the prior knowledge of multiple conformations representing the variation of the pocket, or on a known bounding box for the ligand. Here we described a general induced fit docking protocol that requires only one initial pocket conformation and identifies most of the correct ligand positions as the lowest score. We expanded a previously used diverse "cross-docking" benchmark to thirty ligand-protein pairs extracted from different crystal structures. The algorithm systematically scans pairs of neighbouring side chains, replaces them by alanines, and docks the ligand to each `gapped' version of the pocket. All docked positions are scored, refined with original side chains and flexible backbone and re-scored. In the optimal version of the protocol pairs of residues were replaced by alanines and only one best scoring conformation was selected from each `gapped' pocket for refinement. The optimal SCARE (SCan Alanines and REfine) protocol identifies a near native conformation (under 2 Å RMSD) as the lowest rank for 80% of pairs if the docking bounding box is defined by the predicted pocket envelope, and for as many as 90% of the pairs if the bounding box is derived from the known answer with ˜5 Å margin as used in most previous publications. The presented fully automated algorithm takes about 2 h per pose of a single processor time, requires only one pocket structure and no prior knowledge about the binding site location. Furthermore, the results for conformationally conserved pockets do not deteriorate due to substantial increase of the pocket variability.

  20. Essential oil of Artemisia vestita exhibits potent in vitro and in vivo antibacterial activity: Investigation of the effect of oil on biofilm formation, leakage of potassium ions and survival curve measurement

    PubMed Central

    YANG, CHANG; HU, DONG-HUI; FENG, YAN

    2015-01-01

    The aim of the present study was to investigate the chemical composition of the essential oil of Artemisia vestita and to determine the antibacterial activity of the essential oil and its two major components, grandisol and 1,8-cineole, against certain respiratory infection-causing bacterial strains, in vitro and in vivo. The chemical composition of the essential oil was analyzed using gas chromatography-mass spectrometry. A micro-well dilution method was used to determine the minimum inhibition concentration (MIC) values of the essential oil and its major constituents. A model of Streptococcus pyogenes infection in mice was used to determine its in vivo activities. Lung and blood samples were obtained to assess bacterial cell counts. Toxicity evaluation of the essential oil and its components was completed by performing biochemical analysis of the serum, particularly monitoring aspartate transaminase, alanine transaminase, urea and creatinine. The essential oil exhibited potent antibacterial activity, whereas the two major constituents were less potent. The essential oil exhibited MIC values between 20 and 80 μg/ml, while the values of the two constituents were between 130 and 200 μg/ml. Scanning electron microscopy results demonstrated that the essential oil inhibited biofilm formation and altered its architecture. Survival curves indicated that the essential oil led to a reduction in the viability of different bacteria. The essential oil also induced significant leakage of potassium ions from S. pyogenes. The essential oil (100 μg/mouse) and grandisol (135 μg/mouse) significantly reduced the number of viable bacterial cells in the lungs (P<0.01). However, intake of 100 μg/mouse of essential oil or grandisol 135 μg/mouse once or twice each day for 9 days did not produce any toxic effects in the mice. In conclusion, the in vitro and in vivo results suggested that the essential oil of A. vestita and one of its major constituents, grandisol, can significantly

  1. Paralogous ALT1 and ALT2 Retention and Diversification Have Generated Catalytically Active and Inactive Aminotransferases in Saccharomyces cerevisiae

    PubMed Central

    Peñalosa-Ruiz, Georgina; Aranda, Cristina; Ongay-Larios, Laura; Colon, Maritrini; Quezada, Hector; Gonzalez, Alicia

    2012-01-01

    Background Gene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase. Principal Findings Our results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function. Conclusions ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1

  2. Inhibition study of alanine aminotransferase enzyme using sequential online capillary electrophoresis analysis.

    PubMed

    Liu, Lina; Chen, Yuanfang; Yang, Li

    2014-12-15

    We report the study of several inhibitors on alanine aminotransferase (ALT) enzyme using sequential online capillary electrophoresis (CE) assay. Using metal ions (Na(+) and Mg(2+)) as example inhibitors, we show that evolution of the ALT inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time. The inhibition mechanism and kinetic constants of ALT inhibition with succinic acid and two traditional Chinese medicines were derived from the sequential online CE assay. Our study could provide valuable information about the inhibition reactions of ALT enzyme.

  3. Evaluation of alanine as a reference dosimeter for therapy level dose comparisons in megavoltage electron beams

    NASA Astrophysics Data System (ADS)

    McEwen, Malcolm; Sharpe, Peter; Vörös, Sándor

    2015-04-01

    When comparing absorbed dose standards from different laboratories (e.g. National Measurement Institutes, NMIs, for Key or Supplementary comparisons) it is rarely possible to carry out a direct comparison of primary standard instruments, and therefore some form of transfer detector is required. Historically, air-filled, unsealed ionization chambers have been used because of the long history of using these instruments, very good stability over many years, and ease of transport. However, the use of ion chambers for therapy-level comparisons is not without its problems. Findings from recent investigations suggest that ion chambers are prone to non-random variations, they are not completely robust to standard courier practices, and failure at any step in a comparison can render all measurements potentially useless. An alternative approach is to identify a transfer system that is insensitive to some of these concerns—effectively a dosimeter that is inexpensive, simple to use, robust, but with sufficient precision and of a size relevant to the disseminated quantity in question. The alanine dosimetry system has been successfully used in a number of situations as an audit dosimeter and therefore the purpose of this investigation was to determine whether alanine could also be used as the transfer detector for dosimetric comparisons, which require a lower value for the measurement uncertainty. A measurement protocol was developed for comparing primary standards of absorbed dose to water in high-energy electron beams using alanine pellets irradiated in a water-equivalent plastic phantom. A trial comparison has been carried out between three NMIs and has indicated that alanine is a suitable alternative to ion chambers, with the system used achieving a precision of 0.1%. Although the focus of the evaluation was on the performance of the dosimeter, the comparison results are encouraging, showing agreement at the level of the combined uncertainties (~0.6%). Based on this

  4. GMXPBSA 2.1: A GROMACS tool to perform MM/PBSA and computational alanine scanning

    NASA Astrophysics Data System (ADS)

    Paissoni, C.; Spiliotopoulos, D.; Musco, G.; Spitaleri, A.

    2015-01-01

    GMXPBSA 2.1 is a user-friendly suite of Bash/Perl scripts for streamlining MM/PBSA calculations on structural ensembles derived from GROMACS trajectories, to automatically calculate binding free energies for protein-protein or ligand-protein complexes [R.T. Bradshaw et al., Protein Eng. Des. Sel. 24 (2011) 197-207]. GMXPBSA 2.1 is flexible and can easily be customized to specific needs and it is an improvement of the previous GMXPBSA 2.0 [C. Paissoni et al., Comput. Phys. Commun. (2014), 185, 2920-2929]. Additionally, it performs computational alanine scanning (CAS) to study the effects of ligand and/or receptor alanine mutations on the free energy of binding. Calculations require only for protein-protein or protein-ligand MD simulations. GMXPBSA 2.1 performs different comparative analyses, including a posteriori generation of alanine mutants of the wild-type complex, calculation of the binding free energy values of the mutant complexes and comparison of the results with the wild-type system. Moreover, it compares the binding free energy of different complex trajectories, allowing the study of the effects of non-alanine mutations, post-translational modifications or unnatural amino acids on the binding free energy of the system under investigation. Finally, it can calculate and rank relative affinity to the same receptor utilizing MD simulations of proteins in complex with different ligands. In order to dissect the different MM/PBSA energy contributions, including molecular mechanic (MM), electrostatic contribution to solvation (PB) and nonpolar contribution to solvation (SA), the tool combines two freely available programs: the MD simulations software GROMACS [S. Pronk et al., Bioinformatics 29 (2013) 845-854] and the Poisson-Boltzmann equation solver APBS [N.A. Baker et al., Proc. Natl. Acad. Sci. U.S.A 98 (2001) 10037-10041]. All the calculations can be performed in single or distributed automatic fashion on a cluster facility in order to increase the

  5. Conserved aspartic acid 233 and alanine 231 are not required for poliovirus polymerase function in replicons

    PubMed Central

    Freistadt, Marion S; Eberle, Karen E

    2007-01-01

    Nucleic acid polymerases have similar structures and motifs. The function of an aspartic acid (conserved in all classes of nucleic acid polymerases) in motif A remains poorly understood in RNA-dependent RNA polymerases. We mutated this residue to alanine in a poliovirus replicon. The resulting mutant could still replicate, although at a reduced level. In addition, mutation A231C (also in motif A) yielded high levels of replication. Taken together these results show that poliovirus polymerase conserved residues D233 and A231 are not essential to poliovirus replicon function. PMID:17352827

  6. Beta-alanine and beta-aminoisobutyric acid levels in two siblings with dihydropyrimidinase deficiency.

    PubMed

    van Kuilenburg, A B P; Stroomer, A E M; Bosch, A M; Duran, M

    2008-06-01

    Dihydropyrimidinase (DHP) deficiency is an inborn error of the pyrimidine degradation pathway, affecting the hydrolytic ring opening of the dihydropyrimidines. In two siblings with a complete DHP deficiency and a variable clinical presentation, a normal concentration of beta-alanine and strongly decreased levels of beta-aminoisobutyric acid were observed in plasma, urine and CSF. No major differences were observed for the concentrations of the beta-amino acids in plasma and urine between the symptomatic and asymptomatic sibling. Thus, the relevance of the shortage of beta-aminoisobutyric acid for the onset of a clinical phenotype in patients with DHP deficiency remains to be established.

  7. Preclinical evaluation of a urokinase plasminogen activator receptor-targeted nanoprobe in rhesus monkeys

    PubMed Central

    Chen, Yushu; Gong, Li; Gao, Ning; Liao, Jichun; Sun, Jiayu; Wang, Yuqing; Wang, Lei; Zhu, Pengjin; Fan, Qing; Wang, Yongqiang Andrew; Zeng, Wen; Mao, Hui; Yang, Lily; Gao, Fabao

    2015-01-01

    Purpose To translate a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator receptor-targeted magnetic iron oxide (IO) nanoparticles (uPAR-targeted human ATF-IONPs) into clinical applications, we conducted a pilot study to evaluate the toxicity and pharmacokinetics of this nanoparticle in normal rhesus monkeys. Methods We assessed the changes in the following: magnetic resonance imaging (MRI) signals from pretreatment stage to 14 days posttreatment, serum iron concentrations from 5 minutes posttreatment to 12 weeks posttreatment, routine blood examination and serum chemistry analysis results from pretreatment stage to 12 weeks after administration, and results of staining of the liver with Perls’ Prussian Blue and hematoxylin–eosin at 24 hours and 3 months posttreatment in two rhesus monkeys following an intravenous administration of the targeted nanoparticles either with a polyethylene glycol (ATF-PEG-IONP) or without a PEG (ATF-IONP) coating. Results The levels of alkaline phosphatase, alanine transaminase, and direct bilirubin in the two monkeys increased immediately after the administration of the IONPs but returned to normal within 20 days and stayed within the normal reference range 3 months after the injection. The creatinine levels of the two monkeys stayed within the normal range during the study. In addition, red blood cells, white blood cells, hemoglobin level, and platelets remained normal during the 3 months of the study. Conclusion All of the results suggest that a transient injury in terms of normal organ functions, but no microscopic necrotic lesions, was observed at a systemic delivery dose of 5 mg/kg of iron equivalent concentration in the acute phase, and that no chronic toxicity was found 3 months after the injection. Therefore, we conclude that uPAR-targeted IONPs have the potential to be used as receptor-targeted MRI contrasts as well as theranostic agents for the detection and treatment of

  8. The effect of β-N-methylamino-L-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum.

    PubMed

    Esterhuizen-Londt, M; Pflugmacher, S; Downing, T G

    2011-04-01

    Cyanobacteria are known to produce bioactive secondary metabolites such as hepatotoxins, cytotoxins and neurotoxins. The newly recognized neurotoxin β-N-methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid found in the majority of cyanobacterial genera tested. Evidence that exists for implication of BMAA in neurodegenerative disorders relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. Uptake and accumulation of free BMAA by various non-symbiotic organisms, including aquatic macrophytes, has been documented but to date limited evidence of ecotoxicology exists. We therefore investigated the effect of BMAA on the oxidative stress responses of the macrophyte, Ceratophyllum demersum. Markers for oxidative stress in this study are the antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase and glutathione reductase. We found that BMAA had an inhibitory effect on all the oxidative stress response enzymes tested in plants exposed to BMAA. However enzymes not related to oxidative stress response were not affected by BMAA in in vitro experiments. Binding studies in the presence of BMAA showed reduced enzyme specific activity over time compared to the control. This study shows that BMAA causes oxidative stress indirectly as it inhibits antioxidant enzymes required to combat reactive oxygen species that cause damage to cells. Further investigations are required to fully understand the inhibitory effect of BMAA on these enzymes.

  9. Verification of the pure alanine in PMMA tube dosimeter applicability for dosimetry of radiotherapy photon beams: a feasibility study.

    PubMed

    Al-Karmi, Anan M; Ayaz, Ali Asghar H; Al-Enezi, Mamdouh S; Abdel-Rahman, Wamied; Dwaikat, Nidal

    2015-09-01

    Alanine dosimeters in the form of pure alanine powder in PMMA plastic tubes were investigated for dosimetry in a clinical application. Electron paramagnetic resonance (EPR) spectroscopy was used to measure absorbed radiation doses by detection of signals from radicals generated in irradiated alanine. The measurements were performed for low-dose ranges typical for single-fraction doses often used in external photon beam radiotherapy. First, the dosimeters were irradiated in a solid water phantom to establish calibration curves in the dose range from 0.3 to 3 Gy for 6 and 18 MV X-ray beams from a clinical linear accelerator. Next, the dosimeters were placed at various locations in an anthropomorphic pelvic phantom to measure the dose delivery of a conventional four-field box technique treatment plan to the pelvis. Finally, the doses measured with alanine dosimeters were compared against the doses calculated with a commercial treatment planning system (TPS). The results showed that the alanine dosimeters have a highly sensitive dose response with good linearity and no energy dependence in the dose range and photon beams used in this work. Also, a fairly good agreement was found between the in-phantom dose measurements with alanine dosimeters and the TPS dose calculations. The mean value of the ratios of measured to calculated dose values was found to be near unity. The measured points in the in-field region passed dose-difference acceptance criterion of 3% and those in the penumbral region passed distance-to-agreement acceptance criterion of 3 mm. These findings suggest that the pure alanine powder in PMMA tube dosimeter is a suitable option for dosimetry of radiotherapy photon beams.

  10. Free-energy profile along an isomerization pathway: Conformational isomerization in alanine dipeptide

    NASA Astrophysics Data System (ADS)

    Lee, In-Ho

    2013-02-01

    The free-energy profile for the conformational isomerization process in alanine dipeptide is presented in atomistic detail by using an action-derived molecular dynamics (ADMD) method and replica-exchange molecular dynamics (REMD) method. First, by employing ADMD, a dynamic isomerization pathway model of the alanine dipeptide with two available low-energy conformations, C7 ax and C7 eq , is determined. The pathway model is chosen to be the reaction coordinate, so the isomerization process is characterized by the ADMD step index, which is not an a-priori reaction coordinate as found in conventional studies of molecular conformational changes. Second, by employing the REMD method, the free-energy profile is calculated as a function of temperature. This couple of procedures is a quite natural protocol for conformational isomerization process simulations, irrespective of the arbitrary selection of the reaction coordinate. The alliance between the two simulation methods, ADMD and REMD, is demonstrated to have a great synergy effect on understanding the conformational changes in molecules.

  11. Advancements in accuracy of the alanine dosimetry system. Part 2. The influence of the irradiation temperature

    NASA Astrophysics Data System (ADS)

    Nagy, Vitaly; Puhl, James M.; Desrosiers, Marc F.

    2000-01-01

    Systematic measurements of the temperature coefficient for alanine electron paramagnetic resonance (EPR) response have been performed for irradiation in the temperature range (10-50)°C and in the absorbed dose range (1-100) kGy at the dose rate 9.5 kGy/h. During the 60Co rad -ray irradiation, rad - L-alanine dosimeters were kept in a sealed aluminum holder that provided an effective heat exchange with the temperature-controlled environment. The time between the irradiation and signal measurements was standardized, and a reference sample fixed in the resonant cavity was used to correct the signals for small variations in the spectrometer sensitivity. The temperature coefficient for each dose was determined from approximately 30 experimental points processed by the weighted least-squares technique after the necessary statistical tests were done. The temperature coefficients thus determined were considerably lower than previously reported. The dose dependence of the temperature coefficient features a minimum at (20-30) kGy (about 0.135%/K) with higher values at 1 kGy (0.17%/K) and at 100 kGy ((0.175-0.19) %/K). With the exception of very high doses, no significant distinction was found between the temperature coefficients of Bruker and NIST dosimeters, which differ in shape and binder content.

  12. A single glycine-alanine exchange directs ligand specificity of the elephant progestin receptor.

    PubMed

    Wierer, Michael; Schrey, Anna K; Kühne, Ronald; Ulbrich, Susanne E; Meyer, Heinrich H D

    2012-01-01

    The primary gestagen of elephants is 5α-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems.

  13. Mechano-responsive gelation of water by a short alanine-derivative.

    PubMed

    Reddy M, Amarendar; Srivastava, Aasheesh

    2014-07-21

    We report the design of a structurally concise alanine derivative (Ala-hyd) that has a rotationally flexible aromatic N-protecting group for alanine and a hydrazide functionality at its carboxylic end. Ala-hyd requires mechanical agitation (physically stirring, vortexing or sonicating) to form supramolecular hydrogels at medium concentrations (0.4-0.8 wt%). At higher concentrations (>0.8 wt%), it spontaneously gelates water on undisturbed cooling of the hot solution, while at lower concentrations (<0.4 wt%), only turbid suspensions were formed upon agitation. In the <0.8 wt% regime, hydrogelation by Ala-hyd is modulated by its concentration as well as by the extent of applied mechanical agitation. Turbidimetry and fluorescence spectroscopy indicate enhanced self-assembly of Ala-hyd upon agitation, and FTIR studies point towards stronger hydrogen bonds in the resulting assemblies. Since Ala-hyd requires mechanical agitation to undergo self-assembly, its aqueous sols exhibited mild shear-thickening behaviour in buffered as well as salt-free conditions. During shearing, the formation of an entangled mesh of long, helical nanofibers coincided with the maximum in the bulk shear viscosity. pH-dependent rheological investigations indicate that protonation of the amine unit (pKa = 8.9) of hydrazide diminishes the self-assembly propensity of this compound. The self-assembly of Ala-hyd can thus be modulated through mechanical as well as chemical cues.

  14. Quality by design development of brivanib alaninate tablets: degradant and moisture control strategy.

    PubMed

    Badawy, Sherif I F; Lin, Judy; Gokhale, Madhushree; Desai, Sachin; Nesarikar, Vishwas V; LaMarche, Keirnan R; Subramanian, Ganeshkumar A; Narang, Ajit S

    2014-07-20

    A quality by design approach was applied to the development of brivanib alaninate tablets. Brivanib alaninate, an ester pro-drug, undergoes hydrolysis to its parent compound, BMS-540215. The shelf-life of the tablets is determined by the rate of the hydrolysis reaction. Hydrolysis kinetics in the tablets was studied to understand its dependence on temperature and humidity. The BMS-540215 amount versus time profile was simulated using a kinetic model for the formation of BMS-540215 as function of relative humidity in the environment and a sorption-desorptiom moisture transfer model for the relative humidity inside the package. The combined model was used to study the effect of initial tablet water content on the rate of degradation and to identify a limit for initial tablet water content that results in acceptable level of the degradant at the end of shelf-life. A strategy was established for the moisture and degradant control in the tablet based on the understanding of its stability behavior and mathematical models. The control strategy includes a specification limit on the tablet water content and manufacturing process controls that achieve this limit at the time of tablet release testing.

  15. Ibalizumab-human CD4 receptor interaction: computational alanine scanning molecular dynamics studies.

    PubMed

    Su, Zhi-Yuan

    2014-01-01

    Antibody drugs are used in the treatment of many chronic diseases. Recently, however, patients and doctors have encountered problems with drug resistance, and improving the affinity of antibody drugs has therefore become a pressing issue. Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). In this study, we sought to identify the key residues of the complementaritydetermining regions (CDRs) of ibalizumab. Virtual alanine mutations (complementarity-determining regions of ibalizumab) were also studied using solvated interaction energies derived from molecular dynamics and the explicit water model. Using 1,000 nanosecond molecular dynamic simulations, we identified six residues: Tyr50 [HCDR2], Tyr53 [HCDR3], Asp58 [HCDR2], Glu95 [HCDR2], and Arg95 [LCDR3]. The Robetta alanine-scanning mutagenesis method and crystallographic information were used to verify our simulations. Our simulated binding affinity of -17.33 kcal/mol is close to the experimentally determined value of -16.48 kcal/mol. Our findings may be useful for protein engineering the structure of the ibalizumab-human CD4 receptor complex. Moreover, the six residues that we identified may play a significant role in the development of bioactive antibody analogues.

  16. Determinants of Alanine Dipeptide Conformational Equilibria on Graphene and Hydroxylated Derivatives.

    PubMed

    Poblete, Horacio; Miranda-Carvajal, Ingrid; Comer, Jeffrey

    2017-03-24

    Understanding the interaction of carbon nanomaterials with proteins is essential for determining the potential effects of these materials on health and in the design of biotechnology based on them. Here we leverage explicit-solvent molecular simulation and multidimensional free-energy calculations to investigate how adsorption to carbon nanomaterial surfaces affects the conformational equilibrium of alanine dipeptide, a widely used model of protein backbone structure. We find that the two most favorable structures of alanine dipeptide on graphene (or large carbon nanotubes) correspond to the two amide linkages lying in the same plane, flat against the surface, rather than the nonplanar α-helix-like and β-sheet-like conformations that predominate in aqueous solution. On graphenic surfaces, the latter conformations are metastable and most often correspond to amide-π stacking of the N-terminal amide. The calculations highlight the key role of amide-π interactions in determining the conformational equilibrium. Lesser but significant contributions from hydrogen bonding to the high density interfacial water layer or to the hydroxy groups of hydroxylated graphene also define the most favorable conformations. This work should yield insight on the influence of carbon nanotubes, graphene, and their functionalized derivatives on protein structure.

  17. Alanine substitutions of noncysteine residues in the cysteine-stabilized αβ motif

    PubMed Central

    Yang, Ying-Fang; Cheng, Kuo-Chang; Tsai, Ping-Hsing; Liu, Chung-Cheng; Lee, Tian-Ren; Ping-Chiang Lyu

    2009-01-01

    The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized αβ motif (CSαβ) consists of an α-helix and an antiparallel triple-stranded β-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering. PMID:19533758

  18. Modifications of the acyl-d-alanyl-d-alanine terminus affecting complex-formation with vancomycin

    PubMed Central

    Nieto, M.; Perkins, H. R.

    1971-01-01

    Vancomycin forms complexes with peptides terminating in d-alanyl-d-alanine that are analogous to the biosynthetic precursors of bacterial mucopeptides. The specificity of complex-formation has been studied by means of many synthetic peptides, prepared by both solid-phase and conventional methods. The following conclusions can be drawn: (a) three amide linkages are required to form a stable complex; (b) the terminal carboxyl group must be free; (c) the carboxyl terminal and subterminal residues must be either glycine or of the d-configuration; (d) the size of the side chain