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Sample records for alanine transaminase activity

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

    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.

  2. 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.

  3. 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

  4. 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.

  5. Long-term day-and-night rotating shift work poses a barrier to the normalization of alanine transaminase.

    PubMed

    Lin, Yu-Cheng; Hsieh, I-Chun; Chen, Pau-Chung

    2014-05-01

    To evaluate the impact of day-and-night rotating shift work (RSW) on liver health, we performed a retrospective analysis of the association between long-term RSW exposure and the normalization of plasma alanine transaminase (ALT) levels over a five-year period. The data from physical examinations, blood tests, abdominal sonographic examinations, personal histories, and occupational records were collected from a cohort of workers in a semiconductor manufacturing company. The sample population was divided into three subgroups for analysis, according to self-reported shift work status over the five-year interval: persistent daytime workers, workers exposed intermittently to RSW (i-RSW), and workers exposed persistently to RSW (p-RSW). Records were analyzed for 1196 male workers with an initial mean age of 32.5 years (SD 6.0 years), of whom 821 (68.7%) were identified as rotating shift workers, including 374 i-RSW (31.3%) and 447 p-RSW workers (37.4%). At the beginning of the follow-up, 275 were found to have elevated ALT (e-ALT): 25.1% daytime workers, 23.0% i-RSW workers, and 21.3% p-RSW workers (p = 0.098). Of those with e-ALT at the beginning, 101 workers showed normalized serum ALT levels at the end of five-year follow-up: 40 (10.7%) of 375 daytime workers, 32 (8.6%) of 374 i-RSW workers, and 29 (6.5%) of 447 p-RSW workers (p = 0.016). Compared with the workers having persistent e-ALT at the end of follow-up, the workers normalized serum ALT levels had significantly lesser exposures to RSW during follow-up. By performing multivariate logistic regression analyses, and comparing with the persistent daytime co-workers, after controlling for confounding variables (age, occupational factors, educational levels, lifestyle factors, metabolic syndrome, hepatovirus infection, and fatty liver), analysis indicated that the workers exposed to p-RSW were 46% less likely (OR, 0.54; 95% CI, 0.30-0.95; p = 0.03) to attain normal ALT levels within a five-year interval

  6. [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.

  7. Alanine transaminase (ALT) blood test

    MedlinePlus

    ... RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods . 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap 21. Pratt DS. Liver chemistry and function tests. In: Feldman M, Friedman LS, Brandt LJ, ...

  8. The Effect of Berberis Vulgaris Extract on Transaminase Activities in Non-Alcoholic Fatty Liver Disease

    PubMed Central

    Iloon Kashkooli, Raziye; Najafi, Seyed Saeed; Sharif, Farkhondeh; Hamedi, Azadeh; Hoseini Asl, Mohammad Kazem; Najafi Kalyani, Majid; Birjandi, Mehdi

    2015-01-01

    Background: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disorder in western countries and an important cause of liver cirrhosis, as well as liver failure. Up to now, 20‒40% of the individuals suffer from this disorder and its prevalence is estimated around 5‒30% in Asia. The NAFLD is one of the most prevalent causes for increases in liver enzymes and has a close relationship with obesity, dyslipidemia, hypertension, and type II diabetes. However, no definite treatment has been identified for it yet. Objectives: The present study aimed to investigate the effect of berberis vulgaris extract in inducing changes in liver enzymes levels. Patients and Methods: The present clinical trial was conducted on 80 patients, including 32 males (40%) and 48 females (60%), who were randomly assigned into two groups of case and control. All the patients had ultrasound evidence of lipid accumulation in the liver and increases in liver enzymes. The case group received two capsules (750 mg) containing berberis vulgaris extract every day for 3 months, while the control group was treated with placebo. The weight, liver transaminases levels and lipid profiles of the two groups were assessed before, during, and after the study. Results: In the case group, the mean serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) decreased from 49 to 27.48 and 48.22 to 29.8 u/L, respectively, which was statistically significant compared to the control group (P < 0.001, P < 0.001). In the control group, the mean of ALT and AST decreased from 50.4 to 46.8 and 45.7 to 44.9 u/L, respectively. The difference was not statistically significant. In addition, a significant decrease was observed in weight, triglycerides, and cholesterol, while no significant change was found in fasting blood sugar, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL). Conclusions: Considering the significant decrease in the liver enzymes

  9. D-alanine incorporation into macromolecules and effects of D-alanine deprivation on active transport in Bacillus subtilis.

    PubMed

    Clark, V L; Young, F E

    1978-03-01

    An auxotroph of Bacillus subtilis 168 unable to synthesize D-alanine loses the ability to support endogenously energized transport when deprived of D-alanine. Revertants of the mutant retain transport activity. The loss of transport is specific for substrates taken up by active transport; substrates taken up by group translocation are transported at normal rates. The loss of transport can be retarded by pretreatment of the cells with inhibitors of protein synthesis. Since the loss of transport could be due to an alteration in a D-alanine-containing polymer, we investigated the incorporation of D-[14C]alanine into macromolecules. The major D-alanine-containing polymers in B. subtilis are peptidoglycan and teichoic acid, with 4 to 6% of the D-[14C]alanine label found in trypsin-soluble material. Whereas the peptidoglycan and teichoic acid undergo turnover, the trypsin-soluble material does not. Treatment of the trypsin-soluble material with Pronase releases free D-alanine. Analysis of acid-hydrolyzed trypsin-soluble material indicated that approximately 75% of the radioactivity is present as D-alanine, with the remainder present as L-alanine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified D-[14C]alanine-labeled membranes indicated the presence of two peaks of radioactivity (molecular weights, 230,000 and 80,000) that could be digested by trypsin. The results suggest that D-alanine may be covalently bound to cellular proteins.

  10. 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

  11. Eating a healthy lunch improves serum alanine aminotransferase activity

    PubMed Central

    2013-01-01

    Background Nutritional guidance and diet control play important roles in the treatment of obesity and non-alcoholic fatty liver. However, in Japan, nutritional guidance is difficult to provide in practice. Therefore, we evaluated the effects of providing the ‘once-a-day’ intervention of a healthy lunch on various metabolic parameters. Methods For a 1-month preparatory period, 10 subjects generally consumed the lunches that were provided by the worksite cafeteria. This was followed by a 1-week washout period, after which, the subjects consumed healthy, low-calorie, well-balanced lunches for a 1-month test period. After the preparatory and test periods, blood samples were obtained from all subjects. The serum levels of indices relevant to metabolic syndrome and fatty liver were measured. Results Serum alanine aminotransferase activity significantly decreased by 20.3% after the healthy intervention. However, the indices of metabolic syndrome did not significantly change. Analysis of the relationship between serum alanine aminotransferase activity and nutrient content indicated that the improvement of serum alanine aminotransferase status was due to the higher vegetable content and lower animal-source protein of the meals provided. Conclusions In summary, the ‘once-a-day’ intervention of providing a healthy lunch improved serum alanine aminotransferase status. A diet high in vegetables and low in animal-based protein is important in maintaining a healthy condition. PMID:24034595

  12. 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.

  13. 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. PMID:19165747

  14. 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.

  15. Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17.

    PubMed

    Shin, J-S; Yun, H; Jang, J-W; Park, I; Kim, B-G

    2003-06-01

    A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date. PMID:12687298

  16. 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

  17. Active site model of (R)-selective ω-transaminase and its application to the production of D-amino acids.

    PubMed

    Park, Eul-Soo; Dong, Joo-Young; Shin, Jong-Shik

    2014-01-01

    ω-Transaminase (ω-TA) is one of the important biocatalytic toolkits owing to its unique enzyme property which enables the transfer of an amino group between primary amines and carbonyl compounds. In addition to preparation of chiral amines, ω-TA reactions have been exploited for the asymmetric synthesis of L-amino acids using (S)-selective ω-TAs. However, despite the availability of (R)-selective ω-TAs, catalytic utility of the ω-TAs has not been explored for the production of D-amino acids. Here, we investigated the substrate specificity of (R)-selective ω-TAs from Aspergillus terreus and Aspergillus fumigatus and demonstrated the asymmetric synthesis of D-amino acids from α-keto acids. Substrate specificity toward D-amino acids and α-keto acids revealed that the two (R)-selective ω-TAs possess strict steric constraints in the small binding pocket that precludes the entry of a substituent larger than an ethyl group, which is reminiscent of (S)-selective ω-TAs. Molecular models of the active site bound to an external aldimine were constructed and used to explain the observed substrate specificity and stereoselectivity. α-Methylbenzylamine (α-MBA) showed the highest amino donor reactivity among five primary amines (benzylamine, α-MBA, α-ethylbenzylamine, 1-aminoindan, and isopropylamine), leading us to employ α-MBA as an amino donor for the amination of 5 reactive α-keto acids (pyruvate, 2-oxobutyrate, fluoropyruvate, hydroxypyruvate, and 2-oxopentanoate) among 17 ones tested. Unlike the previously characterized (S)-selective ω-TAs, the enzyme activity of the (R)-selective ω-TAs was not inhibited by acetophenone (i.e., a deamination product of α-MBA). Using racemic α-MBA as an amino donor, five D-amino acids (D-alanine, D-homoalanine, D-fluoroalanine, D-serine, and D-norvaline) were synthesized with excellent product enantiopurity (enantiomeric excess >99.7 %).

  18. Studies on the mechanism of acetaldehyde-mediated inhibition of rat liver transaminases.

    PubMed

    Solomon, L R

    1987-09-30

    Incubation of mitochondria-depleted rat liver homogenates with 5 mmol/l acetaldehyde at 37 degrees C for 1 h inhibited both aspartate and alanine aminotransferases by 30%. Inhibition was prevented by decreasing temperature to 4 degrees C or by preincubating homogenates with cyanate but was unaffected by cyanamide and methylpyrazole which block acetaldehyde oxidation and reduction respectively. Cyanate-sensitive acetaldehyde-mediated inhibition of purified porcine heart transaminases was also demonstrated in the presence of rat liver homogenate but not in Tris/sucrose medium. Moreover, porcine transaminases were inhibited by trichloroacetic acid extracts of rat liver homogenates previously incubated with acetaldehyde but not by extracts of homogenates incubated with both acetaldehyde and cyanate. These findings suggest that acetaldehyde-mediated transaminase inhibition requires further non-oxidative metabolism of acetaldehyde. Since transaminase activities were not restored by addition of pyridoxal 5'-phosphate to the assay systems, acetaldehyde-induced transaminase inhibition does not appear to be mediated by displacement or depletion of this B6 coenzyme. PMID:3677417

  19. Regulation of Transaminase C Synthesis in Escherichia coli: Conditional Leucine Auxotrophy

    PubMed Central

    McGilvray, Derek; Umbarger, H. E.

    1974-01-01

    The regulation of synthesis of the valine-alanine-α-aminobutyrate transaminase (transaminase C) was studied in Escherichia coli mutants lacking the branched-chain amino acid transaminase (transaminase B). An investigation was made of two strains, CU2 and CU2002, each carrying the same transaminase B lesion but exhibiting different growth responses on a medium supplemented with branched-chain amino acids. Both had the absolute isoleucine requirement characteristic of ilvE auxotrophs, but growth of strain CU2 was stimulated by valine, whereas that of strain CU2002 was markedly inhibited by valine. Strain CU2002 behaved like a conditional leucine auxotroph in that the inhibition by valine was reversed by leucine. Results of enzymatic studies showed that synthesis of transaminase C was repressed by valine in strain CU2002 but not in strain CU2. Inhibition by valine in strain CU2002 appears to be the combined effect of repression on transaminase C synthesis and valine-dependent feedback inhibition of α-acetohydroxy acid synthase activity, causing α-ketoisovalerate (and hence leucine) limitation. The ilvE markers of strains CU2 and CU2002 were each transferred by transduction to a wild-type genetical background. All ilvE recombinants from both crosses resembled strain CU2002 and were inhibited by valine in the presence of isoleucine. Thus, strain CU2 carries an additional lesion that allows it to grow on a medium containing isoleucine plus valine. It is concluded that conditional leucine auxotrophy is characteristic of mutants carrying an ilvE lesion alone. PMID:4616947

  20. Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications.

    PubMed Central

    Bertoldi, Mariarita; Cellini, Barbara; Paiardini, Alessandro; Di Salvo, Martino; Borri Voltattorni, Carla

    2003-01-01

    To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with L- and D-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429 nm and of a band absorbing at 498 nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, k (cat) and K (m), for L-alanine are 1.05+/-0.03 s(-1) and 10+/-1 mM respectively, whereas those for D-alanine are 1.4+/-0.1 s(-1) and 10+/-1 mM. During the reaction of cystalysin with L- or D-alanine, a time-dependent loss of beta-elimination activity occurs concomitantly with the conversion of the pyridoxal 5'-phosphate (PLP) coenzyme into pyridoxamine 5'-phosphate (PMP). The catalytic efficiency of the half-transamination of L-alanine is found to be 5.3x10(-5) mM(-1) x s(-1), 5-fold higher when compared with that of D-alanine. The partition ratio between racemization and half-transamination reactions is 2.3x10(3) for L-alanine and 1.4x10(4) for D-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with p K values of 6.5-6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of beta-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4' of both (4' S)- and (4' R)-[4'-(3)H]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites of L- and D-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed. PMID:12519070

  1. 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.

  2. The effect of Morus alba leaves extract and powder on resistin levels and liver transaminase enzymes activities in diabetes.

    PubMed

    Salemi, Z; Barzin Tond, S; Fallah, S; Shojaii, A; Seifi, M

    2016-01-01

    The current study was designed to investigate the changes of the resistin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels of diabetic rats after treatment with Morus alba leaves flavonoid extract (MLE) and Morus alba leaves powder (MLP). Thirty male wistar rats in five groups including control and diabetic groups were included. Diabetic groups consisted of diabetic control, sham and treated group with MLE and MLP. Type 2 diabetes was induced in rats by administration of streptozotocin (STZ) and - nicotinamide. The serum concentrations of resistin and insulin in the study groups were identified by ELISA. ALT and AST activities were assayed by spectrophotometer. For the first time, it was shown that the uptake of MLE and MLP by diabetic rats could significantly decrease the serum fasting blood sugar (FBS), resistin levels and enzymes activity of ALT and AST and increases the concentration of serum insulin significantly (P<0.05). in comparison with the sham group and diabetic control. The results showed that there was no significant difference between the anti-diabetic and inflammatory properties of MLE and MLP. In this study, the possible protective effect of MLE and MLP administration was evaluated against destructive effect of STZ on liver and pancreas function in diabetic rats. The results showed that these effects may play an important role in the regulating of adipokines secretion such as resistin and insulin secretion which are involved in the control of diabetes and obesity. MLE and MLP treatment could be useful agents in combination with other therapies in diabetes improvement. PMID:27262814

  3. [Leucine and alanine aminopeptidase activity in the organs of cattle, sheep and swine].

    PubMed

    Goranov, Kh

    1982-01-01

    Studied was the activity of leucine-aminopeptidase and alanine-aminopeptidase in fresh tissue homogenates of liver, spleen, kidney, heart, pancreas, femoral muscle, stomach (rumen), small intestine, and lung taken from 8 cattle, sheep, and pigs. Both enzymes showed ubiquity. Leucine-aminopeptidase exhibited highest activity in the spleen of pigs and the kidney of sheep and cattle. The kidneys of all investigated animal species showed 10 to 15 times higher alanine-aminopeptidase activity than the remaining organs. This pointed to the relative ubiquity of the enzyme with special reference to kidneys.

  4. Evidence for the generation of transaminase inhibitor(s) during ethanol metabolism by rat liver homogenates: a potential mechanism for alcohol toxicity.

    PubMed

    Solomon, L R

    1987-08-01

    Since ethanol consumption decreases hepatic aminotransferase activities in vivo, mechanisms of ethanol-mediated transaminase inhibition were explored in vitro using mitochondria-depleted rat liver homogenates. When homogenates were incubated at 37 degrees with 50 mM ethanol for 1 hr, alanine aminotransferase decreased by 20%, while aspartate aminotransferase was unchanged. After 2 hr, aspartate aminotransferase decreased by 20% and by 3 hr, alanine and aspartate aminotransferases were decreased by 31 and 23%, respectively. Levels of acetaldehyde generated during ethanol oxidation were 525 +/- 47 microM at 1 hr, 855 +/- 14 microM at 2 hr, and 1293 +/- 140 microM at 3 hr. Although inhibition of alcohol oxidation with methylpyrazole or cyanide markedly decreased ethanol-mediated transaminase inhibition, neither incubation with acetate nor generation of reducing equivalents by oxidation of lactate, malate, xylitol, or sorbitol altered the activity of either enzyme. However, semicarbazide, an aldehyde scavenger, prevented inhibition of both aminotransferases by ethanol. Moreover, incubation with 5 mM acetaldehyde for 1 hr inhibited alanine and aspartate aminotransferases by 36 and 26%, respectively. Cyanamide, an aldehyde dehydrogenase inhibitor, had little effect on ethanol-mediated transaminase inhibition. Thus, metabolism of ethanol by rat liver homogenates produces transaminase inhibition similar to that described in vivo and this effect requires acetaldehyde generation but not acetaldehyde oxidation. Since addition of pyridoxal 5'-phosphate to assay mixes did not reverse ethanol effects, aminotransferase inhibition does not result from displacement of vitamin B6 coenzymes.

  5. Evidence for the generation of transaminase inhibitor(s) during ethanol metabolism by rat liver homogenates: a potential mechanism for alcohol toxicity.

    PubMed

    Solomon, L R

    1987-08-01

    Since ethanol consumption decreases hepatic aminotransferase activities in vivo, mechanisms of ethanol-mediated transaminase inhibition were explored in vitro using mitochondria-depleted rat liver homogenates. When homogenates were incubated at 37 degrees with 50 mM ethanol for 1 hr, alanine aminotransferase decreased by 20%, while aspartate aminotransferase was unchanged. After 2 hr, aspartate aminotransferase decreased by 20% and by 3 hr, alanine and aspartate aminotransferases were decreased by 31 and 23%, respectively. Levels of acetaldehyde generated during ethanol oxidation were 525 +/- 47 microM at 1 hr, 855 +/- 14 microM at 2 hr, and 1293 +/- 140 microM at 3 hr. Although inhibition of alcohol oxidation with methylpyrazole or cyanide markedly decreased ethanol-mediated transaminase inhibition, neither incubation with acetate nor generation of reducing equivalents by oxidation of lactate, malate, xylitol, or sorbitol altered the activity of either enzyme. However, semicarbazide, an aldehyde scavenger, prevented inhibition of both aminotransferases by ethanol. Moreover, incubation with 5 mM acetaldehyde for 1 hr inhibited alanine and aspartate aminotransferases by 36 and 26%, respectively. Cyanamide, an aldehyde dehydrogenase inhibitor, had little effect on ethanol-mediated transaminase inhibition. Thus, metabolism of ethanol by rat liver homogenates produces transaminase inhibition similar to that described in vivo and this effect requires acetaldehyde generation but not acetaldehyde oxidation. Since addition of pyridoxal 5'-phosphate to assay mixes did not reverse ethanol effects, aminotransferase inhibition does not result from displacement of vitamin B6 coenzymes. PMID:3663401

  6. Relation of circulating liver transaminase concentrations to risk of new-onset atrial fibrillation.

    PubMed

    Sinner, Moritz F; Wang, Na; Fox, Caroline S; Fontes, João D; Rienstra, Michiel; Magnani, Jared W; Vasan, Ramachandran S; Calderwood, Audrey H; Pencina, Michael; Sullivan, Lisa M; Ellinor, Patrick T; Benjamin, Emelia J

    2013-01-15

    Heart failure, a strong risk factor for atrial fibrillation (AF), is often accompanied by elevated liver transaminases. The aim of this study was to test the hypothesis that elevated transaminases are associated with the risk for incident AF in the community. A total of 3,744 participants (mean age 65 ± 10 years, 56.8% women) from the Framingham Heart Study Original and Offspring cohorts, free of clinical heart failure, were studied. Cox proportional-hazards models adjusted for standard AF risk factors (age, gender, body mass index, systolic blood pressure, electrocardiographic PR interval, antihypertensive treatment, smoking, diabetes, valvular heart disease, and alcohol consumption) were examined to investigate associations between baseline serum transaminase levels (alanine transaminase and aspartate transaminase) and the incidence of AF over up to 10 years (29,099 person-years) of follow-up. During follow-up, 383 subjects developed AF. The 2 transaminases were significantly associated with greater risk for incident AF (hazard ratio expressed per SD of natural logarithmically transformed biomarker: alanine transaminase hazard ratio 1.19, 95% confidence interval 1.07 to 1.32, p = 0.002; aspartate transaminase hazard ratio 1.12, 95% confidence interval 1.01 to 1.24, p = 0.03). The associations between transaminases and AF remained consistent after the exclusion of participants with moderate to severe alcohol consumption. However, when added to known risk factors for AF, alanine transaminase and aspartate transaminase only subtly improved the prediction of AF. In conclusion, elevated transaminase concentrations are associated with increased AF incidence. The mechanisms by which higher mean transaminase concentrations are associated with incident AF remain to be determined. PMID:23127690

  7. 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.

  8. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    PubMed

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

    2014-11-01

    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

  9. 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

  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

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

    2014-11-01

    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.

  11. Site-directed mutagenesis provides insight into racemization and transamination of alanine catalyzed by Treponema denticola cystalysin.

    PubMed

    Cellini, Barbara; Bertoldi, Mariarita; Paiardini, Alessandro; D'Aguanno, Simona; Voltattorni, Carla Borri

    2004-08-27

    In addition to alpha, beta-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Calpha/C4' of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys --> Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the k(cat)/K(m) is reduced 3.5-fold for alpha, beta-elimination, whereas it is reduced 300-400-fold for racemization. Y123F has approximately 18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face. PMID:15210695

  12. Different hydroxyl radical scavenging activity of water-soluble beta-alanine C60 adducts.

    PubMed

    Sun, Tao; Jia, Zhishen; Xu, Zhude

    2004-04-01

    Three C(60) derivatives [C(60) (NHCH(2)CH(2)COONa)(n)(H)(n)], n=1, 5, 9] (A, B, C) with different additional number of beta-alanine were synthesized by the control of relative amount of C(60) and beta-alanine added. Hydroxyl radical scavenging activity of the adducts was evaluated in a copper-catalyzed Haber-Weiss reaction by chemiluminescence technology. The 50% inhibition concentrations (IC(50)'s) of A, B, and C were 147.2 micromol/L, 76.3 micromol/L, and 96.2 micromol/L, respectively. The difference should be closely related to the numbers of residual C=C bonds in C(60), steric effect and electron-withstanding effect of amino group especially.

  13. 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.

  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. PMID:26168032

  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. [Asymmetric synthesis of aromatic L-amino acids catalyzed by transaminase].

    PubMed

    Xia, Wenna; Sun, Yu; Min, Cong; Han, Wei; Wu, Sheng

    2012-11-01

    Aromatic L-Amino acids are important chiral building blocks for the synthesis of many drugs, pesticides, fine chemicals and food additives. Due to the high activity and steroselectivity, enzymatic synthesis of chiral building blocks has become the main research direction in asymmetric synthesis field. Guided by the phylogenetic analysis of transaminases from different sources, two representative aromatic transaminases TyrB and Aro8 in type I subfamily, from the prokaryote Escherichia coli and eukaryote Saccharomyces cerevisia, respectively, were applied for the comparative study of asymmetric transamination reaction process and catalytic efficiency of reversely converting keto acids to the corresponding aromatic L-amino acid. Both TyrB and Aro8 could efficiently synthesize the natural aromatic amino acids phenylalanine and tyrosine as well as non-natural amino acid phenylglycine. The chiral HPLC analysis showed the produced amino acids were L-configuration and the e.e value was 100%. L-alanine was the optimal amino donor, and the transaminase TyrB and Aro8 could not use D-amino acids as amino donor. The optimal molar ratio of amino donor (L-alanine) and amino acceptor (aromatic alpha-keto acids) was 4:1. Both of the substituted group on the aromatic ring and the length of fatty acid carbon chain part in the molecular structure of aromatic substrate alpha-keto acid have the significant impact on the enzyme-catalyzed transamination efficiency. In the experiments of preparative-scale transamination synthesis of L-phenylglycine, L-phenylalanine and L-tyrosine, the specific production rate catalyzed by TryB were 0.28 g/(g x h), 0.31 g/(g x h) and 0.60 g/(g x h) and the specific production rate catalyzed by Aro8 were 0.61 g/(g x h), 0.48 g/(g x h) and 0.59 g/(g x h). The results obtained here were useful for applying the transaminases to asymmetric synthesis of L-amino acids by reversing the reaction balance in industry.

  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. PMID:27159046

  19. [Activity of alanine aminopeptidase in blood and in urine of smoking and non-smoking smelters].

    PubMed

    Bizoń, Anna; Stasiak, Karolina; Milnerowicz, Halina

    2010-01-01

    The human body is constantly exposed to xenobiotics. This will include exogenous substances from environmental pollution such as heavy metals and lifestyle such as smoking, which may lead to impaired functioning of many organs. The liver and kidney are the critical organs in the case of a long-term occupational or environmental exposure to heavy metals and tobacco smoke. In diagnostics of liver and kidney damage useful are the methods which determine the activity of enzymes such as alanine aminopeptidase (AAP). AAP is a marker for early detection of acute kidney damage, and presence of AAP derive mainly from proximal tubular brush-border. Activity of AAP in urine allows to assess the damage resulting from the nephrotoxic exposure to heavy metals. In the serum AAP is mainly from hepatic. Activity of AAP may be useful to identify liver cancer. The investigation was shown, that AAP activity in the blood is used to detect hepatic cholestasis and congestive jaundice. The aim of present study was to assess the influence of occupational exposure of copper-foundry workers to heavy metals (arsenic, cadmium, lead) on activity of alanine aminopeptidase in blood and urine. The investigations were performed in blood and urine of 166 subjects: 101 male copper smelters and 65 non-exposed male subjects. The study protocol was approved by Local Bioethics Committee of Wroclaw Medical University (KB No: 469/2008). The data on smoking which had been obtained from a direct personal interview were verified by determination of serum cotinine concentrations. Biological material collected from the control group and smelters was divided into subgroups of nonsmokers and smokers. The concentrations of lead and cadmium were determined in whole blood, whilst the level of arsenic and cadmium were determined in urine using FAAS method (Flame Atomic Absorption Spectrometry) in the acetylate flame on the SOLAAR M6. The activity of AA was determined in blood and in urine. The results showed a 9-fold

  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. Diurnal Variation in Serum Alanine Aminotransferase Activity in the United States Population

    PubMed Central

    Everhart, James E.

    2012-01-01

    Goals & Background Serum alanine aminotransferase (ALT) activity has been reported to be greater in the afternoon than the early morning, but data are scarce. We examined diurnal variation of ALT in a national population-based sample. Study Participants in the 1999–2008 U.S. National Health and Nutrition Examination Survey were randomly assigned to morning (AM) (n=4,474 adolescents, 11,235 adults) or afternoon/evening (PM) (n=4,887 adolescents, 11,735 adults) examinations. We examined ALT distributions graphically and compared both geometric mean ALT and the prevalence of elevated ALT, defined as >31 IU/L for adolescent boys, >24 IU/L for adolescent girls, >43 IU/L for adult men and >30 IU/L for adult women, between AM and PM examination groups. Results The examination groups were similar with the exception in the AM group of a longer fasting time and slightly higher prevalence of diabetes among adolescents and viral hepatitis B among adult women. ALT distributions were similar between examination sessions among the four groups. Among adolescents and men, neither mean ALT nor prevalence of abnormal ALT differed by examination group. Among women, mean ALT was statistically significantly, but minimally higher in the PM (19.6 IU/L) than the AM group (19.1 IU/L; p=0.009). Among one subgroup, women with chronic viral hepatitis, there was a higher prevalence of abnormal ALT in the PM (p=0.018 in unadjusted analysis). Adjusting for liver injury risk factors had little effect on the difference in mean ALT. Conclusions In general, clinically significant diurnal variation in ALT activity was not found in the U.S. population. PMID:23164687

  2. Upper Limits of Normal for Alanine Aminotransferase Activity in the United States Population

    PubMed Central

    Ruhl, Constance E.; Everhart, James E.

    2011-01-01

    Background & Rationale Alanine aminotransferase (ALT) is an important test for liver disease, yet there is no generally accepted upper limit of normal (ULN) in the United States. Furthermore, the ability of ALT to differentiate persons with and without liver disease is uncertain. We examined cut-offs for ALT for their ability to discriminate between persons with positive hepatitis C virus (HCV) RNA and those at low risk for liver injury in the U.S. population. Methods Among adult participants in the 1999–2008 U.S. National Health and Nutrition Examination Survey, 259 were positive for serum HCV RNA and 3,747 were at low risk for liver injury (negative HCV RNA and hepatitis B surface antigen, low alcohol consumption, no evidence of diabetes, normal body mass index and waist circumference). Serum ALT activity was measured centrally. Results Maximum correct classification was achieved at ALT=29 IU/L for men (88% sensitivity, 83% specificity) and 22 (89% sensitivity, 82% specificity) for women. The cut-off for 95% sensitivity was an ALT=24 IU/L (70% specificity) for men and 18 (63% specificity) for women. The cut-off for 95% specificity was an ALT=44 IU/L (64% sensitivity) for men and 32 (59% sensitivity) for women. The area under the curve was 0.929 for men and 0.915 for women. If the cut-offs with the best correct classification were applied to the entire population, 36.4% of men and 28.3% of women would have had abnormal ALT. Conclusion ALT discriminates persons infected with HCV from those at low risk of liver disease, but would be considered elevated in a large proportion of the U.S. population. PMID:21987480

  3. Reduced Transaminase B (IlvE) Activity Caused by the Lack of yjgF Is Dependent on the Status of Threonine Deaminase (IlvA) in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Schmitz, George; Downs, Diana M.

    2004-01-01

    The YjgF/YER057c/UK114 family is a highly conserved class of proteins that is represented in the three domains of life. Thus far, a biochemical function demonstrated for these proteins in vivo or in vitro has yet to be defined. In several organisms, strains lacking a YjgF homolog have a defect in branched-chain amino acid biosynthesis. This study probes the connection between yjgF and isoleucine biosynthesis in Salmonella enterica. In strains lacking yjgF the specific activity of transaminase B, catalyzing the last step in the synthesis of isoleucine, was reduced. In the absence of yjgF, transaminase B activity could be restored by inhibiting threonine deaminase, the first enzymatic step in isoleucine biosynthesis. Strains lacking yjgF showed an increased sensitivity to sulfometruron methyl, a potent inhibitor of acetolactate synthase. Based on work described here and structural reports in the literature, we suggest a working model in which YjgF has a role in protecting the cell from toxic effects of imbalanced ketoacid pools. PMID:14729707

  4. 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.

  5. 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. PMID:25090957

  6. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  7. Gamma radiation affects active electrolyte transport by rabbit ileum. II. Correlation of alanine and theophylline response with morphology

    SciTech Connect

    Gunter-Smith, P.J.

    1989-03-01

    The response of ileal segments isolated from rabbits to an actively transported amino acid and a secretagogue was evaluated following exposure to 10 Gy whole-body gamma irradiation. The ability of ileal segments to respond to the actively transported amino acid, alanine, was not significantly diminished until 96 h postexposure. Decreased responsiveness to the secretagogue, theophylline, occurred earlier at 72 h. These effects did not appear to be accounted for by decreased food intake of irradiated animals alone. Examination of intestinal morphological changes with respect to these changes in electrolyte transport revealed that decreased amino acid transport coincides with loss of intestinal villi. Although a morphological correlate of decreased secretory response was not as striking as that for absorption, the theophylline response appeared to decline concomitant with the appearance of increased mitotic activity in the intestinal crypts. The results of this study indicate that, following a dose of 10 Gy, the inability of these tissues to respond to amino acids is due to a loss of mature villus absorptive cells subsequent to denudation of the intestinal mucosa. There appeared to be little impairment of cell membrane transport processes for alanine. In contrast, the decreased secretory response could not be correlated with the disappearance of any one cell type and perhaps results from increased proliferation in the crypts at the expense of differentiation.

  8. Gamma-radiation affects active electrolyte transport by rabbit ileum. 2. Correlation of alanine and theophylline response with morphology

    SciTech Connect

    Gunter-Smith, P.J.

    1989-01-01

    The response of ileal segments isolated from rabbits to an actively transported amino acid and a secretagogue was evaluated following exposure to 10-Gy whole-body gamma irradiation. The ability of ileal segments to respond to the actively transported amino acid, alanine, was not significantly diminished until 96 h postexposure. Decreased responsiveness to the secretagogue, theophylline, occurred earlier at 72 h. These effects did not appear to be accounted for by decreased food intake of irradiated animals alone. Examination of intestinal morphological changes with respect to these changes in electrolyte transport revealed that decreased amino acid transport coincides with loss of intestinal villi. Although a morphological correlate of decreased secretory response was not as striking as that for absorption, the theophylline response appeared to decline concomitant with the appearance of increased mitotic activity in the intestinal crypts. The result of this study indicate that, following a dose of 10 Gy, the inability of these tissues to respond to amino acids is due to a loss of mature villus absorptive cells subsequent to denudation of the intestinal mucosa. There appeared to be little impairment of cell membrane transport processes for alanine. In contrast, the decreased secretory response could not be correlated with the disappearance of any one cell type and perhaps results from increased proliferation in the crypts at the expense of differentiation.

  9. 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.

  10. Reduced Photoinhibition under Low Irradiance Enhanced Kacip Fatimah (Labisia pumila Benth) Secondary Metabolites, Phenyl Alanine Lyase and Antioxidant Activity

    PubMed Central

    Ibrahim, Mohd Hafiz; Jaafar, Hawa Z.E.

    2012-01-01

    A randomized complete block design experiment was designed to characterize the relationship between production of total flavonoids and phenolics, anthocyanin, photosynthesis, maximum efficiency of photosystem II (Fv/Fm), electron transfer rate (Fm/Fo), phenyl alanine lyase activity (PAL) and antioxidant (DPPH) in Labisia pumila var. alata, under four levels of irradiance (225, 500, 625 and 900 μmol/m2/s) for 16 weeks. As irradiance levels increased from 225 to 900 μmol/m2/s, the production of plant secondary metabolites (total flavonoids, phenolics and antocyanin) was found to decrease steadily. Production of total flavonoids and phenolics reached their peaks under 225 followed by 500, 625 and 900 μmol/m2/s irradiances. Significant positive correlation of production of total phenolics, flavonoids and antocyanin content with Fv/Fm, Fm/Fo and photosynthesis indicated up-regulation of carbon-based secondary metabolites (CBSM) under reduced photoinhibition on the under low light levels condition. At the lowest irradiance levels, Labisia pumila extracts also exhibited a significantly higher antioxidant activity (DPPH) than under high irradiance. The improved antioxidative activity under low light levels might be due to high availability of total flavonoids, phenolics and anthocyanin content in the plant extract. It was also found that an increase in the production of CBSM was due to high PAL activity under low light, probably signifying more availability of phenylalanine (Phe) under this condition. PMID:22754297

  11. Elevation of retinol levels and suppression of alanine aminotransferase activity in the liver of taurine-deficient kittens.

    PubMed

    Lehmann, A; Knutsson, L; Bosaeus, I

    1990-10-01

    In taurine-deficient cats, the secretion of bile acids is impaired, and this impairment may reduce intestinal uptake of lipophilic vitamins. It was therefore hypothesized that retinol deficiency is involved in the generation of retinal lesions in taurine-deficient kittens. To this end, the concentration of retinol in plasma and liver was determined in taurine-deficient kittens. Further, the effects of taurine deficiency on amino acid concentrations of heart, liver and kidney were investigated. To see whether taurine deficiency adversely affects the liver, hepatic enzymes were measured in plasma and liver of kittens suffering from taurine deficiency. In addition, liver morphology, growth and food intake were studied. Taurine was the only amino acid whose concentration was consistently decreased in plasma of the experimental group. Unexpectedly, retinol level was increased in plasma and liver from taurine-depleted kittens. Several alterations were noted in amino acid concentrations in liver and kidney, but not in heart. Plasma alanine aminotransferase activity was diminished, probably reflecting decreased activity in the liver. Perivenular steatosis was found in both groups. Controls grew linearly, in contrast to deficient animals, which nevertheless consumed more food. The results demonstrate that retinol deficiency is not involved in taurine-deficiency retinopathy. Moreover, taurine is required for linear growth of juvenile cats and for the maintenance of hepatic and renal pools of certain amino acids. PMID:2213246

  12. Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase.

    PubMed

    Tang, Zhiwen; Liu, Pei; Ma, Changbei; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Lv, Xiaoyuan

    2011-04-01

    We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD(+)) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD(+) related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD(+), resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD(+) with high selectivity against other NAD(+) analogs. This novel assay can also provide a convenient and robust way to analyze NAD(+) in biological samples such as cell lysate. As NAD(+) plays an essential role in many biochemical processes, this method can be used to investigate NAD(+) related life processes. For instance, the effect of calorie restriction on the intracellular NAD(+) level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD(+) assay holds wide applicability as an analytical tool in biochemical and biomedical research.

  13. 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

  14. Comparison of N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase activities for evaluation of microangiopathy in diabetes mellitus.

    PubMed

    Shimojo, N; Kitahashi, S; Naka, K; Fujii, A; Okuda, K; Tanaka, S; Fujii, S

    1987-03-01

    The activities of urinary N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) were measured in 207 diabetic patients and 57 healthy controls, and the relationship of these enzymes to different stages of diabetic microangiopathy was studied. Diabetics with clinical proteinuria had higher urinary NAG and AAP (17.7 +/- 1.9 and 42.8 +/- 4.9 U/g creatinine, mean +/- SE, respectively) than healthy controls (1.8 +/- 0.1 and 10.0 +/- 0.4) or diabetics without proteinuria. Among diabetics without proteinuria, NAG excretion in those with retinopathy was slightly higher than in those without (6.4 +/- 0.5 v 5.4 +/- 0.4), and AAP in those with retinopathy was significantly higher than in those without (23.0 +/- 1.5 v 17.4 +/- 0.8, P less than 0.01). Urinary albumin measured by radioimmunoassay and lysozyme in diabetics with retinopathy but without proteinuria was higher than those without retinopathy (P less than 0.001 and P less than 0.01). The increase in albumin was the greatest in diabetics with long duration of the disease (greater than or equal to 8 years); however, NAG and AAP increased more significantly in those with high hemoglobin A1c than in patients with long duration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2881186

  15. 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).

  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. β-Alanine supplementation.

    PubMed

    Hoffman, Jay R; Emerson, Nadia S; Stout, Jeffrey R

    2012-01-01

    β-Alanine is rapidly developing as one of the most popular sport supplements used by strength/power athletes worldwide. The popularity of β-alanine stems from its unique ability to enhance intramuscular buffering capacity and thereby attenuating fatigue. This review will provide an overview of the physiology that underlies the mechanisms of action behind β-alanine, examine dosing schemes, and examine the studies that have been conducted on the efficacy of this supplement. In addition, the effect that β-alanine has on body mass changes or whether it can stimulate changes in aerobic capacity also will be discussed. The review also will begin to explore the potential health benefits that β-alanine may have on older adult populations. Discussion will examine the potential adverse effects associated with this supplement as well as the added benefits of combining β-alanine with creatine.

  18. The snakehead Channa asiatica accumulates alanine during aerial exposure, but is incapable of sustaining locomotory activities on land through partial amino acid catabolism.

    PubMed

    Chew, Shit F; Wong, Mei Y; Tam, Wai L; Ip, Yuen K

    2003-02-01

    The freshwater snakehead Channa asiatica is an obligatory air-breather that resides in slow-flowing streams and in crevices near riverbanks in Southern China. In its natural habitat, it may encounter bouts of aerial exposure during the dry seasons. In the laboratory, the ammonia excretion rate of C. asiatica exposed to terrestrial conditions in a 12 h:12 h dark:light regime was one quarter that of the submerged control. Consequently, the ammonia contents in the muscle, liver and plasma increased significantly, and C. asiatica was able to tolerate quite high levels of ammonia in its tissues. Urea was not the major product of ammonia detoxification in C. asiatica, which apparently did not possess a functioning ornithine urea cycle. Rather, alanine increased fourfold to 12.6 micromol g(-1) in the muscle after 48 h of aerial exposure. This is the highest level known in adult teleosts exposed to air or an ammonia-loading situation. The accumulated alanine could account for 70% of the deficit in ammonia excretion during this period, indicating that partial amino acid catabolism had occurred. This would allow the utilization of certain amino acids as energy sources and, at the same time, maintain the new steady state levels of ammonia in various tissues, preventing them from rising further. There was a reduction in the aminating activity of glutamate dehydrogenase from the muscle and liver of specimens exposed to terrestrial conditions. Such a phenomenon has not been reported before and could, presumably, facilitate the entry of alpha-ketoglutarate into the Krebs cycle instead of its amination to glutamate, as has been suggested elsewhere. However, in contrast to mudskippers, C. asiatica was apparently unable to reduce the rates of proteolysis and amino acid catabolism, because the reduction in nitrogenous excretion during 48 h of aerial exposure was completely balanced by nitrogenous accumulation in the body. Alanine accumulation also occurred in specimens exposed to

  19. A Highly Active and Negatively Charged Streptococcus pyogenes Lysin with a Rare d-Alanyl-l-Alanine Endopeptidase Activity Protects Mice against Streptococcal Bacteremia

    PubMed Central

    Lood, Rolf; Raz, Assaf; Molina, Henrik; Euler, Chad W.

    2014-01-01

    Bacteriophage endolysins have shown great efficacy in killing Gram-positive bacteria. PlyC, a group C streptococcal phage lysin, represents the most efficient lysin characterized to date, with a remarkably high specificity against different streptococcal species, including the important pathogen Streptococcus pyogenes. However, PlyC is a unique lysin, in terms of both its high activity and structure (two distinct subunits). We sought to discover and characterize a phage lysin active against S. pyogenes with an endolysin architecture distinct from that of PlyC to determine if it relies on the same mechanism of action as PlyC. In this study, we identified and characterized an endolysin, termed PlyPy (phage lysin from S. pyogenes), from a prophage infecting S. pyogenes. By in silico analysis, PlyPy was found to have a molecular mass of 27.8 kDa and a pI of 4.16. It was active against a majority of group A streptococci and displayed high levels of activity as well as binding specificity against group B and C streptococci, while it was less efficient against other streptococcal species. PlyPy showed the highest activity at neutral pH in the presence of calcium and NaCl. Surprisingly, its activity was not affected by the presence of the group A-specific carbohydrate, while the activity of PlyC was partly inhibited. Additionally, PlyPy was active in vivo and could rescue mice from systemic bacteremia. Finally, we developed a novel method to determine the peptidoglycan bond cleaved by lysins and concluded that PlyPy exhibits a rare d-alanyl-l-alanine endopeptidase activity. PlyPy thus represents the first lysin characterized from Streptococcus pyogenes and has a mechanism of action distinct from that of PlyC. PMID:24637688

  20. 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.

  1. Methylmalonic semialdehyde dehydrogenase deficiency: demonstration of defective valine and beta-alanine metabolism and reduced malonic semialdehyde dehydrogenase activity in cultured fibroblasts

    SciTech Connect

    Gray, R.G.; Pollitt, R.J.; Webley, J.

    1987-08-01

    Intact cultured fibroblasts from a child with a new metabolic disorder, thought to be due to a deficiency of methylmalonic semialdehyde dehydrogenase, produced labeled CO/sub 2/ normally from (1-/sup 14/C)valine but not from (2-/sup 14/C)valine. CO/sub 2/ production from labeled beta-alanine was also much reduced, confirming the suspicion that malonic semialdehyde dehydrogenase is also deficient in this condition. An assay for malonic semialdehyde dehydrogenase in cell homogenates showed low activity but it was impossible to assess the degree of reduction.

  2. REVERSAL OF d-CYCLOSERINE INHIBITION OF BACTERIAL GROWTH BY ALANINE

    PubMed Central

    Zygmunt, Walter A.

    1962-01-01

    Zygmunt, Walter A. (Mead Johnson & Co., Evansville, Ind.). Reversal of d-cycloserine inhibition of bacterial growth by alanine. J. Bacteriol. 84:154–156. 1962.—Reversal of the antibacterial activity of d-4-amino-3-isoxazolidone by alanine in bacterial cultures actively growing on chemically defined media was compared in cultures requiring exogenous alanine and those capable of its synthesis. dl-Alanine was the most effective reversal agent in Pediococcus cerevisiae, an alanine-requiring organism, and d-alanine was effective in Escherichia coli and Staphylococcus aureus, organisms synthesizing alanine. With all three cultures, l-alanine was the least effective reversal agent. PMID:16561951

  3. 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…

  4. β-Alanine supplementation and military performance.

    PubMed

    Hoffman, Jay R; Stout, Jeffrey R; Harris, Roger C; Moran, Daniel S

    2015-12-01

    During sustained high-intensity military training or simulated combat exercises, significant decreases in physical performance measures are often seen. The use of dietary supplements is becoming increasingly popular among military personnel, with more than half of the US soldiers deployed or garrisoned reported to using dietary supplements. β-Alanine is a popular supplement used primarily by strength and power athletes to enhance performance, as well as training aimed at improving muscle growth, strength and power. However, there is limited research examining the efficacy of β-alanine in soldiers conducting operationally relevant tasks. The gains brought about by β-alanine use by selected competitive athletes appears to be relevant also for certain physiological demands common to military personnel during part of their training program. Medical and health personnel within the military are expected to extrapolate and implement relevant knowledge and doctrine from research performed on other population groups. The evidence supporting the use of β-alanine in competitive and recreational athletic populations suggests that similar benefits would also be observed among tactical athletes. However, recent studies in military personnel have provided direct evidence supporting the use of β-alanine supplementation for enhancing combat-specific performance. This appears to be most relevant for high-intensity activities lasting 60-300 s. Further, limited evidence has recently been presented suggesting that β-alanine supplementation may enhance cognitive function and promote resiliency during highly stressful situations.

  5. Vertebrate Acyl CoA synthetase family member 4 (ACSF4-U26) is a β-alanine-activating enzyme homologous to bacterial non-ribosomal peptide synthetase.

    PubMed

    Drozak, Jakub; Veiga-da-Cunha, Maria; Kadziolka, Beata; Van Schaftingen, Emile

    2014-03-01

    Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.

  6. Production of chiral β-amino acids using ω-transaminase from Burkholderia graminis.

    PubMed

    Mathew, Sam; Bea, Hanseop; Nadarajan, Saravanan Prabhu; Chung, Taeowan; Yun, Hyungdon

    2015-02-20

    Optically pure β-amino acids are of high pharmacological significance since they are used as key ingredients in many physiologically active compounds. Despite a number of enzymatic routes to these compounds, an efficient synthesis of β-amino acids continues to pose a major challenge for researchers. ω-Transaminase has emerged as an important class of enzymes for generating amine compounds. However, only a few ω-transaminases have been reported so far which show activity towards aromatic β-amino acids. In this study, (S)-ω-transaminase from Burkholderia graminis C4D1M has been functionally characterized and used for the production of chiral aromatic β-amino acids via kinetic resolution. The enzyme showed a specific activity of 3.1 U/mg towards rac-β-phenylalanine at 37°C. The Km and Kcat values of this enzyme towards rac-β-phenylalanine with pyruvate as the amino acceptor were 2.88 mM and 91.57 min(-1) respectively. Using this enzyme, racemic β-amino acids were kinetically resolved to produce (R)-β-amino acids with an excellent enantiomeric excess (> 99%) and ∼ 50% conversion. Additionally, kinetic resolution of aromatic β-amino acids was performed using benzaldehyde as a cheap amino acceptor.

  7. Deracemization of mexiletine biocatalyzed by omega-transaminases.

    PubMed

    Koszelewski, Dominik; Pressnitz, Desiree; Clay, Dorina; Kroutil, Wolfgang

    2009-11-01

    (S)- as well as (R)-mexiletine [1-(2,6-dimethylphenoxy)-2-propanamine], a chiral orally effective antiarrhythmic agent, was prepared by deracemization starting from the commercially available racemic amine using omega-transaminases in up to >99% ee and conversion with 97% isolated yield by a one-pot two-step procedure. The absolute configuration could be easily switched to the other enantiomer, just by switching the order of the applied transaminases. The cosubstrate pyruvate needed in the first oxidative step was recycled by using an amino acid oxidase. PMID:19785441

  8. β-alanine biosynthesis in Methanocaldococcus jannaschii.

    PubMed

    Wang, Yu; Xu, Huimin; White, Robert H

    2014-08-01

    One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosynthetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic archaeon Methanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the β-alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for β-alanine biosynthesis, we have established the pathway for the formation of β-alanine in this organism after experimentally eliminating other known and proposed pathways to β-alanine from malonate semialdehyde, l-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of β-alanine in cell extracts of M. jannaschii. Unlike other prokaryotes where the enzyme producing β-alanine from l-aspartate is a pyruvoyl-containing l-aspartate decarboxylase (PanD), the enzyme in M. jannaschii is a pyridoxal phosphate (PLP)-dependent l-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for methanofuran biosynthesis. A Km of ∼0.80 mM for l-aspartate with a specific activity of 0.09 μmol min(-1) mg(-1) at 70°C for the decarboxylation of l-aspartate was measured for the recombinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo.

  9. Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis.

    PubMed

    Knäuper, V; Docherty, A J; Smith, B; Tschesche, H; Murphy, G

    1997-03-17

    Analysis of the hinge region of neutrophil collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme. The mutagenesis of the amino acid residues in the P1' position of the two autoproteolytically cleaved peptide bonds (Leu243 and Ile248) to Ala showed that the mutant enzymes were more resistant to autoproteolysis. However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala239-Ile240 peptide bond and the half-life of the active enzyme was increased by 50%. In contrast, mutagenesis of Pro247 --> Ala (P1 of the minor cleavage site Pro247-Ile248) lead to increased susceptibility of the enzyme to autoproteolysis. However, when the other P1 position Gly242 was altered to Ala no effect on stability was observed. The analysis of the ability of the mutant active enzymes to hydrolyse 14C-type I collagen was assessed and our results demonstrate that the hinge sequence motif of neutrophil collagenase is important for collagenolytic activity. The alteration of the Gly242-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro247 sequence motif to Gly242-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro247 showed that the collagenolytic activity was reduced by 68.4%. In addition, mutagenesis of the downstream sequence motif Pro247-Thr-Gly-Pro-Ser-Thr-Pro-Lys-Pro258 to Pro247-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro258 had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%. When the Pro residues in the hinge motif (Pro247, Pro250, Pro253 and Pro256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme.

  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. Alanine racemase from the acidophile Acetobacter aceti.

    PubMed

    Francois, Julie A; Kappock, T Joseph

    2007-01-01

    Acetobacter aceti converts ethanol to acetic acid, and survives acetic acid exposure by tolerating cytoplasmic acidification. Alanine racemase (Alr) is a pyridoxal 5' phosphate (PLP) -dependent enzyme that catalyzes the interconversion of the d- and l-isomers of alanine and has a basic pH optimum. Since d-alanine is essential for peptidoglycan biosynthesis, Alr must somehow function in the acidic cytoplasm of A. aceti. We report the partial purification of native A. aceti Alr (AaAlr) and evidence that it is a rather stable enzyme. The C-terminus of AaAlr has a strong resemblance to the ssrA-encoded protein degradation signal, which thwarted initial protein expression experiments. High-activity AaAlr forms lacking a protease recognition sequence were expressed in Escherichia coli and purified. Biophysical and enzymological experiments confirm that AaAlr is intrinsically acid-resistant, yet has the catalytic properties of an ordinary Alr.

  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. 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. PMID:25815820

  14. 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.

  15. 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. PMID:10930696

  16. Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates with Alanine Aminotransferase Serum Activity

    PubMed Central

    Welker, Martin-Walter; Reichert, David; Susser, Simone; Sarrazin, Christoph; Martinez, Yolanda; Herrmann, Eva; Zeuzem, Stefan; Piiper, Albrecht; Kronenberger, Bernd

    2012-01-01

    Aim Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity. Patients and Methods Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry. Results Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027). Conclusion CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis. PMID:22355327

  17. 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.

  18. 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.

  19. The Chaperoning Activity of Amino-oxyacetic Acid on Folding-Defective Variants of Human Alanine:Glyoxylate Aminotransferase Causing Primary Hyperoxaluria Type I.

    PubMed

    Oppici, Elisa; Montioli, Riccardo; Dindo, Mirco; Maccari, Laura; Porcari, Valentina; Lorenzetto, Antonio; Chellini, Sara; Voltattorni, Carla Borri; Cellini, Barbara

    2015-10-16

    The rare disease Primary Hyperoxaluria Type I (PH1) results from the deficit of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene frequently leading to protein misfolding. Pharmacological chaperone (PC) therapy is a newly developed approach for misfolding diseases based on the use of small molecule ligands able to promote the correct folding of a mutant enzyme. In this report, we describe the interaction of amino-oxyacetic acid (AOA) with the recombinant purified form of two polymorphic species of AGT, AGT-Ma and AGT-Mi, and with three pathogenic variants bearing previously identified folding defects: G41R-Ma, G170R-Mi, and I244T-Mi. We found that for all these enzyme AOA (i) forms an oxime at the active site, (ii) behaves as a slow, tight-binding inhibitor with KI values in the nanomolar range, and (iii) increases the thermal stability. Furthermore, experiments performed in mammalian cells revealed that AOA acts as a PC by partly preventing the intracellular aggregation of G41R-Ma and by promoting the correct peroxisomal import of G170R-Mi and I244T-Mi. Based on these data, we carried out a small-scale screening campaign. We identified four AOA analogues acting as AGT inhibitors, even if only one was found to act as a PC. The possible relationship between the structure and the PC activity of these compounds is discussed. Altogether, these results provide the proof-of-principle for the feasibility of a therapy with PCs for PH1-causing variants bearing folding defects and provide the scaffold for the identification of more specific ligands. PMID:26161999

  20. 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.

  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. Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity.

    PubMed

    McCoy, Andrea J; Maurelli, Anthony T

    2005-07-01

    Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D-amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D-alanyl-D-alanine (D-Ala-D-Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics-based approach to demonstrate and characterize the D-Ala-D-Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full-length murC-ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible ara promoter and transformed into a D-Ala-D-Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliDeltaddlADeltaddlB mutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydial murC-ddl thus demonstrating functional ligase activity. Domain mapping of the full-length fusion protein and site-directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala. Chlamydia growth is inhibited by D-cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D-Ala and D-Ala-D-Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.

  3. Ingesting a preworkout supplement containing caffeine, creatine, β-alanine, amino acids, and B vitamins for 28 days is both safe and efficacious in recreationally active men.

    PubMed

    Kendall, Kristina L; Moon, Jordan R; Fairman, Ciaran M; Spradley, Brandon D; Tai, Chih-Yin; Falcone, Paul H; Carson, Laura R; Mosman, Matt M; Joy, Jordan M; Kim, Michael P; Serrano, Eric R; Esposito, Enrico N

    2014-05-01

    The purpose of this study was to determine the safety and efficacy of consuming a preworkout supplement (SUP) containing caffeine, creatine, β-alanine, amino acids, and B vitamins for 28 days. We hypothesized that little to no changes in kidney and liver clinical blood markers or resting heart rate and blood pressure (BP) would be observed. In addition, we hypothesized that body composition and performance would improve in recreationally active males after 28 days of supplementation. In a double-blind, placebo-controlled study, participants were randomly assigned to ingest one scoop of either the SUP or placebo every day for 28 days, either 20 minutes before exercise or ad libitum on nonexercise days. Resting heart rate and BP, body composition, and fasting blood samples were collected before and after supplementation. Aerobic capacity as well as muscular strength and endurance were also measured. Significant (P < .05) main effects for time were observed for resting heart rate (presupplementation, 67.59 ± 7.90 beats per minute; postsupplementation, 66.18 ± 7.63 beats per minute), systolic BP (presupplementation, 122.41 ± 11.25 mm Hg; postsupplementation, 118.35 ± 11.58 mm Hg), blood urea nitrogen (presupplementation, 13.12 ± 2.55 mg/dL; postsupplementation, 15.24 ± 4.47 mg/dL), aspartate aminotransferase (presupplementation, 34.29 ± 16.48 IU/L; postsupplementation, 24.76 ± 4.71 IU/L), and alanine aminotransferase (presupplementation, 32.76 ± 19.72 IU/L; postsupplementation, 24.88 ± 9.68 IU/L). Significant main effects for time were observed for body fat percentage (presupplementation, 15.55% ± 5.79%; postsupplementation, 14.21% ± 5.38%; P = .004) and fat-free mass (presupplementation, 70.80 ± 9.21 kg; postsupplementation, 71.98 ± 9.27 kg; P = .006). A significant decrease in maximal oxygen consumption (presupplementation, 47.28 ± 2.69 mL/kg per minute; postsupplementation, 45.60 ± 2.81 mL/kg per minute) and a significant increase in percentage of

  4. Ingesting a preworkout supplement containing caffeine, creatine, β-alanine, amino acids, and B vitamins for 28 days is both safe and efficacious in recreationally active men.

    PubMed

    Kendall, Kristina L; Moon, Jordan R; Fairman, Ciaran M; Spradley, Brandon D; Tai, Chih-Yin; Falcone, Paul H; Carson, Laura R; Mosman, Matt M; Joy, Jordan M; Kim, Michael P; Serrano, Eric R; Esposito, Enrico N

    2014-05-01

    The purpose of this study was to determine the safety and efficacy of consuming a preworkout supplement (SUP) containing caffeine, creatine, β-alanine, amino acids, and B vitamins for 28 days. We hypothesized that little to no changes in kidney and liver clinical blood markers or resting heart rate and blood pressure (BP) would be observed. In addition, we hypothesized that body composition and performance would improve in recreationally active males after 28 days of supplementation. In a double-blind, placebo-controlled study, participants were randomly assigned to ingest one scoop of either the SUP or placebo every day for 28 days, either 20 minutes before exercise or ad libitum on nonexercise days. Resting heart rate and BP, body composition, and fasting blood samples were collected before and after supplementation. Aerobic capacity as well as muscular strength and endurance were also measured. Significant (P < .05) main effects for time were observed for resting heart rate (presupplementation, 67.59 ± 7.90 beats per minute; postsupplementation, 66.18 ± 7.63 beats per minute), systolic BP (presupplementation, 122.41 ± 11.25 mm Hg; postsupplementation, 118.35 ± 11.58 mm Hg), blood urea nitrogen (presupplementation, 13.12 ± 2.55 mg/dL; postsupplementation, 15.24 ± 4.47 mg/dL), aspartate aminotransferase (presupplementation, 34.29 ± 16.48 IU/L; postsupplementation, 24.76 ± 4.71 IU/L), and alanine aminotransferase (presupplementation, 32.76 ± 19.72 IU/L; postsupplementation, 24.88 ± 9.68 IU/L). Significant main effects for time were observed for body fat percentage (presupplementation, 15.55% ± 5.79%; postsupplementation, 14.21% ± 5.38%; P = .004) and fat-free mass (presupplementation, 70.80 ± 9.21 kg; postsupplementation, 71.98 ± 9.27 kg; P = .006). A significant decrease in maximal oxygen consumption (presupplementation, 47.28 ± 2.69 mL/kg per minute; postsupplementation, 45.60 ± 2.81 mL/kg per minute) and a significant increase in percentage of

  5. 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.

  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. 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

  8. 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

    2015-01-01

    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

  9. 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

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

    PubMed

    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

  11. Alteration of substrate specificity of alanine dehydrogenase

    PubMed Central

    Fernandes, Puja; Aldeborgh, Hannah; Carlucci, Lauren; Walsh, Lauren; Wasserman, Jordan; Zhou, Edward; Lefurgy, Scott T.; Mundorff, Emily C.

    2015-01-01

    The l-alanine dehydrogenase (AlaDH) has a natural history that suggests it would not be a promising candidate for expansion of substrate specificity by protein engineering: it is the only amino acid dehydrogenase in its fold family, it has no sequence or structural similarity to any known amino acid dehydrogenase, and it has a strong preference for l-alanine over all other substrates. By contrast, engineering of the amino acid dehydrogenase superfamily members has produced catalysts with expanded substrate specificity; yet, this enzyme family already contains members that accept a broad range of substrates. To test whether the natural history of an enzyme is a predictor of its innate evolvability, directed evolution was carried out on AlaDH. A single mutation identified through molecular modeling, F94S, introduced into the AlaDH from Mycobacterium tuberculosis (MtAlaDH) completely alters its substrate specificity pattern, enabling activity toward a range of larger amino acids. Saturation mutagenesis libraries in this mutant background additionally identified a double mutant (F94S/Y117L) showing improved activity toward hydrophobic amino acids. The catalytic efficiencies achieved in AlaDH are comparable with those that resulted from similar efforts in the amino acid dehydrogenase superfamily and demonstrate the evolvability of MtAlaDH specificity toward other amino acid substrates. PMID:25538307

  12. 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.

  13. 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.

  14. 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.

  15. Field evaluation of a prototype paper-based point-of-care fingerstick transaminase test.

    PubMed

    Pollock, Nira R; McGray, Sarah; Colby, Donn J; Noubary, Farzad; Nguyen, Huyen; Nguyen, The Anh; Khormaee, Sariah; Jain, Sidhartha; Hawkins, Kenneth; Kumar, Shailendra; Rolland, Jason P; Beattie, Patrick D; Chau, Nguyen V; Quang, Vo M; Barfield, Cori; Tietje, Kathy; Steele, Matt; Weigl, Bernhard H

    2013-01-01

    Monitoring for drug-induced liver injury (DILI) via serial transaminase measurements in patients on potentially hepatotoxic medications (e.g., for HIV and tuberculosis) is routine in resource-rich nations, but often unavailable in resource-limited settings. Towards enabling universal access to affordable point-of-care (POC) screening for DILI, we have performed the first field evaluation of a paper-based, microfluidic fingerstick test for rapid, semi-quantitative, visual measurement of blood alanine aminotransferase (ALT). Our objectives were to assess operational feasibility, inter-operator variability, lot variability, device failure rate, and accuracy, to inform device modification for further field testing. The paper-based ALT test was performed at POC on fingerstick samples from 600 outpatients receiving HIV treatment in Vietnam. Results, read independently by two clinic nurses, were compared with gold-standard automated (Roche Cobas) results from venipuncture samples obtained in parallel. Two device lots were used sequentially. We demonstrated high inter-operator agreement, with 96.3% (95% C.I., 94.3-97.7%) agreement in placing visual results into clinically-defined "bins" (<3x, 3-5x, and >5x upper limit of normal), >90% agreement in validity determination, and intraclass correlation coefficient of 0.89 (95% C.I., 0.87-0.91). Lot variability was observed in % invalids due to hemolysis (21.1% for Lot 1, 1.6% for Lot 2) and correlated with lots of incorporated plasma separation membranes. Invalid rates <1% were observed for all other device controls. Overall bin placement accuracy for the two readers was 84% (84.3%/83.6%). Our findings of extremely high inter-operator agreement for visual reading-obtained in a target clinical environment, as performed by local practitioners-indicate that the device operation and reading process is feasible and reproducible. Bin placement accuracy and lot-to-lot variability data identified specific targets for device

  16. 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.

  17. Magnetic catechol-chitosan with bioinspired adhesive surface: preparation and immobilization of ω-transaminase.

    PubMed

    Ni, Kefeng; Zhou, Xu; Zhao, Li; Wang, Hualei; Ren, Yuhong; Wei, Dongzhi

    2012-01-01

    The magnetic chitosan nanocomposites have been studied intensively and been used practically in various biomedical and biological applications including enzyme immobilization. However, the loading capacity and the remained activity of immobilized enzyme based on existing approaches are not satisfied. Simpler and more effective immobilization strategies are needed. Here we report a simple catechol modified protocol for preparing a novel catechol-chitosan (CCS)-iron oxide nanoparticles (IONPs) composites carrying adhesive moieties with strong surface affinity. The ω-transaminase (ω-TA) was immobilized onto this magnetic composite via nucleophilic reactions between catechol and ω-TA. Under optimal conditions, 87.5% of the available ω-TA was immobilized on the composite, yielding an enzyme loading capacity as high as 681.7 mg/g. Furthermore, the valuation of enzyme activity showed that ω-TA immobilized on CCS-IONPs displayed enhanced pH and thermal stability compared to free enzyme. Importantly, the immobilized ω-TA retained more than 50% of its initial activity after 15 repeated reaction cycles using magnetic separation and 61.5% of its initial activity after storage at 4°C in phosphate buffered saline (PBS) for 15 days. The results suggested that such adhesive magnetic composites may provide an improved platform technology for bio-macromolecules immobilized.

  18. Magnetic catechol-chitosan with bioinspired adhesive surface: preparation and immobilization of ω-transaminase.

    PubMed

    Ni, Kefeng; Zhou, Xu; Zhao, Li; Wang, Hualei; Ren, Yuhong; Wei, Dongzhi

    2012-01-01

    The magnetic chitosan nanocomposites have been studied intensively and been used practically in various biomedical and biological applications including enzyme immobilization. However, the loading capacity and the remained activity of immobilized enzyme based on existing approaches are not satisfied. Simpler and more effective immobilization strategies are needed. Here we report a simple catechol modified protocol for preparing a novel catechol-chitosan (CCS)-iron oxide nanoparticles (IONPs) composites carrying adhesive moieties with strong surface affinity. The ω-transaminase (ω-TA) was immobilized onto this magnetic composite via nucleophilic reactions between catechol and ω-TA. Under optimal conditions, 87.5% of the available ω-TA was immobilized on the composite, yielding an enzyme loading capacity as high as 681.7 mg/g. Furthermore, the valuation of enzyme activity showed that ω-TA immobilized on CCS-IONPs displayed enhanced pH and thermal stability compared to free enzyme. Importantly, the immobilized ω-TA retained more than 50% of its initial activity after 15 repeated reaction cycles using magnetic separation and 61.5% of its initial activity after storage at 4°C in phosphate buffered saline (PBS) for 15 days. The results suggested that such adhesive magnetic composites may provide an improved platform technology for bio-macromolecules immobilized. PMID:22815930

  19. 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.

  20. A novel pattern of transaminase elevation associated with autologous transplant for neuroblastoma.

    PubMed

    Guinan, Eva C; Kalish, Leslie A; Berry, Winter S; McDaniel, Samuel; Lehmann, Leslie E; Diller, Lisa R

    2006-09-01

    To determine the pattern and degree of hepatic transaminitis experienced by children undergoing autologous transplantation for neuroblastoma. Sixty-four children with high-risk neuroblastoma received an autologous transplant with cyclophosphamide, etoposide, and carboplatin conditioning. Forty-eight went on to receive a second transplant with M and TBI conditioning. Charts were reviewed for evidence of hepatic regimen-related toxicity. A high rate of transaminitis was observed after both regimens. In each transplant, there was an early period of transaminitis during conditioning, from which patients recovered, followed by a second period of transaminase elevation. The degree of elevation was not associated with age, whether the administered dose was calculated based on a per kg or per M(2) basis or the presence of regimen-related severe mucositis. Elevated transaminases at admission were not associated with maximal hepatotoxicity during the first transplant although there was an association in the second transplant. However, the magnitude of transaminase elevation was less in the second transplant. VOD occurred in one and three patients in transplants 1 and 2, respectively. Both conditioning regimens were associated with an early and late elevation of transaminases without significant cholestasis. This biphasic pattern of transaminitis has not been reported previously. The high prevalence of transaminase elevation at time of both transplants was not associated with an increased incidence of VOD. We conclude that elevated transaminases should not preclude proceeding to a first or second autologous transplant with these regimens.

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

    PubMed

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

    2015-08-01

    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.

  2. Electron microscope studies of glutamic oxalacetic transaminase in rat liver cell.

    PubMed

    Lee, S H; Torack, R M

    1968-12-01

    Liver tissue of the rat, fixed in glutaraldehyde and formaldehyde, was incubated in a medium which consisted of 20 mML-aspartic acid, 2 mM alpha-ketoglutaric acid, 50 mM imidazole and 6 mM lead nitrate at pH 7.2-7.4. The electron-opaque precipitates, due to glutamic oxalacetic transaminase activity in liver cells, were found to be localized to the cristae and surface membranes of the mitochondria, the limiting membrane of the microbodies, and the nuclear membrane. Sucrose storage and trauma resulted in altered morphology and diminished final product intensity in mitochondria, but the microbody enzyme activity disappeared completely under these conditions. These distinctive responses of enzymatic activity are considered to indicate a difference in either the enzyme protein or its membrane attachment to these two sites. The use of a buffered dehydrating ethanol series to prepare tissue blocks for electron microscopy appeared to result in more precise intracellular localization of enzymatic reaction product.

  3. Effects of insulin and phorbol esters on MARCKS (myristoylated alanine-rich C-kinase substrate) phosphorylation (and other parameters of protein kinase C activation) in rat adipocytes, rat soleus muscle and BC3H-1 myocytes.

    PubMed Central

    Arnold, T P; Standaert, M L; Hernandez, H; Watson, J; Mischak, H; Kazanietz, M G; Zhao, L; Cooper, D R; Farese, R V

    1993-01-01

    To evaluate the question of whether or not insulin activates protein kinase C (PKC), we compared the effects of insulin and phorbol esters on the phosphorylation of the PKC substrate, i.e. myristoylated alanine-rich C-kinase substrate (MARCKS). In rat adipocytes, rat soleus muscle and BC3H-1 myocytes, maximally effective concentrations of insulin and phorbol esters provoked comparable, rapid, 2-fold (on average), non-additive increases in the phosphorylation of immunoprecipitable MARCKS. These effects of insulin and phorbol esters on MARCKS phosphorylation in intact adipocytes and soleus muscles were paralleled by similar increases in the phosphorylation of an exogenous, soluble, 85 kDa PKC substrate (apparently a MARCKS protein) during incubation of post-nuclear membrane fractions in vitro. Increases in the phosphorylation of this 85 kDa PKC substrate in vitro were also observed in assays of both plasma membranes and microsomes obtained from rat adipocytes that had been treated with insulin or phorbol esters. These insulin-induced increases in PKC-dependent phosphorylating activities of adipocyte plasma membrane and microsomes were associated with increases in membrane contents of diacylglycerol, PKC-beta 1 and PKC-beta 2. Our findings suggest that insulin both translocates and activates PKC in rat adipocytes, rat soleus muscles and BC3H-1 myocytes. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 Figure 9 Figure 10 PMID:8216211

  4. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase.

    PubMed

    Schumann, Gerhard; Bonora, Roberto; Ceriotti, Ferruccio; Férard, Georges; Ferrero, Carlo A; Franck, Paul F H; Gella, F Javier; Hoelzel, Wieland; Jørgensen, Poul Jørgen; Kanno, Takashi; Kessner, Art; Klauke, Rainer; Kristiansen, Nina; Lessinger, Jean-Marc; Linsinger, Thomas P J; Misaki, Hideo; Panteghini, Mauro; Pauwels, Jean; Schiele, Françoise; Schimmel, Heinz G; Weidemann, Gerhard; Siekmann, Lothar

    2002-07-01

    This paper is the fourth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of Gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on in Appendix 2.

  5. Functional Characterization of Alanine Racemase from Schizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

    PubMed Central

    Uo, Takuma; Yoshimura, Tohru; Tanaka, Naotaka; Takegawa, Kaoru; Esaki, Nobuyoshi

    2001-01-01

    Schizosaccharomyces pombe has an open reading frame, which we named alr1+, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1+ gene in Escherichia coli and purified the gene product (Alr1p), with an Mr of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent Km and Vmax values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of l-alanine, respectively. S. pombe uses d-alanine as a sole nitrogen source, but deletion of the alr1+ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for l-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but not d-alanine as a sole nitrogen source. Moreover, d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1+ gene enabled S. cerevisiae to grow efficiently on d-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine. PMID:11244061

  6. Neuroprotection by glutamate oxaloacetate transaminase in ischemic stroke: an experimental study.

    PubMed

    Campos, Francisco; Sobrino, Tomás; Ramos-Cabrer, Pedro; Argibay, Bárbara; Agulla, Jesús; Pérez-Mato, María; Rodríguez-González, Raquel; Brea, David; Castillo, José

    2011-06-01

    As ischemic stroke is associated with an excessive release of glutamate into the neuronal extracellular space, a decrease in blood glutamate levels could provide a mechanism to remove it from the brain tissue, by increasing the brain-blood gradient. In this regard, the ability of glutamate oxaloacetate transaminase (GOT) to metabolize glutamate in blood could represent a potential neuroprotective tool for ischemic stroke. This study aimed to determine the neuroprotective effects of GOT in an animal model of cerebral ischemia by means of a middle cerebral arterial occlusion (MCAO) following the Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines. In this animal model, oxaloacetate-mediated GOT activation inhibited the increase of blood and cerebral glutamate after MCAO. This effect is reflected in a reduction of infarct size, smaller edema volume, and lower sensorimotor deficits with respect to controls. Magnetic resonance spectroscopy confirmed that the increase of glutamate levels in the brain parenchyma after MCAO is inhibited after oxaloacetate-mediated GOT activation. These findings show the capacity of the GOT to remove glutamate from the brain by means of blood glutamate degradation, and suggest the applicability of this enzyme as an efficient and novel neuroprotective tool against ischemic stroke.

  7. 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

  8. 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

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

    2016-06-01

    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

  9. 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.

  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. Continuous colorimetric screening assays for the detection of specific L- or D-α-amino acid transaminases in enzyme libraries.

    PubMed

    Heuson, Egon; Petit, Jean-Louis; Debard, Adrien; Job, Aurélie; Charmantray, Franck; de Berardinis, Véronique; Gefflaut, Thierry

    2016-01-01

    In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of L- or D-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays' quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since L-CSA and D-CSA allowed the exclusive detection of L-TA and D-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable L- or D-homophenylalanine. PMID:26452497

  12. Continuous colorimetric screening assays for the detection of specific L- or D-α-amino acid transaminases in enzyme libraries.

    PubMed

    Heuson, Egon; Petit, Jean-Louis; Debard, Adrien; Job, Aurélie; Charmantray, Franck; de Berardinis, Véronique; Gefflaut, Thierry

    2016-01-01

    In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of L- or D-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays' quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since L-CSA and D-CSA allowed the exclusive detection of L-TA and D-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable L- or D-homophenylalanine.

  13. Levels of the glutamic oxaloacetic transaminase of erythrocytes of pregnant women and of cord bloods of newborn infants.

    PubMed

    Kishi, H; Azuma, J; Williams, R H; Watanabe, T; Folkers, K; Stahl, M L

    1978-01-01

    The mean basal specific activity (S.A.) of the glutamic oxaloacetic transaminase of erythrocytes (EGOT) for a group of 64 pregnant women was lower (p less than 0.001) than the value for the cord bloods of newborn infants, and lower (p less than 0.001) than the value for adults who had a top limit of S.A. of EGOT. In establishing the top limit of the S.A., it is important that the mean basal S.A. of the cord bloods from 49 newborn infants was identical to the mean basal S.A. of adults who had an adequate supplement of pyridoxine. There were no differences in the mean basal S.A.'s of the cord bloods between asymptomatic mothers and mothers who had anemia, edema, hypertension, proteinuria and glucosuria. An infant may be born with a top limit of S.A. which is non-deficient in pyridoxal 5'-phosphate, but a mother can have a low level of the transaminase, and which is deficient in the coenzyme. PMID:276565

  14. 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.

  15. 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.

  16. 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

  17. The peroxisome proliferator-activated receptor α agonist, AZD4619, induces alanine aminotransferase-1 gene and protein expression in human, but not in rat hepatocytes: Correlation with serum ALT levels.

    PubMed

    Thulin, Petra; Bamberg, Krister; Buler, Marcin; Dahl, Björn; Glinghammar, Björn

    2016-09-01

    Alanine aminotransferase (ALT) in serum is the standard biomarker for liver injury. We have previously described a clinical trial with a novel selective peroxisome proliferator-activated receptor α (PPARα) agonist (AZD4619), which unexpectedly caused increased serum levels of ALT in treated individuals without any other evidence of liver injury. We pinpointed a plausible mechanism through which AZD4619 could increase serum ALT levels; namely through the PPARα-specific activation of the human ALT1 gene at the transcriptional level. In the present study, we present data from the preceding rat toxicity study, demonstrating that AZD4619 had no effect on rat serum ALT activity levels, and further experiments were performed to elucidate the mechanisms responsible for this species-related difference. Our results revealed that AZD4619 increased ALT1 protein expression in a dose-dependent manner in human, but not in rat primary hepatocytes. Cloning of the human and rat ALT1 promoters into luciferase vectors confirmed that AZD4619 induced only the human, but not the rat ALT1 gene promoter in a dose-dependent manner. In PPARα-GAL4 reporter gene assays, AZD4619 was >100-fold more potent on the human vs. rat PPARα levels, explaining the differences in induction of the ALT1 gene between the species at the concentration range tested. These data demonstrate the usefulness of the human and rat ALT1 reporter gene assays for testing future drug candidates at the preclinical stage. In drug discovery projects, these assays elucidate whether elevations in ALT levels observed in vivo or in the clinic are due to metabolic effects rather than a toxic event in the liver. PMID:27430334

  18. 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.

  19. 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. PMID:10080917

  20. Expression, purification and preliminary crystallographic studies of human glutamate oxaloacetate transaminase 1 (GOT1).

    PubMed

    Jiang, Xiuping; Chang, Haiyang; Zhou, Yong

    2015-09-01

    Glutamate oxaloacetate transaminase (GOT) catalyzes the reversible reaction of l-aspartate and α-ketoglutarate into oxaloacetate and L-glutamate and plays a key role in carbon and nitrogen metabolism in all organisms. In human tissues, GOTs are pyridoxal 5'-phosphate-dependent (PLP) enzymes which exist in cytoplasm and mitochondrial forms, GOT1 and GOT2, respectively. GOT1 expression correlates with the growth of several tumors because cancer cells can utilize the amino acid glutamine to fuel anabolic processes, and therefore, GOT1 represents a new therapeutic target in cancer. In this work, human GOT1 was expressed in Escherichia coli periplasmic space, and purified by a combination of His-tag immobilized metal-ion affinity chromatography and anion exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 37 °C and a pH of 7.5. Cations such as Na(+), K(+) and Mg(2+) slightly inhibited the activity of recombinant human GOT1, while Zn(2+), Mn(2+), Cu(2+), Ni(2+), Co(2+) and Ca(2+) had stronger inhibitory effects. Crystals of human GOT1 were grown using the hanging-drop vapor diffusion method at 4°C with 0.1M Bis-Tris pH 6.0% and 21% (w/v) PEG 3350. The crystals diffracted to 2.99Å resolution and belonged to space group P43212 with the unit cell parameters a = b = 93.4, c = 107.4Å, α = β = γ = 90°. PMID:26003525

  1. Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth.

    PubMed

    Feng, Zhengyu; Cáceres, Nancy E; Sarath, Gautam; Barletta, Raúl G

    2002-09-01

    NAD(H)-dependent L-alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald in Mycobacterium smegmatis, we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence of L- or D-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with L-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.

  2. 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. PMID:24576951

  3. 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.

  4. Induction of arginase and ornithine transaminase in the fission yeast Schizosaccharomyces pombe.

    PubMed Central

    Benítez, T; Farrar, L

    1980-01-01

    The induction of arginase and ornithine transaminase in the fission yeast Schizosaccharomyces pombe requires the absence of ammonia and the presence of the inducer arginine. It seems that immediate arginase degradation is initiated by starved cells or ones from which arginine has been removed. PMID:7430074

  5. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false DL-Alanine. 172.540 Section 172.540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD ADDITIVES....540 DL-Alanine. DL-Alanine (a racemic mixture of D- and L-alanine; CAS Reg. No. 302-72-7) may...

  6. A novel low molecular weight alanine aminotransferase from fasted rat liver.

    PubMed

    Vedavathi, M; Girish, K S; Kumar, M Karuna

    2006-01-01

    Alanine is the most effective precursor for gluconeogenesis among amino acids, and the initial reaction is catalyzed by alanine aminotransferase (AlaAT). Although the enzyme activity increases during fasting, this effect has not been studied extensively. The present study describes the purification and characterization of an isoform of AlaAT from rat liver under fasting. The molecular mass of the enzyme is 17.7 kD with an isoelectric point of 4.2; glutamine is the N-terminal residue. The enzyme showed narrow substrate specificity for L-alanine with Km values for alanine of 0.51 mM and for 2-oxoglutarate of 0.12 mM. The enzyme is a glycoprotein. Spectroscopic and inhibition studies showed that pyridoxal phosphate (PLP) and free -SH groups are involved in the enzymatic catalysis. PLP activated the enzyme with a Km of 0.057 mM. PMID:16487061

  7. 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.

  8. Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea

    SciTech Connect

    Sellinger, M.; Ballatori, N.; Boyer, J.L. )

    1991-02-01

    In mammalian hepatocytes the L-alanine carrier contains a sulfhydryl group that is essential for its activity and is inhibited by mercurials. In hepatocytes of the evolutionarily primitive little skate (Raja erinacea), HgCl2 inhibits Na(+)-dependent alanine uptake and Na+/K(+)-ATPase and increase K+ permeability. To distinguish between direct effects of HgCl2 on the Na(+)-alanine cotransporter and indirect effects on membrane permeability, (3H)alanine transport was studied in plasma membrane vesicles. (3H)Alanine uptake was stimulated by an out-to-in Na+ but not K+ gradient and was saturable confirming the presence of Na(+)-alanine cotransport in liver plasma membranes from this species. Preincubation of the vesicles with HgCl2 for 5 min reduced initial rates of Na(+)-dependent but not Na(+)-independent alanine uptake in a dose-dependent manner (10-200 microM). In the presence of equal concentrations of NaCl or KCl inside and outside of the vesicles, 75 microM HgCl2 directly inhibited sodium-dependent alanine-(3H)alanine exchange, demonstrating that HgCl2 directly affected the alanine cotransporter. Inhibition of Na(+)-dependent alanine uptake by 30 microM HgCl2 was reversed by dithiothreitol (1 mM). HgCl2 (10-30 microM) also increased initial rates of 22Na uptake (at 5 sec), whereas 22Na uptake rates were decreased at HgCl2 concentrations greater than 50 microM. Higher concentrations of HgCl2 (100-200 microM) produced nonspecific effects on vesicle integrity. These studies indicate that HgCl2 inhibits Na(+)-dependent alanine uptake in skate hepatocytes by three different concentration-dependent mechanisms: direct interaction with the transporters, dissipation of the driving force (Na+ gradient), and loss of membrane integrity.

  9. Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus A20 Gene: Temperature-Sensitive Mutants Have a DNA-Minus Phenotype and Are Defective in the Production of Processive DNA Polymerase Activity

    PubMed Central

    Punjabi, Almira; Boyle, Kathleen; DeMasi, Joseph; Grubisha, Olivera; Unger, Beth; Khanna, Marilyn; Traktman, Paula

    2001-01-01

    Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168–175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298–12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 and tsA20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the tsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA− phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication. PMID:11711621

  10. Alanine as an end product during fermentation of monosaccharides by Clostridium strain P2.

    PubMed

    Orlygsson, J; Anderson, R; Svensson, B H

    1995-11-01

    The thermophilic Clostridium P2 was isolated from a semi-continuously fed reactor with high ammonium concentration. This bacterium formed substantial amounts of L-alanine as a major fermentation product from glucose, fructose and mannose. Low amounts of acetate, butyrate, carbon dioxide and hydrogen were also formed. A high partial pressure of hydrogen inhibited the degradation of the monosaccharides, whereas hydrogen removal, in the form of methanogenesis was found to be stimulatory. However, the amount of alanine produced per mole of hexose degraded did not change. Hexose degradation and alanine production were favoured by high ammonium concentrations. Nuclear magnetic resonance spectroscopy studies provided strong evidence that an active Embden-Meyerhof-Parnas pathway existed and that alanine was produced via an amination of pyruvate.

  11. 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...

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. Infrared Spectroscopy of Alanine in Solid Parahydrogen

    NASA Astrophysics Data System (ADS)

    Toh, Shin Yi; Wong, Ying-Tung Angel; Djuricanin, Pavle; Momose, Takamasa

    2014-06-01

    Amino acids are the building blocks of biological molecules, and thus the investigation of their physical and chemical properties would allow for further understanding of their functions in biological systems. In addition, the existence of amino acids in interstellar space has been discussed for many years, but it is still under intense debate. The effect of UV radiation on amino acids is one of the keys for their search in interstellar space, where strong UV radiation exists. In this experiment, conformational compositions of alpha and beta alanine and their UV photolysis were investigated via matrix-isolation FTIR spectroscopy and quantum chemical calculations. Solid parahydrogen was used as the matrix, which provides higher resolution spectra than other noble gas matrices. We have identified several stable conformers for both alpha and beta alanine in solid parahydrogen. A clear correlation between conformational ratio and sublimation temperature was found for beta alanine. Furthermore, it was found that UV photolysis of alanine yields not only its conformational changes, but also photodissociation into a CO2 molecule and fragments. Observed spectra and their analysis will be discussed in relation to interstellar chemistry.

  17. [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.

  18. Compound-specific nitrogen isotope analysis of D-alanine, L-alanine, and valine: application of diastereomer separation to delta15N and microbial peptidoglycan studies.

    PubMed

    Takano, Yoshinori; Chikaraishi, Yoshito; Ogawa, Nanako O; Kitazato, Hiroshi; Ohkouchi, Naohiko

    2009-01-01

    We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

  19. Experimental and computational thermochemical study of α-alanine (DL) and β-alanine.

    PubMed

    da Silva, Manuel A V Ribeiro; da Silva, Maria das Dores M C Ribeiro; Santos, Ana Filipa L O M; Roux, Maria Victoria; Foces-Foces, Concepción; Notario, Rafael; Guzmán-Mejía, Ramón; Juaristi, Eusebio

    2010-12-16

    This paper reports an experimental and theoretical study of the gas phase standard (p° = 0.1 MPa) molar enthalpies of formation, at T = 298.15 K, of α-alanine (DL) and β-alanine. The standard (p° = 0.1 MPa) molar enthalpies of formation of crystalline α-alanine (DL) and β-alanine were calculated from the standard molar energies of combustion, in oxygen, to yield CO2(g), N2(g), and H2O(l), measured by static-bomb combustion calorimetry at T = 298.15 K. The vapor pressures of both amino acids were measured as function of temperature by the Knudsen effusion mass-loss technique. The standard molar enthalpies of sublimation at T = 298.15 K was derived from the Clausius−Clapeyron equation. The experimental values were used to calculate the standard (p° = 0.1 MPa) enthalpy of formation of α-alanine (DL) and β-alanine in the gaseous phase, Δ(f)H(m)°(g), as −426.3 ± 2.9 and −421.2 ± 1.9 kJ·mol(−1), respectively. Standard ab initio molecular orbital calculations at the G3 level were performed. Enthalpies of formation, using atomization reactions, were calculated and compared with experimental data. Detailed inspections of the molecular and electronic structures of the compounds studied were carried out.

  20. 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

  1. Secretion of d-alanine by Escherichia coli.

    PubMed

    Katsube, Satoshi; Sato, Kazuki; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2016-07-01

    Escherichia coli has an l-alanine export system that protects the cells from toxic accumulation of intracellular l-alanine in the presence of l-alanyl-l-alanine (l-Ala-l-Ala). When a DadA-deficient strain was incubated with 6.0 mM l-Ala-l-Ala, we detected l-alanine and d-alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with d-amino acid oxidase resulted in the disappearance of a signal corresponding to d-alanine. Additionally, the culture supernatant enabled a d-alanine auxotroph to grow without d-alanine supplementation, confirming that the signal detected by HPLC was authentic d-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of d-alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from l-alanine to d-alanine enhanced d-alanine secretion. When high-density DadA-deficient cells preloaded with l-Ala-l-Ala were treated with 20 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both l-alanine and d-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the l-alanine and d-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the l-alanine exporter AlaE accumulated [3H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [3H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports d-alanine and that this function is most likely modulated by proton electrochemical potential. PMID:27166225

  2. 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

  3. 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...

  4. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false 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...

  5. 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.

  6. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false 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...

  7. 21 CFR 172.540 - DL-Alanine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false 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...

  8. 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.

  9. Biochemical characterization of alanine racemase--a spore protein produced by Bacillus anthracis.

    PubMed

    Kanodia, Shivani; Agarwal, Shivangi; Singh, Priyanka; Agarwal, Shivani; Singh, Preeti; Bhatnagar, Rakesh

    2009-01-31

    Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent K(m) of 3 mM for L-alanine, and a V(max) of 295 micromoles/min/mg, with the optimum activity occurring at 37 degrees C and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5'-phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a K(i) of 160 microM and 30 mM, respectively. [BMB reports 2009; 42(1): 47-52]. PMID:19192393

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

    PubMed

    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

  11. 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

  12. Earthworms accumulate alanine in response to drought.

    PubMed

    Holmstrup, Martin; Slotsbo, Stine; Henriksen, Per G; Bayley, Mark

    2016-09-01

    Earthworms have ecologically significant functions in tropical and temperate ecosystems and it is therefore important to understand how these animals survive during drought. In order to explore the physiological responses to dry conditions, we simulated a natural drought incident in a laboratory trial exposing worms in slowly drying soil for about one month, and then analyzed the whole-body contents of free amino acids (FAAs). We investigated three species forming estivation chambers when soils dry out (Aporrectodea tuberculata, Aporrectodea icterica and Aporrectodea longa) and one species that does not estivate during drought (Lumbricus rubellus). Worms subjected to drought conditions (< -2MPa) substantially increased the concentration of FAAs and in particular alanine that was significantly upregulated in all tested species. Alanine was the most important FAA reaching 250-650μmolg(-1) dry weight in dehydrated Aporrectodea species and 300μmolg(-1) dry weight in L. rubellus. Proline was only weakly upregulated in some species as were a few other FAAs. Species forming estivation chambers (Aporrectodea spp.) did not show a better ability to conserve body water than the non-estivating species (L. rubellus) at the same drought level. These results suggest that the accumulation of alanine is an important adaptive trait in drought tolerance of earthworms in general. PMID:27107492

  13. Correlation between liver cell necrosis and circulating alanine aminotransferase after ischaemia/reperfusion injuries in the rat liver.

    PubMed

    Knudsen, Anders R; Andersen, Kasper J; Hamilton-Dutoit, Stephen; Nyengaard, Jens R; Mortensen, Frank V

    2016-04-01

    Circulating liver enzymes such as alanine transaminase are often used as markers of hepatocellular damage. Ischaemia/reperfusion (I/R) injury is an inevitable consequence of prolonged liver ischaemia. The aim of this study was to examine the correlation between liver enzymes and volume of liver cell necrosis after ischaemia/reperfusion injuries, using design-unbiased stereological methods. Forty-seven male Wistar rats were subjected to 1 h of partial liver ischaemia, followed by either 4 or 24 h of reperfusion. Within each group, one-third of animals were subjected to ischaemic preconditioning and one-third to ischaemic postconditioning. At the end of reperfusion, blood and liver samples were collected for analysis. The volume of necrotic liver tissue was subsequently correlated to circulating markers of I/R injury. Correlation between histological findings and circulating markers was performed using Pearson's correlation coefficient. Alanine transferase peaked after 4 h of reperfusion; however, at this time-point, only mild necrosis was observed, with a Pearson's correlation coefficient of 0.663 (P = 0.001). After 24 h of reperfusion, alanine aminotransferase was found to be highly correlated to the degree of hepatocellular necrosis R = 0.836 (P = 0.000). Furthermore, alkaline phosphatase (R = 0.806) and α-2-macroglobulin (R = 0.655) levels were also correlated with the degree of necrosis. We show for the first time that there is a close correlation between the volume of hepatocellular necrosis and alanine aminotransferase levels in a model of I/R injury. This is especially apparent after 24 h of reperfusion. Similarly, increased levels of alkaline phosphatase and α-2-macroglobulin are correlated to the volume of liver necrosis. PMID:27292534

  14. ald of Mycobacterium tuberculosis encodes both the alanine dehydrogenase and the putative glycine dehydrogenase.

    PubMed

    Giffin, Michelle M; Modesti, Lucia; Raab, Ronald W; Wayne, Lawrence G; Sohaskey, Charles D

    2012-03-01

    The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown.

  15. 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

  16. Glial cells transform glucose to alanine, which fuels the neurons in the honeybee retina.

    PubMed

    Tsacopoulos, M; Veuthey, A L; Saravelos, S G; Perrottet, P; Tsoupras, G

    1994-03-01

    The retina of honeybee drone is a nervous tissue with a crystal-like structure in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments. The phosphorylation of glucose and its subsequent incorporation into glycogen occur in glia, whereas O2 consumption (QO2) occurs in the photoreceptors. Experimental evidence showed that glia phosphorylate glucose and supply the photoreceptors with metabolic substrates. We aimed to identify these transferred substrates. Using ion-exchange and reversed-phase HPLC and gas chromatography-mass spectrometry, we demonstrated that more than 50% of 14C(U)-glucose entering the glia is transformed to alanine by transamination of pyruvate with glutamate. In the absence of extracellular glucose, glycogen is used to make alanine; thus, its pool size in isolated retinas is maintained stable or even increased. Our model proposes that the formation of alanine occurs in the glia, thereby maintaining the redox potential of this cell and contributing to NH3 homeostasis. Alanine is released into the extracellular space and is then transported into photoreceptors using an Na(+)-dependent transport system. Purified suspensions of photoreceptors have similar alanine aminotransferase activity as glial cells and transform 14C-alanine to glutamate, aspartate, and CO2. Therefore, the alanine entering photoreceptors is transaminated to pyruvate, which in turn enters the Krebs cycle. Proline also supplies the Krebs cycle by making glutamate and, in turn, the intermediate alpha-ketoglutarate. Light stimulation caused a 200% increase of QO2 and a 50% decrease of proline and of glutamate. Also, the production of 14CO2 from 14C-proline was increased. The use of these amino acids would sustain about half of the light-induced delta QO2, the other half being sustained by glycogen via alanine formation. The use of proline meets a necessary anaplerotic function in the Krebs cycle, but implies high NH3 production. The results showed

  17. 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

  18. Isolation and characterization of cytosolic alanine aminotransferase isoforms from starved rat liver.

    PubMed

    Vedavathi, M; Girish, K S; Kumar, M Karuna

    2004-12-01

    Alanine is the most effective precursor for gluconeogenesis among amino acids and the initial reaction is catalyzed by alanine aminotransferases (AlaATs). It is a less extensively studied enzyme under starvation and known to that the enzyme activity increases in liver under starvation. The present study describes the purification and characterization of two isoforms of alanine aminotransferases from starved male rat liver under starvation. The molecular mass of isoforms was found to be 17.7 and 112.2 kDa with isoelectric points of 4.2 and 5.3 respectively for AlaAT I and AlaAT II. Both the enzymes showed narrow substrate specificity for L-alanine with different Km for alanine and 2-oxoglutarate. Both the enzymes were glycoprotein in nature. Inhibition, modification and spectroscopic studies showed that both PLP and free-SH groups are directly involved in the enzymatic catalysis. PLP activated both the enzymes with a Km 0.057 mM and 0.2 mM for AlaAT I and II respectively. PMID:15663181

  19. Biosynthesis of d-Alanyl-Lipoteichoic Acid: Characterization of Ester-Linked d-Alanine in the In Vitro-Synthesized Product

    PubMed Central

    Childs, Warren C.; Neuhaus, Francis C.

    1980-01-01

    d-Alanyl-lipoteichoic acid (d-alanyl-LTA) contains d-alanine ester residues which control the ability of this polyer to chelate Mg2+. In Lactobacillus casei a two-step in vitro reaction sequence catalyzed by the d-alanine-activating enzyme and d-alanine:membrane acceptor ligase incorporates d-alanine into membrane acceptor. In this paper we provide additional evidence that the in vitro system catalyzes the covalent incorporation of d-[14C]alanine into membrane acceptor which is the poly([3H]glycerol phosphate) moiety of d-alanyl-LTA. This conclusion was supported by the observation that the d-[14C]alanine and [3H]glycerol labels of the partially purified product were co-precipitated by antiserum containing globulins specific for poly(glycerol phosphate). The isolation of d-[14C]alanyl-[3H]glycerol from d-[14C]alanine·[3H]glycerol-labeled d-alanyl-LTA synthesized in the in vitro system indicated that the d-alanine was linked to the poly(glycerol phosphate) chain of the LTA. A comparison of the reactivities of the d-alanine residues of d-alanyl-glycerol and d-alanyl-LTA supported the conclusion that the incorporated residue of d-alanine was attached by an ester linkage. Thus, the data indicated that the in vitro system catalyzes the incorporation of d-alanine covalently linked by ester linkages to the glycerol moieties of the poly(glycerol phosphate) chains of d-alanyl-LTA. New procedures are presented for the partial purification of d-alanyl-LTA with a high yield of ester-linked d-alanine and for the sequential degradation of the poly(glycerol phosphate) moiety substituted with d-alanine of d-alanyl-LTA with phosphodiesterase II/phosphatase from Aspergillus niger. PMID:6772629

  20. Antioxidant activity of white rice, brown rice and germinated brown rice (in vivo and in vitro) and the effects on lipid peroxidation and liver enzymes in hyperlipidaemic rabbits.

    PubMed

    Mohd Esa, Norhaizan; Abdul Kadir, Khairul-Kamilah; Amom, Zulkhairi; Azlan, Azrina

    2013-11-15

    Antioxidant activity of different rice extract and the effect on the levels of antioxidant enzyme activity, superoxide dismutase (SOD) and glutathione peroxidase (GPx), vitamin E, lipid peroxidation and liver enzymes in hyperlipidaemia rabbits were investigated. Germinated brown rice (GBR) has the highest antioxidant activity compared to white rice (WR) and brown rice (BR). All rice grains increased the activity of SOD and GPx. However, vitamin E levels increased only in the groups that received the BR and GBR diets. The reduction of lipid peroxidation levels and activity of hepatic enzymes (alanine transferase, ALT and aspartate transaminase, AST) were only significantly observed in the GBR group. In conclusion, GBR supplementation has the greatest impact on increasing antioxidant enzyme activity and vitamin E level and on reducing lipid peroxidation in hypercholesterolaemia rabbit, thereby preventing the formation of atherosclerotic plaques. Furthermore, GBR diet can also reduce the level of hepatic enzymes. PMID:23790918

  1. Alanine Aminotransferase-Old Biomarker and New Concept: A Review

    PubMed Central

    Liu, Zhengtao; Que, Shuping; Xu, Jing; Peng, Tao

    2014-01-01

    Measurement of serum alanine aminotransferase (ALT) is a common, readily available, and inexpensive laboratory assay in clinical practice. ALT activity is not only measured to detect liver disease, but also to monitor overall health. ALT activity is influenced by various factors, including viral hepatitis, alcohol consumption, and medication. Recently, the impact of metabolic abnormalities on ALT variation has raised concern due to the worldwide obesity epidemic. The normal ranges for ALT have been updated and validated considering the metabolic covariates in the various ethnic districts. The interaction between metabolic and demographic factors on ALT variation has also been discussed in previous studies. In addition, an extremely low ALT value might reflect the process of aging, and frailty in older adults has been raised as another clinically significant feature of this enzyme, to be followed with additional epidemiologic investigation. Timely updated, comprehensive, and systematic introduction of ALT activity is necessary to aid clinicians make better use of this enzyme. PMID:25013373

  2. Studies on the influence of combustion exhaust gases and the products of their reaction with ammonia on the living organism. II. The influence on aspartate aminotransferase (AspAT) and alanine aminotransferase (AiAt) activities in the liver of guinea pig.

    PubMed

    Lewandowska-Tokarz, A; Stanosek, J; Ludyga, K; Kochanski, L

    1981-01-01

    The behaviour of aspartate aminotransferase (AspAT) an alanine aminotransferase (AIAT) in the whole homogenate and subcellular liver fractions of guinea pigs exposed to combustion exhaust gases and the neutralization products of these gases is presented in this paper. In the liver of animals exposed to the chronic action of combustion exhaust gases a decrease of both enzyme activities in the whole homogenate as well as in the subcellular fractions could be noted. Statistically significant changes are shown by AspAT. In the group of animals subjected to the action of neutralization products an increase of AIAT activity was observed. The activity of AspAT still shows a decrease, but less distinct in comparison with group I. An exception here is the mitochondrial fraction in which the AspAT activity is distinctly increased.

  3. 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.

  4. Hepatoprotective activity of bacoside A against N-nitrosodiethylamine-induced liver toxicity in adult rats.

    PubMed

    Janani, Panneerselvam; Sivakumari, Kanakarajan; Parthasarathy, Chandrakesan

    2009-10-01

    N-Nitrosodiethylamine (DEN) is a notorious carcinogen, present in many environmental factors. DEN induces oxidative stress and cellular injury due to enhanced generation of reactive oxygen species; free radical scavengers protect the membranes from DEN-induced damage. The present study was designed to evaluate the protective effect of bacoside A (the active principle isolated from Bacopa monniera Linn.) on carcinogen-induced damage in rat liver. Adult male albino rats were pretreated with 15 mg/kg body weight/day of bacoside A orally (for 14 days) and then intoxicated with single necrogenic dose of N-nitrosodiethylamine (200 mg/kg bodyweight, intraperitonially) and maintained for 7 days. The liver weight, lipid peroxidation (LPO), and activity of serum marker enzymes (aspartate transaminases, alanine transaminases, lactate dehydrogenase, alkaline phosphatase, and gamma-glutamyl transpeptidase) were markedly increased in carcinogen-administered rats, whereas the activities of marker enzymes were near normal in bacoside A-pretreated rats. Activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutatione-S-transferase, and reduced glutathione) in liver also decreased in carcinogen-administered rats, which were significantly elevated in bacoside A-pretreated rats. It is concluded that pretreatment of bacoside A prevents the elevation of LPO and activity of serum marker enzymes and maintains the antioxidant system and thus protects the rats from DEN-induced hepatotoxicity.

  5. 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.

  6. 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

  7. Sebelipase alfa over 52 weeks reduces serum transaminases, liver volume and improves serum lipids in patients with lysosomal acid lipase deficiency

    PubMed Central

    Valayannopoulos, Vassili; Malinova, Vera; Honzík, Tomas; Balwani, Manisha; Breen, Catherine; Deegan, Patrick B.; Enns, Gregory M.; Jones, Simon A.; Kane, John P.; Stock, Eveline O.; Tripuraneni, Radhika; Eckert, Stephen; Schneider, Eugene; Hamilton, Gavin; Middleton, Michael S.; Sirlin, Claude; Kessler, Bruce; Bourdon, Christopher; Boyadjiev, Simeon A.; Sharma, Reena; Twelves, Chris; Whitley, Chester B.; Quinn, Anthony G.

    2014-01-01

    Background and aims Lysosomal Acid Lipase Deficiency is an autosomal recessive enzyme deficiency resulting in lysosomal accumulation of cholesteryl esters and triglycerides. LAL-CL04, an ongoing extension study, investigates the long-term effects of sebelipase alfa, a recombinant human lysosomal acid lipase. Methods Sebelipase alfa (1 mg/kg or 3 mg/kg) was infused every-other-week to eligible subjects. Safety and tolerability assessments, including liver function, lipid profiles and liver volume assessment, were carried out at regular intervals. Results 216 infusions were administered to eight adult subjects through Week 52 during LAL-CL04. At Week 52, mean alanine aminotransferase and aspartate aminotransferase were normal with mean change from baseline of −58% and −40%. Mean change for low density lipoprotein, total cholesterol, triglyceride and high-density lipoprotein were −60%, −39%, −36%, and +29%, respectively. Mean liver volume by magnetic resonance imaging and hepatic proton density fat fraction decreased (12% and 55%, respectively). Adverse events were mainly mild and unrelated to sebelipase alfa. Infusion-related reactions were uncommon: three events of moderate severity were reported in two subjects; one patient's event was suggestive of hypersensitivity-like reaction, but additional testing did not confirm this, and the subject has successfully re-started sebelipase alfa. Of samples tested to date, no anti-drug antibodies have been detected. Conclusions Long-term dosing with sebelipase alfa in Lysosomal Acid Lipase-Deficient patients is well tolerated and produces sustained reductions in transaminases, improvements in serum lipid profile and reduction in hepatic fat fraction. A randomized, placebo-controlled phase 3 trial in children and adults is underway (ARISE: NCT01757184). PMID:24993530

  8. 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. PMID:26175657

  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. Vesicular GABA transporter (VGAT) transports β-alanine.

    PubMed

    Juge, Narinobu; Omote, Hiroshi; Moriyama, Yoshinori

    2013-11-01

    Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β-alanine as a substrate. Proteoliposomes containing purified VGAT transport β-alanine using Δψ but not ΔpH as a driving force. The Δψ-driven β-alanine uptake requires Cl(-). VGAT also facilitates Cl(-) uptake in the presence of β-alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β-alanine uptake. These findings indicated that VGAT transports β-alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β-alanine transporter. Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In the present study, we showed that proteoliposomes containing purified VGAT transport β-alanine using Δψ as a driving force. VGAT also facilitates Cl(-) uptake. Our findings indicated that VGAT functions as a vesicular β-alanine transporter.

  11. [A 42-year-old farmer with nonspecific leucocytosis and elevated transaminases. Acute septic reaction in Coxiella burnetii infection].

    PubMed

    Hempel, U; Schäffler, A; Salzberger, B; Rümmele, P; Schölmerich, J

    2008-06-01

    We report about a 42-year-old farmer with leucocytosis, elevation of transaminases and liver cirrhosis as an underlying condition. The diagnosis of Q fever hepatitis was made through liver biopsy and serology. Under therapy with doxycycline, transaminases initially increased again; after switching to ciprofloxacin, the patient could be discharged 3 weeks after admission. Q fever is caused by Coxiella burnetii. The most frequent acute manifestation is a self-limiting flu-like illness. Chronic Q fever mostly presents as endocarditis. The diagnosis is made through histology ("doughnut" granulomas), PCR, serology (acute: anti-phase II antibodies, chronic: anti-phase I antibodies) and culture. Standard therapy is doxycycline.

  12. Sulfation of Lithocholate as a Possible Modifier of Chenodeoxycholic Acid-induced Elevations of Serum Transaminase in Patients with Gallstones

    PubMed Central

    Marks, J. W.; Sue, S. O.; Pearlman, B. J.; Bonorris, G. G.; Varady, P.; Lachin, J. M.; Schoenfield, L. J.

    1981-01-01

    Chenodeoxycholic acid (CDC), through its metabolite, lithocholic acid (LC), is hepatotoxic in certain species. The cause of elevations of serum transaminase in 25% of humans ingesting CDC, however, is unknown, but also may be due to LC. Because efficient hepatic sulfation of LC may protect against hepatic injury, the aim of this study was to determine if sulfation of LC might modify CDC-induced elevations of transaminase. Pretreatment sulfation fraction (SF) was estimated in 63 randomly selected patients with gallstones in a double-blind randomized trial of CDC, 750 mg/d, 375 mg/d, or placebo; in 27 of these, SF was repeated at 1 or 2 yr. In four other patients, the SF was measured at 2 yr only. Serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase were determined monthly for 3 mo and then every 3 or 4 mo; an elevation of transaminase was defined as > 150% of the normal upper limit in asymptomatic patients. 10 μCi of 3H-glyco-LC (sp act 84 mCi/mol) was ingested 10-12 h before fasting duodenal biliary drainage. Bile acids in bile were separated by thin-layer chromatography. The SF was estimated as a percentage of total radioactivity (scintillation counting) in sulfated glyco-LC. The standard deviation for replicate SF determinations (n = 311) was 2.1% The pretreatment SF (mean 60.7±1.7 SEM) correlated inversely with age (r = 0.336, P < 0.005) and directly with the obesity index (r = 0.495, P > 0.001), but was independent of sex. The SF, remeasured at 1 or 2 yr, did not change significantly with time or CDC. Among CDC-treated patients, elevations of transaminase occurred in 75% of patients with a SF < 45% vs. 11% with a SF > 45% (P < 0.001). In conclusion, a SF < 45% occurred in patients with gallstones who had a high probability of developing elevated serum transaminase when treated with CDC. Thus, sulfation of lithocholate may modify CDC-induced elevations of serum transaminase. Images PMID:7298846

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

    DOEpatents

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

    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

    DOEpatents

    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. Subcellular localization and expression of multiple tomato γ-aminobutyrate transaminases that utilize both pyruvate and glyoxylate

    PubMed Central

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

    2009-01-01

    γ-Aminobutyric acid transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, three GABA-T isoforms were identified in the tomato (Solanum lycopersicum L.) plant. The deduced amino acid sequences of the three isoforms are highly similar over most of their coding regions with the exception of their N-terminal regions. Transient expression of the individual full-length GABA-T isoforms fused to the green fluorescent protein in tobacco suspension-cultured cells revealed their distinct subcellular localizations to the mitochondrion, plastid or cytosol, and that the specific targeting of the mitochondrion- and plastid-localized isoforms is mediated by their predicted N-terminal presequences. Removal of the N-terminal targeting presequences from the mitochondrion and plastid GABA-T isoforms yielded good recovery of the soluble recombinant proteins in Escherichia coli when they were co-expressed with the GroES/EL molecular chaperone complex. Activity assays indicated that all three recombinant isoforms possess both pyruvate- and glyoxylate-dependent GABA-T activities, although the mitochondrial enzyme has a specific activity that is significantly higher than that of its plastid and cytosolic counterparts. Finally, differential expression patterns of the three GABA-T isoforms in reproductive tissues, but not vegetative tissues, suggest unique roles for each enzyme in developmental processes. Overall, these findings, together with recent information about rice and pepper GABA-Ts, indicate that the subcellular distribution of GABA-T in the plant kingdom is highly variable. PMID:19470656

  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. Alanine aminotransferase controls seed dormancy in barley.

    PubMed

    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

  18. 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.

  19. 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.

  20. 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

  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. PMID:26713162

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

    PubMed Central

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

    1990-01-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABPPM) 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 with a Ka approximately 1.2-1.4 x 10(7) M-1. Sinusoidally enriched liver plasma membranes and purified h-FABPPM have GOT enzymatic activity; the relative specific activities (units/mg) of the membranes and purified protein suggest that h-FABPPM constitutes 1-2% of plasma membrane protein in the rat hepatocyte. Monospecific rabbit antiserum against h-FABPPM 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 [3H]oleate but not that of [35S]sulfobromophthalein or [14C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABPPM 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-FABPPM and mGOT are closely related. Images PMID:2185471

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

    PubMed

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

    2014-01-09

    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.

  4. Persistent GABAA/C responses to gabazine, taurine and beta-alanine in rat hypoglossal motoneurons.

    PubMed

    Chesnoy-Marchais, D

    2016-08-25

    In hypoglossal motoneurons, a sustained anionic current, sensitive to a blocker of ρ-containing GABA receptors, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and insensitive to bicuculline, was previously shown to be activated by gabazine. In order to better characterize the receptors involved, the sensitivity of this atypical response to pentobarbital (30μM), allopregnanolone (0.3μM) and midazolam (0.5μM) was first investigated. Pentobarbital potentiated the response, whereas the steroid and the benzodiazepine were ineffective. The results indicate the involvement of hybrid heteromeric receptors, including at least a GABA receptor ρ subunit and a γ subunit, accounting for the pentobarbital-sensitivity. The effects of the endogenous β amino acids, taurine and β-alanine, which are released under various pathological conditions and show neuroprotective properties, were then studied. In the presence of the glycine receptor blocker strychnine (1μM), both taurine (0.3-1mM) and β-alanine (0.3mM) activated sustained anionic currents, which were partly blocked by TPMPA (100μM). Thus, both β amino acids activated ρ-containing GABA receptors in hypoglossal motoneurons. Bicuculline (20μM) reduced responses to taurine and β-alanine, but small sustained responses persisted in the presence of both strychnine and bicuculline. Responses to β-alanine were slightly increased by allopregnanolone, indicating a contribution of the bicuculline- and neurosteroid-sensitive GABAA receptors underlying tonic inhibition in these motoneurons. Since sustained activation of anionic channels inhibits most mature principal neurons, the ρ-containing GABA receptors permanently activated by taurine and β-alanine might contribute to some of their neuroprotective properties under damaging overexcitatory situations. PMID:27246441

  5. Studies on the effect of long-term exposure to nitrogen dioxide on serum and liver proteins level and enzyme activity in guinea pigs.

    PubMed

    Drozdz, M; Kucharz, E; Ludyga, K; Molska-Drozda, T

    1976-01-01

    Forty male guinea pigs were exposed to nitrogen dioxide in a concentration of 2 mg/m3, 8 hours daily for a period of 180 days. Forty male animals were used as a control group. The following changes were found in intoxicated animals: the decrease of total protein and seromucoid concentration in blood serum and the decrease of total protein, perchloric acid-soluble proteins, protein-bound hexosamines and sialic acids content, in liver tissue. Electrophoretic examination of the serum proteins showed the increase of alpha 1- and beta 2-globulins and the decrease of albumin concentration. Changes in the level of glycoproteins fractions and protein-bound carbohydrates in blood serum were described also. Estimation of enzymes activity showed the decrease of alanine and aspartate transaminase activity in blood serum caused by the strong decrease of the cytoplasmic fraction of these enzymes. However the simultaneous increase of the mitochondrial fraction of transaminases activity was observed. The decrease of the activity of choline esterase was found also. Similar changes of enzymes activity were found in liver tissue. Histopathological studies were done for the further clearing the influenze of nitrogen dioxide on serum and liver proteins concentration and enzymes activity. It was found that after long-term exposure to nitrogen dioxide the destruction processes may be observed in the liver. The possible mechanism of the nitrogen dioxide-induced damage of protein metabolism is discussed.

  6. Beta-alanine as a small molecule neurotransmitter.

    PubMed

    Tiedje, K E; Stevens, K; Barnes, S; Weaver, D F

    2010-10-01

    This review discusses the role of beta-alanine as a neurotransmitter. Beta-alanine is structurally intermediate between alpha-amino acid (glycine, glutamate) and gamma-amino acid (GABA) neurotransmitters. In general, beta-alanine satisfies a number of the prerequisite classical criteria for being a neurotransmitter: beta-alanine occurs naturally in the CNS, is released by electrical stimulation through a Ca(2+) dependent process, has binding sites, and inhibits neuronal excitability. beta-Alanine has 5 recognized receptor sites: glycine co-agonist site on the NMDA complex (strychnine-insensitive); glycine receptor site (strychnine sensitive); GABA-A receptor; GABA-C receptor; and blockade of GAT protein-mediated glial GABA uptake. Although beta-alanine binding has been identified throughout the hippocampus, limbic structures, and neocortex, unique beta-alaninergic neurons with no GABAergic properties remain unidentified, and it is impossible to discriminate between beta-alaninergic and GABAergic properties in the CNS. Nevertheless, a variety of data suggest that beta-alanine should be considered as a small molecule neurotransmitter and should join the ranks of the other amino acid neurotransmitters. These realizations open the door for a more comprehensive evaluation of beta-alanine's neurochemistry and for its exploitation as a platform for drug design.

  7. Diet-induced changes in serum transaminase and triglyceride levels in healthy adult men. Role of sucrose and excess calories.

    PubMed

    Porikos, K P; Van Itallie, T B

    1983-10-01

    Serum transaminase levels were measured as part of a study on the physiologic control of food intake. Twenty-one men, 15 nonobese and six obese, were housed on a metabolic ward for 30 days where they received ad libitum a baseline diet of conventional foods containing 25 to 30 percent of total calories as sucrose for 18 days and a calorically diluted diet containing less than 10 percent sucrose for 12 days. Serum glutamic pyruvic transaminase (SGPT) and serum glutamic oxaloacetic transaminase (SGOT) levels rose significantly when subjects consumed the baseline diet and returned to their original levels on the calorically diluted aspartame-sweetened diet. Markedly abnormal transaminase levels developed in two subjects on the baseline diet and they had to be discharged from the study. Correlations between various components of the diet and enzyme changes suggested that both surplus calories and a high sucrose intake played a role in the elevation of enzyme levels. Serum triglyceride levels also showed a significant reduction when the subjects were switched from the baseline to the calorically diluted diet. In a second study designed to test systematically the role of sucrose on SGPT and SGOT levels and on serum triglyceride levels, six nonobese subjects received a carefully controlled liquid diet, relatively high in linoleic acid content, containing 50 percent of total calories as either sucrose or maltose. In comparison with the first study, sucrose had a smaller but still significant effect on the levels of both enzymes, but there was no significant effect on triglyceride levels.

  8. Effect of β-alanine treatment on mitochondrial taurine level and 5-taurinomethyluridine content

    PubMed Central

    2010-01-01

    Background The β-amino acid, taurine, is a nutritional requirement in some species. In these species, the depletion of intracellular stores of taurine leads to the development of severe organ dysfunction. The basis underlying these defects is poorly understood, although there is some suggestion that oxidative stress may contribute to the abnormalities. Recent studies indicate that taurine is required for normal mitochondrial protein synthesis and normal electron transport chain activity; it is known that defects in these events can lead to severe mitochondrial oxidative stress. The present study examines the effect of taurine deficiency on the first step of mitochondrial protein synthesis regulation by taurine, namely, the formation of taurinomethyluridine containing tRNA. Methods Isolated rat cardiomyocytes were rendered taurine deficient by incubation with medium containing the taurine transport inhibitor, β-alanine. The time course of cellular and mitochondrial taurine depletion was measured. The primer extension method was employed to evaluate the effect of β-alanine treatment on taurinomethyluridine content of tRNALeu. The protein levels of ND6 were also determined by Western blot analysis. Results β-alanine caused a time-dependent decrease in cellular taurine content, which were reduced in half after 48 hrs of incubation. The amount of taurine in the mitochondria was considerably less than that in the cytosol and was unaffected by β-alanine treatment. Approximately 70% of the tRNALeu in the untreated cell lacked taurinomethyluridine and these levels were unchanged following β-alanine treatment. Protein content of ND6, however, was significantly reduced after 48 hours incubation with β-alanine. Conclusions The taurine levels of the cytosol and the mitochondria are not directly coupled. The β-alanine-mediated reduction in taurine levels is too small to affect taurinomethyluridine levels. Nonetheless, it interferes with mitochondrial protein synthesis

  9. Mechanisms of itch evoked by β-alanine.

    PubMed

    Liu, Qin; Sikand, Parul; Ma, Chao; Tang, Zongxiang; Han, Liang; Li, Zhe; Sun, Shuohao; LaMotte, Robert H; Dong, Xinzhong

    2012-10-17

    β-Alanine, a popular supplement for muscle building, induces itch and tingling after consumption, but the underlying molecular and neural mechanisms are obscure. Here we show that, in mice, β-alanine elicited itch-associated behavior that requires MrgprD, a G-protein-coupled receptor expressed by a subpopulation of primary sensory neurons. These neurons exclusively innervate the skin, respond to β-alanine, heat, and mechanical noxious stimuli but do not respond to histamine. In humans, intradermally injected β-alanine induced itch but neither wheal nor flare, suggesting that the itch was not mediated by histamine. Thus, the primary sensory neurons responsive to β-alanine are likely part of a histamine-independent itch neural circuit and a target for treating clinical itch that is unrelieved by anti-histamines.

  10. Use of β-alanine as an ergogenic aid.

    PubMed

    Derave, Wim

    2013-01-01

    Despite the large variety of so-called ergogenic supplements used by the sporting community, only few of them are effectively supported by scientific proof. One of the recent evidence-based supplements that entered the market is β-alanine. β-Alanine is the rate-limiting precursor for the synthesis of the dipeptide carnosine (β-alanyl-L-histidine) in human muscle. The chronic daily ingestion of β-alanine can markedly elevate muscle carnosine content, which results in improved exercise capacity, especially in sports that include high-intensity exercise episodes. The use of β-alanine is exponentially growing in recent years. This chapter aims to (1) discuss the scientific basis and physiological background of β-alanine and its synthesis product carnosine, and (2) translate these scientific findings to practical applications in sports.

  11. The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance

    PubMed Central

    2010-01-01

    Background GABA (γ-aminobutyric acid) is a non protein amino acid that has been reported to accumulate in a number of plant species when subjected to high salinity and many other environmental constraints. However, no experimental data are to date available on the molecular function of GABA and the involvement of its metabolism in salt stress tolerance in higher plants. Here, we investigated the regulation of GABA metabolism in Arabidopsis thaliana at the metabolite, enzymatic activity and gene transcription levels upon NaCl stress. Results We identified the GABA transaminase (GABA-T), the first step of GABA catabolism, as the most responsive to NaCl. We further performed a functional analysis of the corresponding gene POP2 and demonstrated that the previously isolated loss-of-function pop2-1 mutant was oversensitive to ionic stress but not to osmotic stress suggesting a specific role in salt tolerance. NaCl oversensitivity was not associated with overaccumulation of Na+ and Cl- but mutant showed a slight decrease in K+. To bring insights into POP2 function, a promoter-reporter gene strategy was used and showed that POP2 was mainly expressed in roots under control conditions and was induced in primary root apex and aerial parts of plants in response to NaCl. Additionally, GC-MS- and UPLC-based metabolite profiling revealed major changes in roots of pop2-1 mutant upon NaCl stress including accumulation of amino acids and decrease in carbohydrates content. Conclusions GABA metabolism was overall up-regulated in response to NaCl in Arabidopsis. Particularly, GABA-T was found to play a pivotal function and impairment of this step was responsible for a decrease in salt tolerance indicating that GABA catabolism was a determinant of Arabidopsis salt tolerance. GABA-T would act in salt responses in linking N and C metabolisms in roots. PMID:20122158

  12. 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. PMID:26315099

  13. 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.

  14. Hepatoprotective activity of aqueous extract of Portulaca oleracea in combination with lycopene in rats

    PubMed Central

    Anusha, M.; Venkateswarlu, M.; Prabhakaran, V.; Taj, S. Shareen; Kumari, B. Pushpa; Ranganayakulu, D.

    2011-01-01

    Objective: To investigate the hepatoprotective activity of the aqueous extract of the aerial parts of Portulaca oleracea (P. oleracea) in combination with lycopene against carbon tetrachloride induced hepatotoxicity in rats. Materials and Methods: Hepatotoxicity was induced in male Wistar rats by intraperitoneal injection of carbon tetrachloride (0.1 ml/kg b.w for 14 days). The aqueous extract of P. oleracea in combination with lycopene (50 mg/kg b.w) was administered to the experimental animals at two selected doses for 14 days. The hepatoprotective activity of the combination was evaluated by the liver function marker enzymes in the serum [aspartate transaminases (AST), alanine transaminases (ALT), alkaline phosphatase (Alk.P), total bilirubin (TB), total protein (TP) and total cholesterol (TC)], pentobarbitone induced sleeping time (PST) and histopathological studies of liver. Results: Both the treatment groups showed hepatoprotective effect against carbon tetrachloride induced hepatotoxicity by significantly restoring the levels of serum enzymes to normal which was comparable to that of silymarin group. Besides, the results obtained from PST and histopathological results also support the study. Conclusions: The oral administration of P. oleracea in combination with lycopene significantly ameliorates CCl4 hepatotoxicity in rats. PMID:22022001

  15. 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

  16. Correlation of In Vivo and In Vitro Assay Results for Assessment of Free Radical Scavenging Activity of Green Tea Nutraceuticals.

    PubMed

    Abd-ElSalam, Heba-Alla H; Al-Ghobashy, Medhat A; Al-Shorbagy, Muhammad; Nassar, Noha; Zaazaa, Hala E; Ibrahim, Mohamed A

    2016-07-01

    Green tea (GT)-derived catechins; epigallocatechin gallate (EGCG) in particular are commonly used nutraceuticals for their free-radical scavenging activity (FRSA). The influence of photodegradation on the protective power of GT nutracenticals against oxidative stress was thoroughly explored. Photodegradation of GT extracts was carried out and monitored using orthogonal stability-indicating testing protocol; in vitro and in vivo assays. Total polyphenol content (TPC) and FRSA were determined spectrophotometrically while EGCG was selectively monitored using SPE-HPLC. In vivo assessment of photodegraded samples was investigated via measuring a number of biomarkers for hepatic oxidative stress and apoptosis (caspase-3, inducible nitric oxide synthase, nitric oxide, mitogen-activated protein kinase, glutathione, thiobarbituric acid reactive substances, nuclear factor kappa beta, and nuclear factor erythroid 2-related factor) as well as liver damage (alanine transaminase and aspartate transaminase) in serum of rats previously subjected to oxidative stress. Results showed complete degradation of EGCG in photodegraded green tea samples with no correlation with either TPC or FRSA. On the other hand, in vivo assay results revealed not only loss of activity but formation of harmful pro-oxidants. Photostability was found crucial for the protective effect of GT extract against lead acetate insult. Results confirmed that careful design of quality control protocols requires correlation of chemical assays to bioassays to verify efficacy, stability, and most importantly safety of nutraceuticals.

  17. A mutant of Escherichia coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase IA.

    PubMed Central

    Nishimura, Y; Suzuki, H; Hirota, Y; Park, J T

    1980-01-01

    A mutant of Escherichia coli defective in penicillin-binding protein 5 activity was isolated. The mutation (pfv) was shown to be located at 14.0 min on the E. coli chromosome map. Loss of penicillin-binding protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to beta-lactam antibiotics. We conclude that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity and that the enzyme activity, in vivo, protects E. coli cells from killing by low inhibitory concentrations of beta-lactam antibiotics. PMID:6995448

  18. 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.

  19. 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.

  20. β-Alanine supplementation for athletic performance: an update.

    PubMed

    Bellinger, Phillip M

    2014-06-01

    β-alanine supplementation has become a common practice among competitive athletes participating in a range of different sports. Although the mechanism by which chronic β-alanine supplementation could have an ergogenic effect is widely debated, the popular view is that β-alanine supplementation augments intramuscular carnosine content, leading to an increase in muscle buffer capacity, a delay in the onset of muscular fatigue, and a facilitated recovery during repeated bouts of high-intensity exercise. β-alanine supplementation appears to be most effective for exercise tasks that rely heavily on ATP synthesis from anaerobic glycolysis. However, research investigating its efficacy as an ergogenic aid remains equivocal, making it difficult to draw conclusions as to its effectiveness for training and competition. The aim of this review was to update, summarize, and critically evaluate the findings associated with β-alanine supplementation and exercise performance with the most recent research available to allow the development of practical recommendations for coaches and athletes. A critical review of the literature reveals that when significant ergogenic effects have been found, they have been generally shown in untrained individuals performing exercise bouts under laboratory conditions. The body of scientific data available concerning highly trained athletes performing single competition-like exercise tasks indicates that this type of population receives modest but potentially worthwhile performance benefits from β-alanine supplementation. Recent data indicate that athletes may not only be using β-alanine supplementation to enhance sports performance but also as a training aid to augment bouts of high-intensity training. β-alanine supplementation has also been shown to increase resistance training performance and training volume in team-sport athletes, which may allow for greater overload and superior adaptations compared with training alone. The ergogenic

  1. Glutamic oxaloacetic transaminase isozymes from rat liver. Purification and physicochemical characterization.

    PubMed

    Huynh, Q K; Sakakibara, R; Watanabe, T; Wada, H

    1980-07-01

    Glutamic oxaloacetic transaminase isozymes were purified simultaneously to homogeneity from rat liver with high yields. Three subforms of mitochondrial isozyme and three subforms of cytosolic isozyme were separated by chromatography on CM-Sephadex and electrophoresis on polyacrylamide gel. The general enzymatic properties of the purified isozymes such as their kinetic parameters, isoelectric points, molecular weights, amino acid compositions, NH2-terminal amino acid sequences and COOH-terminal amino acids were determined. Most of these properties of the isozymes are similar to those of the corresponding isozymes from other sources, such as rat brain and pig and human heart. In amino acid compositions, cytosolic isozyme from rat liver has more proline and glycine and less arginine, threonine and leucine than pig heart cytosolic isozyme; the mitochondrial isozyme has more glutamic acid and glycine and less serine than the corresponding pig heart isozyme. The NH2-terminal amino acid sequences of GOT isozymes from rat liver were identical with those of the GOT isozymes from pig heart up to the 10th residues except for the 5th residues. The subforms of mitochondrial isozyme from rat liver were generated on storage at 4 degrees C for 4-8 weeks.

  2. 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

  3. Relationships of Cardiorespiratory Fitness with Metabolic Risk Factors, Inflammation, and Liver Transaminases in Overweight Youths

    PubMed Central

    Bouglé, Dominique; Zunquin, Gautier; Sesbouë, Bruno; Sabatier, Jean-Pierre

    2010-01-01

    The aim of this study was to assess the relationships of fatness and fitness with metabolic risk factors, including liver transaminases and inflammation in obese youth, taking in account gender, age, and pubertal stage. 241 children were studied (135 girls), age 11.9 ± 2.2 years (x ± SD), Body Mass Index z score 5.4 ± 2.7. For girls, VO2max was significantly associated with insulin (P = .001), Insulin resistance (HOMA-IR) (P = .005), and ALT (P = .012); a relationship was displayed between fibrinogen and age and % fat mass (FM) (P = .008); for boys, relationships were found between VO2max and diastolic blood pressure and triglycerides; independent associations were also found between age and insulin, HOMA-IR and HDL cholesterol; fibrinogen and sedimentation rate were related (P ≤ .004) with %FM. Their relationships are observed from young age and increase with the continuous increase of factors. This supports the need to treat overweight as soon as it is detected; improving CRF is one of the ways which could be used to prevent the complications of obesity. PMID:20652084

  4. Preparation and Characterisation of Pva Doped with Beta Alanine

    NASA Astrophysics Data System (ADS)

    Bhuvaneshwari, R.; Karthikeyan, S.; Rajeswari, N.; Selvasekarapandian, S.; Sanjeeviraja, C.

    2013-07-01

    Pure PVA has been doped with different amount of β - alanine. Film has been prepared by Solution Casting Technique using water as a solvent. The Complex formation between the PVA and β - alanine has been confirmed by FTIR. The Pure PVA conductivity is in the order 10-10 Scm-1 at ambient temperature. The conductivity has been found to increase to the order 10-6 when doped with 10% β - alanine. In this paper characterization of a PVA doped with β-ala has been studied using XRD, FTIR, AC impedance analysis and the results are reported.

  5. Comparative Evaluation of Hepatoprotective Activities of Geniposide, Crocins and Crocetin by CCl4-Induced liver Injury in Mice

    PubMed Central

    Chen, Ping; Chen, Yang; Wang, Yarong; Cai, Shining; Deng, Liang; Liu, Jia; Zhang, Hao

    2016-01-01

    Iridoid glycosides (mainly geniposide) and crocetin derivatives (crocins) are the two major active constituents in Gardenia jasminoides Ellis. In the present study, geniposide, crocins, crocin-1 and crocetin were separated from gardenia chromatographically. Then, mice were orally administrated with geniposide (400 mg/kg b.w.), crocins (400 mg/kg b.w.), crocin-1 (400 mg/kg b.w.) and crocetin (140 mg/kg b.w.) once daily for 7 days with CCl4. Hepatoprotective properties were evaluated by biochemical parameters: Administration of geniposide, crocins, crocin-1and crocetin significantly lowered serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) levels in CCl4-treated mice. The reduced glutathione (GSH) levels and antioxidant enzymes (SOD and CAT) activities were also increased by geniposide, crocins, crocin-1 and crocetin. Histopathological examination of livers showed that these components reduced deformability, irregular arrangement and rupture of hepatocyte in CCl4-treated mice. These biochemical results and liver histopathological assessment demonstrated that geniposide, crocetin derivatives and crocetin show comparative beneficial effects on CCl4-induced liver damage via induction of antioxidant defense. Therefore, contents of geniposide and crocetin derivatives should be both considered for hepatoprotective efficacy of Gardenia jasminoides Ellis. PMID:26902084

  6. 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.

  7. Comparative Evaluation of Hepatoprotective Activities of Geniposide, Crocins and Crocetin by CCl4-Induced liver Injury in Mice.

    PubMed

    Chen, Ping; Chen, Yang; Wang, Yarong; Cai, Shining; Deng, Liang; Liu, Jia; Zhang, Hao

    2016-03-01

    Iridoid glycosides (mainly geniposide) and crocetin derivatives (crocins) are the two major active constituents in Gardenia jasminoides Ellis. In the present study, geniposide, crocins, crocin-1 and crocetin were separated from gardenia chromatographically. Then, mice were orally administrated with geniposide (400 mg/kg b.w.), crocins (400 mg/kg b.w.), crocin-1 (400 mg/kg b.w.) and crocetin (140 mg/kg b.w.) once daily for 7 days with CCl4. Hepatoprotective properties were evaluated by biochemical parameters: Administration of geniposide, crocins, crocin-1and crocetin significantlylowered serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) levels in CCl4-treated mice. The reduced glutathione (GSH) levels and antioxidant enzymes (SOD and CAT) activities were also increased by geniposide, crocins, crocin-1 and crocetin. Histopathological examination of livers showed that these components reduced deformability, irregular arrangement and rupture of hepatocyte in CCl4-treated mice. These biochemical results and liver histopathological assessment demonstrated that geniposide, crocetin derivatives and crocetin show comparative beneficialeffects on CCl4-induced liver damage via induction of antioxidant defense. Therefore, contents of geniposide and crocetin derivatives should be both considered for hepatoprotective efficacyof Gardenia jasminoides Ellis. PMID:26902084

  8. 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. PMID:26687753

  9. 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.

  10. Dose response of alanine detectors irradiated with carbon ion beams

    SciTech Connect

    Herrmann, Rochus; Jaekel, Oliver; Palmans, Hugo; Sharpe, Peter; Bassler, Niels

    2011-04-15

    Purpose: The dose response of the alanine detector shows a dependence on particle energy and type when irradiated with ion beams. The purpose of this study is to investigate the response behavior of the alanine detector in clinical carbon ion beams and compare the results to model predictions. Methods: Alanine detectors have been irradiated with carbon ions with an energy range of 89-400 MeV/u. The relative effectiveness of alanine has been measured in this regime. Pristine and spread out Bragg peak depth-dose curves have been measured with alanine dosimeters. The track structure based alanine response model developed by Hansen and Olsen has been implemented in the Monte Carlo code FLUKA and calculations were compared to experimental results. Results: Calculations of the relative effectiveness deviate less than 5% from the measured values for monoenergetic beams. Measured depth-dose curves deviate from predictions in the peak region, most pronounced at the distal edge of the peak. Conclusions: The used model and its implementation show a good overall agreement for quasimonoenergetic measurements. Deviations in depth-dose measurements are mainly attributed to uncertainties of the detector geometry implemented in the Monte Carlo simulations.

  11. Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis.

    PubMed

    Maguire, Sarah E; Rhoades, Seth; Chen, Wen-Feng; Sengupta, Arjun; Yue, Zhifeng; Lim, Jason C; Mitchell, Claire H; Weljie, Aalim M; Sehgal, Amita

    2015-08-14

    Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD(+)/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.

  12. 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.

  13. 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.

  14. 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. PMID:26894530

  15. 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

  16. A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase.

    PubMed

    Mustata, Gabriela Iurcu; Briggs, James M

    2002-12-01

    We report a new structure-based strategy for the identification of novel inhibitors. This approach has been applied to Bacillus stearothermophilus alanine racemase (AlaR), an enzyme implicated in the biosynthesis of the bacterial cell wall. The enzyme catalyzes the racemization of L- and D-alanine using pyridoxal 5'-phosphate (PLP) as a cofactor. The restriction of AlaR to bacteria and some fungi and the absolute requirement for D-alanine in peptidoglycan biosynthesis make alanine racemase a suitable target for drug design. Unfortunately, known inhibitors of alanine racemase are not specific and inhibit the activity of other PLP-dependent enzymes, leading to neurological and other side effects. This article describes the development of a receptor-based pharmacophore model for AllaR, taking into account receptor flexibility (i.e. a 'dynamic' pharmacophore model). In order to accomplish this, molecular dynamics (MD) simulations were performed on the full AlaR dimer from Bacillus stearothermophilus (PDB entry, 1 sft) with a D-alanine molecule in one active site and the non-covalent inhibitor, propionate, in the second active site of this homodimer. The basic strategy followed in this study was to utilize conformations of the protein obtained during MD simulations to generate a dynamic pharmacophore model using the property mapping capability of the LigBuilder program. Compounds from the Available Chemicals Directory that fit the pharmacophore model were identified and have been submitted for experimental testing. The approach described here can be used as a valuable tool for the design of novel inhibitors of other biomolecular targets.

  17. L-alanine uptake in membrane vesicles from Mytilus edulis gills

    SciTech Connect

    Pajor, A.M.; Wright, S.H.

    1986-03-05

    Previous studies have shown that gills from M. edulis can accumulate L-alanine from seawater by a saturable process specific for ..cap alpha..-neutral amino acids. This uptake occurs against chemical gradients in excess of 10/sup 6/ to 1. To further characterize this uptake, membrane vesicles were prepared from M. edulis gill tissue by differential centrifugation. Enrichments of putative enzyme markers (relative to that in combined initial fractions) were as follows: ..gamma..-Glutamyltranspeptidase, 25-30x; Alkaline Phosphatase, 5-6x; K/sup +/-dependent para-Nitrophenyl Phosphatase, 3-5x; Succinate Dehydrogenase 0.1-0.2x. These results suggest that the preparation is enriched in plasma membranes, although histochemical studies will be needed to verify this. The time course of /sup 14/C-L-alanine uptake in the presence of inwardly-directed Na/sup +/ gradient showed a transient overshoot (3-5 fold) at 10 minutes which decreased to equilibrium after six hours. The size of the overshoot and early uptake rates depended on the size of the inwardly-directed Na/sup +/ gradient. No overshoot was seen in the presence of inwardly-directed gradients of LiCl or choline-Cl, or with equilibrium concentrations NaCl or mannitol. A reduced overshoot was seen with a gradient of NaSCN. A small overshoot was seen with an inwardly-directed gradient of KCl. Transport of L-alanine included saturable and diffusive components. Uptake of 6 ..mu..M L-alanine was inhibited more than 80% by 100 ..mu..M ..cap alpha..-zwitterionic amino acids (alanine, leucine, glycine); by 30 to 75% by proline, aspartate and lysine; and less than 20% by a ..beta..-amino acid, taurine. The results of these experiments agree with those from intact gill studies and support the hypothesis that L-alanine is transported into gill epithelial cells by a secondary active transport process involving Na/sup +/.

  18. Comparative effects of petrol and diesel on enzyme activity in Tympanotonus fuscatus after sublethal exposure.

    PubMed

    Edori, O S; Festus, C; Edori, E S

    2014-04-01

    Pollution of the aquatic environment by petroleum and its products is common the world over. This study is aimed at examining sublethal effects of petrol and diesel on enzymes in Tympanotonus fuscatus namely: Aspartate Transaminase (AST) (E.C. 2.6.1.1), Alanine Transaminase (ALT) (E.C. 2.6.2.2) and Alkaline Phosphatase (ALP) (E.C. 3.1.3.1) activity after exposure. The periwinkles were exposed to 10.40, 15.60, 21.00, 26.00 ml L(-1) and a control. The organs were removed on the sixth day and were prepared for enzymatic analysis. Enzyme activities were compared to the control value and between the toxicants. The effects of the toxicants on AST activity in the muscle and viscera were significantly different (p > 0.05) from the control value (137.50 ± 15.10 IU L(-1)). AST activity were raised more in petrol concentrations than the diesel concentrations in the muscle. The reverse was the case in the viscera at 15.60 ml L(-1) (227.50 ± 24.75 IU L(-1)). ALT activity in the muscle were not significant (p > 0.05) between the toxicant media. In the viscera, significant differences (p > 0.05) were observed in some of the concentrations with petrol showing higher activity. ALP activity in the muscle were not significant (p > 0.05) in both media, but were more elicited in the diesel concentrations. In the viscera, the activities of ALP were more pronounced in the petrol concentrations and were significant (p > 0.05) at the higher diesel concentrations. The exposure of Tympanotonus fuscatus to petrol and diesel concentrations caused changes in the enzymatic activities in the organism with those of petrol more pronounced than those of the diesel.

  19. On the existence of "L-threonine formate", "L-alanine lithium chloride" and "bis L-alanine lithium chloride" crystals.

    PubMed

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

    2013-03-15

    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.

  20. Post-Irradiation Study of the Alanine Dosimeter

    PubMed Central

    Desrosiers, Marc F.

    2014-01-01

    Post-irradiation stability of high-dose dosimeters has traditionally been an important measurement influence quantity. Though the exceptional stability of the alanine dosimeter response with time has rendered this factor a non-issue for routine work, the archival quality of the alanine dosimeter has not been characterized. Here the alanine pellet dosimeter response is measured up to seven years post-irradiation for a range of absorbed doses. This long-term study is accompanied by an examination of the environmental influence quantities (e.g., ambient light) on the relatively short-term (3–4 month) stability of both pellet and film commercial dosimeters. Both dosimeter types demonstrated exceptional stability in the short term and proved to be relatively insensitive to common influence quantities. The long-term data revealed a complex dose-dependent response trend. PMID:26601033

  1. Morphosynthesis of alanine mesocrystals by pH control.

    PubMed

    Ma, Yurong; Cölfen, Helmut; Antonietti, Markus

    2006-06-01

    Crystallization of DL-alanine is studied as a single polymorph model case to analyze the different modes of crystallization of polar organic molecules in absence of any structure directing additives. Depending on supersaturation, which is controlled either by temperature or by pH, and in the absence of additives, crystallization by mesoscale assembly of nanoparticles is found over a wide range of conditions, leading to so-called mesocrystals. This supplements the classical molecule-based crystallization mechanism, which is identified at lower supersaturations and at pH values away from the isoelectric point (IEP). The resulting alanine crystals are characterized by SEM, XRD, and single-crystal analysis. Time-resolved conductivity measurements and dynamic light scattering of the reaction solutions reveal information about precursor structures and reaction kinetics. A formation mechanism is proposed for the alanine mesocrystals. PMID:16771332

  2. 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.

  3. Tolerance of Arc repressor to multiple-alanine substitutions.

    PubMed

    Brown, B M; Sauer, R T

    1999-03-01

    Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with Tms equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure. PMID:10051581

  4. [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.

  5. 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.

  6. Differential regulation of alanine aminotransferase homologues by abiotic stresses in wheat (Triticum aestivum L.) seedlings.

    PubMed

    Kendziorek, Maria; Paszkowski, Andrzej; Zagdańska, Barbara

    2012-06-01

    Wheat (Triticum aestivum L.) seedlings contain four alanine aminotransferase (AlaAT) homologues. Two of them encode AlaAT enzymes, whereas two homologues act as glumate:glyoxylate aminotransferase (GGAT). To address the function of the distinct AlaAT homologues a comparative examination of the changes in transcript level together with the enzyme activity and alanine and glutamate content in wheat seedlings subjected to low oxygen availability, nitrogen and light deficiency has been studied. Shoots of wheat seedlings were more tolerant to hypoxia than the roots as judging on the basis of enzyme activity and transcript level. Hypoxia induced AlaAT1 earlier in roots than in shoots, while AlaAT2 and GGAT were unaffected. The increase in AlaAT activity lagged behind the increase in alanine content. Nitrogen deficiency has little effect on the activity of GGAT. In contrast, lower activity of AlaAT and the level of mRNA for AlaAT1 and AlaAT2 in wheat seedlings growing on a nitrogen-free medium seems to indicate that AlaAT is regulated by the availability of nitrogen. Both AlaAT and GGAT activities were present in etiolated wheat seedlings but their activity was half of that observed in light-grown seedlings. Exposure of etiolated seedlings to light caused an increase in enzyme activities and up-regulated GGAT1. It is proposed that hypoxia-induced AlaAT1 and light-induced peroxisomal GGAT1 appears to be crucial for the regulation of energy availability in plants grown under unfavourable environmental conditions. Key message In young wheat seedlings, both AlaAT and GGAT are down-regulated by nitrogen deficiency, whereas AlaAT1 is upregulated by hypoxia and GGAT1 by light.

  7. 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.

  8. 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

  9. 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

    2015-02-01

    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.

  10. 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.

  11. Effects of β-alanine administration on selected parameters of oxidative stress and phosphoryltransfer network in cerebral cortex and cerebellum of rats.

    PubMed

    Gemelli, Tanise; de Andrade, Rodrigo Binkowski; Rojas, Denise Bertin; Bonorino, Nariélle Ferner; Mazzola, Priscila Nicolao; Tortorelli, Lucas Silva; Funchal, Cláudia; Filho, Carlos Severo Dutra; Wannmacher, Clovis Milton Duval

    2013-08-01

    β-Alanine is a β-amino acid derivative of the degradation of pyrimidine uracil and precursor of the oxidative substrate acetyl-coenzyme A (acetyl-CoA). The accumulation of β-alanine occurs in β-alaninemia, an inborn error of metabolism. Patients with β-alaninemia may develop neurological abnormalities whose mechanisms are far from being understood. In this study we evaluated the effects of β-alanine administration on some parameters of oxidative stress and on creatine kinase, pyruvate kinase, and adenylate kinase in cerebral cortex and cerebellum of 21-day-old rats. The animals received three peritoneal injections of β-alanine (0.3 mg /g of body weight) and the controls received the same volume (10 μL/g of body weight) of saline solution (NaCl 0.85 %) at 3 h intervals. CSF levels of β-alanine increased five times, achieving 80 μM in the rats receiving the amino acid. The results of β-alanine administration in the parameters of oxidative stress were similar in both tissues studied: reduction of superoxide dismutase activity, increased oxidation of 2',7'-dihydrodichlorofluorescein, total content of sulfhydryl and catalase activity. However, the results of the phosphoryltransfer network enzymes were similar in all enzymes, but different in the tissues studied: the β-alanine administration was able to inhibit the enzyme pyruvate kinase, cytosolic creatine kinase, and adenylate kinase activities in cerebral cortex, and increase in cerebellum. In case this also occurs in the patients, these results suggest that oxidative stress and alteration of the phosphoryltransfer network may be involved in the pathophysiology of β-alaninemia. Moreover, the ingestion of β-alanine to improve muscular performance deserves more attention in respect to possible side-effects.

  12. 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

  13. Influence of alanine uptake on Staphylococcus aureus surface charge and its susceptibility to two cationic antibacterial agents, nisin and low molecular weight chitosan.

    PubMed

    Chen, Li-Chen; Chiang, Wen-Dee; Chen, Wei-Chiuan; Chen, Hui-Huang; Huang, Yao-Wen; Chen, Wei-Jung; Lin, Shih-Bin

    2012-12-15

    Low molecular weight chitosan (LMWC) and nisin, recognized as cationic antibacterial agents (CAAs), inhibit bacterial growth by interacting with the anionically charged cell wall. In this study, alanine uptake significantly reduced the anionic cell surface charge, as determined by the zeta potential (ZP) measurements, of Staphylococcus aureus, resulting from the incorporation of d-alanine into the cell wall. Minimum inhibitory concentration (MIC) tests and growth inhibition curves revealed that LMWC and nisin possessed inverse antibacterial activity against three strains of S. aureus, depending on the strains' net charge. A twofold reduction in the MIC value of nisin was obtained against S. aureus, inoculated in a 1.0% d- or l-alanine-augmented trypticase soy broth medium. A flocculation test demonstrated that neutralizing the anionic surface charge using d-alanine reduced the adsorption of S. aureus onto LMWC. Furthermore, the reduced surface net charge could enhance the colonization capacity of S. aureus on glass. PMID:22980819

  14. 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.

  15. 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).

  16. 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.

  17. 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.

  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. Beta-alanine supplementation in high-intensity exercise.

    PubMed

    Harris, Roger C; Sale, Craig

    2012-01-01

    Glycolysis involves the oxidation of two neutral hydroxyl groups on each glycosyl (or glucosyl) unit metabolised, yielding two carboxylic acid groups. During low-intensity exercise these, along with the remainder of the carbon skeleton, are further oxidised to CO(2) and water. But during high-intensity exercise a major portion (and where blood flow is impaired, then most) is accumulated as lactate anions and H(+). The accumulation of H(+) has deleterious effects on muscle function, ultimately impairing force production and contributing to fatigue. Regulation of intracellular pH is achieved over time by export of H(+) out of the muscle, although physicochemical buffers in the muscle provide the first line of defence against H(+) accumulation. In order to be effective during high-intensity exercise, buffers need to be present in high concentrations in muscle and have pK(a)s within the intracellular exercise pH transit range. Carnosine (β-alanyl-L-histidine) is ideal for this role given that it occurs in millimolar concentrations within the skeletal muscle and has a pK(a) of 6.83. Carnosine is a cytoplasmic dipeptide formed by bonding histidine and β-alanine in a reaction catalysed by carnosine synthase, although it is the availability of β-alanine, obtained in small amounts from hepatic synthesis and potentially in greater amounts from the diet that is limiting to synthesis. Increasing muscle carnosine through increased dietary intake of β-alanine will increase the intracellular buffering capacity, which in turn might be expected to increase high-intensity exercise capacity and performance where this is pH limited. In this study we review the role of muscle carnosine as an H(+) buffer, the regulation of muscle carnosine by β-alanine, and the available evidence relating to the effects of β-alanine supplementation on muscle carnosine synthesis and the subsequent effects of this on high-intensity exercise capacity and performance.

  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. 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. PMID:27362442

  2. Alanine radicals, part 3: properties of the components contributing to the EPR spectrum of X-irradiated alanine dosimeters.

    PubMed

    Malinen, Eirik; Heydari, Mojgan Z; Sagstuen, Einar; Hole, Eli O

    2003-01-01

    The amino acid l-alpha-alanine has attracted considerable interest for use in radiation dosimetry and has been formally accepted as a secondary standard for high-dose and transfer dosimetry. Recent results have shown that the alanine EPR spectrum consists of contributions from three different radicals. A set of benchmark spectra describing the essential spectral features of these three radical components was used for reconstructions of the experimental spectra. In the present work, these basis spectra have been used to investigate the differential effects of variations in radiation doses and microwave power, as well as the dependence upon temperature annealing and UV illumination. The results presented here, based solely on relatively low-energy (60-80 keV) X rays, indicate that the three components behave very similarly with respect to radiation dose at room temperature. However, with respect to the thermal annealing/fading behavior and microwave power saturation properties, the three species behave significantly differently. It is concluded that even if it is now realized that three different radicals contribute to the composite EPR alanine spectrum, this has a minor impact on the established protocols for present-day applications (high-dose) of EPR/alanine dosimetry. However, some care should be exercised when e.g. constructing calibration curves, since fading and power saturation behavior may vary over the dose range in question. New results from UV-illumination experiments suggest a possible procedure for experimental spectral separation of the EPR signals due to the three radicals.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. ESR kinetics study of the decay of low-temperature radicals in glycine and. beta. -alanine

    SciTech Connect

    Smith, C.J.; Poole, C.P. Jr.; Farach, H.A.

    1981-01-15

    Monocrystals of glycine and ..beta..-alanine have been observed by electron spin resonance (ESR) after x-irradiation near 77 /sup 0/K. The rate constants of the decay of one signal in each have been determined over the ranges 127 /sup 0/--148 /sup 0/K and 98 /sup 0/--188 /sup 0/K, respectively. Both obey first-order kinetics with activation energies of approx.7.0 and approx.2.6 kcal/mole, respectively, and pre-exponential factors of 6.4 x 10/sup 8/ and 15 Hz, respectively.

  8. 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.

  9. Study on the EPR/dosimetric properties of some substituted alanines

    NASA Astrophysics Data System (ADS)

    Gancheva, Veselka; Sagstuen, Einar; Yordanov, Nicola D.

    2006-02-01

    Polycrystalline phenyl-alanine and perdeuterated L- α-alanine ( L- α-alanine-d 4) were studied as potential high-energy radiation-sensitive materials (RSM) for solid state/EPR dosimetry. It was found that phenyl-alanine exhibits a linear dose response in the dose region 0.1-17 kGy. However, phenyl-alanine is about 10 times less sensitive to γ-irradiation than standard L- α-alanine irradiated at the same doses. Moreover, the EPR response from phenyl-alanine is unstable and, independent of the absorbed dose, decreases by about 50% within 20 days after irradiation upon storage at room temperature. γ-irradiated polycrystalline perdeuterated L- α-alanine (CD 3CD(NH 2)COOH) has not previously been studied at room temperature by EPR spectroscopy. The first part of the present analysis was with respect to the structure of the EPR spectrum. By spectrum simulations, the presence of at least two radiation induced free radicals, R 1=CH 3C •(H)COOH and R 2=H 3N +-C •(CH 3)COO -, was confirmed very clearly. Both these radicals were suggested previously from EPR and ENDOR studies of standard alanine crystals. The further investigations into the potential use of alanine-d 4 as RSM, after choosing optimal EPR spectrometer settings parameters for this purpose, show that it is ca. two times more sensitive than standard L- α-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. Accumulation of D- vs. L-isomers of alanine and leucine in rat prostatic adenocarcinoma

    SciTech Connect

    Conti, P.S.; Schmall, B.; Bigler, R.E.; Zanzonico, P.B.; Kleinert, E.; Whitmore, W.F. Jr.

    1985-05-01

    It has been reported that tumor tissue may accumulate some D-amino acids preferentially over the L-isomers. In order to investigate the potential use of carbon-11 labeled amino acid isomers for in vivo tumor studies with positron emission tomography in patients, the tissue distributions of alanine and leucine, substrates for the A-type and L-type amino acid transport systems, respectively, were studied in Copenhagen rates bearing the Dunning R3327G prostatic adenocarcinoma. The authors have previously reported differences in the accumulation of A-type vs. L-type amino acids in rat prostatic adenocarcinoma and normal tissues. All compounds were labeled with C-14 in the carboxyl position with specific activities of 30.0-56.6 mCi/mmol. Higher levels of C-14 activity (Relative Concentration (RC)=dpm found per gm tissue + dpm inject per gm animal mass) were observed in tumor tissue using D-alanine (0.71) compared to L- (0.21) or DL-alanine (0.27) at 45 min post-injection. While tumor/prostate and tumor/liver ratios were above 2 for all three substrates, tumor/blood and tumor/muscle were above one for only the D-isomer. Comparisons made with D-, L-, and DL-leucine also demonstrated a higher level of RC in tumor tissue with the D-isomer (0.84) vs. the L-(0.66) and DL-leucine (0.63). In this case, however, tumor/blood, tumor/prostate, and tumor/muscle ratios were above one for all three substrates, while tumor/liver ratios were below one. These results support the observation of a preferential accumulation of D-amino acids in tumor tissue over the natural L-isomers. Observed differences in the accumulation of the isomers in normal tissues are discussed.

  12. Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

    PubMed

    Park, Duck Hwan; Mirabella, Rossana; Bronstein, Philip A; Preston, Gail M; Haring, Michel A; Lim, Chun Keun; Collmer, Alan; Schuurink, Robert C

    2010-10-01

    Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

  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. PMID:27509858

  14. 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.

  15. Alanine-dependent reactions of 5'-deoxypyridoxal in water.

    PubMed

    Go, Maybelle K; Richard, John P

    2008-12-01

    The non-enzymatic reaction of 5'-deoxypyridoxal (DPL) with l-alanine in water at 25 degrees C was investigated. DPL reacts with alanine to form an imine, which then undergoes deprotonation at the alpha-amino carbon of alanine to form a resonance delocalized DPL-stabilized carbanion. At early reaction times the only detectable products are pyruvate and the dimeric species formed by addition of the alpha-pyridine stabilized carbanion to DPL. No Claisen-type products of addition of the alpha-amino carbanion to DPL, as was previously reported to form from the reaction between DPL and glycine [K. Toth, T.L. Amyes, J.P. Richard, J.P.G. Malthouse, M.E. Ni Beilliu, J. Am. Chem. Soc. 126 (2004) 10538-10539], are observed. The electrophile reacts instead at the alpha-pyridyl carbon. This dimer is in chemical equilibrium with reactants. At longer reaction times about 50% of DPL is converted to 5'-deoxypyridoxamine, the thermodynamically favored product of formal transamination of DPL.

  16. Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH.

    PubMed

    Abee, T; van der Wal, F J; Hellingwerf, K J; Konings, W N

    1989-09-01

    The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986). The results presented in this paper offer an explanation for these findings. Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system. The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3). The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system. Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles. This indicates that the H+/lactose symport system is active without electron transfer. Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport. Uptake of TMG and AIB is regulated by the internal pH. The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB. At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8. In a previous study, we found that K+ plays an essential role in regulating the internal pH (T. Abee, K. J. Hellingwerf, and W

  17. Role of p38 mitogen-activated protein kinase in Kupffer cell secretion of the proinflammatory cytokines after burn trauma.

    PubMed

    Chen, Xu-Lin; Xia, Zhao-Fan; Wei, Duo; Han, Sheng; Ben, Dao-Feng; Wang, Guang-Qing

    2003-09-01

    This study was designed to investigate the role of p38 mitogen-activated protein (MAP) kinase on Kupffer cells (KCs) secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and hepatic injury following burn trauma. Sprague-Dawley rats were randomized into four groups: (1) sham burn rats given vehicle, (2) sham burn rats given the p38 MAP kinase inhibitor SB203580 (10mg/kg i.v., 15min and 12h after sham burn), (3) rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and (4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24h post-burn to examine plasma aspartate transaminase (AST) and alanine transaminase (ALT) and KCs were isolated. The KCs secretion of TNF-alpha and IL-1beta and p38 MAP kinase activity (by Western blot analysis) were also examined. These studies showed by more significant activation of p38 MAP kinase in KCs harvested from burn rats than from shams. Burn trauma resulted in hepatic dysfunction and promoted KCs secretion of TNF-alpha and IL-1beta. SB203580 inhibited p38 MAP kinase activity, reduced KCs secretion of proinflammatory cytokines, and alleviated burn-mediated hepatic dysfunction. These data suggest p38 MAP kinase activation is one important aspect of the signaling event that may mediate the KCs secretion of proinflammatory cytokines TNF-alpha and IL-1beta following burn trauma.

  18. 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

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

    PubMed

    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

  20. 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.

  1. 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.

  2. Biosynthesis of prodigiosin by non-proliferating wild-type Serratia marcescens and mutants deficient in catabolism of alanine, histidine, and proline.

    PubMed

    Lim, D V; Qadri, S M; Nichols, C; Williams, R P

    1977-01-01

    Mutants of Serratia marcescens Nima, designated as Aut, Hut, or Put, did not utilize L-alanine, L-histidine, or L-proline, respectively, as a sole carbon source but did utilize other amino acids or glycerol as carbon sources. The bacteria were permeable to alanine, histidine, and proline but lacked the enzymes responsible for degradation of these amino acids. The Aut mutant contained no L-alanine dehydrogenase activity, whereas the Hut and Put mutants contained only 7 and 4% of the histidase and proline oxidase activities, respectively, found in the wild-type strain. Rates of oxygen uptake and protein synthesis were significantly lower when the mutants were incubated in the presence of amino acids they could not degrade. Studies of L-[14C]alanine, L-[14C]histidine, and L-[14C]proline incorporation into prodigiosin synthesized by these mutants and the wild-type strain revealed that proline was incorporated intact, whereas all of alanine except the carboxyl group was incorporated into the pigment molecule. Histidine did not enter prodigiosin directly. These data suggested that the presence of unique biosynthetic pathways, independent of primary metabolism, leads to formation of prodigiosin from specific amino acids.

  3. Effect of Syzygium calophyllifolium Walp. seed extract on transaminases and phosphatases in alloxan induced diabetic rats

    PubMed Central

    Gurusamy, K.; Kokilavani, R; Teepa, K. S. Ananta

    2007-01-01

    The antidiabetic effect of an aqueous seed extract of Syzygium calophyllifolium Walp. was studied in alloxan induced diabetic rats. Changes in serum and tissue AST, ALT LDH, ACP and ALP, enzymes activities in alloxan induced rats were studied and found to be reversed by the oral administration of S.calophyllifolium seed extract. PMID:22557266

  4. Hepatoprotective activity of Achyrocline satureioides(Lam) D. C.

    PubMed

    Kadarian, C; Broussalis, A M; Miño, J; Lopez, P; Gorzalczany, S; Ferraro, G; Acevedo, C

    2002-01-01

    Aerial parts of Achyrocline satureioides(Lam) D. C. (Asteraceae) are used in folk medicine as infusions or decoctions for the management of several diseases including gastrointestinal and hepatic disorders. These data and the presence of flavonoids and caffeoyl derivatives have led us to study its hepatoprotective and choleretic activities. The hepatoprotective activity was evaluated in the bromobenzene- (BB-) induced hepatotoxicity model in mice through the measurement of the serum levels of alanine-aminotransferase (ALT) and aspartate transaminase (AST), thiobarbituric acid reacting substances (TBARS) and glutathione levels. The aqueous extract of the aerial parts of A. satureioides administered before BB, at the dose of 300 mg kg(-1)p.o., demonstrated significant inhibition (P< 0.01) in the BB increase of liver ALT and AST and in the BB-induced increase of liver TBARS content. Also it was able to significantly increase (P< 0.05) the depleted levels of liver glutathione. In addition, at the same dose, a significant increase (P< 0.01) in the bile flow of rats was found. The results obtained with the aqueous extract of A. satureioides support its use in popular medicine as a hepatoprotective and digestive agent, and the effects might be mediated through the antioxidant and choleretic activities.

  5. 6-Arylpyrido[2,3-d]pyrimidines as Novel ATP-Competitive Inhibitors of Bacterial D-Alanine:D-Alanine Ligase

    PubMed Central

    Škedelj, Veronika; Arsovska, Emilija; Tomašić, Tihomir; Kroflič, Ana; Hodnik, Vesna; Hrast, Martina; Bešter-Rogač, Marija; Anderluh, Gregor; Gobec, Stanislav; Bostock, Julieanne; Chopra, Ian; O'Neill, Alex J.; Randall, Christopher; Zega, Anamarija

    2012-01-01

    Background ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl. Methodology/Principal Findings Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido[2,3-d]pyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido[2,3-d]pyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli. Conclusions/Significance We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety. PMID:22876277

  6. Ergogenic effects of β-alanine and carnosine: proposed future research to quantify their efficacy.

    PubMed

    Caruso, John; Charles, Jessica; Unruh, Kayla; Giebel, Rachel; Learmonth, Lexis; Potter, William

    2012-07-01

    β-alanine is an amino acid that, when combined with histidine, forms the dipeptide carnosine within skeletal muscle. Carnosine and β-alanine each have multiple purposes within the human body; this review focuses on their roles as ergogenic aids to exercise performance and suggests how to best quantify the former's merits as a buffer. Carnosine normally makes a small contribution to a cell's total buffer capacity; yet β-alanine supplementation raises intracellular carnosine concentrations that in turn improve a muscle's ability to buffer protons. Numerous studies assessed the impact of oral β-alanine intake on muscle carnosine levels and exercise performance. β-alanine may best act as an ergogenic aid when metabolic acidosis is the primary factor for compromised exercise performance. Blood lactate kinetics, whereby the concentration of the metabolite is measured as it enters and leaves the vasculature over time, affords the best opportunity to assess the merits of β-alanine supplementation's ergogenic effect. Optimal β-alanine dosages have not been determined for persons of different ages, genders and nutritional/health conditions. Doses as high as 6.4 g day(-1), for ten weeks have been administered to healthy subjects. Paraesthesia is to date the only side effect from oral β-alanine ingestion. The severity and duration of paraesthesia episodes are dose-dependent. It may be unwise for persons with a history of paraesthesia to ingest β-alanine. As for any supplement, caution should be exercised with β-alanine supplementation.

  7. Acute, sub-chronic oral toxicity studies and evaluation of antiulcer activity of Sooktyn in experimental animals

    PubMed Central

    Chandra, Phool; Sachan, Neetu; Kishore, Kamal; Ghosh, Ashoke Kumar

    2012-01-01

    Sooktyn (SKN), mineralo-herbal drug which is being used largely by the patients for its extremely good therapeutic value to treat the gastric ulcers. The present study was undertaken to evaluate the toxicity studies and antiulcer activity of SKN. Acute and sub-chronic toxicities were studied in male and female Wistar rats. A single acute SKN of 2 000 mg/kg was administered by oral gavage for acute toxicity. Sub-chronic doses were 400 and 800 mg/kg/day. The major toxicological end points examined included animal body weight and food intake, selected tissue weights, and detailed gross necropsy. In addition, we examined blood elements: hematocrit, hemoglobin concentration, erythrocyte count, total leukocyte count and MCH, MCHC and platelets as well as biochemical parameters: urea, sugar, alanine transaminase, aspartate transaminase, alkaline phosphatase, total proteins, and creatinine. Also, anti-ulcer activity was carried out by employing indomethacin, ethanol, pylorus ligation, and hypothermic-stress-induced ulcer models. LD50 may be greater than 2 000 mg/kg (orally) for SKN and there were no signs of toxicity on 28 days sub-chronic oral administration of 400 and 800 mg/kg of SKN in rats on the basis of blood elements and biochemical parameters. The ulcer indices decrease in all ulcer models with 66.62%, 61.24%, 80.18%, and 74.76% in indomethacin, ethanol, pylorus ligation, and hypothermic-stress-induced ulcer models, respectively. The results suggest that SKN has no signs of toxicity at 2 000 mg/kg body weight of rats orally; sub-chronically. The drug is safe and has antiulcer activity. PMID:22837960

  8. Performance effects of acute β-alanine induced paresthesia in competitive cyclists.

    PubMed

    Bellinger, Phillip M; Minahan, Clare L

    2016-01-01

    β-alanine is a common ingredient in supplements consumed by athletes. Indeed, athletes may believe that the β-alanine induced paresthesia, experienced shortly after ingestion, is associated with its ergogenic effect despite no scientific mechanism supporting this notion. The present study examined changes in cycling performance under conditions of β-alanine induced paresthesia. Eight competitive cyclists (VO2max = 61.8 ± 4.2 mL·kg·min(-1)) performed three practices, one baseline and four experimental trials. The experimental trials comprised a 1-km cycling time trial under four conditions with varying information (i.e., athlete informed β-alanine or placebo) and supplement content (athlete received β-alanine or placebo) delivered to the cyclist: informed β-alanine/received β-alanine, informed placebo/received β-alanine, informed β-alanine/received placebo and informed placebo/received placebo. Questionnaires were undertaken exploring the cyclists' experience of the effects of the experimental conditions. A possibly likely increase in mean power was associated with conditions in which β-alanine was administered (±95% CL: 2.2% ± 4.0%), but these results were inconclusive for performance enhancement (p = 0.32, effect size = 0.18, smallest worthwhile change = 56% beneficial). A possibly harmful effect was observed when cyclists were correctly informed that they had ingested a placebo (-1.0% ± 1.9%). Questionnaire data suggested that β-alanine ingestion resulted in evident sensory side effects and six cyclists reported placebo effects. Acute ingestion of β-alanine is not associated with improved 1-km TT performance in competitive cyclists. These findings are in contrast to the athlete's "belief" as cyclists reported improved energy and the ability to sustain a higher power output under conditions of β-alanine induced paresthesia.

  9. Structural Insight into the Inhibition of Human Kynurenine Aminotransferase I/Glutamine Transaminase K

    SciTech Connect

    Han, Q.; Robinson, H; Cai, T; Tagle, D; Li, J

    2009-01-01

    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 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.

  10. 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.

  11. 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

  12. Use of the entire spectrum of irradiated alanine for dosimetry.

    PubMed

    Dolo, J M; Moignau, F

    2005-02-01

    Alanine is an amino acid commonly used in ESR dosimetry as a reference detector. The classic approach for the measurement of irradiated samples is to determine the amplitude of the central peak of the first derivative spectrum. It is generally considered that this technique represents the best and most reproducible solution for achieving an accurate proportionality between the concentration of free radicals inside the resonant cavity, characterized by the amplitude, and the dose. It is also accepted that this central peak corresponds to the free radical CH3CHCOO-. The hyperfine structure of this radical in the spectrum shows five main peaks with the approximate ratios 1:4:6:4:1 as regards coupling. This paper presents another approach featuring analysis of the entire spectrum: (i) ratios of identified peaks, (ii) ratio variation vs time with regard to several parameters affecting fading. These variations in the alanine spectrum are probably correlated with the variation of the concentrations of different free radical species. These variations and their positions in the spectrum are very important constraints that increase the uncertainty of this type of measurement.

  13. 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.

  14. 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.

  15. 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.

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

    PubMed Central

    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. PMID:26218070

  17. Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production▿

    PubMed Central

    Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Pozo-Dengra, Joaquín; Tessaro, Davide; Servi, Stefano; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier

    2009-01-01

    An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcarAt) has been characterized. βcarAt is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn2+, Ni2+, and Co2+. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcarAt also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcarAt is able to produce monosubstituted β2- and β3-amino acids, showing better catalytic efficiency (kcat/Km) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcarAt an outstanding candidate for application in the biotechnology industry. PMID:19011069

  18. Potential application of N-carbamoyl-beta-alanine amidohydrolase from Agrobacterium tumefaciens C58 for beta-amino acid production.

    PubMed

    Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Pozo-Dengra, Joaquín; Tessaro, Davide; Servi, Stefano; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier

    2009-01-01

    An N-carbamoyl-beta-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (beta car(At)) has been characterized. Beta car(At) is most active at 30 degrees C and pH 8.0 with N-carbamoyl-beta-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn(2+), Ni(2+), and Co(2+). The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-alpha-, -beta-, -gamma-, and -delta-amino acids, with the greatest catalytic efficiency for N-carbamoyl-beta-alanine. Beta car(At) also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the beta-amino acids taurine and ciliatine, respectively. Beta car(At) is able to produce monosubstituted beta(2)- and beta(3)-amino acids, showing better catalytic efficiency (k(cat)/K(m)) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-beta-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make beta car(At) an outstanding candidate for application in the biotechnology industry.

  19. 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.

  20. Synthesis of thiocarbamide derivatives of. beta. -alanine and aminalon and study of their antiradiation action

    SciTech Connect

    Golubev, A.A.; Shlykov, Yu.V.; Mandrugin, A.A.; Semenenko, M.N.; Fedoseev, V.M.; Dontsova, G.V.; Rakhmanina, O.N.; Konstantinova, M.M.

    1987-01-01

    The authors have synthesized the carboxyl derivatives of S-(2-Amino-ethyl)isothiourea (AET) and S-(3-Aminopropyl)isothiourea (3-APT), the dihydrobromides of S-(1-carboxy-2-aminoethyl)- and S-(1-carboxy-3-aminopropyl)isothioureas, and studied their toxicity and antiradiation effectiveness. Their work is based on data for the high radiation-protective activity of the corresponding aminoalkylisothioureas and on the fact that the introduction of the carboxylic group into the molecule influences the pharmacological properties of a compound. The starting compound for the preparation of S(1-carboxy-2-aminoethyl)isothiourea was beta-alanine. Mice were used in the investigations and in determining the toxicity of the compounds, the amounts of LD/sub 16/, LD/sub 50/, and LD/sub 84/ are given. In studying the anti-radiation activity, the mice were subjected to the action of /sup 60/Co-gamma rays.

  1. (S)-Styryl-α-alanine used to probe the intermolecular mechanism of an intramolecular MIO-aminomutase.

    PubMed

    Wanninayake, Udayanga; Deporre, Yvonne; Ondari, Mark; Walker, Kevin D

    2011-11-22

    A Taxus canadensis phenylalanine aminomutase (TcPAM) catalyzes the isomerization of (S)-α- to (R)-β-phenylalanine, making (E)-cinnamate (~10%) as a byproduct at steady state. A currently accepted mechanism for TcPAM suggests that the amino group is transferred from the substrate to a prosthetic group comprised of an amino acid triad in the active site and then principally rebinds to the carbon skeleton of the cinnamate intermediate to complete the α-β isomerization. In contrast, when (S)-styryl-α-alanine is used as a substrate, TcPAM produces (2E,4E)-styrylacrylate as the major product (>99%) and (R)-styryl-β-alanine (<1%). Comparison of the rates of conversion of the natural substrate (S)-α-phenylalanine and (S)-styryl-α-alanine to their corresponding products (k(cat) values of 0.053 ± 0.001 and 0.082 ± 0.002 s(-1), respectively) catalyzed by TcPAM suggests that the amino group resides in the active site longer than styrylacrylate. To demonstrate this principle, inhibition constants (K(I)) for selected acrylates ranging from 0.6 to 106 μM were obtained, and each had a lower K(I) compared to that of (2E,4E)-styrylacrylate (337 ± 12 μM). Evaluation of the inhibition constants and the rates at which both the α/β-amino acids (between 7 and 80% yield) and styrylacrylate were made from a corresponding arylacrylate and styryl-α-alanine, respectively, by TcPAM catalysis revealed that the reaction progress was largely dependent on the K(I) of the acrylate. Bicyclic amino donor substrates also transferred their amino groups to an arylacrylate, demonstrating for the first time that ring-fused amino acids are productive substrates in the TcPAM-catalyzed reaction.

  2. 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.

  3. (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

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

    PubMed

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

    2009-11-01

    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.

  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. 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.

  7. Use of alanine-silicone pellets for electron paramagnetic resonance gamma dosimetry

    SciTech Connect

    Flores, J.; Galindo, S. )

    1991-03-01

    Silicone is proposed as an alternative binding substance in the production of D-L alanine pellets used in electron paramagnetic resonance (EPR) dosimetry of gamma rays. The dosimeters are manufactured at room temperature, making the production simple. Examination by EPR silicone-alanine pellets irradiated with 60Co gamma rays in the dose range 10 to 10(6) Gy shows that the proposed silicone binder does not affect typical alanine dose-response curves. Thermal stability of the pellets below 40 degrees C is good, but their pre-dose EPR signal amplitude is slightly higher than for nonirradiated alanine.

  8. Effect of β-alanine supplementation on high-intensity exercise performance.

    PubMed

    Harris, Roger C; Stellingwerff, Trent

    2013-01-01

    Carnosine is a dipeptide of β-alanine and L-histidine found in high concentrations in skeletal muscle. Combined with β-alanine, the pKa of the histidine imidazole ring is raised to ∼6.8, placing it within the muscle intracellular pH high-intensity exercise transit range. Combination with β-alanine renders the dipeptide inert to intracellular enzymic hydrolysis and blocks the histidinyl residue from participation in proteogenesis, thus making it an ideal, stable intracellular buffer. For vegetarians, synthesis is limited by β-alanine availability; for meat-eaters, hepatic synthesis is supplemented with β-alanine from the hydrolysis of dietary carnosine. Direct oral β-alanine supplementation will compensate for low meat and fish intake, significantly raising the muscle carnosine concentration. This is best achieved with a sustained-release formulation of β-alanine to avoid paresthesia symptoms and decreasing urinary spillover. In humans, increased levels of carnosine through β-alanine supplementation have been shown to increase exercise capacity and performance of several types, particularly where the high-intensity exercise range is 1-4 min. β-Alanine supplementation is used by athletes competing in high-intensity track and field cycling, rowing, swimming events and other competitions.

  9. Effect of β-alanine supplementation on high-intensity exercise performance.

    PubMed

    Harris, Roger C; Stellingwerff, Trent

    2013-01-01

    Carnosine is a dipeptide of β-alanine and L-histidine found in high concentrations in skeletal muscle. Combined with β-alanine, the pKa of the histidine imidazole ring is raised to ∼6.8, placing it within the muscle intracellular pH high-intensity exercise transit range. Combination with β-alanine renders the dipeptide inert to intracellular enzymic hydrolysis and blocks the histidinyl residue from participation in proteogenesis, thus making it an ideal, stable intracellular buffer. For vegetarians, synthesis is limited by β-alanine availability; for meat-eaters, hepatic synthesis is supplemented with β-alanine from the hydrolysis of dietary carnosine. Direct oral β-alanine supplementation will compensate for low meat and fish intake, significantly raising the muscle carnosine concentration. This is best achieved with a sustained-release formulation of β-alanine to avoid paresthesia symptoms and decreasing urinary spillover. In humans, increased levels of carnosine through β-alanine supplementation have been shown to increase exercise capacity and performance of several types, particularly where the high-intensity exercise range is 1-4 min. β-Alanine supplementation is used by athletes competing in high-intensity track and field cycling, rowing, swimming events and other competitions. PMID:23899755

  10. Some liver functions in the Oriental hornet (Vespa orientalis) are performed in its cuticle: exposure to UV light influences these activities.

    PubMed

    Plotkin, Marian; Volynchik, Stanislav; Itzhaky, Dganit; Lis, Monica; Bergman, David J; Ishay, Jacob S

    2009-06-01

    The Oriental hornet Vespa orientalis (Hymenoptera, Vespinae) coordinates its daily activities (e.g. flights out of the nest associated with digging activities and removal of the dug soil from the nest) with the amount of insolation. Thus, the stronger the insolation, the more intense the flight activity and vise versa. The hornet's cuticle bears a few yellow stripes interposed among brown parts of the gastral cuticle. These yellow stripes are composed of two elements, namely, a transparent cuticle and underneath it a layer of yellow granules. When the hornets are exposed to UV light, the layer containing the yellow granules is less active than in hornets kept in the dark. This diminished activity entails a lower production of glucose as well as of several enzymes prevalent also in the liver of mammals, like creatine kinase, alanine aminotransferase, aspartate transaminase. Thus solar irradiation stimulates and produces a change in the metabolic activities of the hornet. The fact that hornets link their flight activity with the insolation leads us to speculate that the sun contributes energetically to the hornet's activity.

  11. 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.

  12. Adiponectin-derived active peptide ADP355 exerts anti-inflammatory and anti-fibrotic activities in thioacetamide-induced liver injury

    PubMed Central

    Wang, Huafeng; Zhang, Huan; Zhang, Zimu; Huang, Biao; Cheng, Xixi; Wang, Dan; la Gahu, Zha; Xue, Zhenyi; Da, Yurong; Li, Daiqing; Yao, Zhi; Gao, Fei; Xu, Aimin; Zhang, Rongxin

    2016-01-01

    Adiponectin is an adipocyte-derived circulating protein with beneficial effects on injured livers. Adiponectin-deficient (adipo(−/−)) mice develop enhanced liver fibrosis, suggesting that adiponectin could be a therapeutic target for liver injury. In the present study, we investigated the protective role of ADP355, an adiponectin-based active short peptide, in thioacetamide (TAA)-induced acute injury and chronic liver fibrosis in mice. ADP355 remarkably reduced TAA-induced necroinflammation and liver fibrosis. ADP355 treatment increased liver glycogen, decreased serum alanine transaminase and alkaline phosphatase activity, and promoted body weight gain, hyper-proliferation and hypo-apoptosis. In addition, ADP355 administration suppressed the TAA-induced activation of hepatic stellate cells and macrophages in the liver. These were associated with the inactivation of TGF-β1/SMAD2 signaling and the promotion of AMPK and STAT3 signaling. Sensitivity of adipo(−/−) mice to chronic liver injury was decreased with ADP355. In conclusion, ADP355 could mimic adiponectin’s action and may be suitable for the preclinical or clinical therapy of chronic liver injury. PMID:26777428

  13. Utility of Ultrasound, Transaminases, and Visual Inspection to Assess Nonalcoholic Fatty Liver Disease in Bariatric Surgery Patients

    PubMed Central

    Petrick, Anthony; Wood, G. Craig; Still, Christopher D.; Strodel, William E.; Gabrielsen, John; Rolston, David; Chu, Xin; Argyropoulos, George; Ibele, Anna; Gerhard, Glenn S.

    2016-01-01

    Background Nonalcoholic fatty liver disease (NAFLD) is common in adults with extreme obesity and can impact long-term health and survival. Liver biopsy is the only accurate test for diagnosis and staging, but is invasive and costly. Non-invasive testing offers an attractive alternate, but the overall accuracy remains a significant issue. This study was conducted to determine the accuracy and clinical utility of preoperative ultrasound and liver transaminase levels, as well as intra-operative hepatic visual inspection, for assessing presence of NAFLD as confirmed by hepatic histology. Methods Data was collected prospectively from 580 morbidly obese adult patients who underwent Roux-en-Y gastric bypass surgery with intraoperative wedge biopsy between January 2004 and February 2009. Complete data for ultrasound, ALT and AST levels, and documented visual inspection was available for 513 patients. Results The prevalence of NAFLD was 69 % and that of NASH was 32 %. The individual non-invasive clinical assessments demonstrated low sensitivity, specificity, and accuracy for detecting the presence of steatosis, steatohepatitis, or fibrosis. The combination of normal or abnormal results for all tests improved predictive utility. Abnormal tests with all three assessments had a sensitivity of 95–98 % and a specificity of 28–48 % for major histologic findings in NAFLD/NASH. Normal tests with all three assessments had a sensitivity of 12–22 % and a specificity of 89–97 % for major histologic findings in NAFLD/NASH. Conclusions Although individual clinical tests for NAFLD have limited accuracy, the use of combined clinical tests may prove useful. PMID:26003548

  14. 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.

  15. Effects of beta-alanine supplementation on sprint endurance.

    PubMed

    Jagim, Andrew R; Wright, Glenn A; Brice, A Glenn; Doberstein, Scott T

    2013-02-01

    Recent research has shown that beta-alanine (BA) supplementation can increase intramuscular carnosine levels. Carnosine is an intramuscular buffer, and it has been linked to improvements in performance, specifically during bouts of high-intensity exercise that are likely limited by muscle acidosis. Therefore, the purpose of this study was to examine the effect of BA supplementation on sprint endurance at 2 different supramaximal intensities. Twenty-one anaerobically trained (rugby players [n = 4], wrestlers [n = 11], and recreationally strength trained athletes [n = 6]) college-aged men participated in a double-blind, placebo controlled study. The subjects performed an incremental VO2max test and 2 sprint to exhaustion tests set at 115 and 140% of their VO2max on a motorized treadmill before (PRE) and after (POST) a 5-week supplementation period. During this time, the subjects ingested either a BA supplement or placebo (PLA) with meals. The subjects ingested 4 g·d(-1) of BA or PLA during the first week and 6 g·d(-1) the following 4 weeks. Capillary blood samples were taken before and after each sprint to determine blood lactate response to the sprint exercise. No significant group (BA, PLA) × intensity (115%, 140%; p = 0.60), group by time (PRE, POST; p = 0.72), or group × intensity × time (p = 0.74) interactions were observed for time to exhaustion. In addition, similar nonsignificant observations were made for lactate response to the sprints (group × intensity, p = 0.43; group × time, p = 0.33, group × intensity × time, p = 0.56). From the results of this study, it was concluded that beta-alanine supplementation did not have a significant effect on sprint endurance at supramaximal intensities.

  16. Radiation dose measurements with alanine/agarose gel and thin alanine films around a 192Ir brachytherapy source, using ESR spectroscopy.

    PubMed

    Olsson, S; Bergstrand, E S; Carlsson, A K; Hole, E O; Lund, E

    2002-04-21

    Alanine/agarose gel and alanine films in stacks have been used for measurements of absorbed dose around an HDR 192Ir source in a vaginal cylinder-applicator, with and without a 180 degrees tungsten shield. The gel and the films were analysed by means of ESR spectroscopy and calibrated against an ion chamber in a 4 MV photon beam to obtain absolute dose values. The gel serves as both dosimeter and phantom material, and the thin (130 microm) films are used to achieve an improved spatial resolution in the dose estimations. Experimental values were compared with Monte Carlo simulations using two different codes. Results from the measurements generally agree with the simulations to within 5%, for both the alanine/agarose gel and the alanine films.

  17. 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.

  18. 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.

  19. 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

  20. 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.

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

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  3. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  4. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  5. 40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)(4), (c)(4) (where N = 100). The requirement of 40 CFR 721.91(a)(4) that the amount of the substance... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical...

  6. 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

  7. 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.

  8. Thermal decomposition behavior of potassium and sodium jarosite synthesized in the presence of methylamine and alanine

    SciTech Connect

    J. Michelle Kotler; Nancy W. Hinman; C. Doc Richardson; Jill R. Scott

    2010-10-01

    Biomolecules, methylamine and alanine, found associated with natural jarosite samples peaked the interest of astrobiologists and planetary geologists. How the biomolecules are associated with jarosite remains unclear although the mechanism could be important for detecting biosignatures in the rock record on Earth and other planets. A series of thermal gravimetric experiments using synthetic K-jarosite and Na-jarosite were conducted to determine if thermal analysis could differentiate physical mixtures of alanine and methylamine with jarosite from samples where the methylamine or alanine was incorporated into the synthesis procedure. Physical mixtures and synthetic experiments with methylamine and alanine could be differentiated from one another and from the standards by thermal analysis for both the K-jarosite and Na-jarosite end-member suites. Changes included shifts in on-set temperatures, total temperature changes from on-set to final, and the presence of indicator peaks for methylamine and alanine in the physical mixture experiments.

  9. 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

  10. β-Alanine supplementation slightly enhances repeated plyometric performance after high-intensity training in humans.

    PubMed

    Carpentier, Alain; Olbrechts, Naïma; Vieillevoye, Stéphanie; Poortmans, Jacques R

    2015-07-01

    β-Alanine (BA) supplementation has become an ergogenic aid amongst competitive athletes to augment intramuscular carnosine content, leading to higher buffer capacity and exercise performance. We investigated 27 regularly trained young males and females who were randomly allocated either to placebo (PL) or BA ingestion for 8 weeks. Every single day, BA or PL (4.0-5.6 g day(-1)) supplements were ingested by participants and associated with a strong plyometric high-intensity training (two sessions per week during the 8 weeks). Before and after training, maximal jump heights were recorded during squat jump (SJ) and countermovement jump (CMJ) and an index of fatigue was recorded as a mean height of 45 consecutive CMJ. Blood lactate was measured at rest, after completing the fatigue test and every 5 min thereafter up to 30 min recovery. After plyometric training, SJ and CMJ were increased, respectively, by 8.8 and 6.4 % in PL group and 9.9 and 11.0 % in BA group (p < 0.01, no difference between groups). Blood lactate reached a maximal value of 9.4 ± 1.6 mmol l(-1) in PL group, and 10.3 ± 1.3 mmol l(-1) in BA group, with a slight better performance in the fatigue test (+8.6 %, p ≤ 0.01) for BA group as compared to PL group. To conclude, 2-month β-alanine supplementation resulted in a slight improvement of explosive force after 45 maximal consecutive jumps in young athletes. However, the practical adequacy of supplementation remains questionable in an active and healthy population.

  11. Calibration of helical tomotherapy machine using EPR/alanine dosimetry

    SciTech Connect

    Perichon, Nicolas; Garcia, Tristan; Francois, Pascal; Lourenco, Valerie; Lesven, Caroline; Bordy, Jean-Marc

    2011-03-15

    Purpose: Current codes of practice for clinical reference dosimetry of high-energy photon beams in conventional radiotherapy recommend using a 10x10 cm{sup 2} square field, with the detector at a reference depth of 10 cm in water and 100 cm source to surface distance (SSD) (AAPM TG-51) or 100 cm source-to-axis distance (SAD) (IAEA TRS-398). However, the maximum field size of a helical tomotherapy (HT) machine is 40x5 cm{sup 2} defined at 85 cm SAD. These nonstandard conditions prevent a direct implementation of these protocols. The purpose of this study is twofold: To check the absorbed dose in water and dose rate calibration of a tomotherapy unit as well as the accuracy of the tomotherapy treatment planning system (TPS) calculations for a specific test case. Method: Both topics are based on the use of electron paramagnetic resonance (EPR) using alanine as transfer dosimeter between the Laboratoire National Henri Becquerel (LNHB) {sup 60}Co-{gamma}-ray reference beam and the Institut Curie's HT beam. Irradiations performed in the LNHB reference {sup 60}Co-{gamma}-ray beam allowed setting up the calibration method, which was then implemented and tested at the LNHB 6 MV linac x-ray beam, resulting in a deviation of 1.6% (at a 1% standard uncertainty) relative to the reference value determined with the standard IAEA TRS-398 protocol. Results: HT beam dose rate estimation shows a difference of 2% with the value stated by the manufacturer at a 2% standard uncertainty. A 4% deviation between measured dose and the calculation from the tomotherapy TPS was found. The latter was originated by an inadequate representation of the phantom CT-scan values and, consequently, mass densities within the phantom. This difference has been explained by the mass density values given by the CT-scan and used by the TPS which were not the true ones. Once corrected using Monte Carlo N-Particle simulations to validate the accuracy of this process, the difference between corrected TPS

  12. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    PubMed

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina. PMID:8158142

  13. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    PubMed

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.

  14. Tb(3+)-triggered luminescence in a supramolecular gel and its use as a fluorescent chemoprobe for proteins containing alanine.

    PubMed

    Jung, Sung Ho; Kim, Ka Young; Woo, Dong Kyun; Lee, Shim Sung; Jung, Jong Hwa

    2014-11-01

    A tetracarboxylic acid-appended thiacalix[4]arene-based ligand with Tb(3+) formed a supramolecular gel which showed novel fluorogenic sensor capability for probing alanine and proteins containing alanine.

  15. Energy landscapes and global thermodynamics for alanine peptides

    NASA Astrophysics Data System (ADS)

    Somani, Sandeep; Wales, David J.

    2013-09-01

    We compare different approaches for computing the thermodynamics of biomolecular systems. Techniques based on parallel replicas evolving via molecular dynamics or Monte Carlo simulations produce overlapping histograms for the densities of states. In contrast, energy landscape methods employ a superposition partition function constructed from local minima of the potential energy surface. The latter approach is particularly powerful for systems exhibiting broken ergodicity, and it is usually implemented using a harmonic normal mode approximation, which has not been extensively tested for biomolecules. The present contribution compares these alternative approaches for small alanine peptides modelled using the CHARMM and AMBER force fields. Densities of states produced from canonical sampling using multiple temperature replicas provide accurate reference data to evaluate the effect of the harmonic normal mode approximation in the superposition calculations. This benchmarking lays foundations for the application of energy landscape methods to larger biomolecules. It will also provide well characterised model systems for developing enhanced sampling methods, and for the treatment of anharmonicity corresponding to individual local minima.

  16. 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.

  17. Folding simulations of alanine-based peptides with lysine residues.

    PubMed Central

    Sung, S S

    1995-01-01

    The folding of short alanine-based peptides with different numbers of lysine residues is simulated at constant temperature (274 K) using the rigid-element Monte Carlo method. The solvent-referenced potential has prevented the multiple-minima problem in helix folding. From various initial structures, the peptides with three lysine residues fold into helix-dominated conformations with the calculated average helicity in the range of 60-80%. The peptide with six lysine residues shows only 8-14% helicity. These results agree well with experimental observations. The intramolecular electrostatic interaction of the charged lysine side chains and their electrostatic hydration destabilize the helical conformations of the peptide with six lysine residues, whereas these effects on the peptides with three lysine residues are small. The simulations provide insight into the helix-folding mechanism, including the beta-bend intermediate in helix initiation, the (i, i + 3) hydrogen bonds, the asymmetrical helix propagation, and the asymmetrical helicities in the N- and C-terminal regions. These findings are consistent with previous studies. PMID:7756550

  18. 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

  19. 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

  20. 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.

  1. 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.

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

    PubMed Central

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

    2008-01-01

    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 Å 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 (Cα atom RMSD of 0.53 Å) 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. PMID:18829434

  3. 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.

  4. 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.

  5. 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.

  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. 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

  8. 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.

  9. Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae

    PubMed Central

    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. PMID:24616885

  10. 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.

  11. 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.

  12. Alanine-aminotransferase: an early marker for insulin resistance?

    PubMed

    Salazar, Martin R; Carbajal, Horacio A; Curciarello, Jose O; Aizpurua, Marcelo; Adrover, Raul E; Riondet, Beatriz

    2007-01-01

    In a population-based sample, after excluding alcohol consumption, hepatotoxic drugs and hepatitis B and C infected, we investigated if alanine-aminotransferase (ALT) was associated with metabolic syndrome and insulin resistance, and if this association was caused by non-alcoholic fatty liver disease (NAFLD). The sample (432 female and 119 male) was divided into two ALT thresholds corresponding to the 50th and 75th percentiles (P) (female > or = 15 and > or = 19 U/L; male > or = 17 and > or = 23 U/I, respectively). Blood pressure, body mass index, waist circumference, cholesterol, HDL cholesterol (HDLc), triglyceride (TG), TG/HDLc ratio, glycemia and homeostasis model assessment of insulin resistance (HOMA-IR) were compared between those above and below each ALT threshold. Female placed above the 50th P of ALT had higher levels of TG/HDLc ratio (p=0.029), glycemia (p=0.028), and homeostasis model assessment of insulin resistance, (p=0.045), and above the 75th P had higher SBP (p=0.036), DBP (p=0.018), TG (p=0.024), TG/HDLc ratio (p=0.028), glycemia (p=0.004) and HOMA-IR (p=0.0014). Male placed above the 50th P of ALT had higher BMI (p=0.017) and TG/HDLc ratio (p=0.048), and above the 75th P had lower values of HDLc (p=0.042). Only 16.5% of women and 14.5% of men, above the 75th P of ALT, showed an increase in liver brightness in the echography. This work shows in woman an early association of ALT with TG/HDLc ratio and HOMA-IR. Since the last two are independent predictors of cardiovascular risk, attention should be drawn to ALT values near the upper limit of the normal range even in the absence of NAFLD and obesity. PMID:17593595

  13. 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.

  14. 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

  15. 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. PMID:19864064

  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. 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%.

  18. Beta-alanine (Carnosyn™) supplementation in elderly subjects (60-80 years): effects on muscle carnosine content and physical capacity.

    PubMed

    del Favero, Serena; Roschel, Hamilton; Solis, Marina Y; Hayashi, Ana P; Artioli, Guilherme G; Otaduy, Maria Concepción; Benatti, Fabiana B; Harris, Roger C; Wise, John A; Leite, Cláudia C; Pereira, Rosa M; de Sá-Pinto, Ana L; Lancha-Junior, Antonio Herbert; Gualano, Bruno

    2012-07-01

    The aim of this study was to investigate the effects of beta-alanine supplementation on exercise capacity and the muscle carnosine content in elderly subjects. Eighteen healthy elderly subjects (60-80 years, 10 female and 4 male) were randomly assigned to receive either beta-alanine (BA, n=12) or placebo (PL, n=6) for 12 weeks. The BA group received 3.2 g of beta-alanine per day (2×800 mg sustained-release Carnosyn™ tablets, given 2 times per day). The PL group received 2× (2×800 mg) of a matched placebo. At baseline (PRE) and after 12 weeks (POST-12) of supplementation, assessments were made of the muscle carnosine content, anaerobic exercise capacity, muscle function, quality of life, physical activity and food intake. A significant increase in the muscle carnosine content of the gastrocnemius muscle was shown in the BA group (+85.4%) when compared with the PL group (+7.2%) (p=0.004; ES: 1.21). The time-to-exhaustion in the constant-load submaximal test (i.e., TLIM) was significantly improved (p=0.05; ES: 1.71) in the BA group (+36.5%) versus the PL group (+8.6%). Similarly, time-to-exhaustion in the incremental test was also significantly increased (p=0.04; ES 1.03) following beta-alanine supplementation (+12.2%) when compared with placebo (+0.1%). Significant positive correlations were also shown between the relative change in the muscle carnosine content and the relative change in the time-to-exhaustion in the TLIM test (r=0.62; p=0.01) and in the incremental test (r=0.48; p=0.02). In summary, the current data indicate for the first time, that beta-alanine supplementation is effective in increasing the muscle carnosine content in healthy elderly subjects, with subsequent improvement in their exercise capacity.

  19. Effect of beta-alanine supplementation on repeated sprint performance during the Loughborough Intermittent Shuttle Test.

    PubMed

    Saunders, Bryan; Sale, Craig; Harris, Roger C; Sunderland, Caroline

    2012-07-01

    The aim of this study was to examine the effect of β-alanine supplementation on repeated sprint performance during an intermittent exercise protocol designed to replicate games play. Sixteen elite and twenty non-elite game players performed the Loughborough Intermittent Shuttle Test (LIST) on two separate occasions. Trials were separated by 4 weeks of supplementation with either β-alanine (BA) or maltodextrin (MD). There was no deterioration in sprint times from Set 1 to Set 6 of the LIST in either group prior to supplementation (elite: P=0.92; non-elite: P=0.12). Neither BA nor MD supplementation affected sprint times. Blood lactate concentrations were elevated during exercise in both groups, with no effect of supplementation. β-Alanine supplementation did not significantly improve sprint performance during the LIST. Neither group showed a performance decrement prior to supplementation, which might have masked any benefit from increased muscle buffering capacity due to β-alanine supplementation.

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

    SciTech Connect

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

    1987-07-15

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

  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. Alanine blends for ESR measurements of thermal neutron fluence in a mixed radiation field.

    PubMed

    Marrale, M; Brai, M; Gennaro, G; Triolo, A; Bartolotta, A; D'Oca, M C; Rosi, G

    2007-01-01

    In this paper, the results of a study on the electron spin resonance (ESR) dosimetry to measure thermal neutron fluence in a mixed radiation field (neutron and photons) are presented. The ESR responses of alanine dosemeters with different additives are compared. In particular, the (10)B-acid boric and the Gd-oxide were chosen to enhance the sensitivity of alanine dosemeters to thermal neutrons. Irradiations were carried out inside the thermal column of the TAPIRO reactor of the ENEA center, Casaccia Rome. The main results are a greater neutron sensitivity and a smaller lowest detectable fluence for the dosemeters with gadolinium than for dosemeters of alanine with (10)B, which is well known to be much more sensitive to thermal neutrons than simple alanine.

  3. An automated system for the measurement of alanine/EPR dosimeters

    PubMed

    Sharpe; Sephton

    2000-05-01

    NPL for several years has offered mailed reference dosimetry services based on alanine/EPR dosimeters, both at industrial and therapy dose levels. Compared to other methods of reference dosimetry, operator involvement in alanine/EPR has been found to be relatively high, and contributes significantly to the overall economics of the process. Commercially available sample changers are not suitable for high accuracy applications, and it has proved necessary to develop a dedicated automation system to handle NPL alanine dosimeter pellets. In this paper we describe an automatic sample changer for placing and retrieving alanine pellets into and out of the cavity of a standard research grade EPR spectrometer. Up to 32 pellets can be held in each removable sample tray. The sample changer software has been interfaced into the spectrometer control software to enable complete automation of the measurement process, including the optimization of spectrometer settings and rotation of the sample within the cavity.

  4. Role of the omega loop in specificity determination in subsite 2 of the D-alanine:D-alanine (D-lactate) ligase from Leuconostoc mesenteroides: a molecular docking study.

    PubMed

    Neuhaus, Francis C

    2011-09-01

    The synthesis of D-ala-D-lactate in Leuconostoc mesenteroides is catalyzed by D-alanine:D-alanine (D-lactate) ligase (ADP). The ability to assemble this depsipeptide as well as D-ala-D-ala provides a mechanism for the organism's intrinsic resistance to vancomycin. Mutation of Phe261 to Tyr261 in the Ω-loop of this ligase showed a complete loss of the ability to make D-ala-D-lactate (Park and Walsh, J. Biol. Chem. 272 (1997) 9210-9214). Phe261 is a key specificity determinant in the α-helical cap of the Ω-loop when folded into the closed conformation. A molecular docking study of the closed ligase using AutoDock 4.2 defines additional specificity constraints promoted by the Ω-loop capping the catalytic center. Attaining productive orientations of D-lactate with favorable ligation chemistry requires the flexibilities of Phe261 and Arg301 in the docking protocol. These are in addition to the optimization of van der Waals contacts with Lys260, Met326, and Ser327. The location of Phe261 and Lys260 in the α-helical cap of the Ω-loop over subsite 2 is an essential part of the folding process ensuring depsipeptide formation in the hydrophobic environment of the catalytic center. The importance of the F261Y mutation suggests that the hydroxyl of Tyr261 plays an instrumental role in determining non-productive docking orientations of D-lactate. Two of these are presented: (A) D-lactate-OH as an H-bond donor to the Tyr261-OH; (B) D-lactate as an H-bond donor to the phosphoryl of the intermediate D-alanyl phosphate, and the D-lactate-COO- as an H-bond acceptor for the Tyr261-OH. Neither orientation, A or B, show the bifurcated H-bonding with Arg301 recently proposed for the activation of the nucleophilic D-lactate for D-ala-D-lactate formation. Insights into the role of the Ω-loop and its K(F/Y) signature provide additional background for inhibitor design targeted to subsite 2 of the D-alanine:D-alanine (D-X) ligases.

  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. Oligomerization of glycine and alanine catalyzed by iron oxides: implications for prebiotic chemistry.

    PubMed

    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.

  7. Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

    PubMed Central

    Chen, Yanling; Gollnick, Paul

    2008-01-01

    SUMMARY 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 (AT3) 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 (AT12) structure. Using alanine-scanning mutagenesis we found four residues, all located on the “top” region of AT3, which 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. PMID:18334255

  8. EPR dosimetry of radiotherapy photon beams in inhomogeneous media using alanine films

    NASA Astrophysics Data System (ADS)

    Helge Østerås, Bjørn; Olaug Hole, Eli; Rune Olsen, Dag; Malinen, Eirik

    2006-12-01

    In the current work, EPR (electron paramagnetic resonance) dosimetry using alanine films (134 µm thick) was utilized for dose measurements in inhomogeneous phantoms irradiated with radiotherapy photon beams. The main phantom material was PMMA, while either Styrofoam or aluminium was introduced as an inhomogeneity. The phantoms were irradiated to a maximum dose of about 30 Gy with 6 or 15 MV photons. The performance of the alanine film dosimeters was investigated and compared to results from ion chamber dosimetry, Monte Carlo simulations and radiotherapy treatment planning calculations. It was found that the alanine film dosimeters had a linear dose response above approximately 5 Gy, while a background signal obscured the response at lower dose levels. For doses between 5 and 60 Gy, the standard deviation of single alanine film dose estimates was about 2%. The alanine film dose estimates yielded results comparable to those from the Monte Carlo simulations and the ion chamber measurements, with absolute differences between estimates in the order of 1 15%. The treatment planning calculations exhibited limited applicability. The current work shows that alanine film dosimetry is a method suitable for estimating radiotherapeutical doses and for dose measurements in inhomogeneous media.

  9. EPR dosimetry of radiotherapy photon beams in inhomogeneous media using alanine films.

    PubMed

    Osterås, Bjørn Helge; Hole, Eli Olaug; Olsen, Dag Rune; Malinen, Eirik

    2006-12-21

    In the current work, EPR (electron paramagnetic resonance) dosimetry using alanine films (134 microm thick) was utilized for dose measurements in inhomogeneous phantoms irradiated with radiotherapy photon beams. The main phantom material was PMMA, while either Styrofoam or aluminium was introduced as an inhomogeneity. The phantoms were irradiated to a maximum dose of about 30 Gy with 6 or 15 MV photons. The performance of the alanine film dosimeters was investigated and compared to results from ion chamber dosimetry, Monte Carlo simulations and radiotherapy treatment planning calculations. It was found that the alanine film dosimeters had a linear dose response above approximately 5 Gy, while a background signal obscured the response at lower dose levels. For doses between 5 and 60 Gy, the standard deviation of single alanine film dose estimates was about 2%. The alanine film dose estimates yielded results comparable to those from the Monte Carlo simulations and the ion chamber measurements, with absolute differences between estimates in the order of 1-15%. The treatment planning calculations exhibited limited applicability. The current work shows that alanine film dosimetry is a method suitable for estimating radiotherapeutical doses and for dose measurements in inhomogeneous media. PMID:17148820

  10. Allele-specific characterization of alanine: glyoxylate aminotransferase variants associated with primary hyperoxaluria.

    PubMed

    Lage, Melissa D; Pittman, Adrianne M C; Roncador, Alessandro; Cellini, Barbara; Tucker, Chandra L

    2014-01-01

    Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.

  11. 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

  12. NAD(+)-aminoaldehyde dehydrogenase candidates for 4-aminobutyrate (GABA) and β-alanine production during terminal oxidation of polyamines in apple fruit.

    PubMed

    Zarei, Adel; Trobacher, Christopher P; Shelp, Barry J

    2015-09-14

    The last step of polyamine catabolism involves the oxidation of 3-aminopropanal or 4-aminobutanal via aminoaldehyde dehydrogenase. In this study, two apple (Malus x domestica) AMADH genes were selected (MdAMADH1 and MdAMADH2) as candidates for encoding 4-aminobutanal dehydrogenase activity. Maximal activity and catalytic efficiency were obtained with NAD(+) and 3-aminopropanal, followed by 4-aminobutanal, at pH 9.8. NAD(+) reduction was accompanied by the production of GABA and β-alanine, respectively, when 4-aminobutanal and 3-aminopropanal were utilized as substrates. MdAMADH2 was peroxisomal and MdAMADH1 cytosolic. These findings shed light on the potential role of apple AMADHs in 4-aminobutyrate and β-alanine production.

  13. Liver stiffness in the hepatitis B virus carrier: A non-invasive marker of liver disease influenced by the pattern of transaminases

    PubMed Central

    Oliveri, Filippo; Coco, Barbara; Ciccorossi, Pietro; Colombatto, Piero; Romagnoli, Veronica; Cherubini, Beatrice; Bonino, Ferruccio; Brunetto, Maurizia Rossana

    2008-01-01

    AIM: To investigate the usefulness of transient elastography by Fibroscan (FS), a rapid non-invasive technique to evaluate liver fibrosis, in the management of chronic hepatitis B virus (HBV) carriers. METHODS: In 297 consecutive HBV carriers, we studied the correlation between liver stiffness (LS), stage of liver disease and other factors potentially influencing FS measurements. In 87 chronic hepatitis B (CHB) patients, we monitored the FS variations according to the spontaneous or treatment-induced variations of biochemical activity during follow-up. RESULTS: FS values were 12.3 ± 3.3 kPa in acute hepatitis, 10.3 ± 8.8 kPa in chronic hepatitis, 4.3 ± 1.0 kPa in inactive carriers and 4.6 ± 1.2 kPa in blood donors. We identified the cut-offs of 7.5 and 11.8 kPa for the diagnosis of fibrosis ≥ S3 and cirrhosis respectively, showing 93.9% and 86.5% sensitivity, 88.5% and 96.3% specificity, 76.7% and 86.7% positive predictive value (PPV), 97.3% and 96.3% negative predictive value (NPV) and 90.1% and 94.2% diagnostic accuracy. At multivariate analysis in 171 untreated carriers, fibrosis stage (t = 13.187, P < 0.001), active vs inactive HBV infection (t = 6.437, P < 0.001), alanine aminotransferase (ALT) (t = 4.740, P < 0.001) and HBV-DNA levels (t = -2.046, P = 0.042) were independently associated with FS. Necroinflammation score (t = 2.158, > 10/18 vs ≤ 10/18, P = 0.035) and ALT levels (t = 3.566, P = 0.001) were independently associated with LS in 83 untreated patients without cirrhosis and long-term biochemical remission (t = 4.662, P < 0.001) in 80 treated patients. During FS monitoring (mean follow-up 19.9 ± 7.1 mo) FS values paralleled those of ALT in patients with hepatitis exacerbation (with 1.2 to 4.4-fold increases in CHB patients) and showed a progressive decrease during antiviral therapy. CONCLUSION: FS is a non-invasive tool to monitor liver disease in chronic HBV carriers, provided that the pattern of biochemical activity is taken into account

  14. A Micro-Platinum Wire Biosensor for Fast and Selective Detection of Alanine Aminotransferase.

    PubMed

    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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. Design and development of new class of Mycobacterium tuberculosisl-alanine dehydrogenase inhibitors.

    PubMed

    Reshma, Rudraraju Srilakshmi; Saxena, Shalini; Bobesh, Karyakulam Andrews; Jeankumar, Variam Ullas; Gunda, Saritha; Yogeeswari, Perumal; Sriram, Dharmarajan

    2016-09-15

    Mycobacterium tuberculosisl-alanine dehydrogenase (MTB l-AlaDH) is one of the important drug targets for treating latent/persistent tuberculosis. In this study we used crystal structure of the MTB l-AlaDH bound with cofactor NAD(+) as a structural framework for virtual screening of our in-house database to identified new classes of l-AlaDH inhibitor. We identified azetidine-2,4-dicarboxamide derivative as one of the potent inhibitor with IC50 of 9.22±0.72μM. Further lead optimization by synthesis leads to compound 1-(isonicotinamido)-N(2),N(4)-bis(benzo[d]thiazol-2-yl)azetidine-2,4-dicarboxamide (18) with l-AlaDH IC50 of 3.83±0.12μM, 2.0log reduction in nutrient starved dormant MTB model and MIC of 11.81μM in actively replicative MTB. PMID:27477207

  20. Ir-192 HDR transit dose and radial dose function determination using alanine/EPR dosimetry

    NASA Astrophysics Data System (ADS)

    Guzmán Calcina, Carmen S.; de Almeida, Adelaide; Oliveira Rocha, José R.; Abrego, Felipe Chen; Baffa, Oswaldo

    2005-03-01

    Source positioning close to the tumour in high dose rate (HDR) brachytherapy is not instantaneous. An increment of dose will be delivered during the movement of the source in the trajectory to its static position. This increment is the transit dose, often not taken into account in brachytherapeutic treatment planning. The transit dose depends on the prescribed dose, number of treatment fractions, velocity and activity of the source. Combining all these factors, the transit dose can be 5% higher than the prescribed absorbed dose value (Sang-Hyun and Muller-Runkel, 1994 Phys. Med. Biol. 39 1181 8, Nath et al 1995 Med. Phys. 22 209 34). However, it cannot exceed this percentage (Nath et al 1995). In this work, we use the alanine-EPR (electron paramagnetic resonance) dosimetric system using analysis of the first derivative of the signal. The transit dose was evaluated for an HDR system and is consistent with that already presented for TLD dosimeters (Bastin et al 1993 Int. J. Radiat. Oncol. Biol. Phys. 26 695 702). Also using the same dosimetric system, the radial dose function, used to evaluate the geometric dose degradation around the source, was determined and its behaviour agrees better with those obtained by Monte Carlo simulations (Nath et al 1995, Williamson and Nath 1991 Med. Phys. 18 434 48, Ballester et al 1997 Med. Phys. 24 1221 8, Ballester et al 2001 Phys. Med. Biol. 46 N79 90) than with TLD measurements (Nath et al 1990 Med. Phys. 17 1032 40).

  1. 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.

  2. 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.

  3. 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. PMID:20570592

  4. The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: β-alanine recycles through the retina.

    PubMed

    Borycz, Janusz; Borycz, Jolanta A; Edwards, Tara N; Boulianne, Gabrielle L; Meinertzhagen, Ian A

    2012-04-15

    Flies recycle the photoreceptor neurotransmitter histamine by conjugating it to β-alanine to form β-alanyl-histamine (carcinine). The conjugation is regulated by Ebony, while Tan hydrolyses carcinine, releasing histamine and β-alanine. In Drosophila, β-alanine synthesis occurs either from uracil or from the decarboxylation of aspartate but detailed roles for the enzymes responsible remain unclear. Immunohistochemically detected β-alanine is present throughout the fly's entire brain, and is enhanced in the retina especially in the pseudocone, pigment and photoreceptor cells of the ommatidia. HPLC determinations reveal 10.7 ng of β-alanine in the wild-type head, roughly five times more than histamine. When wild-type flies drink uracil their head β-alanine increases more than after drinking l-aspartic acid, indicating the effectiveness of the uracil pathway. Mutants of black, which lack aspartate decarboxylase, cannot synthesize β-alanine from l-aspartate but can still synthesize it efficiently from uracil. Our findings demonstrate a novel function for pigment cells, which not only screen ommatidia from stray light but also store and transport β-alanine and carcinine. This role is consistent with a β-alanine-dependent histamine recycling pathway occurring not only in the photoreceptor terminals in the lamina neuropile, where carcinine occurs in marginal glia, but vertically via a long pathway that involves the retina. The lamina's marginal glia are also a hub involved in the storage and/or disposal of carcinine and β-alanine.

  5. β-alanine improves punch force and frequency in amateur boxers during a simulated contest.

    PubMed

    Donovan, Tim; Ballam, Tim; Morton, James P; Close, Graeme L

    2012-10-01

    The aim of this study was to test the hypothesis that ß-alanine supplementation improves punch power and frequency in amateur boxers during a simulated contest. Sixteen amateur boxers (each approximately 6 yr experience) were assigned to ß-alanine (n = 8; 1.5 g 4 times/d for 4 wk) or placebo supplementation (n = 8) after initially being assessed for baseline punch performance. Before and after the supplementation period, all boxers completed a simulated contest consisting of 3 × 3-min rounds (interspersed with 60-s rests) on a punching bag (with a force transducer attached). Each round involved performing 2 min 50 s standardized punching (standardized jab, cross combination) based on notation analysis, whereas the last 10 s involved maximal-output punching (standardized jab, cross combination), during which time punch force and frequency were recorded. Postcontest blood lactate was significantly increased in the ß-alanine group (presupplementation 9.5 ± 0.9 mmol/L, postsupplementation 12.6 ± 0.5 mmol/L, p < .05), whereas the placebo group showed no change (presupplementation 8 ± 2.8 mmol/L, postsupplementation 7.0 ± 2.7 mmol/L; p > .05). During the 10-s maximal-output punching, changes in mean punch force (ß-alanine 20 ± 1.01 kg, placebo 1 ± 1 kg) and punch frequency (ß-alanine 5 ± 4, placebo -2 ± 3) were greater (p < .05) in the ß-alanine-supplemented group. The authors conclude that ß-alanine supplementation improves punching performance in amateur boxers and suggest that this supplementation protocol may also prove ergogenic for other combat-related sports.

  6. Effects of β-alanine supplementation on exercise performance: a meta-analysis.

    PubMed

    Hobson, R M; Saunders, B; Ball, G; Harris, R C; Sale, C

    2012-07-01

    Due to the well-defined role of β-alanine as a substrate of carnosine (a major contributor to H+ buffering during high-intensity exercise), β-alanine is fast becoming a popular ergogenic aid to sports performance. There have been several recent qualitative review articles published on the topic, and here we present a preliminary quantitative review of the literature through a meta-analysis. A comprehensive search of the literature was employed to identify all studies suitable for inclusion in the analysis; strict exclusion criteria were also applied. Fifteen published manuscripts were included in the analysis, which reported the results of 57 measures within 23 exercise tests, using 18 supplementation regimes and a total of 360 participants [174, β-alanine supplementation group (BA) and 186, placebo supplementation group (Pla)]. BA improved (P=0.002) the outcome of exercise measures to a greater extent than Pla [median effect size (IQR): BA 0.374 (0.140-0.747), Pla 0.108 (-0.019 to 0.487)]. Some of that effect might be explained by the improvement (P=0.013) in exercise capacity with BA compared to Pla; no improvement was seen for exercise performance (P=0.204). In line with the purported mechanisms for an ergogenic effect of β-alanine supplementation, exercise lasting 60-240 s was improved (P=0.001) in BA compared to Pla, as was exercise of >240 s (P=0.046). In contrast, there was no benefit of β-alanine on exercise lasting <60 s (P=0.312). The median effect of β-alanine supplementation is a 2.85% (-0.37 to 10.49%) improvement in the outcome of an exercise measure, when a median total of 179 g of β-alanine is supplemented.

  7. 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.

  8. Short one-pot chemo-enzymatic synthesis of L-lysine and L-alanine diblock co-oligopeptides.

    PubMed

    Fagerland, Jenny; Finne-Wistrand, Anna; Numata, Keiji

    2014-03-10

    Amphiphilic diblock co-oligopeptides are interesting and functional macromolecular materials for biomedical applications because of their self-assembling properties. Here, we developed a synthesis method for diblock co-oligopeptides by using chemo-enzymatic polymerization, which was a relatively short (30 min) and efficient reaction (over 40% yield). Block and random oligo(L-lysine-co-L-alanine) [oligo(Lys-co-Ala)] were synthesized using activated papain as enzymatic catalyst. The reaction time was optimized according to kinetic studies of oligo(L-alanine) and oligo(L-lysine). Using (1)H NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we confirmed that diblock and random co-oligopeptides were synthesized. Optical microscopy further revealed differences in the crystalline morphology between random and block co-oligopeptides. Plate-like, hexagonal, and hollow crystals were formed due to the strong impact of the monomer distribution and pH of the solution. The different crystalline structures open up interesting possibilities to form materials for both tissue engineering and controlled drug/gene delivery systems.

  9. ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex

    PubMed Central

    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/. PMID:25685322

  10. Temperature related alterations in the acidic alanine-rich "A" protein from the 50S ribosomal particle of the extreme halophile, Halobacterium cutirubrum.

    PubMed

    Strom, A R; Oda, G; Hasnain, S; Yaguchi, M; Visentin, L P

    1975-09-15

    50-S ribosomal subunits from the extreme halophilic bacterium, Halobacterium cutirubrum, contain an alanine-rich acidic "A" protein which resembles the L7--L12 multimer (Kaltschmidt and Wittmann, 1970) found in the 50-S ribosomal subunit of Escherichia coli cells. The protein contains 24 mole % alanine and is devoid of histidine, tryptophan and cysteine. Unlike E. coli which has two forms of the "A" protein distinguished solely by the acetylation state of the serine amino terminus. H. cutirubrum 50-S subunits contain only one unsubstituted form of the "A" protein in vivo. However, during purification of ribosomes from cells grown between 25 and 37 degrees C the latter "A" protein undergoes rapid, specific, in vitro enzymatic alteration at its carboxy-terminal end. When the halophile is grown in the temperature range of 40 to 42 degrees C the cleaving enzyme is not active and only one form of the "A" protein is found on the ribosomes.

  11. 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. PMID:12495589

  12. 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.

  13. Purification and characterization of alanine dehydrogenase from a cyanobacterium, Phormidium lapideum.

    PubMed

    Sawa, Y; Tani, M; Murata, K; Shibata, H; Ochiai, H

    1994-11-01

    Alanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240 kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH2 terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD+, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The Km values were 5.0 mM for L-alanine and 0.04 mM for NAD+, 0.33 mM for pyruvate, 60.6 mM for NH4+ (pH 8.7), and 0.02 mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP. PMID:7896761

  14. Theoretical and experimental study of valence photoelectron spectrum of D,L-alanine amino acid.

    PubMed

    Farrokhpour, H; Fathi, F; De Brito, A Naves

    2012-07-01

    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.

  15. 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

    2016-02-01

    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 [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.

  16. 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.

  17. 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.

  18. Conformational composition and population analysis of β-alanine isolated in solid parahydrogen

    NASA Astrophysics Data System (ADS)

    Angel Wong, Y. T.; Toh, Shin Y.; Djuricanin, Pavle; Momose, Takamasa

    2015-04-01

    The conformational composition and the change in conformational ratio induced by UV irradiation of β-alanine have been investigated using solid parahydrogen FT-IR matrix isolation spectroscopy for the first time. In order to assign the observed spectra, the vibrational wavenumbers and intensities of the eleven lowest energy β-alanine conformers were calculated at the B3LYP/aug-cc-pVTZ level of theory. In-situ UV photo-irradiation of β-alanine in solid parahydrogen was used to assist the spectral assignment. Out of the eleven lowest energy conformers, conformers I, II, III, IV, and VII were identified in the solid parahydrogen matrix, with conformer III observed in a matrix environment for the first time. Argon matrix FT-IR spectra of β-alanine were also recorded for comparison and only four conformers, conformers I, II, IV and VII, were found, as reported previously. Conformational changes to higher energy structures were observed when β-alanine was irradiated with UV radiation. These changes were more pronounced in parahydrogen matrices than in argon matrices, indicating the usefulness of solid parahydrogen matrix isolation spectroscopy for the conformational study of amino acids.

  19. UV-induced isomerization of β-alanine isolated in argon matrices

    NASA Astrophysics Data System (ADS)

    Stepanian, Stepan G.; Ivanov, Alexander Yu.; Smyrnova, Daryna A.; Adamowicz, Ludwik

    2012-10-01

    We have employed low-temperature matrix-isolation FTIR spectroscopy, the density functional theory and ab initio calculations at the MP2 and CCSD(T) levels of theory to determine the conformational composition of the simplest β-amino acid, β-alanine. UV irradiation and thermal annealing of the samples together with the FTIR spectra of deuterated β-alanine were used to separate bands of different conformers. A detailed study of the potential energy surface of β-alanine obtained at the MP2/aug-cc-pVDZ level of theory reveals twenty β-alanine conformers, but only five of them may exist in matrices due to their sufficiently high relative stabilities and low energy barriers separating them from each other. An analysis of the FTIR spectra allows us to confirm the presence of four β-alanine conformers in argon matrices with certainty. Two of them, conformers I and II, have an Nsbnd H⋯O intramolecular H-bond, the third, conformer V, has an N⋯Hsbnd O H-bond, and the fourth, conformer IV, has no intramolecular H-bonds. The relative populations of the conformers determined using the relative Gibbs free energies calculated at the CCSD(T)/CBS level of theory at 420 K are 48.1%, 23.7%, 16.8% and 3.2% for the conformers I, II, IV, and V, respectively. Some trace amount of conformer VII was also detected.

  20. Purification and Characterization of a Novel Aminopeptidase, Plastidial Alanine-Aminopeptidase, from the Cotyledons of Etiolated Sugar Beet Seedlings.

    PubMed

    Amrani, A. E.; Suire, C.; Camara, B.; Gaudillere, J. P.; Couee, I.

    1995-09-01

    During prolonged dark growth of sugar beet (Beta vulgaris L.) seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications, protein loss, and the decrease of carotenoid and chlorophyll accumulation upon illumination. Two plastidial aminopeptidase activities were identified as early markers of this degenerative process (A. El Amrani, I. Couee, J.-P. Carde, J.-P. Gaudillere, P. Raymond [1994] Plant Physiology 106: 1555-1565). The present study focuses on one of these markers and describes the purification to homogeneity and characterization of plastidial alanine-aminopeptidase. This novel aminopeptidase was found to be a metallo-type naphthylamidase particularly active with alanyl, arginyl, and leucyl substrates. Its plastidial location was confirmed by immunofluorescence with polyclonal antibodies against the purified enzyme. Its physico-chemical and enzymic properties are discussed with respect to other higher plant aminopeptidases and to its potential functions during prolonged dark growth.

  1. 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

  2. A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli.

    PubMed Central

    Fan, C; Moews, P C; Shi, Y; Walsh, C T; Knox, J R

    1995-01-01

    Examination of x-ray crystallographic structures shows the tertiary structure of D-alanine:D-alanine ligase (EC 6.3.2.4). a bacterial cell wall synthesizing enzyme, is similar to that of glutathione synthetase (EC 6.32.3) despite low sequence homology. Both Escherichia coli enzymes, which convert ATP to ADP during ligation to produce peptide products, are made of three domains, each folded around a 4-to 6-stranded beta-sheet core. Sandwiched between the beta-sheets of the C-terminal and central domains of each enzyme is a nonclassical ATP-binding site that contains a common set of spatially equivalent amino acids. In each enzyme, two loops are proposed to exhibit a required flexibility that allows entry of ATP and substrates, provides protection of the acylphosphate intermediate and tetrahedral adduct from hydrolysis during catalysis, and then permits release of products. PMID:7862655

  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.

    PubMed

    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. Combined TL and 10B-alanine ESR dosimetry for BNCT.

    PubMed

    Bartolotta, A; D'Oca, M C; Lo Giudice, B; Brai, M; Borio, R; Forini, N; Salvadori, P; Manera, S

    2004-01-01

    The dosimetric technique described in this paper is based on electron spin resonance (ESR) detectors using an alanine-boric compound acid enriched with (10)B, and beryllium oxide thermoluminescent (TL) detectors; with this combined dosimetry, it is possible to discriminate the doses due to thermal neutrons and gamma radiation in a mixed field. Irradiations were carried out inside the thermal column of a TRIGA MARK II water-pool-type research nuclear reactor, also used for Boron Neutron Capture therapy (BNCT) applications, with thermal neutron fluence from 10(9) to 10(14) nth cm(-2). The ESR dosemeters using the alanine-boron compound indicated ESR signals about 30-fold stronger than those using only alanine. Moreover, a negligible correction for the gamma contribution, measured with TL detectors, almost insensitive to thermal neutrons, was necessary. Therefore, a simultaneous analysis of our TL and ESR detectors allows discrimination between thermal neutron and gamma doses, as required in BNCT.

  7. 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.

  8. Combined TL and 10B-alanine ESR dosimetry for BNCT.

    PubMed

    Bartolotta, A; D'Oca, M C; Lo Giudice, B; Brai, M; Borio, R; Forini, N; Salvadori, P; Manera, S

    2004-01-01

    The dosimetric technique described in this paper is based on electron spin resonance (ESR) detectors using an alanine-boric compound acid enriched with (10)B, and beryllium oxide thermoluminescent (TL) detectors; with this combined dosimetry, it is possible to discriminate the doses due to thermal neutrons and gamma radiation in a mixed field. Irradiations were carried out inside the thermal column of a TRIGA MARK II water-pool-type research nuclear reactor, also used for Boron Neutron Capture therapy (BNCT) applications, with thermal neutron fluence from 10(9) to 10(14) nth cm(-2). The ESR dosemeters using the alanine-boron compound indicated ESR signals about 30-fold stronger than those using only alanine. Moreover, a negligible correction for the gamma contribution, measured with TL detectors, almost insensitive to thermal neutrons, was necessary. Therefore, a simultaneous analysis of our TL and ESR detectors allows discrimination between thermal neutron and gamma doses, as required in BNCT. PMID:15353720

  9. A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica.

    PubMed

    Hilbert, F; García-del Portillo, F; Groisman, E A

    1999-04-01

    The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.

  10. Effects of maternal ethanol ingestion on uptake of glucose alanine analogs in fetal rats

    SciTech Connect

    Snyder, A.K.; Singh, S.P.; Pullen, G.L.

    1986-05-01

    The distribution of maternally-derived glucose and alanine has been studied in selected tissues of fetuses from ethanol-fed (EF) rats (30% of caloric intake throughout gestation). Controls received diet without ethanol by pair-feeding (PF) or ad libitum (AF). On the 22nd day of gestation, 2 ..mu..Ci /sup 3/H 2-deoxyglucose (DG) and 1 ..mu..Ci /sup 14/C ..cap alpha..-aminoisobutyric acid (AIB) were administered i.v. to each rat. One hour later, maternal blood, placenta, and fetal blood, liver, lung and brain were sampled for /sup 3/H and /sup 14/C activities. When compared to either control group, the mean /sup 14/C AIB activities of tissues from EF animals were reduced from 19 to 46%, with the greatest effect seen in the brain (3.7 +/- 0.1, 7.2 +/- 0.3 and 6.9 +/- 1.3 dpm/mg in EF, PF and AF fetuses respectively). In addition, the ratios of tissue:plasma /sup 14/C were reduced (p < 0.01 or lower) in the EF fetal tissues and placenta. Maternal ethanol ingestion reduced the /sup 3/H 2-DG content of placenta (p < 0.05) and of brain (38.6 + 1.2, 48.1 +/- 1.2 and 47.2 +/- 1.2 in EF, PF and AF, p < 0.001). Brain weight showed significant positive correlations with AIB content (r = 0.466, p < 0.001) and with 2-DG content (r = 0.267, p < 0.01). Impaired uptake of maternally-derived nutrients may play a significant role in the effects of ethanol in utero.

  11. Vitamin E and changes in serum alanine aminotransferase levels in patients with non-alcoholic steatohepatitis

    PubMed Central

    Hoofnagle, J. H.; Van Natta, M. L.; Kleiner, D. E.; Clark, J. M.; Kowdley, K. V.; Loomba, R.; Neuschwander-Tetri, B. A.; Sanyal, A. J.; Tonascia, J.

    2013-01-01

    SUMMARY Background Non-alcoholic steatohepatitis (NASH) is a common cause of serum alanine aminotransferase (ALT) elevations and chronic liver disease, but it is unclear how well ALT elevations reflect the liver injury. Aim To assess how well changes in ALT elevations reflect improvements in liver histology in response to vitamin E therapy. Methods The vitamin E and placebo arms of the Pioglitazone vs. Vitamin E vs. Placebo in Non-alcoholic Steatohepatitis (PIVENS) trial were reassessed for associations among changes in ALT levels, body weight and liver histology. An ALT response was defined as a decrease to ≤40 U/L and by ≥30% of baseline. Liver biopsies taken before and after treatment were scored for non-alcoholic fatty liver disease activity (NAS) and fibrosis. Results ALT responses were more frequent among vitamin E (48%) than placebo (16%) recipients (P < 0.001). Among vitamin E recipients, ALT responses were associated with decreases in NAS (P < 0.001), but not fibrosis scores (P = 0.34), whereas among placebo recipients, ALT responses were associated with significant decreases in both (P < 0.05). Weight loss (≥2 kg) was also associated with ALT response (P < 0.001), improvements in NAS (P < 0.001) and fibrosis (P < 0.02), but vitamin E had an added effect both with and without weight loss. Weight gain (≥2 kg) was associated with lack of ALT response and worsening NAS and fibrosis scores in patients not on vitamin E. Conclusions Decrease of ALT levels to normal in patients with NASH is usually associated with improved histological activity. Management should stress the value of weight loss and strongly discourage weight gain. Vitamin E can improve both ALT levels and histology with and without weight loss. Clinical Trial Number: NCT00063622. PMID:23718573

  12. Trunk Fat is Associated with Increased Serum Levels of Alanine Aminotransferase in the US

    PubMed Central

    Ruhl, Constance E.; Everhart, James E.

    2010-01-01

    Background & Aims Liver injury is associated with obesity and related measures such as body mass index (BMI) and waist circumference. The relationship between liver injury and body composition has not been evaluated in a population-based study. Methods Using data from a US population-based survey, we examined the contributions of body composition, measured by dual-energy x-ray absorptiometry (DXA), to increased serum alanine aminotransferase (ALT) activity among 11,821 adults without viral hepatitis. Trunk fat, extremity fat, trunk lean, and extremity lean mass were divided by height squared and used to categorize subjects into quintiles; logistic regression odds ratios (OR) were calculated for increased ALT. Results Increased ALT was associated with higher measures of fat and lean mass (p<0.001) after adjustment for alcohol consumption and other liver injury risk factors in separate models for each DXA measure. Trunk fat was associated with increased ALT (p≤0.001) in models also including any 1 of the other 3 measures. Extremity fat was independently inversely associated among women (p<0.001). Trunk and extremity lean mass were not independently related to increased ALT. In models that contained all 4 DXA measures, the OR (95% confidence interval) for increased ALT for the highest, relative to lowest, quintile of trunk fat/height squared was 13.8 (5.4-35.3) for men and 7.8 (3.9-15.8) for women. When BMI, waist circumference, and trunk fat were considered together, only trunk fat remained independently associated with increased ALT. Conclusions Trunk fat is a major body composition determinant of increased ALT, supporting the hypothesis that liver injury can be induced by metabolically active intra-abdominal fat. PMID:20060831

  13. The energy dependence of lithium formate and alanine EPR dosimeters for medium energy x rays

    SciTech Connect

    Waldeland, Einar; Hole, Eli Olaug; Sagstuen, Einar; Malinen, Eirik

    2010-07-15

    Purpose: To perform a systematic investigation of the energy dependence of alanine and lilthium formate EPR dosimeters for medium energy x rays. Methods: Lithium formate and alanine EPR dosimeters were exposed to eight different x-ray beam qualities, with nominal potentials ranging from 50 to 200 kV. Following ionometry based on standards of absorbed dose to water, the dosimeters were given two different doses of approximately 3 and 6 Gy for each radiation quality, with three dosimeters for each dose. A reference series was also irradiated to three different dose levels at a {sup 60}Co unit. The dose to water energy response, that is, the dosimeter reading per absorbed dose to water relative to that for {sup 60}Co {gamma}-rays, was estimated for each beam quality. In addition, the energy response was calculated by Monte Carlo simulations and compared to the experimental energy response. Results: The experimental energy response estimates ranged from 0.89 to 0.94 and from 0.68 to 0.90 for lithium formate and alanine, respectively. The uncertainties in the experimental energy response estimates were typically 3%. The relative effectiveness, that is, the ratio of the experimental energy response to that following Monte Carlo simulations was, on average, 0.96 and 0.94 for lithium formate and alanine, respectively. Conclusions: This work shows that lithium formate dosimeters are less dependent on x-ray energy than alanine. Furthermore, as the relative effectiveness for both lithium formate and alanine were systematically less than unity, the yield of radiation-induced radicals is decreased following x-irradiation compared to irradiation with {sup 60}Co {gamma}-rays.

  14. Relative response of the alanine dosimeter to medium energy x-rays.

    PubMed

    Anton, M; Büermann, L

    2015-08-01

    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. PMID:26216572

  15. Relative response of the alanine dosimeter to medium energy x-rays

    NASA Astrophysics Data System (ADS)

    Anton, M.; Büermann, L.

    2015-08-01

    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.

  16. Relative response of the alanine dosimeter to medium energy x-rays.

    PubMed

    Anton, M; Büermann, L

    2015-08-01

    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.

  17. The effect of β-alanine supplementation on cycling time trials of different length.

    PubMed

    Bellinger, Phillip M; Minahan, Clare L

    2016-10-01

    The varying results reported in response to β-alanine supplementation may be related to the duration and nature of the exercise protocol employed. We investigated the effects of β-alanine supplementation on a wide range of cycling performance tests in order to produce a clear concise set of criteria for its efficacy. Fourteen trained cyclists (Age = 24.8 ± 6.7 years; VO2max = 65.4 ± 10.2 mL·kg·min(-1)) participated in this placebo-controlled, double-blind study. Prior to supplementation, subjects completed two (familiarization and baseline) supramaximal cycling bouts until exhaustion (120% pre-supplementation VO2max) and two 1-, 4- and 10-km cycling time trial (TT). Subjects then supplemented orally for 4 weeks with 6.4 g/d placebo or β-alanine and repeated the battery of performance tests. Blood lactate was measured pre-exercise, post-exercise and 5  min post-exercise. β-alanine supplementation elicited significant increases in time to exhaustion (TTE) (17.6 ± 11.5 s; p = 0.013, effect compared with placebo) and was likely to be beneficial to 4-km TT performance time (-7.8 ± 8.1 s; 94% likelihood), despite not being statistically different (p = 0.060). Performance times in the 1- and 10-km TT were not affected by treatment. For the highly trained cyclists in the current study, β-alanine supplementation significantly extended supramaximal cycling TTE and may have provided a worthwhile improvement to 4-km TT performance. However, 1- and 10-km cycling TT performance appears to be unaffected by β-alanine supplementation.

  18. 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.

  19. Steric effect exerted by the proline residue on the antecedent alanine residue.

    PubMed

    Siemión, I Z; Sobczyk, K; Nawrocka, E

    1982-05-01

    Five model tetrapeptides: Ala-Ala-Ala-Ala, Pro-Ala-Ala-Ala, Ala-Pro-Ala-Ala, Ala-Ala-Pro-Ala and Ala-Ala-Ala-Pro, were synthesized and measured in D2O by 13 C-n.m.r. spectroscopy. The spectra analysis led us to the conclusion that for each model (irrespective of pD) in conformational equilibrium, the predominant conformation is the one in which side methyl of alanine preceding proline residue eclipses alanine carbonyl group. The influence of pD changes in cis-trans isomerism of Ala-Pro amide bond was also investigated. PMID:7118413

  20. Changes in blood metals, hematology and hepatic enzyme activities in lactating cows reared in the vicinity of a lead-zinc smelter.

    PubMed

    Mohajeri, G; Norouzian, M A; Mohseni, M; Afzalzadeh, A

    2014-06-01

    The present study examines blood metals levels and health parameters in dairy cows reared in areas around a Pb-Zn industrial complex located near the city of Zanjan-Iran. Blood samples (n = 27) were collected from cows reared around the smelter. Blood samples (n = 25) were also collected from cows reared in areas where chances of Pb contamination of soil, water and fodder do not exist, to serve as reference. The mean blood Pb level in cows around the smelter (1.09 ± 0.26 µg/mL) was higher than the cows from the reference area (0.72 ± 0.25 µg/mL). Hematocrit (PCV) levels decreased in cows reared in the near of smelter (24.37 % ± 2.22 %) compared to the reference site (27.54 % ± 2.87 %). Hemoglobin (Hb) concentration and mean corpuscular Hb concentration value in cows from the area around the smelter were statistically lower than the cows reared in reference area. Activities of alanine transaminase and aspartate aminotransferase (IU/l) were significantly higher in cows around the smelter as compared to reference animals.

  1. C/EBPα Short-Activating RNA Suppresses Metastasis of Hepatocellular Carcinoma through Inhibiting EGFR/β-Catenin Signaling Mediated EMT

    PubMed Central

    Chen, Xuejiao; Wu, Lili; Liu, Weihui; Habib, Nagy A.; Bie, Ping; Xia, Feng

    2016-01-01

    Hepatocellular carcinoma is associated with high mortality, and tumor metastasis is an important reason for poor prognosis. However, metastasis has not been effectively prevented in clinical therapy and the mechanisms underlying metastasis have not been fully characterized. CCAAT/enhancer-binding protein-α (C/EBPα) is a transcriptional regulator with an essential role in tumor metastasis. We used short-activating RNAs (saRNA) to enhance expression of C/EBPα. Intravenous injection of C/EBPα-saRNA in a nude mouse liver orthotopic xenograft tumor model inhibited intrahepatic and distant metastasis. C/EBPα-saRNA-treated mice showed increased serum levels of albumin and decreased alanine aminotransferase (ALT), glutamic-oxalacetic transaminase (AST), indicating a role of C/EBPα in improving liver function. Migration and invasion were inhibited in hepatoma cell lines transfected with C/EBPα-saRNA. We also observed an inhibition of epithelial-mesenchymal transition (EMT) and suppression of epidermal growth factor receptor (EGFR), EGFR phosphorylation, and β-catenin in C/EBPa-saRNA-transfected cells. Our results suggested that C/EBPα-saRNA successfully inhibited HCC metastasis by inhibiting EGFR/β-catenin signaling pathway mediated EMT in vitro and in vivo. PMID:27050434

  2. 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. PMID:27677549

  3. Expression of rat liver Na+/L-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo.

    PubMed Central

    Palacin, M; Werner, A; Dittmer, J; Murer, H; Biber, J

    1990-01-01

    Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA. Images Fig. 6. PMID:2396979

  4. Structural and Functional Analysis of Interhelical Interactions in the Human Immunodeficiency Virus Type 1 gp41 Envelope Glycoprotein by Alanine-Scanning Mutagenesis

    PubMed Central

    Lu, Min; Stoller, Marisa O.; Wang, Shilong; Liu, Jie; Fagan, Melinda B.; Nunberg, Jack H.

    2001-01-01

    Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention. PMID:11602754

  5. β-Alanine ingestion increases muscle carnosine content and combat specific performance in soldiers.

    PubMed

    Hoffman, Jay R; Landau, Geva; Stout, Jeffrey R; Hoffman, Mattan W; Shavit, Nurit; Rosen, Philip; Moran, Daniel S; Fukuda, David H; Shelef, Ilan; Carmom, Erez; Ostfeld, Ishay

    2015-03-01

    The purpose of this study was to examine the effect of β-alanine (BA) ingestion on tissue carnosine levels and the impact such changes would have on combat specific activity. Eighteen soldiers (19.9 ± 0.8 year) from an elite combat unit were randomly assigned to either a BA or placebo (PL) group. Before and following a 30-day supplementation period carnosine content of the gastrocnemius muscle and brain was determined by proton magnetic resonance spectroscopy. During each testing session, participants performed military relevant tasks that included a 2.5 km run, a 1-min sprint, 50-m casualty carry, repeated 30-m sprints with target shooting, and a 2-min serial subtraction test (SST) to assess cognitive function under stressful conditions. A significant elevation (p = 0.048) in muscle carnosine content was noted in BA compared to PL. Changes in muscle carnosine content was correlated to changes in fatigue rate (r = 0.633, p = 0.06). No changes (p = 0.607) were observed in brain carnosine content. Following supplementation, no differences were noted in 2.5 km run, 1-min sprint, repeated sprint, or marksmanship performance, but participants in BA significantly (p = 0.044) improved their time for the 50-m casualty carry and increased their performance (p = 0.022) in the SST compared to PL. In summary, 30-days of BA ingestion can increase muscle carnosine content and improve aspects of military specific performance. Although cognitive performance was significantly greater in participants consuming BA compared to placebo, current study methods were unable to detect any change in brain carnosine levels, thus, the precise mechanism underlying these effects remains elusive.

  6. 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.

  7. 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

  8. Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice.

    PubMed

    de Souza, Ludmilla Regina; Muehlmann, Luis Alexandre; Matos, Lívia Carneiro; Simón-Vázquez, Rosana; Lacava, Zulmira Guerreiro Marques; De-Paula, Alfredo Maurício Batista; Mosiniewicz-Szablewska, Ewa; Suchocki, Piotr; Morais, Paulo César; González-Fernández, África; Báo, Sônia Nair; Azevedo, Ricardo Bentes

    2015-12-18

    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. PMID:26580675

  9. 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.

  10. New insights into the metabolism of organomercury compounds: Mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine γ-lyase

    PubMed Central

    Bridges, Christy C.; Krasnikov, Boris F.; Joshee, Lucy; Pinto, John T.; Hallen, André; Li, Jianyong; Zalups, Rudolfs K.; Cooper, Arthur J. L.

    2011-01-01

    Anthropogenic practices and recycling in the environment through natural processes result in release of potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-conjugate, such as the L-cysteine (Cys)-S-conjugate of methylmercury (CH3Hg-S-Cys) or inorganic mercury (Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to L-methionine and L-cystine/L-cystathionine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-containing amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing L-amino acids, including L-methionine, L-cystine, and L-cystathionine as substrates. The current study extends this list to include mercuric S-conjugates, and shows that CH3Hg-S-Cys and Cys-S-Hg-S-Cys are substrates and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH3Hg-S-Hcy, are also inhibitors. Finally, we show that HgCl2, CH3Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible inhibitors of rat cystathionine γ-lyase. The present study broadens our knowledge of the biochemistry of mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze transformations of biologically important sulfur-containing amino acids. PMID:22093698

  11. New insights into the metabolism of organomercury compounds: mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine γ-lyase.

    PubMed

    Bridges, Christy C; Krasnikov, Boris F; Joshee, Lucy; Pinto, John T; Hallen, André; Li, Jianyong; Zalups, Rudolfs K; Cooper, Arthur J L

    2012-01-01

    Anthropogenic practices and recycling in the environment through natural processes result in release of potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-conjugate, such as the l-cysteine (Cys)-S-conjugate of methylmercury (CH(3)Hg-S-Cys) or inorganic mercury (Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to l-methionine and l-cystine/l-cystathionine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-containing amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing l-amino acids, including l-methionine, l-cystine, and l-cystathionine as substrates. The current study extends this list to include mercuric S-conjugates, and shows that CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are substrates and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH(3)Hg-S-Hcy, are also inhibitors. Finally, we show that HgCl(2), CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible inhibitors of rat cystathionine γ-lyase. The present study broadens our knowledge of the biochemistry of mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze transformations of biologically important sulfur-containing amino acids.

  12. 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.

  13. AlaScan: A Graphical User Interface for Alanine Scanning Free-Energy Calculations.

    PubMed

    Ramadoss, Vijayaraj; Dehez, François; Chipot, Christophe

    2016-06-27

    Computation of the free-energy changes that underlie molecular recognition and association has gained significant importance due to its considerable potential in drug discovery. The massive increase of computational power in recent years substantiates the application of more accurate theoretical methods for the calculation of binding free energies. The impact of such advances is the application of parent approaches, like computational alanine scanning, to investigate in silico the effect of amino-acid replacement in protein-ligand and protein-protein complexes, or probe the thermostability of individual proteins. Because human effort represents a significant cost that precludes the routine use of this form of free-energy calculations, minimizing manual intervention constitutes a stringent prerequisite for any such systematic computation. With this objective in mind, we propose a new plug-in, referred to as AlaScan, developed within the popular visualization program VMD to automate the major steps in alanine-scanning calculations, employing free-energy perturbation as implemented in the widely used molecular dynamics code NAMD. The AlaScan plug-in can be utilized upstream, to prepare input files for selected alanine mutations. It can also be utilized downstream to perform the analysis of different alanine-scanning calculations and to report the free-energy estimates in a user-friendly graphical user interface, allowing favorable mutations to be identified at a glance. The plug-in also assists the end-user in assessing the reliability of the calculation through rapid visual inspection.

  14. 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.

  15. EPR study of light illumination effects on radicals in gamma-irradiated ?-alanine

    NASA Astrophysics Data System (ADS)

    Ciesielski, B.; Schultka, K.; Penkowski, M.; Sagstuen, E.

    2004-05-01

    Exposure of γ-irradiated L-alanine samples to sunlight and to light from a regular, fluorescent lamp resulted in significant changes in their EPR resonance patterns, both to spectral shapes and intensities. The experimental EPR spectra were numerically decomposed into three components reflecting contributions of three different radicals (R1-R3) generated by ionizing radiation in alanine. The light exposure caused a decay of the measured EPR signal intensity. For similar light intensities and exposure times the decay was much more pronounced in samples illuminated by sunlight than in samples illuminated by the fluorescent lamp. In both cases light-induced decay of R1 radicals was observed. Sunlight illumination resulted in a moderate decay of R2 radicals and in a doubling of the R3 radical population. On the other hand, fluorescent light caused a significant increase of R2 radicals and did not change the amount of R3 radicals. A quantitative analysis of the variations of the three radical contributions to the total EPR spectra upon fluorescent light exposure suggests a net R1→R2 free radical transformation. These effects of light on the alanine dosimetric signal should be taken into account in dosimetry protocols, assuring protection of alanine dosimeters from extended exposure to light.

  16. Uncertainties in alanine/ESR dosimetry at the Physikalisch-Technische Bundesanstalt.

    PubMed

    Anton, Mathias

    2006-11-01

    In radiation therapy, the effect of ionizing radiation is quantified in terms of the absorbed dose to water. Dosimetry with alanine and readout via electron spin resonance (ESR) is a method which is used as a secondary standard by several national metrology institutions. The advantages of the method are the good water-equivalence of the probes, their small size and the very weak dependence of the response on the radiation quality for MV x-rays and high-energy electrons used in radiation therapy. For radiation therapy, a small uncertainty of the applied dose is required. The present publication describes the determination of the uncertainty budget for the alanine/ESR dosimetry system of the Physikalisch-Technische Bundesanstalt (PTB), which relies on the use of a reference sample. A method is also presented which allows a reduction of the influence of fading or other changes of the ESR amplitude of irradiated alanine probes with time. If certain conditions are met which are described in detail, a relative uncertainty of less than 0.5% can be reached for probes irradiated with (60)Co in the 5-25 Gy dose range, including the uncertainty of the primary standard. First results for dose values between 2 Gy and 10 Gy are presented as well. From the high accuracy achievable with alanine dosimetry, we conclude that this method has great potential to solve measurement problems for modern methods of radiation therapy such as intensity modulated radiation therapy (IMRT) or tomotherapy.

  17. Mechanism of inactivation of alanine racemase by beta, beta, beta-trifluoroalanine

    SciTech Connect

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

    1989-01-24

    The alanine racemases are a group of PLP-dependent bacterial enzymes that catalyze the racemization of alanine, providing D-alanine for cell wall synthesis. Inactivation of the alanine racemases from the Gram-negative organism Salmonella typhimurium and Gram-positive organism Bacillus stearothermophilus with beta, beta, beta-trifluoroalanine has been studied. The inactivation occurs with the same rate constant as that for formation of a broad 460-490-nm chromophore. Loss of two fluoride ions per mole of inactivated enzyme and retention of (1-/sup 14/C)trifluoroalanine label accompany inhibition, suggesting a monofluoro enzyme adduct. Partial denaturation (1 M guanidine) leads to rapid return of the initial 420-nm chromophore, followed by a slower (t1/2 approximately 30 min-1 h) loss of the fluoride ion and /sup 14/CO/sub 2/ release. At this point, reduction by NaB/sub 3/H/sub 4/ and tryptic digestion yield a single radiolabeled peptide. Purification and sequencing of the peptide reveals that lysine-38 is covalently attached to the PLP cofactor. A mechanism for enzyme inactivation by trifluoroalanine is proposed and contrasted with earlier results on monohaloalanines, in which nucleophilic attack of released aminoacrylate on the PLP aldimine leads to enzyme inactivation. For trifluoroalanine inactivation, nucleophilic attack of lysine-38 on the electrophilic beta-difluoro-alpha, beta-unsaturated imine provides an alternative mode of inhibition for these enzymes.

  18. 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. PMID:19596338

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. Cadmium inhibition of L-alanine transport into renal brush border membrane vesicles isolated from the winter flounder (Pseudopleuronectes americanus)

    SciTech Connect

    Bevan, C.; Kinne-Saffran, E.; Foulkes, E.C.; Kinne, R.K. )

    1989-12-01

    Using isolated brush border membrane vesicles from the kidney of the winter flounder (Pseudopleuronectes americanus), we have studied the effect of cadmium on L-alanine transport. Pretreatment of vesicles with 0.1 mM Cd{sup 2+} resulted in inhibition of L-alanine uptake in the presence of a NaCl (but not KCl) gradient. Inhibition was due to a specific interaction with the sodium-alanine cotransport system and not a change in the driving forces for alanine transport, since Cd{sup 2+} did not affect sodium-dependent D-glucose uptake. The effect of Cd{sup 2+} on Na{sup +}-alanine cotransport showed mixed-type inhibition which is only partially reversible by EDTA. Cd{sup 2+} uptake itself was shown to be time and temperature dependent, resulting in binding to both sides of the membrane. No direct correlation was possible between inhibition of L-alanine transport and the amount of Cd{sup 2+} taken up by the membranes. Nevertheless, the striking time dependence of the effect of Cd{sup 2+} on sodium-dependent L-alanine uptake and the inability of EDTA to reverse the inhibitory action of Cd{sup 2+} suggest that Cd{sup 2+} inhibits Na+-alanine cotransport at the cytoplasmic side of the membrane.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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. PMID:27002976

  10. Auxin amidohydrolases from Brassica rapa cleave the alanine conjugate of indolepropionic acid as a preferable substrate: a biochemical and modeling approach.

    PubMed

    Savić, Bojana; Tomić, Sanja; Magnus, Volker; Gruden, Kristina; Barle, Katja; Grenković, Renata; Ludwig-Müller, Jutta; Salopek-Sondi, Branka

    2009-09-01

    Two auxin amidohydrolases, BrIAR3 and BrILL2, from Chinese cabbage [Brassica rapa L. ssp. pekinensis (Lour.) Hanelt] were produced by heterologous expression in Escherichia coli, purified, and screened for activity towards N-(indol-3-ylacetyl)-L-alanine (IAA-Ala) and the long-chain auxin-amino acid conjugates, N-[3-(indol-3-yl)propionyl]-L-alanine (IPA-Ala) and N-[4-(indol-3-yl)butyryl]-L-alanine (IBA-Ala). IPA-Ala was shown to be the favored substrate of both enzymes, but BrILL2 was approximately 15 times more active than BrIAR3. Both enzymes cleaved IBA-Ala and IAA-Ala to a lesser extent. The enzyme kinetics were measured for BrILL2 and the obtained parameters suggested similar binding affinities for the long-chain auxin-amino acid conjugates (IPA-Ala and IBA-Ala). The velocity of the hydrolyzing reaction decreased in the order IPA-Ala > IBA-Ala > IAA-Ala. In a root growth bioassay, higher growth inhibition was caused by IPA-Ala and IBA-Ala in comparison with IAA-Ala. Neither these conjugates nor the corresponding free auxins affected the expression of the BrILL2 gene. A modeling study revealed several possible modes of IPA-Ala binding to BrILL2. Based on these results, two possible scenarios for substrate hydrolysis are proposed. In one the metal binding water is activated by the carboxyl group of the substrate itself, and in the other by a glutamate residue from the active site of the enzyme.

  11. 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

  12. 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.

  13. Mechanism-Based Inactivation by Aromatization of the Transaminase BioA Involved in Biotin Biosynthesis in Mycobaterium tuberculosis

    PubMed Central

    Shi, Ce; Geders, Todd W.; Park, Sae Woong; Wilson, Daniel J.; Boshoff, Helena I.; Orisadipe, Abayomi; Barry, Clifton E.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2011-01-01

    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 Å 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. PMID:21988601

  14. Detection of Reduced GABA Synthesis Following Inhibition of GABA Transaminase Using in Vivo Magnetic Resonance Signal of [13C]GABA C1

    PubMed Central

    Yang, Jehoon; Johnson, Christopher; Shen, Jun

    2009-01-01

    Previous in vivo magnetic resonance spectroscopy (MRS) studies of gamma-aminobutyric acid (GABA) synthesis have relied on 13C label incorporation into GABA C2 from [1-13C] or [1,6-13C2]glucose. In this study, the [13C]GABA C1 signal at 182.3 ppm in the carboxylic/amide spectral region of localized in vivo 13C spectra was detected. GABA-transaminase of rat brain was inhibited by administration of gabaculine after pre-labeling of GABA C1 and its metabolic precursors with exogenous [2,5-13C2]glucose. A subsequent isotope chase experiment was performed by infusing unlabeled glucose, which revealed a markedly slow change in the labeling of GABA C1 accompanying the blockade of the GABA shunt. This slow labeling of GABA at elevated GABA concentration was attributed to the relatively small intercompartmental GABA-glutamine cycling flux that constitutes the main route of 13C label loss during the isotope chase. Because this study showed that using low RF power broadband stochastic proton decoupling is feasible at very high field strength, it has important implications for the development of carboxylic/amide 13C MRS methods to study brain metabolism and neurotransmission in human subjects at high magnetic fields. PMID:19540876

  15. Transient elevation of liver transaminase after starting insulin therapy for diabetic ketosis or ketoacidosis in newly diagnosed type 1 diabetes mellitus.

    PubMed

    Takaike, Hiroko; Uchigata, Yasuko; Iwasaki, Naoko; Iwamoto, Yasuhiko

    2004-04-01

    A mild increase of AST and/or ALT is sometimes observed among patients with diabetic ketosis or diabetic ketoacidosis (DKA) after initial insulin treatment, and the exact mechanism is still undefined. Therefore, we compared the clinical background between patients with and without transient elevation of liver transaminase (TELT) at the onset of type 1 diabetes mellitus with diabetic ketosis or DKA. Among 50 patients, 13 patients showed TELT. The TELT group was characterized by higher plasma glucose (P<0.05), higher HbA1c (P<0.05), and higher triglyceride (P<0.01) before insulin therapy than those without TELT. The TELT group needed significantly more insulin (P<0.05) for the treatment. In echo-histogram analysis of three patients with TELT, the increase of liver-kidney contrast after insulin treatment suggested fat deposition to the liver. The fat deposition to the liver might be one of the causes of the mild increase of AST and/or ALT after initial treatment of insulin in diabetic ketosis or DKA.

  16. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by. beta. -Cl-alanine

    SciTech Connect

    Medlock, K.A.; Merrill, A.H. Jr.

    1987-05-01

    Serine palmitoyltransferase (SPT) is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the first committed step of long-chain base (LCB) synthesis. Inhibition of SPT activity and de novo biosynthesis of sphinganine and sphingosine was observed in vitro and in intact Chinese hamster ovary cells (CHO). In vitro studies revealed that inhibition was irreversible and concentration- and time-dependent, which are characteristics of suicide inhibition. Incubation of intact CHO cells with 5 mM ..beta..-Cl-alanine for 15 min completely inhibited SPT activity and LCB synthesis from (/sup 14/C)serine. The concentration dependences of inhibition of SPT activity and LCB formation were identical. There was no loss of viability of recovery of SPT activity over the 2 hour time course of these experiments. The synthesis of several other lipids was not affected by the same treatment. These results establish the association between the activity of SPT and the cellular rate of LCB formation and indicate that ..beta..-Cl-alanine can be used to study alterations in cellular LCB synthesis.

  17. Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice.

    PubMed

    Xie, Jing-Dun; Huang, Yang; Chen, Dong-Tai; Pan, Jia-Hao; Bi, Bing-Tian; Feng, Kun-Yao; Huang, Wan; Zeng, Wei-An

    2015-01-01

    Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided.

  18. 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

    2011-11-01

    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.

  19. Molecular dynamics simulations of the intramolecular proton transfer and carbanion stabilization in the pyridoxal 5'-phosphate dependent enzymes L-dopa decarboxylase and alanine racemase.

    PubMed

    Lin, Yen-Lin; Gao, Jiali; Rubinstein, Amir; Major, Dan Thomas

    2011-11-01

    Molecular dynamics simulations using a combined quantum mechanical and molecular mechanical (QM/MM) potential have been carried out to investigate the internal proton transfer equilibrium of the external aldimine species in l-dopa decarboxylase, and carbanion stabilization by the enzyme cofactor in the active site of alanine racemase. Solvent effects lower the free energy of the O-protonated PLP tautomer both in aqueous solution and in the active site, resulting a free energy difference of about -1 kcal/mol relative to the N-protonated Schiff base in the enzyme. The external aldimine provides the dominant contribution to lowering the free energy barrier for the spontaneous decarboxylation of l-dopa in water, by a remarkable 16 kcal/mol, while the enzyme l-dopa decarboxylase further lowers the barrier by 8 kcal/mol. Kinetic isotope effects were also determined using a path integral free energy perturbation theory on the primary (13)C and the secondary (2)H substitutions. In the case of alanine racemase, if the pyridine ring is unprotonated as that in the active site, there is destabilizing contribution to the formation of the α-carbanion in the gas phase, although when the pyridine ring is protonated the contribution is stabilizing. In aqueous solution and in alanine racemase, the α-carbanion is stabilized both when the pyridine ring is protonated and unprotonated. The computational studies illustrated in this article show that combined QM/MM simulations can help provide a deeper understanding of the mechanisms of PLP-dependent enzymes. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

  20. 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

  1. The alanine detector in BNCT dosimetry: Dose response in thermal and epithermal neutron fields

    SciTech Connect

    Schmitz, T.; Bassler, N.; Blaickner, M.; Ziegner, M.; Hsiao, M. C.; Liu, Y. H.; Koivunoro, H.; Auterinen, I.; Serén, T.; Kotiluoto, P.; Palmans, H.; Sharpe, P.; Langguth, P.; Hampel, G.

    2015-01-15

    Purpose: The response of alanine solid state dosimeters to ionizing radiation strongly depends on particle type and energy. Due to nuclear interactions, neutron fields usually also consist of secondary particles such as photons and protons of diverse energies. Various experiments have been carried out in three different neutron beams to explore the alanine dose response behavior and to validate model predictions. Additionally, application in medical neutron fields for boron neutron capture therapy is discussed. Methods: Alanine detectors have been irradiated in the thermal neutron field of the research reactor TRIGA Mainz, Germany, in five experimental conditions, generating different secondary particle spectra. Further irradiations have been made in the epithermal neutron beams at the research reactors FiR 1 in Helsinki, Finland, and Tsing Hua open pool reactor in HsinChu, Taiwan ROC. Readout has been performed with electron spin resonance spectrometry with reference to an absorbed dose standard in a {sup 60}Co gamma ray beam. Absorbed doses and dose components have been calculated using the Monte Carlo codes FLUKA and MCNP. The relative effectiveness (RE), linking absorbed dose and detector response, has been calculated using the Hansen and Olsen alanine response model. Results: The measured dose response of the alanine detector in the different experiments has been evaluated and compared to model predictions. Therefore, a relative effectiveness has been calculated for each dose component, accounting for its dependence on particle type and energy. Agreement within 5% between model and measurement has been achieved for most irradiated detectors. Significant differences have been observed in response behavior between thermal and epithermal neutron fields, especially regarding dose composition and depth dose curves. The calculated dose components could be verified with the experimental results in the different primary and secondary particle fields. Conclusions: The

  2. Mutation in a D-alanine-D-alanine ligase of Azospirillum brasilense Cd results in an overproduction of exopolysaccharides and a decreased tolerance to saline stress.

    PubMed

    Jofré, Edgardo; Fischer, Sonia; Príncipe, Analía; Castro, Marina; Ferrari, Walter; Lagares, Antonio; Mori, Gladys

    2009-01-01

    Bacteria of the genus Azospirillum are free-living nitrogen-fixing, rhizobacteria that are found in close association with plant roots, where they exert beneficial effects on plant growth and yield in many crops of agronomic importance. Unlike other bacteria, little is known about the genetics and biochemistry of exopolysaccharides in Azospirillum brasilense. In an attempt to characterize genes associated with exopolysaccharides production, we generated an A. brasilense Cd Tn5 mutant that showed exopolysaccharides overproduction, decreased tolerance to saline conditions, altered cell morphology, and increased sensitivity to detergents. Genetic characterization showed that the Tn5 was inserted within a ddlB gene encoding for a d-alanine-d-alanine ligase, and located upstream of the ftsQAZ gene cluster responsible for cell division in different bacteria. Heterologous complementation of the ddlB Tn5 mutant restored the exopolysaccharides production to wild-type levels and the ability to grow in the presence of detergents, but not the morphology and growth characteristics of the wild-type bacteria, suggesting a polar effect of Tn5 on the fts genes. This result and the construction of a nonpolar ddlB mutant provide solid evidence of the presence of transcriptional coupling between a gene associated with peptidoglycan biosynthesis and the fts genes required to control cell division.

  3. Alanine scanning analysis and structure-function relationships of the frog-skin antimicrobial peptide temporin-1Ta.

    PubMed

    Grieco, Paolo; Luca, Vincenzo; Auriemma, Luigia; Carotenuto, Alfonso; Saviello, Maria Rosaria; Campiglia, Pietro; Barra, Donatella; Novellino, Ettore; Mangoni, Maria Luisa

    2011-05-01

    The increasing resistance of bacteria and fungi to the available antibiotic/antimycotic drugs urges for a search for new anti-infective compounds with new modes of action. In line of this, natural CAMPs represent promising and attractive candidates. Special attention has been devoted to frog-skin temporins, because of their short size (10-14 residues long) and their unique features. In particular, temporin-1Ta has the following properties: (i) it is mainly active on Gram-positive bacteria; (ii) it can synergize, when combined with temporin-1Tl, in inhibiting both gram-negative bacterial growth and the toxic effect of LPS; (iii) it preserves biological activity in the presence of serum; and (iv) it is practically not hemolytic. Rational design of CAMPs represents a straightforward approach to obtain a peptide with a better therapeutic index. Here, we used alanine scanning analogs to elucidate the contribution of the side chains of each amino acid residue to the peptide's antimicrobial and hemolytic activity. Beside providing insight into the biophysical attributes and the critical positions within the peptide sequence, which govern the antimicrobial/hemolytic activity of this temporin isoform, our studies assist in optimizing the design of temporin-based lead structures for the production of new anti-infective agents. PMID:21337476

  4. The behaviour of alanine dosimeters at temperatures between 100 and 300 K

    NASA Astrophysics Data System (ADS)

    Sharpe, P. H. G.; Sephton, J. P.; Gouldstone, C. A.

    2009-07-01

    A cryostat has been constructed to enable irradiations in a MDS Nordion Gammacell 220 irradiator to be carried out at selected temperatures between 100 and 300 K. The principle of operation and the performance of this cryostat are described and results are given of a study into the behaviour of alanine dosimeters at cryogenic temperatures. This work extends previously published data to the region between solid CO 2 and liquid N 2 temperatures and has demonstrated complex dose-dependent behaviour. A sharp discontinuity in the effect of temperature on alanine dosimeter response has been found in the region between 150 and 180 K, with no further influence of irradiation temperature on response observed below this point.

  5. 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.

  6. 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.

  7. Chiral effects on helicity studied via the energy landscape of short (d, l)-alanine peptides

    NASA Astrophysics Data System (ADS)

    Neelamraju, Sridhar; Oakley, Mark T.; Johnston, Roy L.

    2015-10-01

    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.

  8. 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.

  9. 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.

  10. 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

    2015-01-01

    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.

  11. Response of the alanine/ESR dosimeter to radiation from an Ir-192 HDR brachytherapy source

    NASA Astrophysics Data System (ADS)

    Anton, M.; Hackel, T.; Zink, K.; von Voigts-Rhetz, P.; Selbach, H.-J.

    2015-01-01

    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.

  12. L-alanine and inosine enhancement of glucose triggering in Bacillus megaterium spores.

    PubMed

    Bédard, J; Lefebvre, G M

    1989-08-01

    Both rate and extent of germination of Bacillus megaterium 14581 (ATCC) spores are considerably augmented when L-alanine and inosine are added to the glucose commonly used as triggering agent for this strain. This enhancement does not arise from heterogeneity in germination requirements of the dormant spore, but is rather a consequence of the combined action of glucose and either or both of the added reagents on a sizeable fraction of spores unable to germinate in glucose alone. Nearly half of the spores that eventually germinate in the mixture of germinants used are either triggered by glucose or are sensitized by it to subsequent triggering by L-alanine and inosine in the first 10 s of imbibition. For a good number of these spores, then, triggering consists of a sequence of separable events. PMID:2510916

  13. 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.

  14. β-alanine suppresses malignant breast epithelial cell aggressiveness through alterations in metabolism and cellular acidity in vitro

    PubMed Central

    2014-01-01

    Background Deregulated energetics is a property of most cancer cells. This phenomenon, known as the Warburg Effect or aerobic glycolysis, is characterized by increased glucose uptake, lactate export and extracellular acidification, even in the presence of oxygen. β-alanine is a non-essential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. Because of this buffering capacity, we investigated the effects of β-alanine on the metabolic cancerous phenotype. Methods Non-malignant MCF-10a and malignant MCF-7 breast epithelial cells were treated with β-alanine at 100 mM for 24 hours. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate (OCR). mRNA of metabolism-related genes was quantified by qRT-PCR with corresponding protein expression quantified by immunoblotting, or by flow cytometry which was verified by confocal microscopy. Mitochondrial content was quantified using a mitochondria-specific dye and measured by flow cytometry. Results Cells treated with β-alanine displayed significantly suppressed basal and peak ECAR (aerobic glycolysis), with simultaneous increase in glucose transporter 1 (GLUT1). Additionally, cells treated with β-alanine exhibited significantly reduced basal and peak OCR (oxidative metabolism), which was accompanied by reduction in mitochondrial content with subsequent suppression of genes which promote mitochondrial biosynthesis. Suppression of glycolytic and oxidative metabolism by β-alanine resulted in the reduction of total metabolic rate, although cell viability was not affected. Because β-alanine treatment reduces extracellular acidity, a constituent of the invasive microenvironment that promotes progression, we investigated the effect of β-alanine on breast cell viability and migration. β-alanine was shown to reduce both cell migration and proliferation

  15. 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.

  16. 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.

  17. The influence of β-alanine supplementation on markers of exercise-induced oxidative stress.

    PubMed

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

    2014-01-01

    β-Alanine (BA) has been linked with oxidative protection. This study evaluated antioxidant properties of BA. Twenty-five men consumed BA or placebo for 4 weeks, and completed a 40-min run to induce oxidative stress. Blood draws were taken to measure 8-isoprostane, total antioxidant capacity, superoxide dismutase, and glutathione. BA had no significant influence on reducing exercise-induced oxidative stress. Confidence intervals suggest a reduction in lipid peroxidation. BA supplementation may have little influence as an antioxidant.

  18. 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. PMID:27215379

  19. 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

  20. 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.

  1. Upper Limits of Normal for Serum Alanine Aminotransferase Levels in Chinese Han Population

    PubMed Central

    Zheng, Ming-Hua; Shi, Ke-Qing; Fan, Yu-Chen; Liu, Wen-Yue; Lin, Xian-Feng; Li, Ling-Fei; Chen, Yong-Ping

    2012-01-01

    Background and Objectives Serum alanine aminotransferase (ALT) activity is the most common tool for the assessment of liver diseases. However, it is not clear whether the current normal ALT range really discriminate patients with or without liver diseases. The present study was to establish a new normal range of ALT and examine its ability to identify patients with hepatitis B or nonalcoholic fatty liver disease (NAFLD) in Chinese Han population. Methods 53037 adults were included in this study from January 1st 2008 to August 31st 2010. The 95th percentile of ALT in population with relative low risk factors for liver diseases was set as the new upper limits of normal ALT in gender-specific manner. Results The 95th percentile levels at low risk factors for liver diseases were achieved at 35 U/L for men and 23 U/L for women. The concordance statistics for detection were 0.873 (95%CI: 0.865–0.881) for HBV and 0.932 (95%CI: 0.927–0.937) for NAFLD in men while 0.857 (95%CI: 0.850–0.864) for HBV and 0.909 (95%CI: 0.903–0.915) for NAFLD in women. The median sensitivity of the current used ALT upper limit (40 U/L) was 6.6% for HBV and 29.7% for NAFLD and median specificity was 98.7% for men and 99.4% for women. Using our new-derived thresholds, the sensitivities ranged from 35.3% to 61.1% and the specificities were 94.8% for men and 94.6% for women. Conclusions Our results suggest that upper limits of ALT 35 U/L for men and 23 U/L for women in Chinese Han population. Re-consideration of normal limits of ALT should be recommended. Trial Registration ChiCTR.org ChiCTR-OCS-11001173 PMID:22962588

  2. Monte Carlo Simulation of the Irradiation of Alanine Coated Film Dosimeters with Accelerated Electrons

    NASA Astrophysics Data System (ADS)

    Uribe, R. M.; Salvat, F.; Cleland, M. R.; Berejka, A.

    2009-03-01

    The Monte Carlo code PENELOPE was used to simulate the irradiation of alanine coated film dosimeters with electron beams of energies from 1 to 5 MeV being produced by a high-current industrial electron accelerator. This code includes a geometry package that defines complex quadratic geometries, such as those of the irradiation of products in an irradiation processing facility. In the present case the energy deposited on a water film at the surface of a wood parallelepiped was calculated using the program PENMAIN, which is a generic main program included in the PENELOPE distribution package. The results from the simulation were then compared with measurements performed by irradiating alanine film dosimeters with electrons using a 150 kW Dynamitron™ electron accelerator. The alanine films were placed on top of a set of wooden planks using the same geometrical arrangement as the one used for the simulation. The way the results from the simulation can be correlated with the actual measurements, taking into account the irradiation parameters, is described. An estimation of the percentage difference between measurements and calculations is also presented.

  3. Characterisation of L-alanine and glycine absorption across the gut of an ancient vertebrate.

    PubMed

    Glover, Chris N; Bucking, Carol; Wood, Chris M

    2011-08-01

    This study utilised an in vitro technique to characterise absorption of two amino acids across the intestinal epithelium of Pacific hagfish, Eptatretus stoutii. Uptake of L-alanine and glycine conformed to Michaelis-Menten kinetics. An uptake affinity (K(m); substrate concentration required to attain a 50% uptake saturation) of 7.0 mM and an uptake capacity (J (max)) of 83 nmol cm(-2) h(-1) were described for L-alanine. The K(m) and J(max) for glycine were 2.2 mM and 11.9 nmol cm(-2) h(-1), respectively. Evidence suggested that the pathways of L-alanine and glycine absorption were shared, and sodium dependent. Further analysis indicated that glycine uptake was independent of luminal pH and proline, but a component of uptake was significantly impaired by 100-fold excesses of threonine or asparagine. The presence of a short-term (24 h) exposure to waterborne glycine, similar in nature to that which may be expected to occur when feeding inside an animal carcass, had no significant impact on gastrointestinal glycine uptake. This may indicate a lack of cross talk between absorptive epithelia. These results are the first published data to describe gastrointestinal uptake of an organic nutrient in the oldest extant vertebrate and may provide potential insight into the evolution of nutrient transport systems.

  4. Conformational study of N-methylated alanine peptides and design of Abeta inhibitor.

    PubMed

    Nandel, Fateh S; Jaswal, Radhika R

    2014-02-01

    N-Methylation increases the proteolytic stability of peptides and leads to improved pharmacological and increased nematicidal property against plant pathogens. In this study, the quantum mechanical and molecular dynamic simulation approaches were used to investigate conformational behavior of peptides containing only N-methylated alanine (NMeAla) residues and N-methylated alanine and alanine residues at alternate positions. The amide bond geometry was found to be trans and the poly NMeAla peptides were shown to populate in the helical structure without hydrogen bond with phi, psi values of - 0, 90 degrees stabilized by carbonyl-carbonyl interactions. Molecular dynamic simulations in water/methanol revealed the formation of beta-strand structure, irrespective of the starting geometry due to the interaction of solvent molecules with the carbonyl groups of peptide backbone. Analysis of simulation results as a function of time suggested that the opening of helical structure without hydrogen bond started from C-terminal. Conformational behavior of peptides containing N-MeAla and Ala was used to design Abeta peptide inhibitor and the model tetrapeptide Ac-Ala-NMeAla-Ala-NHMe in the beta-strand structure was shown to interact with the hydrophobic stretch of Abeta15-42 peptide.

  5. 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.

  6. Monte Carlo Simulation of the Irradiation of Alanine Coated Film Dosimeters with Accelerated Electrons

    SciTech Connect

    Uribe, R. M.; Salvat, F.; Cleland, M. R.; Berejka, A.

    2009-03-10

    The Monte Carlo code PENELOPE was used to simulate the irradiation of alanine coated film dosimeters with electron beams of energies from 1 to 5 MeV being produced by a high-current industrial electron accelerator. This code includes a geometry package that defines complex quadratic geometries, such as those of the irradiation of products in an irradiation processing facility. In the present case the energy deposited on a water film at the surface of a wood parallelepiped was calculated using the program PENMAIN, which is a generic main program included in the PENELOPE distribution package. The results from the simulation were then compared with measurements performed by irradiating alanine film dosimeters with electrons using a 150 kW Dynamitron electron accelerator. The alanine films were placed on top of a set of wooden planks using the same geometrical arrangement as the one used for the simulation. The way the results from the simulation can be correlated with the actual measurements, taking into account the irradiation parameters, is described. An estimation of the percentage difference between measurements and calculations is also presented.

  7. 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. PMID:26369758

  8. 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.

  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. [Hepatitis C and normal transaminases].

    PubMed

    Malé Velázquez, René; Ayuzo del Valle, Cipatli

    2002-10-01

    Since the discovery of hepatitis C virus and its serologic markers, an identification of a subgroup of patients with persistent normal ALT (PNALT) was done. It has been estimated that this group accounts to 25% of patients with chronic hepatitis C. It is not recommended to biopsy or initiate treatment because of the natural history of the disease is benign with slow progression. However, recent studies have shown mild to moderate histologic abnormalities in nearly 80% of the patients. Interferon treatment as monotherapy has not shown significant results and there are not enough data to support combined therapy. It is recommended clinical follow up every 3 to 6 months combined with liver function tests. Biopsy and antiviral treatment should only be considered for patients included in clinical trials.

  11. Alanine aminotransferase 1 (OsAlaAT1) plays an essential role in the regulation of starch storage in rice endosperm.

    PubMed

    Yang, Jungil; Kim, Sung-Ryul; Lee, Sang-Kyu; Choi, Heebak; Jeon, Jong-Seong; An, Gynheung

    2015-11-01

    Alteration of storage substances, in particular the major storage form starch, leads to floury endosperm. Because floury mutants have physical attributes for milling processes, identification and characterization of those mutants are valuable. In this study we identified a floury endosperm mutant caused by a T-DNA insertion in Oryza sativa alanine-aminotransferase1 (OsAlaAT1). OsAlaAT1 is localized in the cytosol and has aminotransferase enzyme activity. The osalaat1 mutant has less amylose and its amylopectin is structurally altered. OsAlaAT1 is predominantly expressed in developing seeds during active starch synthesis. AlaAT catalyzes the interconversion of pyruvate to alanine, and this pathway is activated under low-oxygen conditions. Consistently, OsAlaAT1 is induced by such conditions. Expression of the starch synthesis genes AGPases, OsSSI, OsSSIIa, and OsPPDKB is decreased in the mutant. Thus, our observations suggest that OsAlaAT1 plays an essential role in starch synthesis in developing seeds that are exposed to low concentrations of oxygen. PMID:26475189

  12. Polymorphism in supramolecular chiral structures of R- and S-alanine on Cu(1 1 0)

    NASA Astrophysics Data System (ADS)

    Barlow, S. M.; Louafi, S.; Le Roux, D.; Williams, J.; Muryn, C.; Haq, S.; Raval, R.

    2005-10-01

    A comprehensive study of the local and supramolecular adsorption structures created by the chiral R- and S-enantiomers of alanine on the Cu(1 1 0) surface has been conducted using a multi-technique approach, including reflection absorption infrared spectroscopy (RAIRS), X-ray photoelectron spectroscopy (XPS), low energy electron diffraction (LEED) and scanning tunnelling microscopy (STM). Over the entire 300-470 K temperature range studied, the amino acid is found to adsorb as an alaninate species with a local chiral adsorption motif. However, this singular preference of local chemical form contrasts sharply with the supramolecular organisation at the surface where polymorphism is exhibited. This polymorphic behaviour arises from subtle and dynamic changes in the bonding, orientation and adsorption footprints of individual molecules, leading to alterations in the molecule-metal, intermolecular and metal-metal interactions that dictate self-assembly. Thus, at low coverage, a single disordered phase is observed but at higher coverage, three other temperature dependent phases occur. At room temperature, a two-dimensional equivalent of a 'nematic' phase is constructed from short single- and double-chain chiral assemblies that possess a preferred chiral orientation but no long range periodicity. This 'nematic' phase acts as a precursor to a highly ordered chiral supramolecular assembly, created at 430 K, that consists of regular arrays of size- and shape-defined chiral clusters. This phase possesses global organisational chirality with only one chiral domain observed for each enantiomer. For both the 'nematic' and the highly ordered chiral phase, the organisation for the R-enantiomer is the mirror image of that seen for the S-enantiomer, i.e., there is chirality transfer from the nanoscale to the macroscale. By 470 K, both R- and S-alanine form an achirally organised (3 × 2) structure that appears to be the thermodynamically favoured phase for the alanine/Cu(1 1 0

  13. Promoter scanning of the human COX-2 gene with 8-ring polyamides: unexpected weakening of polyamide-DNA binding and selectivity by replacing an internal N-Me-pyrrole with β-alanine.

    PubMed

    Bashkin, James K; Aston, Karl; Ramos, Joseph P; Koeller, Kevin J; Nanjunda, Rupesh; He, Gaofei; Dupureur, Cynthia M; David Wilson, W

    2013-02-01

    Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal β-alanines as polyamide building blocks in place of N-methylpyrrole; β-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with β-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with β-alanine to generate a β/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that β-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.

  14. Neutron diffraction investigations of L- and D-alanine at different temperatures: The search for structural evidence for parity violation

    SciTech Connect

    Wilson, Chick C.; Ghosh, Minakshi; Johnson, Louise N.; Wang, Wenging

    2005-09-01

    Single crystal neutron diffraction has been used in an investigation of the structures of the amino acids L- and D-alanine. The aim of the study was to look for proposed phase transitions around T{sub c} {approx} 270 K. Measurements of both structures at 295 K and 60 K - the neutron structure of D-alanine being determined for the first time - show no significant structural basis for this phase transition in alanine. Further, confirmatory, investigation of the structure of D-alanine at temperatures of 240, 250, 260 and 300 K also showed no significant changes in bond lengths or angles. We can thus offer no structural support to other physical measurements, which are indicative of the observable effect of parity violation of the electroweak force in these phase transitions.

  15. Painting proteins blue: β-(1-azulenyl)-L-alanine as a probe for studying protein-protein interactions.

    PubMed

    Moroz, Yurii S; Binder, Wolfgang; Nygren, Patrik; Caputo, Gregory A; Korendovych, Ivan V

    2013-01-18

    We demonstrated that β-(1-azulenyl)-L-alanine, a fluorescent pseudoisosteric analog of tryptophan, exhibits weak environmental dependence and thus allows for using weak intrinsic quenchers, such as methionines, to monitor protein-protein interactions while not perturbing them.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. Evidence-based evaluation of potential benefits and safety of beta-alanine supplementation for military personnel.

    PubMed

    Ko, Richard; Low Dog, Tieraona; Gorecki, Dennis K J; Cantilena, Louis R; Costello, Rebecca B; Evans, William J; Hardy, Mary L; Jordan, Scott A; Maughan, Ronald J; Rankin, Janet W; Smith-Ryan, Abbie E; Valerio, Luis G; Jones, Donnamaria; Deuster, Patricia; Giancaspro, Gabriel I; Sarma, Nandakumara D

    2014-03-01

    This Department of Defense-sponsored evidence-based review evaluates the safety and putative outcomes of enhancement of athletic performance or improved recovery from exhaustion in studies involving beta-alanine alone or in combination with other ingredients. Beta-alanine intervention studies and review articles were collected from 13 databases, and safety information was collected from adverse event reporting portals. Due to the lack of systematic studies involving military populations, all the available literature was assessed with a subgroup analysis of studies on athletes to determine if beta-alanine would be suitable for the military. Available literature provided only limited evidence concerning the benefits of beta-alanine use, and a majority of the studies were not designed to address safety. Overall, the strength of evidence in terms of the potential for risk of bias in the quality of the available literature, consistency, directness, and precision did not support the use of beta-alanine by military personnel. The strength of evidence for a causal relation between beta-alanine and paresthesia was moderate.

  2. Effects of beta-alanine supplementation on the onset of neuromuscular fatigue and ventilatory threshold in women.

    PubMed

    Stout, J R; Cramer, J T; Zoeller, R F; Torok, D; Costa, P; Hoffman, J R; Harris, R C; O'Kroy, J

    2007-01-01

    This study examined the effects of 28 days of beta-alanine supplementation on the physical working capacity at fatigue threshold (PWCFT), ventilatory threshold (VT), maximal oxygen consumption (VO2-MAX), and time-to-exhaustion (TTE) in women. Twenty-two women (age+/-SD 27.4+/-6.1 yrs) participated and were randomly assigned to either the beta-alanine (CarnoSyn) or Placebo (PL) group. Before (pre) and after (post) the supplementation period, participants performed a continuous, incremental cycle ergometry test to exhaustion to determine the PWCFT, VT, VO2-MAX, and TTE. There was a 13.9, 12.6 and 2.5% increase (p<0.05) in VT, PWCFT, and TTE, respectively, for the beta-alanine group, with no changes in the PL (p>0.05). There were no changes for VO2-MAX (p>0.05) in either group. Results of this study indicate that beta-alanine supplementation delays the onset of neuromuscular fatigue (PWCFT) and the ventilatory threshold (VT) at submaximal workloads, and increase in TTE during maximal cycle ergometry performance. However, beta-alanine supplementation did not affect maximal aerobic power (VO2-MAX). In conclusion, beta-alanine supplementation appears to improve submaximal cycle ergometry perf