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Sample records for acid alanine aminotransferase

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

    DOEpatents

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

    2011-10-04

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

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

    SciTech Connect

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

    2014-11-18

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

  3. Association between Serum Uric Acid and Elevated Alanine Aminotransferase in the General Population

    PubMed Central

    Chen, Shuang; Guo, Xiaofan; Yu, Shasha; Sun, Guozhe; Yang, Hongmei; Li, Zhao; Sun, Yingxian

    2016-01-01

    Background: Both the serum uric acid (SUA) level and elevated alanine aminotransferase (ALT) are related to metabolic syndrome. However, the association between SUA and elevated ALT has not been elucidated in the general population. The objective of this study was to investigate the association between SUA and elevated ALT in the general population of China; Methods: A total of 11,572 adults (≥35 years of age) participated in this survey. Elevated ALT was defined as >40 U/L. SUA ≥ 7.0 mg/dL in males or ≥6.0 mg/dL in females was defined as hyperuricemia. SUA within the reference range was divided into quartiles, and its associations with elevated ALT were evaluated by logistic regressions; Results: A total of 7.4% participants had elevated ALT. The prevalence of hyperuricemia was 14.9% in males and 7.3% in females. There was a significantly positive dose-response association between SUA levels and the prevalence of elevated ALT. After adjusting for potential confounders, a positive relationship for elevated ALT was observed in subjects with hyperuricemia (odds ratio [OR]: 2.032, 95% confidence interval [CI]: 1.443–2.861 for men; OR: 2.045, 95% CI: 1.221–3.425 for women, both p < 0.05). Within the reference range, the association between SUA and elevated ALT persisted in the fourth quartile (OR: 1.467, 95% CI: 1.063–2.025 for men; OR: 1.721, 95% CI: 1.146–2.585 for women, both p < 0.05); Conclusions: Our results indicated that an increased SUA level, even within the reference range, was independently associated with elevated ALT in Chinese adults. PMID:27563918

  4. 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

  5. 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.

  6. Crystal structures of Aedes aegypti alanine glyoxylate aminotransferase.

    PubMed

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

    2006-12-01

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

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

    PubMed

    Masola, B; Devlin, T M

    1995-12-01

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

  8. Diabetes-linked transcription factor HNF4α regulates metabolism of endogenous methylarginines and β-aminoisobutyric acid by controlling expression of alanine-glyoxylate aminotransferase 2

    PubMed Central

    Burdin, Dmitry V.; Kolobov, Alexey A.; Brocker, Chad; Soshnev, Alexey A.; Samusik, Nikolay; Demyanov, Anton V.; Brilloff, Silke; Jarzebska, Natalia; Martens-Lobenhoffer, Jens; Mieth, Maren; Maas, Renke; Bornstein, Stefan R.; Bode-Böger, Stefanie M.; Gonzalez, Frank; Weiss, Norbert; Rodionov, Roman N.

    2016-01-01

    Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients. PMID:27752141

  9. Diabetes-linked transcription factor HNF4α regulates metabolism of endogenous methylarginines and β-aminoisobutyric acid by controlling expression of alanine-glyoxylate aminotransferase 2.

    PubMed

    Burdin, Dmitry V; Kolobov, Alexey A; Brocker, Chad; Soshnev, Alexey A; Samusik, Nikolay; Demyanov, Anton V; Brilloff, Silke; Jarzebska, Natalia; Martens-Lobenhoffer, Jens; Mieth, Maren; Maas, Renke; Bornstein, Stefan R; Bode-Böger, Stefanie M; Gonzalez, Frank; Weiss, Norbert; Rodionov, Roman N

    2016-10-18

    Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1-6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.

  10. Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I

    PubMed Central

    Pey, Angel L.; Albert, Armando; Salido, Eduardo

    2013-01-01

    Alanine-glyoxylate aminotransferase catalyzes the transamination between L-alanine and glyoxylate to produce pyruvate and glycine using pyridoxal 5′-phosphate (PLP) as cofactor. Human alanine-glyoxylate aminotransferase is a peroxisomal enzyme expressed in the hepatocytes, the main site of glyoxylate detoxification. Its deficit causes primary hyperoxaluria type I, a rare but severe inborn error of metabolism. Single amino acid changes are the main type of mutation causing this disease, and considerable effort has been dedicated to the understanding of the molecular consequences of such missense mutations. In this review, we summarize the role of protein homeostasis in the basic mechanisms of primary hyperoxaluria. Intrinsic physicochemical properties of polypeptide chains such as thermodynamic stability, folding, unfolding, and misfolding rates as well as the interaction of different folding states with protein homeostasis networks are essential to understand this disease. The view presented has important implications for the development of new therapeutic strategies based on targeting specific elements of alanine-glyoxylate aminotransferase homeostasis. PMID:23956997

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

    PubMed Central

    McAllister, Chandra H.; Good, Allen G.

    2015-01-01

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

  12. 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.

  13. Serum Alanine Aminotransferase Levels, Hematocrit Rate and Body Weight Correlations Before and After Hemodialysis Session

    PubMed Central

    Lopes, Edmundo Pessoa; Sette, Luis Henrique B. C.; Sette, Jorge Bezerra C.; Luna, Carlos F.; Andrade, Amaro M.; Moraes, Maviael; Sette, Paulo C. A.; Menezes, Roberto; Cavalcanti, Rui L.; Conceição, Sergio C.

    2009-01-01

    PURPOSE To evaluate alanine aminotransferase levels before and after a hemodialysis session and to correlate these values with the hematocrit rate and weight loss during hemodialysis. PATIENTS AND METHODS The serum alanine aminotransferase levels, hematocrit rate and body weight were measured and correlated before and after a single hemodialysis session for 146 patients with chronic renal failure. An receiver operating characteristic (ROC) curve for the serum alanine aminotransferase levels collected before and after hemodialysis was plotted to identify hepatitis C virus-infected patients. RESULTS The mean weight loss of the 146 patients during hemodialysis was 5.3% (p < 0.001). The mean alanine aminotransferase levels before and after hemodialysis were 18.8 and 23.9 IU/, respectively, denoting a significant 28.1% increase. An equally significant increase of 16.4% in the hematocrit rate also occurred after hemodialysis. The weight loss was inversely correlated with the rise in both the alanine aminotransferase level (r = 0.3; p < 0.001) and hematocrit rate (r = 0.5; p < 0.001). A direct correlation was found between the rise in alanine aminotransferase levels and the hematocrit during the hemodialysis session (r = 0.4; p < 0.001). Based on the ROC curve, the upper limit of the normal alanine aminotransferase level should be reduced by 40% relative to the upper limit of normal if the blood samples are collected before the hemodialysis session or by 60% if blood samples are collected after the session. CONCLUSION In the present study, significant elevations in the serum alanine aminotransferase levels and hematocrit rates occurred in parallel to a reduction in body weight after the hemodialysis session. These findings suggest that one of the factors for low alanine aminotransferase levels prior to hemodialysis could be hemodilution in patients with chronic renal failure. PMID:19841699

  14. Observations of alanine aminotransferase and aspartate aminotransferase in THRIVE studies treated orally with ximelagatran.

    PubMed

    Harenberg, Job; Jörg, Ingrid; Weiss, Christel

    2006-01-01

    Treatment of acute venous thromboembolism (VTE) and prophylaxis of recurrent events has been investigated in the THRIVE (THRombin Inhibitor in Venous Thrombe Embolism) Treatment and the THRIVE III trial using the oral direct thrombin inhibitor ximelagatran. Alanine aminotransferase (ALAT) increased in 9.6% and 6.4% of patients in the THRIVE Treatment and THRIVE III trials, respectively. The authors analysed the time course of the ALAT and in additionally of aspartate aminotransferase (ASAT) in blood from 52 and 23 patients participating in the THRIVE Treatment and the THRIVE III trials in Germany. Analysis of variance for repeated measures and t test were performed. In the THRIVE Treatment trial, ALAT was significantly higher at week 2 for enoxaparin/warfarin (p => .0039, t test) and at months 3 and 6 for ximelagatran (p = .0453, p = .0014, respectively). ASAT and ASAT/ALAT ratio values did not increase and not differ for both groups. In the THRIVE III trial, ALAT and ASAT did not increase and did not differ compared to the comparator placebo. 2 x 36 mg Ximelagatran, induced higher ALAT values at months 3 and 6 compared to 2 x 24 mg ximelagatran (p = .0105, p = .0063, respectively). ASAT did not differ between the two doses of ximelagatran. The ASAT/ALAT ratios were lower at week 2 for enoxaparin/warfarin (t-test, p = .0032) and at month 3 and 6 for 2 x 36 mg versus warfarin or 2 x 24 mg Ximelagatran (p between .0187 and .0002). The authors conclude that ALAT increases dose dependently during therapy with ximelagatran. The less frequent and lower increase of ASAT values compared to ALAT values indicates a nontoxic effect of ximelagatran on liver cells.

  15. Correlation of serum alanine aminotransferase and aspartate aminotransferase with coronary heart disease

    PubMed Central

    Shen, Jianying; Zhang, Jingying; Wen, Jing; Ming, Qiang; Zhang, Ji; Xu, Yawei

    2015-01-01

    Objective: This study aimed to explore the relationship between different risk factors (especially serum alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) and coronary heart disease (CHD). Methods: A total of 610 inpatients were recruited. Initial coronary angiography (CAG) was performed to evaluate the severity of coronary lesions. On the basis of findings from CAG, patients were divided into control group (n=260) and CHD group (n=350). Logistic regression analysis was employed for the evaluation of clinical characteristics and biochemical parameters, aiming to explore the relationship between risk factors (including AST and ALT) and CHD. Results: Results showed type 2 diabetes, hypertension, dyslipidemia, smoking and family history of CHD were clinical risk factors of CHD. Laboratory examinations showed the serum levels of triglycerides, low-density lipoprotein, AST and ALT in CHD group were significantly higher than those in control group (P<0.05). Of these parameters, the AST was 50.98±8.12 U/L in CHD group and 20.14±3.94 U/L in control group (P<0.01); the ALT was 42.31±8.34 U/L in CHD group and 18.25±6.38 U/L in control group (P<0.01). Conclusion: The serum levels of AST and ALT in CHD patients are higher than those in controls. High serum AST and ALT are biochemical markers which can be used to predict the severity of CHD and are also independent risk factors of CHD. PMID:26064360

  16. Inhibition study of alanine aminotransferase enzyme using sequential online capillary electrophoresis analysis.

    PubMed

    Liu, Lina; Chen, Yuanfang; Yang, Li

    2014-12-15

    We report the study of several inhibitors on alanine aminotransferase (ALT) enzyme using sequential online capillary electrophoresis (CE) assay. Using metal ions (Na(+) and Mg(2+)) as example inhibitors, we show that evolution of the ALT inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time. The inhibition mechanism and kinetic constants of ALT inhibition with succinic acid and two traditional Chinese medicines were derived from the sequential online CE assay. Our study could provide valuable information about the inhibition reactions of ALT enzyme.

  17. 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.

  18. Effective disposal of nitrogen waste in blood-fed Aedes aegypti mosquitoes requires alanine aminotransferase

    PubMed Central

    Mazzalupo, Stacy; Isoe, Jun; Belloni, Virginia; Scaraffia, Patricia Y.

    2016-01-01

    To better understand the mechanisms responsible for the success of female mosquitoes in their disposal of excess nitrogen, we investigated the role of alanine aminotransferase (ALAT) in blood-fed Aedes aegypti. Transcript and protein levels from the 2 ALAT genes were analyzed in sucrose- and blood-fed A. aegypti tissues. ALAT1 and ALAT2 exhibit distinct expression patterns in tissues during the first gonotrophic cycle. Injection of female mosquitoes with either double-stranded RNA (dsRNA)-ALAT1 or dsRNA ALAT2 significantly decreased mRNA and protein levels of ALAT1 or ALAT2 in fat body, thorax, and Malpighian tubules compared with dsRNA firefly luciferase-injected control mosquitoes. The silencing of either A. aegypti ALAT1 or ALAT2 caused unexpected phenotypes such as a delay in blood digestion, a massive accumulation of uric acid in the midgut posterior region, and a significant decrease of nitrogen waste excretion during the first 48 h after blood feeding. Concurrently, the expression of genes encoding xanthine dehydrogenase and ammonia transporter (Rhesus 50 glycoprotein) were significantly increased in tissues of both ALAT1- and ALAT2-deficient females. Moreover, perturbation of ALAT1 and ALAT2 in the female mosquitoes delayed oviposition and reduced egg production. These novel findings underscore the efficient mechanisms that blood-fed mosquitoes use to avoid ammonia toxicity and free radical damage.—Mazzalupo, S., Isoe, J., Belloni, V., Scaraffia, P. Y. Effective disposal of nitrogen waste in blood-fed Aedes aegypti mosquitoes requires alanine aminotransferase. PMID:26310269

  19. 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

  20. Identification and expression analyses of the alanine aminotransferase (AlaAT) gene family in poplar seedlings.

    PubMed

    Xu, Zhiru; Ma, Jing; Qu, Chunpu; Hu, Yanbo; Hao, Bingqing; Sun, Yan; Liu, Zhongye; Yang, Han; Yang, Chengjun; Wang, Hongwei; Li, Ying; Liu, Guanjun

    2017-04-05

    Alanine aminotransferase (AlaAT, E.C.2.6.1.2) catalyzes the reversible conversion of pyruvate and glutamate to alanine and α-oxoglutarate. The AlaAT gene family has been well studied in some herbaceous plants, but has not been well characterized in woody plants. In this study, we identified four alanine aminotransferase homologues in Populus trichocarpa, which could be classified into two subgroups, A and B. AlaAT3 and AlaAT4 in subgroup A encode AlaAT, while AlaAT1 and AlaAT2 in subgroup B encode glutamate:glyoxylate aminotransferase (GGAT), which catalyzes the reaction of glutamate and glyoxylate to α-oxoglutarate and glycine. Four AlaAT genes were cloned from P. simonii × P. nigra. PnAlaAT1 and PnAlaAT2 were expressed predominantly in leaves and induced by exogenous nitrogen and exhibited a diurnal fluctuation in leaves, but was inhibited in roots. PnAlaAT3 and PnAlaAT4 were mainly expressed in roots, stems and leaves, and was induced by exogenous nitrogen. The expression of PnAlaAT3 gene could be regulated by glutamine or its related metabolites in roots. Our results suggest that PnAlaAT3 gene may play an important role in nitrogen metabolism and is regulated by glutamine or its related metabolites in the roots of P. simonii × P. nigra.

  1. Identification and expression analyses of the alanine aminotransferase (AlaAT) gene family in poplar seedlings

    PubMed Central

    Xu, Zhiru; Ma, Jing; Qu, Chunpu; Hu, Yanbo; Hao, Bingqing; Sun, Yan; Liu, Zhongye; Yang, Han; Yang, Chengjun; Wang, Hongwei; Li, Ying; Liu, Guanjun

    2017-01-01

    Alanine aminotransferase (AlaAT, E.C.2.6.1.2) catalyzes the reversible conversion of pyruvate and glutamate to alanine and α-oxoglutarate. The AlaAT gene family has been well studied in some herbaceous plants, but has not been well characterized in woody plants. In this study, we identified four alanine aminotransferase homologues in Populus trichocarpa, which could be classified into two subgroups, A and B. AlaAT3 and AlaAT4 in subgroup A encode AlaAT, while AlaAT1 and AlaAT2 in subgroup B encode glutamate:glyoxylate aminotransferase (GGAT), which catalyzes the reaction of glutamate and glyoxylate to α-oxoglutarate and glycine. Four AlaAT genes were cloned from P. simonii × P. nigra. PnAlaAT1 and PnAlaAT2 were expressed predominantly in leaves and induced by exogenous nitrogen and exhibited a diurnal fluctuation in leaves, but was inhibited in roots. PnAlaAT3 and PnAlaAT4 were mainly expressed in roots, stems and leaves, and was induced by exogenous nitrogen. The expression of PnAlaAT3 gene could be regulated by glutamine or its related metabolites in roots. Our results suggest that PnAlaAT3 gene may play an important role in nitrogen metabolism and is regulated by glutamine or its related metabolites in the roots of P. simonii × P. nigra. PMID:28378825

  2. 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

  3. 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

  4. 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.

  5. Association between Elevated Alanine Aminotransferase and Urosepsis in Children with Acute Pyelonephritis

    PubMed Central

    Kim, Dongwan; Lee, Sung Hyun; Ryoo, Eell; Cho, Hye Kyung; Kim, Yun Mi

    2016-01-01

    Purpose The aim of this study is to investigate the association between elevated alanine aminotransferase (ALT) and urosepsis in children with acute pyelonephritis (APN). Methods We retrospectively identified all children who were managed in our hospital with APN during a decade period. In our study a diagnosis of APN was defined as having a positive urine culture and a positive (99m)Tc-dimercaptosuccinic acid scintigraphy. We compared those with elevated ALT and those with normal ALT according to the following variables: age, gender, duration of fever prior to admission, presence of hypotension, C-reactive protein (CRP), creatinine, presence of anemia, white blood cells count, platelet count, blood culture result, and grades of vesicoureteral reflux. In addition, the correlation between elevated ALT and positive blood culture was analyzed in detail. Results A total of 996 children were diagnosed with APN, of which 883 were included in the study. ALT was elevated in 81 children (9.2%). In the analysis of demographic characteristics, the number of children with elevated ALT was higher in children between 0 to 3 months, boys, and in those with positive blood culture (p=0.002, 0.036, and 0.010, respectively). In multivariate analysis of variables associated with positive blood culture, age younger than 3 months, elevated ALT, elevated CRP, and elevated creatinine showed statistical significance (p=0.004, 0.030, 0.043, and 0.044, respectively). Conclusion Our study demonstrates the association between elevated ALT and increased prevalence of urosepsis in addition to elevated CRP, elevated creatinine, and age younger than 3 months in children with APN. PMID:27066449

  6. Alanine Aminotransferase Elevation in Obese Infants and Children: A Marker of Early Onset Non Alcoholic Fatty Liver Disease

    PubMed Central

    Engelmann, Guido; Hoffmann, Georg Friedrich; Grulich-Henn, Juergen; Teufel, Ulrike

    2014-01-01

    Background: Elevated aminotransferases serve as surrogate markers of non-alcoholic fatty liver disease, a feature commonly associated with the metabolic syndrome. Studies on the prevalence of fatty liver disease in obese children comprise small patient samples or focus on those patients with liver enzyme elevation. Objectives: We have prospectively analyzed liver enzymes in all overweight and obese children coming to our tertiary care centre. Patients and Methods: In a prospective study 224 healthy, overweight or obese children aged 1 - 12 years were examined. Body Mass Index-Standard Deviation Score, alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl-transpeptidase were measured. Results: Elevated alanine aminotransferase was observed in 29% of children. 26 % of obese and 30 % of overweight children had liver enzyme elevations. Obese children had significantly higher alanine aminotransferase levels than overweight children (0.9 vs. 0.7 times the Upper Limit of Normal; P = 0.04). Conclusions: Elevation of liver enzymes appears in 29 % obese children in a tertiary care centre. Absolute alanine aminotransferase levels are significantly higher in obese than in overweight children. Even obese children with normal liver enzymes show signs of fatty liver disease as demonstrated by liver enzymes at the upper limit of normal. PMID:24748893

  7. Abdominal obesity validates the association between elevated alanine aminotransferase and newly diagnosed diabetes mellitus.

    PubMed

    Yueh, Chen-Yu; Yang, Yao-Hsu; Sung, Yi-Ting; Lee, Li-Wen

    2014-01-01

    To examine how elevated alanine aminotransferase (ALT) could be associated with newly diagnosed diabetes mellitus. We conducted a cross-sectional analysis on a mass health examination. The odds ratios (ORs) for diabetes mellitus and newly diagnosed diabetes mellitus were compared between people with and without abdominal obesity, together with and without elevated ALT levels. 5499 people were included in this study. Two hundred fifty two (4.6%) fulfilled the diagnosis of diabetes mellitus with 178 (3.2%) undiagnosed before. Metabolic syndrome was vigorously associated with diabetes mellitus and newly diagnosed diabetes mellitus (12.4% vs. 1.4% and 9.0% vs. 0.9%), but elevated ALT alone was not. However, coexisting with obesity, elevated ALTs were robustly associated with diabetes mellitus and newly diagnosed diabetes mellitus. For the incidence of newly diagnosed diabetes mellitus, in comparison to non-obese people with normal ALT (1.7%, OR = 1), obese people especially with elevated ALT levels had significantly higher ORs (obese with ALT ≤ 40 U/L: 4.7%, OR 1.73, 95% CI 1.08-2.77, P 0.023; ALT 41-80 U/L: 6.8%, OR 2.06, 95% CI 1.20-3.55, P 0.009; ALT 81-120 U/L: 8.8%, OR 3.07, 95% CI 1.38-6.84, P 0.006; ALT > 120 U/L: 18.2%, OR 7.44, 95% CI 3.04-18.18, P < 0.001). Abdominal obesity validates the association between elevated alanine aminotransferase and diabetes mellitus and newly diagnosed diabetes mellitus. People with abdominal obesity, especially with coexisting elevated ALT levels should be screened for undiagnosed diabetes mellitus.

  8. Irritable Bowel Syndrome May Be Associated with Elevated Alanine Aminotransferase and Metabolic Syndrome

    PubMed Central

    Lee, Seung-Hwa; Kim, Kwang-Min; Joo, Nam-Seok

    2016-01-01

    Purpose Recent studies have revealed close relationships between hepatic injury, metabolic pathways, and gut microbiota. The microorganisms in the intestine also cause irritable bowel syndrome (IBS). The aim of this study was to examine whether IBS was associated with elevated hepatic enzyme [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)], gamma-glutamyl transferase (γ-GT) levels, and metabolic syndrome (MS). Materials and Methods This was a retrospective, cross-sectional, case-control study. The case and control groups comprised subjects who visited our health promotion center for general check-ups from June 2010 to December 2010. Of the 1127 initially screened subjects, 83 had IBS according to the Rome III criteria. The control group consisted of 260 age- and sex-matched subjects without IBS who visited our health promotion center during the same period. Results Compared to control subjects, patients with IBS showed significantly higher values of anthropometric parameters (body mass index, waist circumference), liver enzymes, γ-GT, and lipid levels. The prevalences of elevated ALT (16.9% vs. 7.7%; p=0.015) and γ-GT (24.1% vs. 11.5%; p=0.037) levels were significantly higher in patients with IBS than in control subjects. A statistically significant difference was observed in the prevalence of MS between controls and IBS patients (12.7% vs. 32.5%; p<0.001). The relationships between elevated ALT levels, MS, and IBS remained statistically significant after controlling for potential confounding factors. Conclusion On the basis of our study results, IBS may be an important condition in certain patients with elevated ALT levels and MS. PMID:26632395

  9. Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase*

    PubMed Central

    Albert, Armando; Yunta, Cristina; Arranz, Rocío; Peña, Álvaro; Salido, Eduardo; Valpuesta, José María; Martín-Benito, Jaime

    2010-01-01

    Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism. PMID:20056599

  10. Histological and Clinical Characteristics of Patients with Chronic Hepatitis C and Persistently Normal Alanine Aminotransferase Levels

    PubMed Central

    Guzman, Grace

    2014-01-01

    Patients with chronic hepatitis C virus (HCV) infection and persistently normal alanine aminotransferase (PNALT) are generally described to have mild liver disease. The aim of this study was to compare clinical and histological features in HCV-infected patients with PNALT and elevated ALT. Patients presenting to the University of Illinois Medical Center, Chicago, who had biopsy proven HCV, an ALT measurement at the time of liver biopsy, at least one additional ALT measurement over the next 12 months, and liver biopsy slides available for review were identified. PNALT was defined as ALT ≤ 30 on at least 2 different occasions over 12 months. Of 1200 patients with HCV, 243 met the study criteria. 13% (32/243) of patients had PNALT while 87% (211/243) had elevated ALT. Significantly more patients with PNALT had advanced fibrosis (F3 and F4) compared to those with elevated ALT (P = 0.007). There was no significant difference in the histology activity index score as well as mean inflammatory score between the two groups. In conclusion, in a well-characterized cohort of patients at a tertiary medical center, PNALT did not distinguish patients with mild liver disease. PMID:24891947

  11. Elevated Aspartate and Alanine Aminotransferase Levels and Natural Death among Patients with Methamphetamine Dependence

    PubMed Central

    Kuo, Chian-Jue; Tsai, Shang-Ying; Liao, Ya-Tang; Conwell, Yeates; Lee, Wen-Chung; Huang, Ming-Chyi; Lin, Shih-Ku; Chen, Chiao-Chicy; Chen, Wei J.

    2012-01-01

    Background Methamphetamine is one of the fastest growing illicit drugs worldwide, causing multiple organ damage and excessive natural deaths. The authors aimed to identify potential laboratory indices and clinical characteristics associated with natural death through a two-phase study. Methods Methamphetamine-dependent patients (n = 1,254) admitted to a psychiatric center in Taiwan between 1990 and 2007 were linked with a national mortality database for causes of death. Forty-eight subjects died of natural causes, and were defined as the case subjects. A time-efficient sex- and age-matched nested case-control study derived from the cohort was conducted first to explore the potential factors associated with natural death through a time-consuming standardized review of medical records. Then the identified potential factors were evaluated in the whole cohort to validate the findings. Results In phase I, several potential factors associated with natural death were identified, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), comorbid alcohol use disorder, and the prescription of antipsychotic drugs. In phase II, these factors were confirmed in the whole cohort using survival analysis. For the characteristics at the latest hospital admission, Cox proportional hazards models showed that the adjusted hazard ratios for natural death were 6.75 (p<0.001) in the group with markedly elevated AST (>80 U/L) and 2.66 (p<0.05) in the group with mildly elevated AST (40–80 U/L), with reference to the control group (<40 U/L). As for ALT, the adjusted hazard ratios were 5.41 (p<0.001), and 1.44 (p>0.05). Comorbid alcohol use disorder was associated with an increased risk of natural death, whereas administration of antipsychotic drugs was not associated with lowered risk. Conclusions This study highlights the necessity of intensive follow-up for those with elevated AST and ALT levels and comorbid alcohol use disorder for preventing excessive natural

  12. A Novel Pathway for Metabolism of the Cardiovascular Risk Factor Homoarginine by alanine:glyoxylate aminotransferase 2

    PubMed Central

    Rodionov, Roman N.; Oppici, Elisa; Martens-Lobenhoffer, Jens; Jarzebska, Natalia; Brilloff, Silke; Burdin, Dmitrii; Demyanov, Anton; Kolouschek, Anne; Leiper, James; Maas, Renke; Cellini, Barbara; Weiss, Norbert; Bode-Böger, Stefanie M.

    2016-01-01

    Low plasma concentrations of L-homoarginine are associated with an increased risk of cardiovascular events, while homoarginine supplementation is protective in animal models of metabolic syndrome and stroke. Catabolism of homoarginine is still poorly understood. Based on the recent findings from a Genome Wide Association Study we hypothesized that homoarginine can be metabolized by alanine:glyoxylate aminotransferase 2 (AGXT2). We purified human AGXT2 from tissues of AGXT2 transgenic mice and demonstrated its ability to metabolize homoarginine to 6-guanidino-2-oxocaproic acid (GOCA). After incubation of HepG2 cells overexpressing AGXT2 with isotope-labeled homoarginine-d4 we were able to detect labeled GOCA in the medium. We injected wild type mice with labeled homoarginine and detected labeled GOCA in the plasma. We found that AGXT2 knockout (KO) mice have higher homoarginine and lower GOCA plasma levels as compared to wild type mice, while the reverse was true for AGXT2 transgenic (Tg) mice. In summary, we experimentally proved the presence of a new pathway of homoarginine catabolism – its transamination by AGXT2 with formation of GOCA and demonstrated that endogenous AGXT2 is required for maintenance of homoarginine levels in mice. Our findings may lead to development of novel therapeutic approaches for cardiovascular pathologies associated with homoarginine deficiency. PMID:27752063

  13. Conversion of cysteine to 3-mercaptopyruvic acid by bacterial aminotransferases.

    PubMed

    Andreeßen, Christina; Gerlt, Vanessa; Steinbüchel, Alexander

    2017-04-01

    3-Mercaptopyruvate (3MPy), a structural analog of 3-mercaptopropionic acid, is a precursor compound for biosynthesis of polythioesters in bacteria. The cost-effectiveness and sustainability of the whole process could be greatly improved by using the cysteine degradation pathway for an intracellular supply of 3MPy. Transamination of cysteine to its corresponding α-keto acid 3MPy is catalyzed by cysteine aminotransferases (CAT). However, CAT activity has so far not been described for bacterial aminotransferases (AT), and it was unknown whether they can be applied for the conversion of cysteine to 3MPy. In this study, we selected eight bacterial aminotransferases based on sequence homology to CAT of Rattus norvegicus (Got1). The aminotransferases included four aspartate aminotransferases (AATs) and four aromatic amino acid aminotransferases (ArATs) from Advenella mimigardefordensis DPN7, Escherichia coli MG1655, Shimwellia blattae ATCC 33430, Ralstonia eutropha H16 and Paracoccus denitrificans PD1222. For a more detailed characterization, all selected AAT or ArAT encoding genes were heterologously expressed in E. coli and purified. CAT activity was detected for all aminotransferases when a novel continuous coupled enzyme assay was applied. Kinetic studies revealed the highest catalytic efficiency of 5.1mM/s for AAT from A. mimigardefordensis. Formation of 3MPy from cysteine could additionally be verified by an optimized approach using derivatization of 3MPy with the Girard T reagent and liquid chromatography-mass spectrometry analyses.

  14. Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1

    PubMed Central

    1990-01-01

    We have previously shown that in some patients with primary hyperoxaluria type 1 (PH1), disease is associated with mistargeting of the normally peroxisomal enzyme alanine/glyoxylate aminotransferase (AGT) to mitochondria (Danpure, C.J., P.J. Cooper, P.J. Wise, and P.R. Jennings. J. Cell Biol. 108:1345-1352). We have synthesized, amplified, cloned, and sequenced AGT cDNA from a PH1 patient with mitochondrial AGT (mAGT). This identified three point mutations that cause amino acid substitutions in the predicted AGT protein sequence. Using PCR and allele-specific oligonucleotide hybridization, a range of PH1 patients and controls were screened for these mutations. This revealed that all eight PH1 patients with mAGT carried at least one allele with the same three mutations. Two were homozygous for this allele and six were heterozygous. In at least three of the heterozygotes, it appeared that only the mutant allele was expressed. All three mutations were absent from PH1 patients lacking mAGT. One mutation encoding a Gly----Arg substitution at residue 170 was not found in any of the control individuals. However, the other two mutations, encoding Pro----Leu and Ile----Met substitutions at residues 11 and 340, respectively, cosegregated in the normal population at an allelic frequency of 5-10%. In an individual homozygous for this allele (substitutions at residues 11 and 340) only a small proportion of AGT appeared to be rerouted to mitochondria. It is suggested that the substitution at residue 11 generates an amphiphilic alpha-helix with characteristics similar to recognized mitochondrial targeting sequences, the full functional expression of which is dependent upon coexpression of the substitution at residue 170, which may induce defective peroxisomal import. PMID:1703535

  15. The aspartate aminotransferase-to-alanine aminotransferase ratio predicts all-cause and cardiovascular mortality in patients with type 2 diabetes

    PubMed Central

    Zoppini, Giacomo; Cacciatori, Vittorio; Negri, Carlo; Stoico, Vincenzo; Lippi, Giuseppe; Targher, Giovanni; Bonora, Enzo

    2016-01-01

    Abstract An increased aspartate aminotransferase-to-alanine aminotransferase ratio (AAR) has been widely used as a marker of advanced hepatic fibrosis. Increased AAR was also shown to be significantly associated with the risk of developing cardiovascular (CV) disease. The aim of this study was to assess the relationship between the AAR and mortality risk in a well-characterized cohort of patients with type 2 diabetes. A cohort of 2529 type 2 diabetic outpatients was followed-up for 6 years to collect cause-specific mortality. Cox regression analyses were modeled to estimate the independent association between AAR and the risk of all-cause and CV mortality. Over the 6-year follow-up period, 12.1% of patients died, 47.5% of whom from CV causes. An increased AAR, but not its individual components, was significantly associated with an increased risk of all-cause (adjusted-hazard risk 1.83, confidence interval [CI] 95% 1.14–2.93, P = 0.012) and CV (adjusted-hazard risk 2.60, CI 95% 1.38–4.90, P < 0.003) mortality after adjustment for multiple clinical risk factors and potential confounding variables. The AAR was independently associated with an increased risk of both all-cause and CV mortality in patients with type 2 diabetes. These findings suggest that an increased AAR may reflect more systemic derangements that are not simply limited to liver damage. Further studies are needed to elucidate the pathophysiological implications of an increased AAR. PMID:27787357

  16. Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview.

    PubMed

    Oppici, Elisa; Montioli, Riccardo; Cellini, Barbara

    2015-09-01

    Liver peroxisomal alanine:glyoxylate aminotransferase (AGT) (EC 2.6.1.44) catalyses the conversion of l-alanine and glyoxylate to pyruvate and glycine, a reaction that allows glyoxylate detoxification. Inherited mutations on the AGXT gene encoding AGT lead to Primary Hyperoxaluria Type I (PH1), a rare disorder characterized by the deposition of calcium oxalate crystals primarily in the urinary tract. Here we describe the results obtained on the biochemical features of AGT as well as on the molecular and cellular effects of polymorphic and pathogenic mutations. A complex scenario on the molecular pathogenesis of PH1 emerges in which the co-inheritance of polymorphic changes and the condition of homozygosis or compound heterozygosis are two important factors that determine the enzymatic phenotype of PH1 patients. All the reported data represent relevant steps toward the understanding of genotype/phenotype correlations, the prediction of the response of the patients to the available therapies, and the development of new therapeutic approaches. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  17. CaAlaAT1 catalyzes the alanine: 2-oxoglutarate aminotransferase reaction during the resistance response against Tobacco mosaic virus in hot pepper.

    PubMed

    Kim, Ki-Jeong; Park, Chang-Jin; An, Jong-Min; Ham, Byung-Kook; Lee, Boo-Ja; Paek, Kyung-Hee

    2005-08-01

    Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P0. To elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P0 and genes specifically up-regulated during the HR were isolated by differential screening. One of the clones, CaAlaAT1 encoding a putative alanine aminotransferase (EC 2.6.1.2) exhibited organ-specific expression pattern and the transcript accumulated abundantly in red (ripe) fruit tissues. CaAlaAT1 transcript was also induced in older leaves during senescence. The expression of CaAlaAT1 gene was increased in the incompatible interaction with TMV-P0 but was not in the compatible interaction with TMV-P1.2. When a strain of Xanthomonas campestris pv. vesicatoria (Xcv) carrying an AvrBs2 gene was infiltrated into the leaves of a pepper cv. ECW 20R carrying Bs2 resistance gene, a marked induction and maintenance of CaAlaAT1 gene expression was observed. The expression of CaAlaAT1 gene was triggered by salicylic acid (SA) and ethylene but not by methyl jasmonate (MeJA). CaAlaAT1 seemed to be localized mostly at the cytosol from the polyethylene glycol (PEG)-mediated transformation experiment. CaAlaAT1 seemed to catalyze alanine: 2-oxoglutarate aminotransferase (AKT) reaction, which was a main activity among the four activities in vitro, during the resistance response against TMV in hot pepper. These results suggest that CaAlaAT1, a protein known to be involved in metabolic reactions, might be one of the components in the plant's defense signal pathway against pathogens.

  18. Ceruloplasmin, a reliable marker of fibrosis in chronic hepatitis B virus patients with normal or minimally raised alanine aminotransferase

    PubMed Central

    Zeng, Da-Wu; Dong, Jing; Jiang, Jia-Ji; Zhu, Yue-Yong; Liu, Yu-Rui

    2016-01-01

    AIM To develop a non-invasive model to evaluate significant fibrosis and cirrhosis by investigating the association between serum ceruloplasmin (CP) levels and liver fibrosis in chronic hepatitis B (CHB) patients with normal or minimally raised alanine aminotransferase (ALT). METHODS Serum samples and liver biopsy were obtained from 193 CHB patients with minimally raised or normal ALT who were randomly divided into a training group (n = 97) and a validation group (n = 96). Liver histology was evaluated by the METAVIR scoring system. Receiver operator characteristic curves were applied to the diagnostic value of CP for measuring liver fibrosis in CHB patients. Spearman rank correlation analyzed the relationship between CP and liver fibrosis. A non-invasive model was set up through multivariate logistic regression analysis. RESULTS Serum CP levels individualized various fibrosis stages via area under the curve (AUC) values. Multivariate analysis revealed that CP levels were significantly related to liver cirrhosis. Combining CP with serum GGT levels, a CG model was set up to predict significant fibrosis and liver cirrhosis in CHB patients with normal or minimally raised ALT. The AUC, sensitivity, specificity, positive predictive value, and negative predictive value were 0.84, 83.1%, 78.6%, 39.6%, and 96.5% to predict liver cirrhosis, and 0.789, 80.26%, 68.38%, 62.25%, and 84.21% to predict significant fibrosis. This model expressed a higher AUC than FIB-4 (age, ALT, aspartate aminotransferase, platelets) and GP (globulin, platelets) models to predict significant fibrosis (P = 0.019 and 0.022 respectively) and revealed a dramatically greater AUC than FIB-4 (P = 0.033) to predict liver cirrhosis. CONCLUSION The present study showed that CP was independently and negatively associated with liver fibrosis. Furthermore, we developed a novel promising model (CG), based on routine serum markers, for predicting liver fibrosis in CHB patients with normal or minimally raised

  19. Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase.

    PubMed

    Shrawat, Ashok K; Carroll, Rebecka T; DePauw, Mary; Taylor, Gregory J; Good, Allen G

    2008-09-01

    Summary Nitrogen is quantitatively the most essential nutrient for plants and a major factor limiting crop productivity. One of the critical steps limiting the efficient use of nitrogen is the ability of plants to acquire it from applied fertilizer. Therefore, the development of crop plants that absorb and use nitrogen more efficiently has been a long-term goal of agricultural research. In an attempt to develop nitrogen-efficient plants, rice (Oryza sativa L.) was genetically engineered by introducing a barley AlaAT (alanine aminotransferase) cDNA driven by a rice tissue-specific promoter (OsAnt1). This modification increased the biomass and grain yield significantly in comparison with control plants when plants were well supplied with nitrogen. Compared with controls, transgenic rice plants also demonstrated significant changes in key metabolites and total nitrogen content, indicating increased nitrogen uptake efficiency. The development of crop plants that take up and assimilate nitrogen more efficiently would not only improve the use of nitrogen fertilizers, resulting in lower production costs, but would also have significant environmental benefits. These results are discussed in terms of their relevance to the development of strategies to engineer enhanced nitrogen use efficiency in crop plants.

  20. Dose-Response Relationship between Alanine Aminotransferase Levels within the Reference Interval and Metabolic Syndrome in Chinese Adults

    PubMed Central

    Wu, Peipei; Chen, Qicai; Chen, Lili; Zhang, Pengpeng; Xiao, Juan; Chen, Xiaoxiao; Liu, Meng

    2017-01-01

    Purpose Elevation in serum alanine aminotransferase (ALT) levels is a biomarker for metabolic syndrome (MS); however, the relationship has not been fully investigated within the reference interval of ALT levels. Our objective was to explore the relationship between serum ALT levels within the reference interval and MS in Chinese adults. Materials and Methods This cross-sectional study included 16028 adults, who attended routine health check-ups at Shengli Oilfield Central Hospital from January 2006 to March 2012. The reference interval of serum ALT level was defined as less than 40 U/L. Logistic regression models and restricted cubic spline were used to evaluate the association of ALT with MS. Results The prevalence of MS in the total population was 13.7% (6.4% for females and 18.4% for males). Multiple logistic regression showed that ALT levels were positively associated with MS after adjustment for potential confounding factors. The odds ratio of MS in the top quartile was 4.830 [95% confidence interval (CI): 2.980–7.829] in females and 3.168 (95% CI: 2.649–3.790) in males, compared with the ALT levels in the bottom quartile. The restricted cubic spline models revealed a positive non-linear dose-response relationship between ALT levels and the risk of MS in women (p for nonlinearity was 0.0327), but a positive linear dose-response relationship in men (p for nonlinearity was 0.0659). Conclusion Serum ALT levels within the reference interval are positively associated with MS in a dose-response manner. Elevated ALT levels, even within the reference interval, may reflect early dysmetabolic changes. PMID:27873509

  1. Prevalence of elevated alanine-aminotransferase (ALT) among US adolescents and associated factors: NHANES 1999-2004

    PubMed Central

    Fraser, Abigail; Longnecker, Matthew P.; Lawlor, Debbie A

    2007-01-01

    Background & aims Non-alcoholic fatty liver disease (NAFLD) is a common cause of liver disease in children and adolescents. The majority of studies of NAFLD in children have been in select populations of the clinically obese. Study aims were to estimate the prevalence of elevated alanine-aminotransferase (ALT, as a marker of NAFLD) in a general contemporary adolescent population and to identify leading risk factors for ALT elevation (> 30 U/L). Methods We analysed data of adolescent participants (age 12-19, N=5586) in NHANES 1999-2004, a representative sample of the civilian non-institutionalized U.S population. Results The prevalence of elevated ALT (>30 U/L) was 7∙4% among white adolescents, 11∙5%, among Mexican Americans, and 6∙0%, among black adolescents. It was prevalent in 12∙4% of males compared to 3∙5% of females. Multivariable associations with elevated ALT were found for sex (OR male versus female = 7∙7, 95%CI: 3∙9, 15∙1), ethnicity (OR black versus white=0∙6, 95%CI: 0∙3, 1∙3; OR Mexican American versus white=1∙6, 95%CI: 1∙0, 2∙6), waist circumference (OR per 1 SD=1∙4, 95%CI: 1∙0, 2∙0), and fasting insulin (OR per 1 SD=1∙ 6, 95%CI: 1∙ 2, 2∙ 1). Age, C-reactive protein and triglycerides were also positively, and socio-economic position inversely associated with elevated ALT. The magnitude of associations with ALT was similar across ethnic groups. Conclusions ALT is associated with waist circumference and insulin resistance even in a young population. These characteristics could be utilized to identify adolescents who may benefit from screening for NAFLD, offering an opportunity to prevent disease progression at an early age. PMID:18054554

  2. Diet and the frequency of the alanine:glyoxylate aminotransferase Pro11Leu polymorphism in different human populations.

    PubMed

    Caldwell, Elizabeth F; Mayor, Lianne R; Thomas, Mark G; Danpure, Christopher J

    2004-11-01

    The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) contains a Pro11Leu polymorphism that decreases its catalytic activity by a factor of three and causes a small proportion to be mistargeted from its normal intracellular location in the peroxisomes to the mitochondria. These changes are predicted to have significant effects on the synthesis and excretion of the metabolic end-product oxalate and the deposition of insoluble calcium oxalate in the kidney and urinary tract. Based on the evolution of AGT targeting in mammals, we have previously hypothesised that this polymorphism would be advantageous for individuals who have a meat-rich diet, but disadvantageous for those who do not. If true, the frequency distribution of Pro11Leu in different extant human populations should have been shaped by their dietary history so that it should be more common in populations with predominantly meat-eating ancestral diets than it is in populations in which the ancestral diets were predominantly vegetarian. In the present study, we have determined frequency of Pro11Leu in 11 different human populations with divergent ancestral dietary lifestyles. We show that the Pro11Leu allelic frequency varies widely from 27.9% in the Saami, a population with a very meat-rich ancestral diet, to 2.3% in Chinese, who are likely to have had a more mixed ancestral diet. FST analysis shows that the differences in Pro11Leu frequency between some populations (particularly Saami vs Chinese) was very high when compared with neutral loci, suggesting that its frequency might have been shaped by dietary selection pressure.

  3. Genetic variations in the alanine-glyoxylate aminotransferase 2 (AGXT2) gene and dimethylarginines levels in rheumatoid arthritis.

    PubMed

    Dimitroulas, Theodoros; Hodson, James; Panoulas, Vasileios F; Sandoo, Aamer; Smith, Jacqueline; Kitas, George

    2017-03-29

    Rheumatoid arthritis (RA) is associated with high rates of cardiovascular events mainly due to coronary and cerebrovascular atherosclerotic disease. Asymmetric (ADMA) and symmetric (SDMA) dimethylarginines are endogenous inhibitors of nitric oxide synthase and have been repeatedly linked with adverse cardiovascular outcomes in the general population and various disease settings. Alanine-glyoxylate aminotransferase 2 (AGTX2) is considered an alternative metabolic pathway contributing to the clearance of dimethylarginines in humans. The aim of the current study was to investigate the effect of specific AGXT-2 gene polymorphisms on circulating levels of ADMA or SDMA in patients with RA. Serum ADMA and SDMA levels were measured in 201 individuals with RA [median age: 67 years (IQR: 59-73), 155 females]. Two single nucleotide polymorphisms (SNPs) in the AGXT-2 gene-rs37369 and rs28305-were genotyped. Distributions of SDMA and ADMA were skewed, hence comparisons across the gene polymorphisms were performed using Kruskal-Wallis tests, and summarized using medians and interquartile ranges. Univariable analysis did not demonstrate a significant difference in the levels of SDMA or ADMA amongst the different genotypic groups of either rs37369AGXT2 (p = 0.800, 0.977) or rs28305AGXT2 (p = 0.463, 0.634). In multivariable analyses, ADMA levels were found to be significantly associated with erythrocyte sedimentation rate and estimated glomerular filtration rate, whilst SDMA levels were significantly associated with estimated glomerular filtration rate and quantitative insulin sensitivity check index. After adjustments for these factors, the relationship between the AGXT2 gene variants and both ADMA and SDMA remained non-significant. Our study in a well-characterized RA population did not show an association between serum concentrations of dimethylarginines and genetic variants of the AGXT2 gene.

  4. ALT (Alanine Aminotransferase) Test

    MedlinePlus

    ... to help recognize heart or muscle injury. ALT values are often compared to the results of other tests such as alkaline phosphatase (ALP) , total protein , and bilirubin to help determine which form of liver disease is present. ALT is often used to monitor the treatment ...

  5. Trihalomethane exposure and biomonitoring for the liver injury indicator, alanine aminotransferase, in the United States population (NHANES 1999–2006)

    PubMed Central

    Burch, James B.; Everson, Todd M.; Seth, Ratanesh K.; Wirth, Michael D.; Chatterjee, Saurabh

    2015-01-01

    Exposure to trihalomethanes (or THMs: chloroform, bromoform, bromodichloromethane, and dibromochloromethane [DBCM]) formed via drinking water disinfection has been associated with adverse reproductive outcomes and cancers of the digestive or genitourinary organs. However, few studies have examined potential associations between THMs and liver injury in humans, even though experimental studies suggest that these agents exert hepatotoxic effects, particularly among obese individuals. This study examined participants in the National Health and Nutrition Examination Survey (1999–2006, N = 2781) to test the hypothesis that THMs are associated with liver injury as assessed by alanine aminotransferase (ALT) activity in circulation. Effect modification by body mass index (BMI) or alcohol consumption also was examined. Associations between blood THM concentrations and ALT activity were assessed using unconditional multiple logistic regression to calculate prevalence odds ratios (ORs) with 95% confidence intervals (CIs) for exposure among cases with elevated ALT activity (men: >40 IU/L, women: >30 IU/L) relative to those with normal ALT, after adjustment for variables that may confound the relationship between ALT and THMs. Compared to controls, cases were 1.35 times more likely (95% CI: 1.02, 1.79) to have circulating DBCM concentrations exceeding median values in the population. There was little evidence for effect modification by BMI, although the association varied by alcohol consumption. Among non-drinkers, cases were more likely than controls to be exposed to DBCM (OR: 3.30, 95% CI: 1.37–7.90), bromoform (OR: 2.88, 95% CI: 1.21–6.81), or brominated THMs (OR: 4.00, 95% CI: 1.31–12.1), but no association was observed among participants with low, or moderate to heavy alcohol consumption. Total THM levels exceeding benchmark exposure limits continue to be reported both in the United States and globally. Results from this study suggest a need for further

  6. SAFETY study: Alanine aminotransferase cutoff values are set too high for reliable detection of pediatric chronic liver disease

    PubMed Central

    Schwimmer, Jeffrey B.; Dunn, Winston; Norman, Gregory J.; Pardee, Perrie E.; Middleton, Michael S.; Kerkar, Nanda; Sirlin, Claude B.

    2010-01-01

    Background & Aims The appropriate alanine aminotransferase (ALT) threshold value to use for diagnosis of chronic liver disease in children is unknown. We sought to develop sex-specific, biology-based, pediatric ALT thresholds. Methods The screening ALT for elevation in today’s youth (SAFETY) study collected observational data from acute care children’s hospitals, the national health and nutrition examination survey (NHANES, 1999–2006), overweight children with and without non-alcoholic fatty liver disease (NAFLD), and children with chronic Hepatitis B virus (HBV) or Hepatitis C virus (HCV) infections. The study compared the sensitivity and specificity of ALT thresholds currently used by children’s hospitals versus study-derived, sex-specific, biology-based, ALT thresholds for detecting children with NAFLD, HCV, or HBV. Results The median upper limit of ALT at children’s hospitals was 53 U/L (range, 30–90). The 95th percentile levels for ALT in healthy weight, metabolically normal, liver disease-free, NHANES pediatric participants were 25.8 U/L (boys) and 22.1 U/L (girls). The concordance statistics of these NHANES-derived thresholds for liver disease detection were 0.85 (95% confidence interval [CI] 0.74–0.96) in boys and 0.91 (95% CI 0.83–0.99) in girls for NAFLD, 0.80 (95% CI 0.70–0.91) in boys and 0.79 (95% CI 0.69–0.89) in girls for HBV, and 0.86 (95% CI 0.77–0.95) in boys and 0.84 (95% CI 0.75–0.93) in girls for HCV. Using current children’s hospitals ALT thresholds, the median sensitivity for detection of NAFLD, HBV, and HCV ranged from 32% to 48%; median specificity was 92% (boys) and 96% (girls). Using NHANES-derived thresholds, the sensitivities were 72% (boys) and 82% (girls); specificities were 79% (boys) and 85% (girls). Conclusions The upper limit of ALT used in children’s hospitals varies widely and is set too high to reliably detect chronic liver disease. Biology-based thresholds provide higher sensitivity and only

  7. Purification and properties of 7, 8-diaminopelargonic acid aminotransferase.

    PubMed

    Stoner, G L; Eisenberg, M A

    1975-06-10

    The enzyme 7, 8-diaminopelargonic acid aminotransferase utilizes S-adenosyl-L-methionine to transaminate the biotin precurson 7-keto-8-aminopelargonic acid and form the next intermediate in the pathway, 7, 8-diaminopelargonic acid. The enzyme has been purified nearly 1000-fold from an extract of a regulatory mutant of Escherichia coli which is derepressed for the enzymes of the biotin operon. The extract was treated with protamine sulfate, ammonium sulfate, and subjected to acid and heat treatments. Subsequently, the enzyme was chromatographed on columns of DEAE-cellulose, phosphocellulose, hydroxylapatite, and two Sephadex G-100. The resulting purified preparation was judged 86% homogeneous by the scanning of of a stained disc gel. The enzymatic activity was associated with the major band in gels run at two different gel concentrations and two different pH values. The cofactor, pyridoxal phosphate, can be resolved from the enzyme in the presence of phosphate buffer after incubation with the amino donor, S-adenosyl-L-methionine. A molecular weight estimation of 94,000 plus or minus 10, 000 has been obtained by gel filtration and sucrose gradient sedimentation studies. Gel electrophoresis in the presence of sodium dodecyl sulfate, shows a single subunit with a molecular weight of 47, 000 plus or minus 3, 000 indicating a dimeric enzyme. A neutral compound was detected in the acidified reaction mixture which was derived from the methionine moiety of S-adenosyl-L-methionine and was present in amounts equivalent to the 7, 8-diaminopelargonic acid produced in the reaction mixture. It is suggested that the keto product of the reaction, i.e. S-adenosyl-2-oxo-4-methylthiobutyric acid, may decompose nonenzymatically under the conditions of the reaction to form 5'-methylthioadenosine and the neutral compound, 2-oxo-3-butenoic acid.

  8. Enzymological and mutational analysis of a complex primary hyperoxaluria type I phenotype involving alanine: Glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting and intraperoxisomal aggregation

    SciTech Connect

    Danpure, C.J.; Purdue, P.E.; Allsop, J.; Lumb, M.J.; Jennings, P.R. ); Scheinman, J.I. ); Mauer, S.M. ); Davidson, N.O. )

    1993-08-01

    Primary hyperoxaluri type 1 (PH1) is a rare autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Three unrelated PH1 patients, who possess a novel complex phenotype, are described. At the enzymological level, this phenotype is characterized by a complete, or nearly complete, absence of AGT catalytic activity and reduced AGT immunoreactivity. Unlike normal individuals in whom the AGT is confined to the peroxisomal matrix, the immunoreactive AGT in these three patients was distributed approximately equally between the peroxisomes and mitochondria. The peroxisomal AGT appeared to be aggregated into amorphous core-like structures in which no other peroxisomal enzymes could be identified. Mutational analysis of the AGT gene showed that two of the three patients were compound heterozygotes for two previously unrecognized point mutations which caused Gly41[yields]Arg and Phe152[yields]Iso amino acid substitutions. The third patient was shown to be a compound heterozygote for the Gly41[yields]Arg mutation and a previously recognized Gly170[yields]Arg mutation. All three patients were homozygous for the Pro11[yields]Leu polymorphism that had been found previously with a high allelic frequency in normal populations. It is suggested the the Phe152[yields]Iso and Gly170[yields]Arg substitutions, which are only eighteen residues apart and located in the same highly conserved internal region of 58 amino acids, might be involved in the inhibition of peroxisomal targeting and/or import of AGT and, in combination with the Pro11[yields]Leu polymorphism, be responsible for its aberrant mitochondrial compartmentalization. On the other hand, the Gly41[yields]Arg substitution, either in combination with the Pro11[yields]Leu polymorphism or by itself, is predicted to be responsible for the intraperoxisomal aggregation of the AGT protein. 50 refs., 8 figs., 4 tabs.

  9. 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.

  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. The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

    PubMed

    Duff, Stephen M G; Rydel, Timothy J; McClerren, Amanda L; Zhang, Wenlan; Li, Jimmy Y; Sturman, Eric J; Halls, Coralie; Chen, Songyang; Zeng, Jiamin; Peng, Jiexin; Kretzler, Crystal N; Evdokimov, Artem

    2012-12-01

    In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins.

  12. Alanine-glyoxylate aminotransferase 2 (AGXT2) polymorphisms have considerable impact on methylarginine and β-aminoisobutyrate metabolism in healthy volunteers.

    PubMed

    Kittel, Anja; Müller, Fabian; König, Jörg; Mieth, Maren; Sticht, Heinrich; Zolk, Oliver; Kralj, Ana; Heinrich, Markus R; Fromm, Martin F; Maas, Renke

    2014-01-01

    Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common AGXT2 single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding AGXT2 SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the AGXT2 SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated AGXT2 SNP rs16899974, which was not significantly linked to the other AGXT2 SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [²H₆]-SDMA as substrate. In vitro, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). In silico modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of AGXT2 affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper

  13. Alanine-glyoxylate aminotransferase 2 (AGXT2) Polymorphisms Have Considerable Impact on Methylarginine and β-aminoisobutyrate Metabolism in Healthy Volunteers

    PubMed Central

    König, Jörg; Mieth, Maren; Sticht, Heinrich; Zolk, Oliver; Kralj, Ana; Heinrich, Markus R.; Fromm, Martin F.; Maas, Renke

    2014-01-01

    Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common AGXT2 single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding AGXT2 SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the AGXT2 SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated AGXT2 SNP rs16899974, which was not significantly linked to the other AGXT2 SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [2H6]-SDMA as substrate. In vitro, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). In silico modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of AGXT2 affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper

  14. Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants.

    PubMed

    de la Torre, Fernando; Cañas, Rafael A; Pascual, M Belén; Avila, Concepción; Cánovas, Francisco M

    2014-10-01

    In the chloroplasts and in non-green plastids of plants, aspartate is the precursor for the biosynthesis of different amino acids and derived metabolites that play distinct and important roles in plant growth, reproduction, development or defence. Aspartate biosynthesis is mediated by the enzyme aspartate aminotransferase (EC 2.6.1.1), which catalyses the reversible transamination between glutamate and oxaloacetate to generate aspartate and 2-oxoglutarate. Plastids contain two aspartate aminotransferases: a eukaryotic-type and a prokaryotic-type bifunctional enzyme displaying aspartate and prephenate aminotransferase activities. A general overview of the biochemistry, regulation, functional significance, and phylogenetic origin of both enzymes is presented. The roles of these plastidic aminotransferases in the biosynthesis of essential amino acids are discussed.

  15. Determination of Alanine Aminotransferase with an Electrochemical Nano Ir-C Biosensor for the Screening of Liver Diseases

    PubMed Central

    Hsueh, Chang-Jung; Wang, Joanne H.; Dai, Liming; Liu, Chung-Chiun

    2011-01-01

    Alanine aminotransaminase (ALT), is an enzyme that normally resides in serum and body tissues, especially in the liver. It is released into the serum as a result of tissue injury; hence the concentration of ALT in the serum may be increased with acute damage to hepatic cells. A single use, disposable biosensor, comprising iridium nano-particle as catalyst dispersed on carbon paste, has been developed for the determination of ALT concentration. The biosensor is based on quantifying H2O2 concentration produced by a serial of ALT enzymatic reactions. It operates well at room temperature in different physiological fluids: phosphate buffer, calf serum and human serum for ALT concentration of 0–544 ng/mL. Experimental results in human serum are compared to those obtained by spectrophotometric assays with excellent agreement. Therefore, the Ir/C biosensor shows good relationship on the dilution of concentrated ALT clinical applications. PMID:25586923

  16. Biochemical and Structural Characterization of a Ureidoglycine Aminotransferase in the Klebsiella pneumoniae Uric Acid Catabolic Pathway

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2010-09-03

    Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an {alpha}-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

  17. Crystal structure of the S187F variant of human liver alanine: glyoxylate [corrected] aminotransferase associated with primary hyperoxaluria type I and its functional implications.

    PubMed

    Oppici, Elisa; Fodor, Krisztian; Paiardini, Alessandro; Williams, Chris; Voltattorni, Carla Borri; Wilmanns, Matthias; Cellini, Barbara

    2013-08-01

    The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5'-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT-pyridoxamine 5'-phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300- to 500-fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results.

  18. Risk factors associated with hepatitis B or C markers or elevated alanine aminotransferase level among blood donors on a tropical island: the Guadeloupe experience.

    PubMed

    Fest, T; Viel, J F; Agis, F; Coffe, C; Dupond, J L; Hervé, P

    1992-10-01

    Donated blood is currently screened for hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen (anti-HBc), antibody to hepatitis C virus (anti-HCV), and alanine aminotransferase (ALT) levels to prevent posttransfusion hepatitis. A prospective study of 2368 blood donors was carried out in Guadeloupe (French West Indies) with a view to determining the risk factors associated with serologic abnormalities. Blood donors included in the study had to complete a questionnaire. Statistical analysis was performed on the data thus obtained: 571 donations (24%) were positive for at least one of the four analyzed markers. The results were that 3.2 percent were positive for HBsAg, 22 percent for anti-HBc, and 0.8 percent for anti-HCV, and 1.4 percent had ALT > or = 45 IU per L. A good correlation was found between anti-HCV and elevated ALT. Transfusion history and two socioeconomic categories (working class, military personnel) were found to be risk factors. Other risk factors were lifelong residence in Guadeloupe (with risk increasing with the number of years), birthplace and current residence in the southern part of the island, and the existence of gastrointestinal discomfort unrelated to viral hepatitis (odds ratio = 2.98). The results of this study illustrate the difficulty of implementing a preventive policy against posttransfusion hepatitis in a tropical area. The unique epidemiologic situation of Guadeloupe as regards hepatitis B virus has led to more restrictive criteria for the acceptance of blood donors.

  19. The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase.

    PubMed

    Mesa-Torres, Noel; Yunta, Cristina; Fabelo-Rosa, Israel; Gonzalez-Rubio, Juana María; Sánchez-Ruiz, José M; Salido, Eduardo; Albert, Armando; Pey, Angel L

    2014-09-15

    Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.

  20. Gly161 mutations associated with Primary Hyperoxaluria Type I induce the cytosolic aggregation and the intracellular degradation of the apo-form of alanine:glyoxylate aminotransferase.

    PubMed

    Oppici, Elisa; Roncador, Alessandro; Montioli, Riccardo; Bianconi, Silvia; Cellini, Barbara

    2013-12-01

    Primary Hyperoxaluria Type I (PH1) is a severe rare disorder of metabolism due to inherited mutations on liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme whose deficiency causes the deposition of calcium oxalate crystals in the kidneys and urinary tract. PH1 is an extremely heterogeneous disease and there are more than 150 disease-causing mutations currently known, most of which are missense mutations. Moreover, the molecular mechanisms by which missense mutations lead to AGT deficiency span from structural, functional to subcellular localization defects. Gly161 is a highly conserved residue whose mutation to Arg, Cys or Ser is associated with PH1. Here we investigated the molecular bases of the AGT deficit caused by Gly161 mutations with expression studies in a mammalian cellular system paired with biochemical analyses on the purified recombinant proteins. Our results show that the mutations of Gly161 (i) strongly reduce the expression levels and the intracellular half-life of AGT, and (ii) make the protein in the apo-form prone to an electrostatically-driven aggregation in the cell cytosol. The coenzyme PLP, by shifting the equilibrium from the apo- to the holo-form, is able to reduce the aggregation propensity of the variants, thus partly decreasing the effect of the mutations. Altogether, these results shed light on the mechanistic details underlying the pathogenicity of Gly161 variants, thus expanding our knowledge of the enzymatic phenotypes leading to AGT deficiency.

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

    PubMed

    Sato, Masaru; Kirimura, Kohtaro; Kino, Kuniki

    2005-06-01

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

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

    PubMed

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

    2000-05-01

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

  3. 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.

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

    PubMed Central

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

    2003-01-01

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

  5. An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds.

    PubMed Central

    Yvon, M; Thirouin, S; Rijnen, L; Fromentier, D; Gripon, J C

    1997-01-01

    The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development. In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds. Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp. cremoris strain was purified and characterized. The enzyme transaminates the aromatic amino acids, leucine, and methionine. It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors. In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate. It is essential for the transformation of aromatic amino acids to flavor compounds. It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa. The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions. PMID:9023921

  6. The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae).

    PubMed Central

    Cronin, V B; Maras, B; Barra, D; Doonan, S

    1991-01-01

    1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5. PMID:1859361

  7. Paralogous ALT1 and ALT2 Retention and Diversification Have Generated Catalytically Active and Inactive Aminotransferases in Saccharomyces cerevisiae

    PubMed Central

    Peñalosa-Ruiz, Georgina; Aranda, Cristina; Ongay-Larios, Laura; Colon, Maritrini; Quezada, Hector; Gonzalez, Alicia

    2012-01-01

    Background Gene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase. Principal Findings Our results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function. Conclusions ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1

  8. Determination of D- and L-alanine concentrations using a pyruvic acid sensor.

    PubMed

    Inaba, Yohei; Hamada-Sato, Naoko; Kobayashi, Takeshi; Imada, Chiaki; Watanabe, Etsuo

    2003-08-01

    The concentrations of D- and L-alanine in bivalves are useful as indicators of environmental pollution. Amino acid oxidase with a low substrate specificity catalyzes the oxidation of various amino acids. Among the various amino acids, pyruvic acid can be generated from alanine only by the catalytic oxidative reaction of this oxidase. Therefore, in this study, the concentrations of D- and L-alanine were determined from the concentration of pyruvic acid, which was determined from the consumption of oxygen based on the oxidative reaction of pyruvate oxidase. From this point of view, there is a very strong possibility that biosensors utilizing enzymes with a low substrate specificity can be developed. The results obtained were as follows. (1) The optimum conditions for the use of pyruvic acid sensor were as follows: temperature of 25 degrees C, pH of 6.8, flow rate of 0.1 ml/min, thiamin diphosphate concentration of 1.5 mM, and injection volume of 50 microl. (2) D-Alanine and L-alanine optimally reacted with D- and L-amino acid oxidase at 30 degrees C, pH 8.2, for 30 min and at 37 degrees C, pH 7.8, for 90 min, respectively. (3) The linear relationships between the concentrations of D- and L-alanine and the output of the sensor were obtained at 3.56-106.8 microg of D-alanine and 5.34-71.3 microg of L-alanine. (4) The concentrations of D- and L-alanine in Meretrix iusoria, Patinopecten yessonsi, and Corbicula leana obtained by the proposed assay were in good agreement with those determined by a conventional method.

  9. [The effect of diet ratio of polyunsaturated fatty acids of omega-3 and omega-6 families on activity of aminotransferases and gamma-glutamyltransferase in rat blood serum].

    PubMed

    Ketsa, O V; Marchenko, M M

    2014-01-01

    The effect of diet fat compositions with various ratio of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) on alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) activities in blood serum of 45 white mongrel rats weighing 90-110 g (9 animals in group) has been investigated. Fat components in the semi-synthetic diet, compiled on the basis of AIN-93 diet, and sources of omega-6 and omega-3 PUFA were presented by sunflower oil, soybean oil and fish oil. It has been shown that four-week inclusion of linoleic acid (LA) and alpha-linolenic acid (alpha-LNA) in a ratio of 7:1 into the diet (soybean oil) as well as use of only omega-6 PUFA (sunflower oil) has lead to an increase in the activity of ALT and GGT in rat blood serum compared to control animals treated with the complex of linolenic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid through the mixture of sunflower oil and fish oil (9:1) with the ratio of omega-6 and omega-3 PUFA 7:1. Along with this, the AST:ALT ratio (de Ritis ratio) was lower (p < 0.05) as compared with the control group of rat, amounting respectively 0.92 +/- 0.08 and 0.79 +/- 0.12 vs 1.26 +/- 0.10. The use of high doses of omega-3 fatty acids (600 mg EPA and 400 mg DHA per kg of animal weight per day coming through fish oil) did not affect the activity of ALT and GGT, but increased AST serum activity (0.47 +/- 0.04 micromoles/min per mg protein) and the de Ritis ratio (2.53 +/- 0.23). The diet deprived with fat increased enzyme activity of ALT, AST and GGT in rat blood serum.

  10. 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.

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

    PubMed

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

    2017-01-01

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

  12. 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

  13. Equine endurance exercise alters serum branched-chain amino acid and alanine concentrations.

    PubMed

    Trottier, N L; Nielsen, B D; Lang, K J; Ku, P K; Schott, H C

    2002-09-01

    Six 2-year-old Arabian horses were used to determine whether 60 km prolonged endurance exercise (approximately 4 h) alters amino acid concentrations in serum and muscle, and the time required for serum amino acid concentrations to return to basal resting values. Blood and muscle samples were collected throughout exercise and during a 3 day recovery period. Isoleucine concentration in muscle tended to increase and leucine and valine did not change due to exercise. Serum alanine concentrations did not increase immediately after exercise, but increased at 24, 48 and 72 h postexercise. Serum isoleucine, leucine, and valine concentrations decreased after exercise and time required to reach pre-exercising concentrations was 48 h. In conclusion, endurance exercise in the horse decreases serum isoleucine, leucine, and valine concentrations, and increases serum alanine concentration. The decrease in serum branched-chain amino acid concentrations did not correspond to a measurable increase in total muscle branched-chain amino acid concentrations.

  14. A comparative study on the growth and characterization of nonlinear optical amino acid crystals: L-alanine (LA) and L-alanine alaninium nitrate (LAAN).

    PubMed

    Aravindan, A; Srinivasan, P; Vijayan, N; Gopalakrishnan, R; Ramasamy, P

    2008-11-15

    A comparative study on the properties of L-alanine and LAAN crystals has been made and discussed. It may be concluded that the protonation of the amino group in the L-alanine molecule is the key factor in increasing the relative SHG efficiency of LAAN. The protonation is justified by the crystal structure analysis, FTIR and photoluminescence studies. The factor group vibrations are compared and found that there is an increase in vibrational modes of LA when reacted with nitric acid forming LAAN.

  15. Distinguishing the cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) from other diamino acids.

    PubMed

    Banack, S A; Metcalf, J S; Spáčil, Z; Downing, T G; Downing, S; Long, A; Nunn, P B; Cox, P A

    2011-04-01

    β-N-methylamino-L-alanine (BMAA) is produced by diverse taxa of cyanobacteria, and has been detected by many investigators who have searched for it in cyanobacterial blooms, cultures and collections. Although BMAA is distinguishable from proteinogenic amino acids and its isomer 2,4-DAB using standard chromatographic and mass spectroscopy techniques routinely used for the analysis of amino acids, we studied whether BMAA could be reliably distinguished from other diamino acids, particularly 2,6-diaminopimelic acid which has been isolated from the cell walls of many bacterial species. We used HPLC-FD, UHPLC-UV, UHPLC-MS, and triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) to differentiate BMAA from the diamino acids 2,6-diaminopimelic acid, N-2(amino)ethylglycine, lysine, ornithine, 2,4-diaminosuccinic acid, homocystine, cystine, tryptophan, as well as other amino acids including asparagine, glutamine, and methionine methylsulfonium.

  16. 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.

  17. Conserved aspartic acid 233 and alanine 231 are not required for poliovirus polymerase function in replicons

    PubMed Central

    Freistadt, Marion S; Eberle, Karen E

    2007-01-01

    Nucleic acid polymerases have similar structures and motifs. The function of an aspartic acid (conserved in all classes of nucleic acid polymerases) in motif A remains poorly understood in RNA-dependent RNA polymerases. We mutated this residue to alanine in a poliovirus replicon. The resulting mutant could still replicate, although at a reduced level. In addition, mutation A231C (also in motif A) yielded high levels of replication. Taken together these results show that poliovirus polymerase conserved residues D233 and A231 are not essential to poliovirus replicon function. PMID:17352827

  18. Beta-alanine and beta-aminoisobutyric acid levels in two siblings with dihydropyrimidinase deficiency.

    PubMed

    van Kuilenburg, A B P; Stroomer, A E M; Bosch, A M; Duran, M

    2008-06-01

    Dihydropyrimidinase (DHP) deficiency is an inborn error of the pyrimidine degradation pathway, affecting the hydrolytic ring opening of the dihydropyrimidines. In two siblings with a complete DHP deficiency and a variable clinical presentation, a normal concentration of beta-alanine and strongly decreased levels of beta-aminoisobutyric acid were observed in plasma, urine and CSF. No major differences were observed for the concentrations of the beta-amino acids in plasma and urine between the symptomatic and asymptomatic sibling. Thus, the relevance of the shortage of beta-aminoisobutyric acid for the onset of a clinical phenotype in patients with DHP deficiency remains to be established.

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

    PubMed Central

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

    1984-01-01

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

  20. Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

    PubMed Central

    Yoshikane, Yu; Yokochi, Nana; Ohnishi, Kouhei; Hayashi, Hideyuki; Yagi, Toshiharu

    2006-01-01

    Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed. PMID:16545075

  1. Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia, Aves and Amphibia.

    PubMed

    Danpure, C J; Fryer, P; Jennings, P R; Allsop, J; Griffiths, S; Cunningham, A

    1994-08-01

    As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes. In the second category (type II), AGT1 was found more evenly distributed in both peroxisomes and mitochondria. In the third category (type III), AGT1 was localized mainly within the mitochondria with much lower, but widely variable, amounts in the peroxisomes. Type I animals include the human, two great apes (gorilla, orangutan), two Old World monkeys (anubis baboon, Japanese macaque), a New World monkey (white-faced Saki monkey), a lago, morph (European rabbit), a bat (Seba's short-tailed fruit bat), two caviomorph rodents (guinea pig, orange-rumped agouti), and two Australian marsupials (koala, Bennett's wallaby). Type II animals include two New World monkeys (common marmoset, cotton-top tamarin), three prosimians (brown lemur, fat-tailed dwarf lemur, pygmy slow loris), five rodents (a hybrid crested porcupine, Colombian ground squirrel, laboratory rat, laboratory mouse, golden hamster), an American marsupial (grey short-tailed opossum), and a bird (raven). Type III animals include the large tree shrew, three insectivores (common Eurasian mole, European hedgehog, house shrew), four carnivores (domestic cat, ocelot, domestic dog, polecat ferret), and an amphibian (common frog). In addition to these categories, some animals (e.g. guinea pig, common frog) possessed significant amounts of cytosolic AGT1. Whereas the subcellular distribution of AGT1 in some orders (e.g. Insectivora and Carnivora) did not appear

  2. CPP-115, a Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Cocaine Addiction

    PubMed Central

    Pan, Yue; Gerasimov, Madina R.; Kvist, Trine; Wellendorph, Petrine; Madsen, Karsten K.; Pera, Elena; Lee, Hyunbeom; Schousboe, Arne; Chebib, Mary; Bräuner-Osborne, Hans; Craft, Cheryl M.; Brodie, Jonathan D.; Schiffer, Wynne K.; Dewey, Stephen L.; Miller, Steven R.; Silverman, Richard B.

    2011-01-01

    Vigabatrin, a GABA aminotransferase (GABA-AT) inactivator, is used to treat infantile spasms and refractory complex partial seizures and is in clinical trials to treat addiction. We evaluated a novel GABA-AT inactivator (CPP-115) and observed that it does not exhibit other GABAergic or off-target activities and is rapidly and completely orally absorbed and eliminated. Using in vivo microdialysis techniques in freely moving rats and micro-PET imaging techniques, CPP-115 produced similar inhibition of cocaine-induced increases in extracellular dopamine and in synaptic dopamine in the nucleus accumbens at 1/300–1/600th the dose of vigabatrin. It also blocks expression of cocaine-induced conditioned place preference at a dose 1/300th that of vigabatrin. Electroretinographic (ERG) responses in rats treated with CPP-115, at doses 20–40 times higher than those needed to treat addiction in rats, exhibited reductions in ERG responses, which were less than the reductions observed in rats treated with vigabatrin at the same dose needed to treat addiction in rats. In conclusion, CPP-115 can be administered at significantly lower doses than vigabatrin, which suggests a potential new treatment for addiction with a significantly reduced risk of visual field defects. PMID:22128851

  3. Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase.

    PubMed

    Pinto, Andrea; Tamborini, Lucia; Pennacchietti, Eugenia; Coluccia, Antonio; Silvestri, Romano; Cullia, Gregorio; De Micheli, Carlo; Conti, Paola; De Biase, Daniela

    2016-01-01

    The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.

  4. Branched-chain amino acid metabolon: interaction of glutamate dehydrogenase with the mitochondrial branched-chain aminotransferase (BCATm).

    PubMed

    Islam, Mohammad Mainul; Nautiyal, Manisha; Wynn, R Max; Mobley, James A; Chuang, David T; Hutson, Susan M

    2010-01-01

    The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

  5. Aspartic acid aminotransferase activity is increased in actively spiking compared with non-spiking human epileptic cortex.

    PubMed Central

    Kish, S J; Dixon, L M; Sherwin, A L

    1988-01-01

    Increased concentration of the excitatory neurotransmitter aspartic acid in actively spiking human epileptic cerebral cortex was recently described. In order to further characterise changes in the aspartergic system in epileptic brain, the behaviour of aspartic acid aminotransferase (AAT), a key enzyme involved in aspartic acid metabolism has now been examined. Electrocorticography performed during surgery was employed to identify cortical epileptic spike foci in 16 patients undergoing temporal lobectomy for intractable seizures. Patients with spontaneously spiking lateral temporal cortex (n = 8) were compared with a non-spiking control group (n = 8) of patients in whom the epileptic lesions were confined to the hippocampus sparing the temporal convexity. Mean activity of AAT in spiking cortex was significantly elevated by 16-18%, with aspartic acid concentration increased by 28%. Possible explanations for the enhanced AAT activity include increased proliferation of cortical AAT-containing astrocytes at the spiking focus and/or a generalised increase in neuronal or extraneuronal metabolism consequent to the ongoing epileptic discharge. It is suggested that the data provide additional support for a disturbance of central excitatory aspartic acid mechanisms in human epileptic brain. PMID:2898010

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

    PubMed Central

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

    2001-01-01

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

  7. Stimulation of [3H] GABA and beta-[3H] alanine release from rat brain slices by cis-4-aminocrotonic acid.

    PubMed

    Chebib, M; Johnston, G A

    1997-02-01

    cis-4-Aminocrotonic acid (CACA; 100 microM), an analogue of GABA in a folded conformation, stimulated the passive release of [3H] GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of beta-[3H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 microM) did not stimulate the passive release of [3H]taurine from slices of cerebellum and spinal cord or of D-[3H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [3H]-taurine from the cerebellum and spinal cord and D-[3H]-aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and beta-alanine release are due to CACA acting as a substrate for a beta-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of beta-[3H]alanine into slices of rat cerebellum and cerebral cortex. The observed Ki for CACA against beta-[3H]alanine uptake in the cerebellum was 750 +/- 60 microM. CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABAc receptor. The effects of CACA on GABA and beta-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, beta-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, beta-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.

  8. Expression of mitochondrial branched-chain aminotransferase and α-keto-acid dehydrogenase in rat brain: implications for neurotransmitter metabolism

    PubMed Central

    Cole, Jeffrey T.; Sweatt, Andrew J.; Hutson, Susan M.

    2012-01-01

    In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron. PMID:22654736

  9. N- Trichloro- and dichloroacetyl amino acids and compounds of amino acids with halogeno acetic acids: 35Cl nuclear quadrupole resonance spectroscopy; crystal structure of N- trichloroacetyl- glycine, - DL-alanine, and - L-alanine

    NASA Astrophysics Data System (ADS)

    Dou, Shi-qi; Kehrer, Armin; Ofial, Armin R.; Weiss, Alarich

    1995-02-01

    The crystal structures of N- trichloroacetyl- glycine ( N- TCA- G), N-trichloroacetyl-dl-alanine ( N-TCA- dl-A ), and N-trichloroacetyl- l-alanine ( N-TCA- l-A ) were determined. In addition, the 35Cl NQR spectra of these N-trichloroacetyl amino acids, of N-trichloroacetyl- l-valine ( N-TCA- l-V ), and of N- dichloroacetyl- glycine and - L-alanine were measured, mostly as a function of temperature. Compounds of glycine and L-alanine with chlorodifluoroacetic acid, of glycine and L-leucine with monochloroacetic acid, of glycine and L-leucine with dichloroacetic acid, and of glycine and L-leucine with trichloroacetic acid were also studied using 35Cl NQR. The structures (in picometres and degrees) were found to be as follows. N- TCA- G: Pna2 1, Z = 8, a = 1641, b = 1002, c = 1018. N-TCA- dl-A : {C2}/{c}, Z = 8, a = 3280, b = 556, c = 1031, β = 96.68. N-TCA- l-A: P1 , Z = 2, a = 967, b = 949, c = 619, α = 74.97, β = 74.20, γ = 61.20. The 35Cl NQR frequencies (ν) were observed in the range 35-41 MHz, and decrease with increasing temperature. Some of the resonances bleach out at a temperature ( Tb) far below the melting temperature; this provides information about the crystal structures at 77 K. No phase transitions were observed by differential thermal analysis between 77 and 295 K. The crystal structures are discussed in connection with the NQR results, and conclusions are drawn about the structures of the compounds for which only 35Cl NQR data are available.

  10. Characterization of the Branched-Chain Amino Acid Aminotransferase Enzyme Family in Tomato1[W][OA

    PubMed Central

    Maloney, Gregory S.; Kochevenko, Andrej; Tieman, Denise M.; Tohge, Takayuki; Krieger, Uri; Zamir, Dani; Taylor, Mark G.; Fernie, Alisdair R.; Klee, Harry J.

    2010-01-01

    Branched-chain amino acids (BCAAs) are synthesized in plants from branched-chain keto acids, but their metabolism is not completely understood. The interface of BCAA metabolism lies with branched-chain aminotransferases (BCAT) that catalyze both the last anabolic step and the first catabolic step. In this study, six BCAT genes from the cultivated tomato (Solanum lycopersicum) were identified and characterized. SlBCAT1, -2, -3, and -4 are expressed in multiple plant tissues, while SlBCAT5 and -6 were undetectable. SlBCAT1 and -2 are located in the mitochondria, SlBCAT3 and -4 are located in chloroplasts, while SlBCAT5 and -6 are located in the cytosol and vacuole, respectively. SlBCAT1, -2, -3, and -4 were able to restore growth of Escherichia coli BCAA auxotrophic cells, but SlBCAT1 and -2 were less effective than SlBCAT3 and -4 in growth restoration. All enzymes were active in the forward (BCAA synthesis) and reverse (branched-chain keto acid synthesis) reactions. SlBCAT3 and -4 exhibited a preference for the forward reaction, while SlBCAT1 and -2 were more active in the reverse reaction. While overexpression of SlBCAT1 or -3 in tomato fruit did not significantly alter amino acid levels, an expression quantitative trait locus on chromosome 3, associated with substantially higher expression of Solanum pennellii BCAT4, did significantly increase BCAA levels. Conversely, antisense-mediated reduction of SlBCAT1 resulted in higher levels of BCAAs. Together, these results support a model in which the mitochondrial SlBCAT1 and -2 function in BCAA catabolism while the chloroplastic SlBCAT3 and -4 function in BCAA synthesis. PMID:20435740

  11. Distribution of messenger RNAs encoding the enzymes glutaminase, aspartate aminotransferase and glutamic acid decarboxylase in rat brain.

    PubMed

    Najlerahim, A; Harrison, P J; Barton, A J; Heffernan, J; Pearson, R C

    1990-05-01

    In situ hybridization histochemistry (ISHH) using synthetic oligonucleotide probes has been used to identify cells containing the mRNAs coding for glutaminase (GluT), aspartate aminotransferase (AspT) and glutamic acid decarboxylase (GAD). The distribution of GAD mRNA confirms previous descriptions and matches the distribution of GAD detected using specific antibodies. AspT mRNA is widely distributed in the brain, but is present at high levels in GABAergic neuronal populations, some that may be glutamatergic, and in a subset of neurons which do not contain significant levels of either GAD or GluT mRNA. Particularly prominent are the neurons of the magnocellular division of the red nucleus, the large cells in the deep cerebellar nuclei and the vestibular nuclei and neurons of the lateral superior olivary nucleus. GluT mRNA does not appear to be present at high levels in all GAD-containing neurons, but is seen prominently in many neuronal populations that may use glutamate as a neurotransmitter, such as neocortical and hippocampal pyramidal cells, the granule cells of the cerebellum and neurons of the dentate gyrus of the hippocampus. The heaviest labelling of GluT mRNA is seen in the lateral reticular nucleus of the medulla. ISHH using probes directed against the mRNAs encoding these enzymes may be an important technique for identifying glutamate and aspartate using neuronal populations and for examining their regulation in a variety of experimental and pathological circumstances.

  12. Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism.

    PubMed

    de la Torre, Fernando; De Santis, Laura; Suárez, María Fernanda; Crespillo, Remedios; Cánovas, Francisco M

    2006-05-01

    In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.

  13. AGXT2: a promiscuous aminotransferase

    PubMed Central

    Rodionov, Roman N.; Jarzebska, Natalia; Weiss, Norbert; Lentz, Steven R.

    2014-01-01

    Alanine-glyoxylate aminotransferase 2 (AGXT2) is a multifunctional mitochondrial aminotransferase that was first identified in 1978. The physiological importance of AGXT2 was largely overlooked for three decades because AGXT2 is less active in glyoxylate metabolism than AGXT1, the enzyme that is deficient in primary hyperoxaluria type I. Recently, several novel functions of AGXT2 have been “rediscovered” in the setting of modern genomic and metabolomic studies. It is now apparent that AGXT2 has multiple substrates and products and that altered AGXT2 activity may contribute to the pathogenesis of cardiovascular, renal, neurological and hematological diseases. This article reviews the biochemical properties and physiological functions of AGXT2, its unique role at the intersection of key mitochondrial pathways, and its potential as a drug target. PMID:25294000

  14. A versatile proline/alanine transporter in the unicellular pathogen Leishmania donovani regulates amino acid homoeostasis and osmotic stress responses.

    PubMed

    Inbar, Ehud; Schlisselberg, Doreen; Suter Grotemeyer, Marianne; Rentsch, Doris; Zilberstein, Dan

    2013-01-15

    Unlike all other organisms, parasitic protozoa of the family Trypanosomatidae maintain a large cellular pool of proline that, together with the alanine pool, serve as alternative carbon sources as well as reservoirs of organic osmolytes. These reflect adaptation to their insect vectors whose haemolymphs are exceptionally rich in the two amino acids. In the present study we identify and characterize a new neutral amino acid transporter, LdAAP24, that translocates proline and alanine across the Leishmania donovani plasma membrane. This transporter fulfils multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates the transport and homoeostasis of glutamate and arginine, none of which are its substrates. Notably, we provide evidence that proline and alanine exhibit different roles in the parasitic response to hypotonic shock; alanine affects swelling, whereas proline influences the rate of volume recovery. On the basis of our data we suggest that LdAAP24 plays a key role in parasite adaptation to its varying environments in host and vector, a phenomenon essential for successful parasitism.

  15. The non-protein amino acid β-N-methylamino-L-alanine in Portuguese cyanobacterial isolates.

    PubMed

    Cervantes Cianca, Rosa C; Baptista, Mafalda S; Lopes, Viviana R; Vasconcelos, Vitor M

    2012-06-01

    The tailor made amino acid β-N-methyl-amino-L-alanine (BMAA) is a neurotoxin produced by cyanobacteria. It has been associated with certain forms of progressive neurodegenerative disease, including sporadic Amyotrophic Lateral Sclerosis and Alzheimer's disease. Some different reports of BMAA in cyanobacterial blooms from lakes, reservoirs, and other water resources have been made by different investigators. We here report the detection of BMAA of both free and protein-bound produced by cyanobacteria, belonging to the Chroococcales, Oscillatoriales and Nostocales ordered. We use a rapid and sensitive HPLC-FD method that utilizes methanol elution and the Waters AQC Tag chemistry. On other hand, we have used three different assay procedures for BMAA extraction from cyanobacteria: Trichloroacetic acid (TCA), Methanol/Acetone and hydrochloric acid (HCl). All assays let successfully detect BMAA in all cyanobacteria samples analyzed. Nevertheless, with TCA and HCl extraction procedures the highest BMAA values, for free as well as protein-bound BMAA were detected. BMAA content could not be related to the taxonomy of the isolates or to their geographical origin, and no correlation between free and protein-bound BMAA concentrations were observed within or between taxonomic groups. These data offer confirmation of the taxonomic and geographic ubiquity of BMAA from naturally occurring populations of cyanobacteria, for the first time reported for estuaries.

  16. Chiral selectivity of amino acid adsorption on chiral surfaces—The case of alanine on Pt

    SciTech Connect

    Franke, J.-H.; Kosov, D. S.

    2015-02-07

    We study the binding pattern of the amino acid alanine on the naturally chiral Pt surfaces Pt(531), Pt(321), and Pt(643). These surfaces are all vicinal to the (111) direction but have different local environments of their kink sites and are thus a model for realistic roughened Pt surfaces. Alanine has only a single methyl group attached to its chiral center, which makes the number of possible binding conformations computationally tractable. Additionally, only the amine and carboxyl group are expected to interact strongly with the Pt substrate. On Pt(531), we study the molecule in its pristine as well as its deprotonated form and find that the deprotonated one is more stable by 0.47 eV. Therefore, we study the molecule in its deprotonated form on Pt(321) and Pt(643). As expected, the oxygen and nitrogen atoms of the deprotonated molecule provide a local binding “tripod” and the most stable adsorption configurations optimize the interaction of this “tripod” with undercoordinated surface atoms. However, the interaction of the methyl group plays an important role: it induces significant chiral selectivity of about 60 meV on all surfaces. Hereby, the L-enantiomer adsorbs preferentially to the Pt(321){sup S} and Pt(643){sup S} surfaces, while the D-enantiomer is more stable on Pt(531){sup S}. The binding energies increase with increasing surface density of kink sites, i.e., they are largest for Pt(531){sup S} and smallest for Pt(643){sup S}.

  17. 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.

  18. Enantioselective hydrogenation of pyruvic acid oxime to alanine on Pd/Alumina

    SciTech Connect

    Borszeky, K.; Mallat, T.; Aeschiman, R.

    1996-06-01

    The chemo- and enantioselective hydrogenation of pyruvic acid oxime have been studied on Pd/alumina, the latter in the presence of the 1,2-amino alcohol type alkaloids ephedrine, cinchonidine, and cinchonine. High yields of racemic alanine (90-98%) were obtained in the absence of alkaloids in polar solvents at 0-45{degrees}C and 10 bar. Enantioselection increased with higher temperature and alkalid: oxime molar ratio. A 1:1 ephedrine: oxime molar ratio afforded the best enantiomeric excess (26%). The presence of alkaloid resulted in a decrease of reaction rate by a factor of up to 140, compared to the racemic hydrogenation. Based on X-ray crystal structure analysis of the alkaloid-pyruvic acid oxime adduct, a mechanism is proposed for the steric course of the reaction. Extended interactions by multiple H bonds between the adsorbed alkaloid-oxime salt units on the Pd surface is assumed to be at the origin of the moderate enantioselectivity and the very low enantioselective hydrogenation rate. 28 refs., 5 figs., 3 tabs.

  19. Beta-alanine-oxalic acid (1:1) hemihydrate crystal: structure, 13C NMR and vibrational properties, protonation character.

    PubMed

    Godzisz, D; Ilczyszyn, M; Ilczyszyn, M M

    2003-03-01

    The crystal structure of beta-alanine-oxalic acid (1:1) hemihydrate complex has been reinvestigated by X-ray diffraction method at 293 K. Formation of monoclinic crystal system belonging to C2/c space group and consisting of semi-oxalate chains, diprotonated beta-alanine dimers and water molecules bonded to both these units is confirmed. New results are obtained for distances in the carboxylic groups and hydrogen bonds. These structural observations are used for protonation degree monitoring on the carboxylic oxygen atoms. They are in accordance with our vibrational study. The 13C NMR spectra provide insights into the solid structure of this complex, character of its hydrogen bonds and the beta-alanine protonation.

  20. Amino Acid Mixture Enriched With Arginine, Alanine, and Phenylalanine Stimulates Fat Metabolism During Exercise.

    PubMed

    Ueda, Keisuke; Nakamura, Yutaka; Yamaguchi, Makoto; Mori, Takeshi; Uchida, Masayuki; Fujita, Satoshi

    2016-02-01

    Although there have been many investigations of the beneficial effects of both exercise and amino acids (AAs), little is known about their combined effects on the single-dose ingestion of AAs for lipid metabolism during exercise. We hypothesize that taking a specific combination of AAs implicated in glucagon secretion during exercise may increase fat metabolism. We recently developed a new mixture, d-AA mixture (D-mix), that contains arginine, alanine, and phenylalanine to investigate fat oxidation. In a double-blind, placebo-controlled crossover study, 10 healthy male volunteers were randomized to ingest either D-mix (3 g/dose) or placebo. Subjects in each condition subsequently performed a physical task that included workload trials on a cycle ergometer at 50% of maximal oxygen consumption for 1 hr. After oral intake of D-mix, maximum serum concentrations of glycerol (9.32 ± 6.29 mg/L and 5.22 ± 2.22 mg/L, respectively; p = .028), free fatty acid level (0.77 ± 0.26 mEq/L and 0.63 ± 0.28 mEq/L, respectively; p = .022), and acetoacetic acid levels (37.9 ± 17.7 μmol/L and 30.3 ± 13.9 μmol/L, respectively; p = .040) were significantly higher than in the placebo groups. The area under the curve for glucagon during recovery was numerically higher than placebo (6.61 ± 1.33 μg/L · min and 6.06 ± 1.23 μg/L · min, respectively; p = .099). These results suggest that preexercise ingestion of D-mix may stimulate fat metabolism. Combined with exercise, the administration of AA mixtures could prove to be a useful nutritional strategy to maximize fat metabolism.

  1. 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.

  2. 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.

  3. Determination of the non protein amino acid β-N-methylamino-l-alanine in estuarine cyanobacteria by capillary electrophoresis.

    PubMed

    Baptista, Mafalda S; Cianca, Rosa C C; Lopes, Viviana R; Almeida, C Marisa R; Vasconcelos, Vitor M

    2011-10-01

    A capillary electrophoretic method for the determination of the amino acid β-N-methylamino-l-alanine (BMAA) was achieved using a fused-silica capillary column (50 cm × 75 μm I.D.) filled with 5 mM sodium tetraborate solution (pH 9), with an applied voltage of 25 kV, at 25 °C. The method was then applied in quantifying BMAA in eighteen strains of lyophilized estuarine cyanobacteria, following amino acid extraction using 0.1 M trichloroacetic acid and 6 M hydrochloric acid, sequentially.

  4. Plasma aminotransferase concentrations in preterm infants.

    PubMed

    Victor, S; Dickinson, H; Turner, M A

    2011-03-01

    The aim of this study was to generate reference ranges for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in preterm infants by describing the observed plasma concentration of these enzymes in babies born between 22 and 36 weeks' gestation. A service evaluation was conducted in babies admitted to two large neonatal intensive care units in the UK. 7006 blood samples from 1860 infants admitted to the two units between 2004 and 2008 were included. Extremely premature infants had high plasma enzyme activities when compared to babies at a later corrected gestational age. This may be due to more severe illness immediately after birth.

  5. Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis

    SciTech Connect

    Feild, M.J.

    1988-01-01

    The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase of Salmonella typhimurium was determined by automated Edman degradation of peptide fragments generated by chemical and enzymatic digestion of S-carboxymethylated and S-pyridylethylated transaminase B. Peptide fragments of transaminase B were generated by treatment of the enzyme with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. Protocols were developed for separation of the peptide fragments by reverse-phase high performance liquid chromatography (HPLC), ion-exchange HPLC, and SDS-urea gel electrophoresis. The enzyme subunit contains 308 amino acid residues and has a molecular weight of 33,920 daltons. The coenzyme-binding site was determined by treatment of the enzyme, containing bound pyridoxal 5-phosphate, with tritiated sodium borohydride prior to trypsin digestion. Monitoring radioactivity incorporation and peptide map comparisons with an apoenzyme tryptic digest, allowed identification of the pyridoxylated-peptide which was isolated by reverse-phase HPLC and sequenced. The coenzyme-binding site is a lysyl residue at position 159. Some peptides were further characterized by fast atom bombardment mass spectrometry.

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

    PubMed

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

    2012-07-05

    In this work, the He-I (21.218 eV) photoelectron spectrum of D,L-alanine in the gas phase is revisited experimentally and theoretically. To support the experiment, the high level ab initio calculations were used to calculate and assign the photoelectron spectra of the four most stable conformers of gaseous alanine, carefully. The symmetry adapted cluster/configuration interaction (SAC-CI) method based on single and double excitation operators (SD-R) and its more accurate version, termed general-R, was used to separately calculate the energies and intensities of the ionization bands of the L- and D-alanine conformers. The intensities of ionization bands were calculated based on the monopole approximation. Also, natural bonding orbital (NBO) calculations were employed for better spectral band assignment. The relative electronic energy, Gibbs free energy, and Boltzmann population ratio of the conformers were calculated at the experimental temperature (403 K) using several theoretical methods. The theoretical photoelectron spectrum of alanine was calculated by summing over the spectra of individual D and L conformers weighted by different population ratios. Finally, the population ratio of the four most stable conformers of alanine was estimated from the experimental photoelectron spectrum using theoretical calculations for the first time.

  7. Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S ,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

    DOE PAGES

    Lee, Hyunbeom; Doud, Emma H.; Wu, Rui; ...

    2015-01-23

    γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently,more » CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. Ultimately, this represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.« less

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

    PubMed

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

    2016-11-18

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

  9. Structural and Functional Characterization of PseC, an Aminotransferase Involved in the Biosynthesis of Pseudaminic Acid, an Essential Flagellar Modification in Helicobacter Pylori

    SciTech Connect

    Schoenhofen,I.; Lunin, V.; Julien, J.; Li, Y.; Ajamian, E.; Matte, A.; Cygler, M.; Brisson, J.; Aubry, A.; et al.

    2006-01-01

    Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. As motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-a-D-GlcNAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-{beta}-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc product, the latter being converted to the external aldimine form in the enzyme's active site. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the {beta}-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and in conjunction with the recent functional characterization of this enzyme, will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

  10. Immunocytoma effect upon circadian variation in murine urinary excretion of beta-aminoisobutyric acid, beta-alanine, phenylalanine and tyrosine.

    PubMed

    Halberg, F; Gehrke, C W; Kuo, K; Nelson, W L; Sothern, R B; Cadotte, L M; Haus, E; Scheving, L E

    1978-01-01

    Under the conditions of disynchronization by the manipulation of both the alternation of light and darkness and the availability and unavailability of food, circadian rhythms characterize the excretion of several amino acids by inbred LOU rats bearing an immunocytoma. Large amplitude rhythms can be demonstrated for urinary beta-aminoisobutyric acid, beta-alanine, phenylalanine and tyrosine. Under the same conditions of disynchronization, control animals excrete the same compounds also with a marked circadian variation but at an invariably lower average rate. These excretory rhythms, along with those demonstrated earlier for polyamines and light-chains, are of interest as potential markers for the chronotherapy of cancer.

  11. Corticosterone, cortisol, triglycerides, aspartate aminotransferase and uric acid plasma concentrations during foie gras production in male mule ducks (Anas platyrhynchos × Cairina moschata).

    PubMed

    Flament, A; Delleur, V; Poulipoulis, A; Marlier, D

    2012-01-01

    1. Corticosterone, cortisol, triglycerides, aspartate aminotransferase (AST) and uric acid (UA) plasma concentration were measured at 8 (7 days after group housing), 12 (after 7 days of force feeding) and 13 weeks of age (at slaughter after 12 days of force feeding), and 45 min after an adrenocorticotrophic hormone (ACTH) stimulation test at 8 weeks of age in 12 male mule ducks in an on-farm experiment. 2. No significant increase of corticosterone was found during the force-feeding period compared with the concentration after housing. 3. Comparison of corticosterone and cortisol values indicates that cortisol can be considered as a reliable acute stress indicator in future routine examinations. 4. Plasma concentrations of triglycerides and aspartate aminotransferase increased progressively from pre-force feeding period to slaughtering. 5. Plasma concentrations of uric acid increased from the start at 8 weeks of age to the mid-force feeding period but no difference was noticed between the mid-force feeding period and slaughtering. 6. It is concluded that acute stress induced by force-feeding is similar at the beginning and end of the commercial production of foie gras.

  12. 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.

  13. Antiretroviral Drugs and Risk of Chronic Alanine Aminotransferase Elevation in Human Immunodeficiency Virus (HIV)-Monoinfected Persons: The Data Collection on Adverse Events of Anti-HIV Drugs Study

    PubMed Central

    Kovari, Helen; Sabin, Caroline A.; Ledergerber, Bruno; Ryom, Lene; Reiss, Peter; Law, Matthew; Pradier, Christian; Dabis, Francois; d'Arminio Monforte, Antonella; Smith, Colette; de Wit, Stephane; Kirk, Ole; Lundgren, Jens D.; Weber, Rainer

    2016-01-01

    Background. Although human immunodeficiency virus (HIV)-positive persons on antiretroviral therapy (ART) frequently have chronic liver enzyme elevation (cLEE), the underlying cause is often unclear. Methods. Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) Study participants without chronic viral hepatitis were observed to the earliest of cLEE (elevated aminotransferase ≥6 months), death, last follow-up, or January 2, 2014. Antiretroviral treatment exposure was categorized as follows: no exposure and ongoing short- and long-term exposure (<2 or ≥2 years) after initiation. Association between development of cLEE and ART exposure was investigated using Poisson regression. Results. Among 21 485 participants observed for 105 413 person-years (PY), 6368 developed cLEE (incidence 6.04/100 PY; 95% confidence interval [CI], 5.89–6.19). Chronic liver enzyme elevation was associated with short-and long-term exposure to didanosine (<2 years rate ratio [RR] = 1.29, 95% CI, 1.11–1.49; >2 years RR = 1.26, 95% CI, 1.13–1.41); stavudine (<2 years RR = 1.51, 95% CI, 1.26–1.81; >2 years RR = 1.17, 95% CI, 1.03–1.32), and tenofovir disoproxil fumarate (<2 years RR = 1.55, 95% CI, 1.40–1.72; >2 years RR = 1.18, 95% CI, 1.05–1.32), but only short-term exposure to nevirapine (<2 years RR = 1.44, 95% CI, 1.29–1.61), efavirenz (<2 years RR = 1.14, 95% CI, 1.03–1.26), emtricitabine (<2 years RR = 1.18, 95% CI, 1.04–1.33), and atazanavir (<2 years RR = 1.20, 95% CI, 1.04–1.38). Chronic liver enzyme elevation was not associated with use of lamivudine, abacavir, and other protease inhibitors. Mortality did not differ between participants with and without cLEE. Conclusions. Although didanosine, stavudine, nevirapine, and efavirenz have been described to be hepatotoxic, we additionally observed a consistent association between tenofovir and cLEE emerging within the first 2 years after drug initiation. This novel tenofovir-cLEE signal should be

  14. The natural non-protein amino acid N-β-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

    PubMed

    Glover, W Broc; Mash, Deborah C; Murch, Susan J

    2014-11-01

    N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

  15. Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (l-Ala-P)

    SciTech Connect

    Au, Kinfai; Ren, Jingshan; Walter, Thomas S.; Harlos, Karl; Nettleship, Joanne E.; Owens, Raymond J.; Stuart, David I.; Esnouf, Robert M.

    2008-05-01

    Structures of BA0252, an alanine racemase from B. anthracis, in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) and determined by X-ray crystallography to resolutions of 2.1 and 1.47 Å, respectively, are described. Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (l-Ala-P) have determined by X-ray crystallo@@graphy to resolutions of 2.1 and 1.47 Å, respectively. Difficulties in crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is d-alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of l-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies.

  16. Synthesis, growth and optical properties of an efficient nonlinear optical single crystal: L-alanine DL-malic acid

    NASA Astrophysics Data System (ADS)

    Kirubagaran, R.; Madhavan, J.

    2015-02-01

    Single crystals of L-alanine DL-malic acid (LADLMA) have been grown from aqueous solution by slow-cooling technique. Powder X-ray diffraction studies reveal the structure of the crystal to be orthorhombic. The nonlinear optical conversion efficiency test was carried out for the grown crystals using the Kurtz powder technique. The third order nonlinear refractive index and the nonlinear absorption coefficient where evaluated by Z-scan measurements. As the material have a negative refractive index it could be used in the protection of optical sensors such as night vision devices.

  17. Determination of β -Cyano-L-alanine, γ -Glutamyl- β -cyano-L-alanine, and Common Free Amino Acids in Vicia sativa (Fabaceae) Seeds by Reversed-Phase High-Performance Liquid Chromatography.

    PubMed

    Megías, Cristina; Cortés-Giraldo, Isabel; Girón-Calle, Julio; Vioque, Javier; Alaiz, Manuel

    2014-01-01

    A method for determination of β-cyano-L-alanine, γ-glutamyl-β-cyano-L-alanine and other free amino acids in Vicia sativa is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification was 0.15 and 0.50 μM, respectively. The method has high intra- (RSD = 0.28-0.31%) and interrepeatability (RSD = 2.76-3.08%) and remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties. The method has been applied to the determination of γ-glutamyl-β-cyano-L-alanine, β-cyano-L-alanine, and common free amino acids in eight wild populations of V. sativa from southwestern Spain.

  18. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

    PubMed Central

    Isola, L M; Zhou, S L; Kiang, C L; Stump, D D; Bradbury, M W; Berk, P D

    1995-01-01

    To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7568234

  19. 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.

  20. Dual-column cation-exchange chromatographic method for beta-aminoisobutyric acid and beta-alanine in biological samples.

    PubMed

    Kuo, K C; Cole, T F; Gehrke, C W; Waalkes, T P; Borek, E

    1978-08-01

    A rapid, automated chromatographic method has been developed for the quantitation of the nucleic acid catabolites beta-aminoisobutyric acid and beta-alanine in urine, serum, and other physiological fluids. The analyses were performed on a modified Beckman 121M amino acid analyzer with dual ion-exchange columns and the use of a single sodium citrate buffer (pH 4.38, 0.20 mol/liter). By carefully matching the elution pattern for the two ion-exchange columns and alternating use of these columns, analyses are completed every 40 min. The chromatography, regeneration, and equilibration of the two columns are precisely programmed, thus the detector sees only the elution of beta-aminoisobutyric acid and beta-alanine alternately from each column. Long-term precision and analytical recovery for the two metabolites in urine were 1.9 and 102%, and 3.3 and 101%, respectively. Their normal physiological values were determined in human serum and urine. Their excretion in the urine was also studied as a function of collection time, to validate a more convenient, less costly method of sampling. This study shows that randomly collected samples are acceptable when the concentration of the two metabolites are expressed in terms of creatinine excretion. In addition, the distribution of the free and conjugated forms of the two metabolites in urine and serum was studied. A preparative method was also developed for the quantitative isolation of beta-amino-isobutyric acid from urine samples. The alternating dual-column technique may be applied to any ion-exchange chromatographic method where many analyses must be performed. This method is currently used in our laboratories for measuring these beta-amino acids in urine and serum of patients with various types of cancers.

  1. Docking and quantitative structure-activity relationship studies for sulfonyl hydrazides as inhibitors of cytosolic human branched-chain amino acid aminotransferase.

    PubMed

    Caballero, Julio; Vergara-Jaque, Ariela; Fernández, Michael; Coll, Deysma

    2009-11-01

    We have performed the docking of sulfonyl hydrazides complexed with cytosolic branched-chain amino acid aminotransferase (BCATc) to study the orientations and preferred active conformations of these inhibitors. The study was conducted on a selected set of 20 compounds with variation in structure and activity. In addition, the predicted inhibitor concentration (IC(50)) of the sulfonyl hydrazides as BCAT inhibitors were obtained by a quantitative structure-activity relationship (QSAR) method using three-dimensional (3D) vectors. We found that three-dimensional molecule representation of structures based on electron diffraction (3D-MoRSE) scheme contains the most relevant information related to the studied activity. The statistical parameters [cross-validate correlation coefficient (Q(2) = 0.796) and fitted correlation coefficient (R(2) = 0.899)] validated the quality of the 3D-MoRSE predictive model for 16 compounds. Additionally, this model adequately predicted four compounds that were not included in the training set.

  2. Synthesis, growth, thermal, optical and mechanical properties of new organic NLO crystal: L-alanine DL-malic acid

    NASA Astrophysics Data System (ADS)

    Jaikumar, D.; Kalainathan, S.; Bhagavanarayana, G.

    2009-12-01

    A new organic nonlinear optical crystal, L-alanine DL-malic acid (LADLMA), has been grown from aqueous solution by the slow cooling technique. L-alanine and DL-malic acid were used in the ratio 2:1 for synthesis. Crystals of size 24×13×8 mm 3 have been obtained in 26 days. Characterizations were carried out to study the structural, optical and mechanical properties of the grown crystals. Single crystal X-ray diffraction analysis shows that they belong to the orthorhombic system. To study the crystalline perfection of the grown crystals, high-resolution X-ray diffraction (HR-XRD) study was carried out. The vibrational frequencies of various functional groups have been derived from FTIR spectrum. Thermal behaviour of the crystal was investigated by TG-DTA analyses. Transmission spectrum has been recorded in the solution state and the cut-off frequency has been determined. Nonlinear optical property of the crystal has been confirmed using the Kurtz powder technique and a study of its second harmonic generation efficiency in comparison with KDP has been made. Knoop hardness test was carried out and its Young's modulus was calculated.

  3. Intramolecular vibrations in low-frequency normal modes of amino acids: L-alanine in the neat solid state.

    PubMed

    Zhang, Feng; Wang, Houng-Wei; Tominaga, Keisuke; Hayashi, Michitoshi

    2015-03-26

    This paper presents a theoretical analysis of the low-frequency phonons of L-alanine by using the solid-state density functional theory at the Γ point. We are particularly interested in the intramolecular vibrations accessing low-frequency phonons via harmonic coupling with intermolecular vibrations. A new mode-analysis method is introduced to quantify the vibrational characteristics of such intramolecular vibrations. We find that the torsional motions of COO(-) are involved in low-frequency phonons, although COO(-) is conventionally assumed to undergo localized torsion. We also find the broad distributions of intramolecular vibrations relevant to important functional groups of amino acids, e.g., the COO(-) and NH3(+) torsions, in the low-frequency phonons. The latter finding is illustrated by the concept of frequency distribution of vibrations. These findings may lead to immediate implications in other amino acid systems.

  4. Formation of [b3 - 1 + cat]+ ions from metal-cationized tetrapeptides containing beta-alanine, gamma-aminobutyric acid or epsilon-aminocaproic acid residues.

    PubMed

    Osburn, Sandra M; Ochola, Sila O; Talaty, Erach R; Van Stipdonk, Michael J

    2008-11-01

    The presence and position of a single beta-alanine (betaA), gamma-aminobutyric acid (gammaABu) or epsilon-aminocaproic acid (Cap) residue has been shown to have a significant influence on the formation of b(n)+ and y(n)+ product ions from a series of model, protonated peptides. In this study, we examined the effect of the same residues on the formation of analogous [b3 - 1 + cat]+ products from metal (Li+, Na+ and Ag+)-cationized peptides. The larger amino acids suppress formation of b3+ from protonated peptides with general sequence AAXG (where X = beta-alanine, gamma-aminobutyric acid or epsilon-aminocaproic acid), presumably because of the prohibitive effect of larger cyclic intermediates in the 'oxazolone' pathway. However, abundant [b3 - 1 + cat]+ products are generated from metal-cationized versions of AAXG. Using a group of deuterium-labeled and exchanged peptides, we found that formation of [b3 - 1 + cat]+ involves transfer of either amide or alpha-carbon position H atoms, and the tendency to transfer the atom from the alpha-carbon position increases with the size of the amino acid in position X. To account for the transfer of the H atom, a mechanism involving formation of a ketene product as [b3 - 1 + cat]+ is proposed.

  5. First principles DFT study of weak C-H…O bonds in crystalline amino acids under pressure-alanine

    NASA Astrophysics Data System (ADS)

    Ramaniah, Lavanya M.; Kamal, C.; Sikka, S. K.

    2013-02-01

    Many crystalline solids containing C-H…O hydrogen bonds display blue shifting of the C-H stretching frequency under pressure. No agreed explanation is available for this. Here, we use first principles density functional theory, to determine the hydrogen atom positions to understand the cause of this blue shift. No neutron diffraction is feasible due to flux limitations for this purpose. As a first case, we have taken up the study of the amino acid, alanine. We find that the C_H_…O bond in it no longer remain isolated under compression as is case at ambient pressure. The hydrogen atom in the bond has now repulsive contacts with other atoms. This results in contraction of the C-H bond length and consequently to blue shifting as is found experimentally.

  6. Efficient optical resolution of amino acid by alanine racemaze chiral analogue supported on mesoporous carbon

    NASA Astrophysics Data System (ADS)

    Jang, D.; Kim, K.; Park, D.; Kim, G.

    2012-09-01

    Optically pure D-amino acids are industrially important chiral building blocks for the synthesis of pharmaceuticals, food ingredients, and drug intermediates. Chemoenzymatic dynamic kinetic-resolution processes have recently been developed for deracemization of amino acids. S-ARCA would be a good candidate for the selective adsorption of D amino acid through the imine formation reaction. The organic phase containing S-ARCA adsorbent, TPPC or Ionic Liquid (as a phase transfer catalyst) in MC were coated on the surfaces of mesoporous carbon C-SBA-15(CMK). The aqueous solution of racemic D/L-amino acid and NaOH were added to the carbon support coated with ARCA. The D/L ratios on ARCA and in solution were determined with increasing reaction time. S-ARCA has a unique property for the selective adsorption of D- amino acid (up to 90% selcetivity) in the racemic mixture. The fixed bed reactor containing ARCA/carbon support was also adopted successfully for the selective separation of amino acid.

  7. Conformation of the phosphate D-alanine zwitterion in bacterial teichoic acid from nuclear magnetic resonance spectroscopy.

    PubMed

    Garimella, Ravindranath; Halye, Jeffrey L; Harrison, William; Klebba, Phillip E; Rice, Charles V

    2009-10-06

    The conformation of d-alanine (d-Ala) groups of bacterial teichoic acid is a central, yet untested, paradigm of microbiology. The d-Ala binds via the C-terminus, thereby allowing the amine to exist as a free cationic NH(3)(+) group with the ability to form a contact ion pair with the nearby anionic phosphate group. This conformation hinders metal chelation by the phosphate because the zwitterion pair is charge neutral. To the contrary, the repulsion of cationic antimicrobial peptides (CAMPs) is attributed to the presence of the d-Ala cation; thus the ion pair does not form in this model. Solid-state nuclear magnetic resonance (NMR) spectroscopy has been used to measure the distance between amine and phosphate groups within cell wall fragments of Bacillus subtilis. The bacteria were grown on media containing (15)N d-Ala and beta-chloroalanine racemase inhibitor. The rotational-echo double-resonance (REDOR) pulse sequence was used to measure the internuclear dipolar coupling, and the results demonstrate (1) the metal-free amine-to-phosphate distance is 4.4 A and (2) the amine-to-phosphate distance increases to 5.4 A in the presence of Mg(2+) ions. As a result, the zwitterion exists in a nitrogen-oxygen ion pair configuration providing teichoic acid with a positive charge to repel CAMPs. Additionally, the amine of d-Ala does not prevent magnesium chelation in contradiction to the prevailing view of teichoic acids in metal binding. Thus, the NMR-based description of teichoic acid structure resolves the contradictory models, advances the basic understanding of cell wall biochemistry, and provides possible insight into the creation of new antibiotic therapies.

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

    PubMed

    Deutch, Charles E

    2004-09-15

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

  9. Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study.

    PubMed Central

    Dugelay, S; Chauvin, M F; Megnin-Chanet, F; Martin, G; Laréal, M C; Lhoste, J M; Baverel, G

    1999-01-01

    Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction. PMID:10477267

  10. Quantitative structure-activity relationship and molecular docking studies of a series of quinazolinonyl analogues as inhibitors of gamma amino butyric acid aminotransferase.

    PubMed

    Abdulfatai, Usman; Uzairu, Adamu; Uba, Sani

    2017-01-01

    Quantitative structure-activity relationship and molecular docking studies were carried out on a series of quinazolinonyl analogues as anticonvulsant inhibitors. Density Functional Theory (DFT) quantum chemical calculation method was used to find the optimized geometry of the anticonvulsants inhibitors. Four types of molecular descriptors were used to derive a quantitative relation between anticonvulsant activity and structural properties. The relevant molecular descriptors were selected by Genetic Function Algorithm (GFA). The best model was validated and found to be statistically significant with squared correlation coefficient (R(2)) of 0.934, adjusted squared correlation coefficient (R(2)adj) value of 0.912, Leave one out (LOO) cross validation coefficient (Q(2)) value of 0.8695 and the external validation (R(2)pred) of 0.72. Docking analysis revealed that the best compound with the docking scores of -9.5 kcal/mol formed hydrophobic interaction and H-bonding with amino acid residues of gamma aminobutyric acid aminotransferase (GABAAT). This research has shown that the binding affinity generated was found to be better than the commercially sold anti-epilepsy drug, vigabatrin. Also, it was found to be better than the one reported by other researcher. Our QSAR model and molecular docking results corroborate with each other and propose the directions for the design of new inhibitors with better activity against GABAAT. The present study will help in rational drug design and synthesis of new selective GABAAT inhibitors with predetermined affinity and activity and provides valuable information for the understanding of interactions between GABAAT and the anticonvulsants inhibitors.

  11. Dipeptide Nanotubes Containing Unnatural Fluorine-Substituted β(2,3)-Diarylamino Acid and L-Alanine as Candidates for Biomedical Applications.

    PubMed

    Bonetti, Andrea; Pellegrino, Sara; Das, Priyadip; Yuran, Sivan; Bucci, Raffaella; Ferri, Nicola; Meneghetti, Fiorella; Castellano, Carlo; Reches, Meital; Gelmi, Maria Luisa

    2015-09-18

    The synthesis and the structural characterization of dipeptides composed of unnatural fluorine-substituted β(2,3)-diarylamino acid and L-alanine are reported. Depending on the stereochemistry of the β amino acid, these dipeptides are able to self-assemble into proteolytic stable nanotubes. These architectures were able to enter the cell and locate in the cytoplasmic/perinuclear region and represent interesting candidates for biomedical applications.

  12. 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

  13. Novel emissive bio-inspired non-proteinogenic coumarin-alanine amino acid: fluorescent probe for polyfunctional systems.

    PubMed

    Oliveira, Elisabete; Capelo, José Luis; Lima, João Carlos; Lodeiro, Carlos

    2012-10-01

    Two new bio-inspired non-proteinogenic compounds L1 and L2, containing coumarin and/or acridine chromophores and bearing as spacer an alanine amino acid were successfully synthesized and fully characterized by elemental analysis, (1)H and (13)C NMR, infrared spectroscopy (KBr discs), melting point, ESI-TOF (electrospray ionization-time of flight-mass), UV-vis absorption and emission spectroscopy, fluorescence quantum yields and lifetime measurements. A relative fluorescence quantum yield of 0.02 was determined for both compounds. In L2 the presence of an intramolecular energy transfer from the coumarin to the acridine unit was observed. L1 and L2 are quite sensitive to the basicity of the environment. At alkaline values both compounds show a strong quenching in the fluorescence emission, attributed to the photoinduced electron transfer (PET). However, both deprotonated forms recover the emission with the addition of Zn(2+), Cd(2+) and Al(3+) metal ions. As multifunctional emissive probes, the titration of L1 and L2 with lanthanides (III), Eu(3+) and Tb(3+) was also explored as new visible bio-probes in the absence and in the presence of liposomes. In a liposomal environment a lower energy transfer was observed.

  14. Growth, structural, spectral, mechanical, thermal and dielectric characterization of phosphoric acid admixtured L-alanine (PLA) single crystals

    NASA Astrophysics Data System (ADS)

    Rose, A. S. J. Lucia; Selvarajan, P.; Perumal, S.

    2011-10-01

    Phosphoric acid admixtured L-alanine (PLA) single crystals were grown successfully by solution method with slow evaporation technique at room temperature. Crystals of size 18 mm × 12 mm × 8 mm have been obtained in 28 days. The grown crystals were colorless and transparent. The solubility of the grown samples has been found out at various temperatures. The lattice parameters of the grown crystals were determined by X-ray diffraction technique. The reflection planes of the sample were confirmed by the powder X-ray diffraction study and diffraction peaks were indexed. Fourier transform infrared (FTIR) studies were used to confirm the presence of various functional groups in the crystals. UV-visible transmittance spectrum was recorded to study the optical transparency of grown crystal. The nonlinear optical (NLO) property of the grown crystal was confirmed by Kurtz-Perry powder technique and a study of its second harmonic generation efficiency in comparison with potassium dihydrogen phosphate (KDP) has been made. The mechanical strength of the crystal was estimated by Vickers hardness test. The grown crystals were subjected to thermo gravimetric and differential thermal analysis (TG/DTA). The dielectric behavior of the sample was also studied.

  15. Growth, structural, spectral, mechanical, thermal and dielectric characterization of phosphoric acid admixtured L-alanine (PLA) single crystals.

    PubMed

    Rose, A S J Lucia; Selvarajan, P; Perumal, S

    2011-10-15

    Phosphoric acid admixtured L-alanine (PLA) single crystals were grown successfully by solution method with slow evaporation technique at room temperature. Crystals of size 18 mm×12 mm×8 mm have been obtained in 28 days. The grown crystals were colorless and transparent. The solubility of the grown samples has been found out at various temperatures. The lattice parameters of the grown crystals were determined by X-ray diffraction technique. The reflection planes of the sample were confirmed by the powder X-ray diffraction study and diffraction peaks were indexed. Fourier transform infrared (FTIR) studies were used to confirm the presence of various functional groups in the crystals. UV-visible transmittance spectrum was recorded to study the optical transparency of grown crystal. The nonlinear optical (NLO) property of the grown crystal was confirmed by Kurtz-Perry powder technique and a study of its second harmonic generation efficiency in comparison with potassium dihydrogen phosphate (KDP) has been made. The mechanical strength of the crystal was estimated by Vickers hardness test. The grown crystals were subjected to thermo gravimetric and differential thermal analysis (TG/DTA). The dielectric behavior of the sample was also studied.

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

    PubMed

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

    2004-11-20

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

  17. High-performance liquid chromatographic determination of beta-alanine, beta-aminoisobutyric acid and gamma-aminobutyric acid in tissue extracts and urine of normal and (aminooxy)acetate-treated rats.

    PubMed

    Abe, T; Kurozumi, Y; Yao, W B; Ubuka, T

    1998-08-07

    A method is described for the simultaneous determination of beta-alanine, beta-aminoisobutyric acid and gamma-aminobutyric acid in biological materials. Amino acids including these beta- and gamma-amino acids were derivatized with 4-dimethylaminoazobenzene-4'-sulfonyl (dabsyl) chloride and dabsyl amino acids formed were separated by reversed-phase high-performance liquid chromatography. Dabsyl derivatives of these beta- and gamma-amino acids were well separated from other dabsyl-amino acids. The method was applied to the determination of these beta- and gamma-amino acids in trichloroacetic acid extracts of various tissues and to the urine of normal rats and those injected with (aminooxy)acetate (AOA). AOA injection (15 mg per kg of body mass) produced remarkable increase in beta-alanine contents in liver, kidney and urine (10.2, 4.6 and 25.7 times, respectively).

  18. Excess of L-alanine in amino acids synthesized in a plasma torch generated by a hypervelocity meteorite impact reproduced in the laboratory

    NASA Astrophysics Data System (ADS)

    Managadze, George G.; Engel, Michael H.; Getty, Stephanie; Wurz, Peter; Brinckerhoff, William B.; Shokolov, Anatoly G.; Sholin, Gennady V.; Terent'ev, Sergey A.; Chumikov, Alexander E.; Skalkin, Alexander S.; Blank, Vladimir D.; Prokhorov, Vyacheslav M.; Managadze, Nina G.; Luchnikov, Konstantin A.

    2016-10-01

    We present a laboratory reproduction of hypervelocity impacts of a carbon containing meteorite on a mineral substance representative of planetary surfaces. The physical conditions of the resulting impact plasma torch provide favorable conditions for abiogenic synthesis of protein amino acids: We identified glycine and alanine, and in smaller quantities serine, in the produced material. Moreover, we observe breaking of alanine mirror symmetry with L excess, which coincides with the bioorganic world. Therefore the selection of L-amino acids for the formation of proteins for living matter could have been the result from plasma processes occurring during the impact meteorites on the surface. This indicates that the plasma torch from meteorite impacts could play an important role in the formation of biomolecular homochirality. Thus, meteorite impacts possibly were the initial stage of this process and promoted conditions for the emergence of a living matter.

  19. 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.

  20. Studies on some kinetic parameters of aminotransferases in tissues of the snail, Pila globosa (Swainson) during malathion intoxication.

    PubMed

    Sahib, I K; Rao, K R

    1988-01-01

    The aspartate and alanine aminotransferases in the tissues of the snail, Pila globosa showed high catalytic potentials (low Km and high Vmax) during malathion exposure in vivo. In vitro addition of different concentrations of malathion did not influence aminotransferase activity. The results are discussed in relation to the regulative influence of the intracellular environment of the cell.

  1. A gene encoding lysine 6-aminotransferase, which forms the beta-lactam precursor alpha-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans.

    PubMed Central

    Coque, J J; Liras, P; Laiz, L; Martín, J F

    1991-01-01

    A gene (lat) encoding lysine 6-aminotransferase was found upstream of the pcbAB (encoding alpha-aminoadipylcysteinyl-valine synthetase) and pcbC (encoding isopenicillin N synthase) genes in the cluster of early cephamycin biosynthetic genes in Nocardia lactamdurans. The lat gene was separated by a small intergenic region of 64 bp from the 5' end of the pcbAB gene. The lat gene contained an open reading frame of 1,353 nucleotides (71.4% G + C) encoding a protein of 450 amino acids with a deduced molecular mass of 48,811 Da. Expression of DNA fragments carrying the lat gene in Streptomyces lividans led to a high lysine 6-aminotransferase activity which was absent from untransformed S. lividans. The enzyme was partially purified from S. lividans(pULBS8) and showed a molecular mass of 52,800 Da as calculated by Sephadex gel filtration and polyacrylamide gel electrophoresis. DNA sequences which hybridized strongly with the lat gene of N. lactamdurans were found in four cephamycin-producing Streptomyces species but not in four other actinomycetes which are not known to produce beta-lactams, suggesting that the gene is specific for beta-lactam biosynthesis and is not involved in general lysine catabolism. The protein encoded by the lat gene showed similarity to ornithine-5-aminotransferases and N-acetylornithine-5-aminotransferases and contained a pyridoxal phosphate-binding consensus amino acid sequence around Lys-300 of the protein. The evolutionary implications of the lat gene as a true beta-lactam biosynthetic gene are discussed. Images PMID:1917857

  2. The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O57 containing an amide of D-galacturonic acid with L-alanine.

    PubMed

    Kocharova, Nina A; Ovchinnikova, Olga G; Bushmarinov, Ivan S; Toukach, Filip V; Torzewska, Agnieszka; Shashkov, Alexander S; Knirel, Yuriy A; Rozalski, Antoni

    2005-03-21

    The O-polysaccharide (O-antigen) was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O57:H29. Studies by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments, showed that the polysaccharide contains an amide of D-galacturonic acid with L-alanine and has the following pentasaccharide repeating unit: [formula: see text

  3. Identification of the roles of individual amino acid residues of the helix E of the major antenna of photosystem II (LHCII) by alanine scanning mutagenesis.

    PubMed

    Liu, Cheng; Rao, Yan; Zhang, Lei; Yang, Chunhong

    2014-10-01

    The functions of the helix E (W97-F105), an amphiphilic lumenal 310 helix of the major antenna of photosystem II (LHCII), are still unidentified. To elucidate the roles of individual amino acid residue of the helix E, alanine scanning mutagenesis has been performed to mutate every residue of this domain to alanine. The influence of every alanine substitution on the structure and function of LHCII has been investigated biochemically and spectroscopically. The results show that all mutations have little impact on the pigment binding and configuration. However, many mutants presented decreased thermo- or photo-stability compared with the wild type, highlighting the significance of this helix to the stability of LHCII. The most critical residue for stability is W97. The mutant W97A yielded very fragile trimeric pigment protein complexes. The structural analysis revealed that the hydrogen bonding and aromatic interactions between W97, F195, F194 and a water molecule contributed greatly to the stability of LHCII. Moreover, Q103A and F105A have been identified to be able to reinforce the tendency of aggregation in vitro. The structural analysis suggested that the enhancement in aggregation formation for Q103A and F105A might be attributed to the changing hydrophobicity of the region.

  4. Amino acids (L-arginine and L-alanine) passivated CdS nanoparticles: Synthesis of spherical hierarchical structure and nonlinear optical properties

    NASA Astrophysics Data System (ADS)

    Talwatkar, S. S.; Tamgadge, Y. S.; Sunatkari, A. L.; Gambhire, A. B.; Muley, G. G.

    2014-12-01

    CdS nanoparticles (NPs) passivated with amino acids (L-alanine and L-arginine) having spherical hierarchical morphology were synthesized by room temperature wet chemical method. Synthesized NPs were characterized by ultraviolet-visible (UV-vis) spectroscopy to study the variation of band gaps with concentration of surface modifying agents. Increase in band gap has been observed with the increase in concentration of surface modifying agents and was found more prominent for CdS NPs passivated with L-alanine. Powder X-ray diffraction (XRD) and transmission electron microscopy (TEM) analysis were carried out for the study of crystal structure and morphology of CdS NPs. The average particle size of CdS NPs calculated from Debye-Scherer formula was found to less than 5 nm and agrees well with those determined from UV-vis spectra and TEM data. Fourier transform infrared (FT-IR) spectroscopy was performed to know the functional groups of the grown NPs. Peaks in FT-IR spectra indicate the formation of CdS NPs and capping with L-alanine and L-arginine. Photoluminescence spectra of these NPs were also studied. Finally, colloidal solution of CdS-PVAc was subjected to Z-scan experiment under low power cw laser illumination to characterize them for third order nonlinear optical properties. CdS-PVAc colloidal solution shows enhanced nonlinear absorption due to RSA and weak FCA on account of two photon absorption processes triggered by thermal effect.

  5. Aminotransferase levels in relation to short-term use of acetaminophen four grams daily in postoperative cardiothoracic patients in the intensive care unit.

    PubMed

    Ahlers, S J G M; Van Gulik, L; Van Dongen, E P A; Bruins, P; Tibboel, D; Knibbe, C A J

    2011-11-01

    A volunteer study suggested that taking paracetamol 4 g daily could result in elevated alanine aminotransferase plasma levels in a substantial proportion of healthy volunteers. The safety of this dose of paracetamol for acute postoperative pain remains controversial. This study aimed to examine the incidence of alanine aminotransferase elevations after short-term use of paracetamol 4 g daily, as part of the standard pain management protocol, for 93 consecutive patients after cardiothoracic surgery. Alanine aminotransferase levels and other liver function tests were measured preoperatively as baseline and once daily after surgery during the intensive care unit stay. Preoperative alanine aminotransferase levels of more than one time the upper limit of normal (ULN >40 U/l) was observed in 11% (n=10) of the patients but none of these baseline alanine aminotransferase levels exceeded three times the ULN (>3 x ULN). The average daily dose of paracetamol administered was 50 mg/kg (SD=16) after surgery. Postoperative alanine aminotransferase levels of >1 x ULN was observed in 17% (n=16), and 4% (n=4) exceeded >3 x ULN The other liver function tests of the latter four patients, including aspartate aminotransferase (range 173 to 5590 U/l), gamma-glutamyltransferase (range 56 to 103 U/l), lactate dehydrogenase (range 376 to 3518 U/l) and the International Normalised Ratio (range 2.0 to 6.6), were all abnormal. These four patients all had right ventricular failure or cardiogenic shock during the postoperative period which could explain the significant rises in alanine aminotransferase after surgery. In conclusion, the incidence of significant alanine aminotransferase elevations after using daily paracetamol as an analgesic agent for cardiac surgery, at a dose of 4 g per day, was low and mostly due to complications after surgery. Our results, albeit still very limited, provided some reassurance about the safety of paracetamol 4 g daily, as a supplementary analgesic agent for

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

    PubMed

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

    2015-08-03

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

  7. Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum.

    PubMed

    Marienhagen, Jan; Kennerknecht, Nicole; Sahm, Hermann; Eggeling, Lothar

    2005-11-01

    Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5'-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with L-glutamate, L-aspartate, and L-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) L-glutamate > 2-aminobutyrate > L-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes L-alanine to aminate 2-oxo-isovalerate, the L-valine precursor, and 2-oxo-butyrate. A second AT active with the L-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for L-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.

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

    PubMed

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

    2009-12-01

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

  9. Electron paramagnetic resonance studies of gamma-irradiated DL-alanine ethyl ester hydrochloride, L-theanine and L-glutamic acid dimethyl ester hydrochloride

    NASA Astrophysics Data System (ADS)

    Başkan, M. Halim; Aydın, Murat

    2013-08-01

    The electron paramagnetic resonance (EPR) of gamma irradiated powders of DL-alanine ethyl ester hydrochloride, L-theanine and L-glutamic acid dimethyl ester hydrochloride were investigated at room temperature. The observed paramagnetic species were attributed to the CH3ĊHCOOC2H5, -CH2ĊHCOOH and -CH2ĊHCOOCH3 radicals, respectively. Hyperfine structure constants and g-values were determined for these three radicals. Some spectroscopic properties and suggestions concerning the possible structure of the radicals were also discussed.

  10. Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons.

    PubMed

    Dadsetan, Sherry; Bak, Lasse K; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Leke, Renata; Schousboe, Arne; Waagepetersen, Helle S

    2011-09-01

    It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5h with [U-(13)C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH(4)Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.

  11. Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S ,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

    SciTech Connect

    Lee, Hyunbeom; Doud, Emma H.; Wu, Rui; Sanishvili, Ruslan; Juncosa, Jose I.; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.

    2015-01-23

    γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. Ultimately, this represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.

  12. The metabolism of the non-proteinogenic amino acid β-N-methylamino-L-alanine (BMAA) in the cyanobacterium Synechocystis PCC6803.

    PubMed

    Downing, Simoné; Downing, Timothy Grant

    2016-06-01

    The neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is produced by cyanobacteria under nitrogen starvation conditions and its metabolism is closely associated with cellular nitrogen control. Very little is known regarding the metabolism or biosynthesis of this amino acid in the producing organisms and current knowledge is limited to the spontaneous formation of carbamate adducts in the presence of aqueous carbon dioxide, the rapid removal of free cellular BMAA upon the addition of ammonia to nitrogen-starved cyanobacterial cultures, and the link between cellular nitrogen status and BMAA synthesis. Data presented here show that exogenous BMAA is readily metabolised by cyanobacteria during which, the primary amino group is rapidly transferred to other cellular amino acids. Furthermore, data suggest that BMAA is metabolised in cyanobacteria via a reversible transamination reaction. This study presents novel data on BMAA metabolism in cyanobacteria and provides the first proposed biosynthetic precursor to BMAA biosynthesis in cyanobacteria.

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

    PubMed Central

    Heaton, M P; Neuhaus, F C

    1992-01-01

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

  14. Inhibitors of alanine racemase enzyme: a review.

    PubMed

    Azam, Mohammed Afzal; Jayaram, Unni

    2016-08-01

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

  15. Amino acid residues in the GerAB protein important in the function and assembly of the alanine spore germination receptor of Bacillus subtilis 168.

    PubMed

    Cooper, Gareth R; Moir, Anne

    2011-05-01

    The paradigm gerA operon is required for endospore germination in response to c-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.

  16. Aspartate aminotransferase (AST) blood test

    MedlinePlus

    ... 2016:chap 73. Read More Acute kidney failure Acute pancreatitis Alanine transaminase (ALT) blood test ALP - blood test Burns Cardiac catheterization Enzyme Heart attack Hemolytic anemia Hepatic Liver cancer - hepatocellular carcinoma Liver ...

  17. A structural insight into the P1S1 binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases

    SciTech Connect

    Węglarz-Tomczak, Ewelina; Berlicki, Łukasz; Pawełczak, Małgorzata; Nocek, Bogusław; Joachimiak, Andrzej; Mucha, Artur

    2016-07-01

    N0 -substituted 1,2-diaminoethylphosphonic acids and 1,2-diaminoethylphosphinic dipeptides were explored to unveil the structural context of the unexpected selectivity of these inhibitors of M1 alanine aminopeptidases (APNs) versus M17 leucine aminopeptidase (LAP). The diaminophosphonic acids were obtained via aziridines in an improved synthetic procedure that was further expanded for the phosphinic pseudodipeptide system. The inhibitory activity, measured for three M1 and one M17 metalloaminopeptidases of different sources (bacterial, human and porcine), revealed several potent compounds (e.g., Ki ¼ 65 nM of 1u for HsAPN). Two structures of an M1 representative (APN from Neisseria meningitidis) in complex with N-benzyl-1,2-diaminoethylphosphonic acid and N-cyclohexyl-1,2- diaminoethylphosphonic acid were determined by the X-ray crystallography. The analysis of these structures and the models of the phosphonic acid complexes of the human ortholog provided an insight into the role of the additional amino group and the hydrophobic substituents of the ligands within the S1 active site region.

  18. Rhodotorulic Acid from Species of Leucosporidium, Rhodosporidium, Rhodotorula, Sporidiobolus, and Sporobolomyces, and a New Alanine-Containing Ferrichrome from Cryptococcus melibiosum

    PubMed Central

    Atkin, C. L.; Neilands, J. B.; Phaff, H. J.

    1970-01-01

    An examination of 142 strains within 19 genera of yeasts and yeastlike organisms for formation of hydroxamic acids in low-iron culture showed production of hydroxamates by two unclassified strains and by 52 strains among the genera Aessosporon (3 of 3 strains), Cryptococcus (1 of 43), Leucosporidium (3 of 11), Rhodosporidium (4 of 4), Rhodotorula (27 of 39), Sporidiobolus (2 of 2), and Sporobolomyces (12 of 13). Crystalline rhodotorulic acid was isolated in amounts sufficient to account for most or all of the measured hydroxamate in culture supernatants of 16 strains representative of the five last-mentioned hydroxamate-producing genera. A new alanine-containing ferrichrome was isolated from one strain of Cryptococcus melibiosum. Rhodotorulic acid was a major metabolic product of many of the positive strains when grown in low-iron media, and iron was shown to repress its synthesis and excretion into the culture medium. The taxonomic significance of production of hydroxamic acids is described in connection with the position of these yeast species in the subclass Heterobasidiomycetidae. PMID:5529038

  19. Protein association of the neurotoxin and non-protein amino acid BMAA (β-N-methylamino-L-alanine) in the liver and brain following neonatal administration in rats.

    PubMed

    Karlsson, Oskar; Jiang, Liying; Andersson, Marie; Ilag, Leopold L; Brittebo, Eva B

    2014-04-07

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) is not an amino acid that is normally found in proteins. Our previous autoradiographic study of (3)H-labeled BMAA in adult mice unexpectedly revealed a tissue distribution similar to that of protein amino acids. The aim of this study was to characterize the distribution of free and protein-bound BMAA in neonatal rat tissues following a short exposure using autoradiographic imaging and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The autoradiographic imaging of (14)C-L-BMAA demonstrated a distinct uptake of radioactivity that was retained following acid extraction in tissues with a high rate of cell turnover and/or protein synthesis. The UHPLC-MS/MS analysis conclusively demonstrated a dose-dependent increase of protein-associated BMAA in neonatal rat tissues. The level of protein-associated BMAA in the liver was more than 10 times higher than that in brain regions not fully protected by the blood-brain barrier which may be due to the higher rate of protein synthesis in the liver. In conclusion, this study demonstrated that BMAA was associated with rat proteins suggesting that BMAA may be misincorporated into proteins. However, protein-associated BMAA seemed to be cleared over time, as none of the samples from adult rats had any detectable free or protein-associated BMAA.

  20. Limiting values of diffusion coefficients of glycine, alanine, [Formula: see text]-amino butyric acid, norvaline and norleucine in a relevant physiological aqueous medium.

    PubMed

    Rodriguez, Diana M; Verissimo, Luis M P; Barros, Marisa C F; Rodrigues, Daniela F S L; Rodrigo, Maria Melia; Esteso, Miguel A; Romero, Carmen M; Ribeiro, Ana C F

    2017-02-01

    The side chain effect on transport in ionic aqueous salt solutions was investigated for [Formula: see text]-amino acids glycine, alanine, [Formula: see text]-amino butyric acid, norvaline, and norleucine --that together define a chemical homologous series based on the length of the characteristic side chain which increases from zero to four carbons, respectively. Binary mutual diffusion coefficients at infinitesimal concentration in aqueous solutions of NaCl (0.15 mol kg (-1)) are measured by means of Taylor dispersion technique for this series and significant differences were found against previous published results for identical systems in pure water. In this way, NaCl effect on the transport of each amino acid is thus assessed and discussed in terms of salting-out effects. Also, solvated Stokes hydrodynamic radii were computed for the series showing comparable results in water and NaCl solution. The new information should prove useful in the design and characterization of transport-controlled systems in physiological and pharmacological studies.

  1. Increased beta-aminoisobutyric acid in rat liver with 6-azauracil and its enantiomer.

    PubMed

    Tamaki, N; Fujimoto, S; Mizutani, N; Mizota, C

    1985-10-21

    When 6-azauracil was subcutaneously injected, beta-aminoisobutyric acid and beta-alanine contents were increased 22 and 61-fold, respectively, in rat liver. Incorporation of [methyl-14C]thymine into beta-aminoisobutyric acid was increased to 42-fold by 6-azauracil treatment. The absolute configuration of this amino acid was proved to be the (R)-form by means of a gas-chromatographic technique. 6-Azauracil inhibited beta-alanine-pyruvate aminotransferase activity with an I50 of approx. 2.5 mM.

  2. Distinguishing the cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) from its structural isomer 2,4-diaminobutyric acid (2,4-DAB).

    PubMed

    Banack, S A; Downing, T G; Spácil, Z; Purdie, E L; Metcalf, J S; Downing, S; Esterhuizen, M; Codd, G A; Cox, P A

    2010-11-01

    The cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) has been associated with certain forms of progressive neurodegenerative disease, including sporadic Amyotrophic Lateral Sclerosis and Alzheimer's disease. Reports of BMAA in cyanobacterial blooms from lakes, reservoirs, and other water resources continue to be made by investigators in a variety of laboratories. Recently it was suggested that during analysis BMAA may be confused with its structural isomer 2,4-diaminobutyric acid (2,4-DAB), or that current detection methods may mistake other compounds for BMAA. We here review the evidence that BMAA can be consistently and reliably separated from 2,4-DAB during reversed-phase HPLC, and that BMAA can be confidently distinguished from 2,4-DAB during triple quadrupole LC-MS/MS analysis by i) different retention times, ii) diagnostic product ions resulting from collision-induced dissociation, and iii) consistent ratios between selected reaction monitoring (SRM) transitions. Furthermore, underivatized BMAA can be separated from 2,4-DAB with an amino acid analyzer with post-column visualization using ninhydrin. Other compounds that may be theoretically confused with BMAA during chloroformate derivatization during GC analysis are distinguished due to their different retention times.

  3. Production of Alanine by Fusarium moniliforme

    PubMed Central

    Carito, Sebastian L.; Pisano, Michael A.

    1966-01-01

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

  4. Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II.

    PubMed

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

    2008-08-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  5. Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II

    PubMed Central

    HAN, Qian; CAI, Tao; TAGLE, Danilo A.; ROBINSON, Howard; LI, Jianyong

    2008-01-01

    Synopsis KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-d-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to α-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested α-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with α-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity. PMID:18620547

  6. Substrate Specificity and Structure of Human aminoadipate aminotransferase/kynurenine aminotransferase II

    SciTech Connect

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

    2009-01-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  7. Substrate Specificity and Structure of Human Aminoadipate Aminotransferase/kynurenine Aminotransferase II

    SciTech Connect

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

    2008-01-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to a-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested a-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with a-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  8. Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence of Corynebacterium glutamicum

    PubMed Central

    Marienhagen, Jan; Kennerknecht, Nicole; Sahm, Hermann; Eggeling, Lothar

    2005-01-01

    Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with l-glutamate, l-aspartate, and l-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) l-glutamate > 2-aminobutyrate > l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes l-alanine to aminate 2-oxo-isovalerate, the l-valine precursor, and 2-oxo-butyrate. A second AT active with the l-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for l-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far. PMID:16267288

  9. The Effect of Artichoke Leaf Extract on Alanine Aminotransferase and Aspartate Aminotransferase in the Patients with Nonalcoholic Steatohepatitis

    PubMed Central

    Rangboo, Vajiheh; Noroozi, Mostafa; Zavoshy, Roza; Rezadoost, Seyed Amirmansoor; Mohammadpoorasl, Asghar

    2016-01-01

    Background. Based on recent basic and clinical investigations, the extract of artichoke (Cynara scolymus) leaf has been revealed to be used for hepatoprotective and cholesterol reducing purposes. We aimed to assess the therapeutic effects of artichoke on biochemical and liver biomarkers in patients with nonalcoholic steatohepatitis (NASH). Methods. In a randomized double blind clinical trial, 60 consecutive patients suffering NASH were randomly assigned to receive Cynara scolymus extract (as 6 tablets per day consisting of 2700 mg extract of the herb) as the intervention group or placebo as the control group for two months. Results. Comparing changes in study markers following interventions showed improvement in liver enzymes. The levels of triglycerides and cholesterol were significantly reduced in the group treated with Cynara scolymus when compared to placebo group. To compare the role of Cynara scolymus use with placebo in changes in study parameters, multivariate linear regression models were employed indicating higher improvement in liver enzymes and also lipid profile particularly triglycerides and total cholesterol following administration of Cynara scolymus in comparison with placebo use. Conclusion. This study sheds light on the potential hepatoprotective activity and hypolipidemic effect of Cynara scolymus in management of NASH. This clinical trial is registered in the IRCT, Iranian Registry of Clinical Trials, by number IRCT2014070218321N1. PMID:27293900

  10. The Effect of Artichoke Leaf Extract on Alanine Aminotransferase and Aspartate Aminotransferase in the Patients with Nonalcoholic Steatohepatitis.

    PubMed

    Rangboo, Vajiheh; Noroozi, Mostafa; Zavoshy, Roza; Rezadoost, Seyed Amirmansoor; Mohammadpoorasl, Asghar

    2016-01-01

    Background. Based on recent basic and clinical investigations, the extract of artichoke (Cynara scolymus) leaf has been revealed to be used for hepatoprotective and cholesterol reducing purposes. We aimed to assess the therapeutic effects of artichoke on biochemical and liver biomarkers in patients with nonalcoholic steatohepatitis (NASH). Methods. In a randomized double blind clinical trial, 60 consecutive patients suffering NASH were randomly assigned to receive Cynara scolymus extract (as 6 tablets per day consisting of 2700 mg extract of the herb) as the intervention group or placebo as the control group for two months. Results. Comparing changes in study markers following interventions showed improvement in liver enzymes. The levels of triglycerides and cholesterol were significantly reduced in the group treated with Cynara scolymus when compared to placebo group. To compare the role of Cynara scolymus use with placebo in changes in study parameters, multivariate linear regression models were employed indicating higher improvement in liver enzymes and also lipid profile particularly triglycerides and total cholesterol following administration of Cynara scolymus in comparison with placebo use. Conclusion. This study sheds light on the potential hepatoprotective activity and hypolipidemic effect of Cynara scolymus in management of NASH. This clinical trial is registered in the IRCT, Iranian Registry of Clinical Trials, by number IRCT2014070218321N1.

  11. Similarities between cysteinesulphinate transaminase and aspartate aminotransferase.

    PubMed

    Recasens, M; Mandel, P

    1979-01-01

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

  12. Synthesis and carbonic anhydrase inhibitory properties of amino acid - coumarin/quinolinone conjugates incorporating glycine, alanine and phenylalanine moieties.

    PubMed

    Küçükbay, F Zehra; Küçükbay, Hasan; Tanc, Muhammet; Supuran, Claudiu T

    2016-12-01

    N-Protected amino acids (Gly, Ala and Phe) were reacted with amino substituted coumarin and quinolinone derivatives, leading to the corresponding N-protected amino acid-coumarin/quinolinone conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against various human (h) isoforms, such as hCA I, hCA II, hCA IV and hCA XII. The quinolinone conjugates were inactive as enzyme inhibitors, whereas the coumarins were ineffective hCA I/II inhibitors (KIs > 50 μM) but were submicromolar hCA IV and XII inhibitors, with inhibition constants ranging between 92 nM and 1.19 μM for hCA IV, and between 0.11 and 0.79 μM for hCA XII. These coumarin derivatives, as many others reported earlier, thus show an interesting selective inhibitory profile for the membrane-bound over the cytosolic CA isoforms.

  13. Rationally Developed Organic Salts of Tolfenamic Acid and Its β-Alanine Derivatives for Dual Purposes as an Anti-Inflammatory Topical Gel and Anticancer Agent.

    PubMed

    Parveen, Rumana; Sravanthi, Bommagani; Dastidar, Parthasarathi

    2017-04-04

    A new series of primary ammonium monocarboxylate (PAM) salts of a nonsteroidal anti-inflammatory drug (NSAID), namely, tolfenamic acid (TA), and its β-alanine derivatives were generated. Nearly 67 % of the salts in the series showed gelling abilities with various solvents, including water (biogenic solvent) and methyl salicylate (typically used for topical gel formulations). Gels were characterized by rheology, electron microscopy, and so forth. Structure-property correlations based on single-crystal and powder XRD data of several gelator and nongelator salts revealed intriguing insights. Studies (in vitro) on an aggressive human breast cancer cell line (MDA-MB-231) with the l-tyrosine methyl ester salt of TA (S7) revealed that the hydrogelator salt was more effective at killing cancer cells than the mother drug TA (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay); displayed better anti-inflammatory activity compared with that of TA (prostaglandin E2 assay); could be internalized within the cancer cells, as revealed by fluorescence microscopy; and inhibited effectively migration of the cancer cells. Thus, the easily accessible ambidextrous gelator salt S7 can be used for two purposes: as an anti-inflammatory topical gel and as an anticancer agent.

  14. Potential transfer of neurotoxic amino acid β-N-methylamino-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines.

    PubMed

    Andersson, Marie; Ersson, Lisa; Brandt, Ingvar; Bergström, Ulrika

    2017-04-01

    β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [(14)C]l-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [(14)C]l-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here, we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [(14)C]l- and [(14)C]d-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [(14)C]l- and [(14)C]d-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [(14)C]l-and [(14)C]d-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [(14)C]l-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant.

  15. 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).

  16. Growth, spectral and optical characterization of a novel nonlinear optical organic material: D-Alanine DL-Mandelic acid single crystal

    NASA Astrophysics Data System (ADS)

    Jayaprakash, P.; Mohamed, M. Peer; Caroline, M. Lydia

    2017-04-01

    An organic nonlinear optical single crystal, D-alanine DL-mandelic acid was synthesized and successfully grown by slow evaporation solution growth technique at ambient temperature using solvent of aqueous solution. The unit cell parameters were assessed from single crystal X-ray diffraction analysis. The presence of diverse functional groups and vibrational modes were identified using Fourier Transform Infra Red and Fourier Transform Raman spectral analyses. The chemical structure of grown crystal has been identified by Nuclear Magnetic Resonance spectroscopic study. Ultraviolet-visible spectral analysis reveal that the crystal has lower cut-off wavelength down to 259 nm, is a key factor to exhibit second harmonic generation signal. The electronic optical band gap and Urbach energy is calculated as 5.31 eV and 0.2425 eV respectively from the UV absorption profile. The diverse optical properties such as, extinction coefficient, reflectance, linear refractive index, optical conductivity was calculated using UV-visible data. The relative second harmonic efficiency of the compound is found to be 0.81 times greater than that of KH2PO4 (KDP). The thermal stability of the grown crystal was studied by thermogravimetric analysis and differential thermal analysis techniques. The luminescence spectrum exhibited two peaks (520 nm, 564 nm) due to the donation of protons from carboxylic acid to amino group. The Vickers microhardness test was carried out employing one of the as-grown hard crystal and there by hardness number (Hv), Meyer's index (n), yield strength (σy), elastic stiffness constant (C11) and Knoop hardness number (HK) were assessed. The dielectric behaviour of the as-grown crystal was analyzed for different temperatures (313 K, 333 K, 353 K, and 373 K) at different frequencies.

  17. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV

    PubMed Central

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

    2010-01-01

    Synopsis Mammalian mitochondrial aspartate aminotransferase (mAspAT) is recently reported to have kynurenine aminotransferase (KAT) activity and plays a role in the biosynthesis of kynurenic acid (KYNA) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen keto acids were tested for the co-substrate specificity of mouse mAspAT and fourteen of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor binding residues of mAspAT are similar to those of other KATs. The substrate binding residues of mAspAT are slightly different from those of other KATs. Our data provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain. PMID:20977429

  18. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV.

    PubMed

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

    2011-10-01

    Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  19. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV

    SciTech Connect

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

    2011-10-01

    Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  20. Investigation of the possible protective role of gallic acid on paraoxanase and arylesterase activities in livers of rats with acute alcohol intoxication.

    PubMed

    Kartkaya, Kazim; Oğlakçi, Ayşegül; Şentürk, Hakan; Bayramoğlu, Gökhan; Canbek, Mediha; Kanbak, Güngör

    2013-04-01

    Gallic acid, a polyphenyl class natural product from gallnut and green tea, is known to be antioxidant, anti-inflammatory and radical scavenger. In this study, we aimed to investigate the possible protective effects of gallic acid on paraoxonase and arylesterase activities in liver exposed to acute alcohol intoxication. Paraoxonase and arylesterase activities in liver tissue and serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase levels were measured. Histological investigations were also made. In our study, we observed a significant increase of serum alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities, which are indicators of liver damage after acute ethanol consumption. Gallic acid therapy has significantly reduced the increase in these biomarkers, indicating a possible hepatoprotective effect of gallic acid. Ethanol consumption caused a significant decrease in liver paraoxonase activity (P < 0.001). Gallic acid treatment partly restored this decreased paraoxonase activity, which resulted from ethanol administration. A gallic acid dose of 100 mg/kg was observed as highest restoring effect for paraoxonase activity (P < 0.05). The activity of arylesterase was decreased in the ethanol group as compared with the control group, but this was not significant. However, 50 mg/kg of gallic acid treatment restored the loss of this activity due to ethanol exposure (P < 0.001). We observed that gallic acid ameliorates the liver damage caused by excessive alcohol consumption in a dose-dependent way. Our results in this study showed that gallic acid might have a protective effect against alcoholic liver disease.

  1. Structural characterization and epitope mapping of the glutamic acid/alanine-rich protein from Trypanosoma congolense: defining assembly on the parasite cell surface.

    PubMed

    Loveless, Bianca C; Mason, Jeremy W; Sakurai, Tatsuya; Inoue, Noboru; Razavi, Morteza; Pearson, Terry W; Boulanger, Martin J

    2011-06-10

    Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.

  2. Targeted deletion of the kynurenine aminotransferase ii gene reveals a critical role of endogenous kynurenic acid in the regulation of synaptic transmission via alpha7 nicotinic receptors in the hippocampus.

    PubMed

    Alkondon, Manickavasagon; Pereira, Edna F R; Yu, Ping; Arruda, Emerson Z; Almeida, Luis E F; Guidetti, Paolo; Fawcett, William P; Sapko, Michael T; Randall, William R; Schwarcz, Robert; Tagle, Danilo A; Albuquerque, Edson X

    2004-05-12

    It has been postulated that endogenous kynurenic acid (KYNA) modulates alpha7* nicotinic acetylcholine receptor (nAChR) and NMDA receptor activities in the brain.a To test this hypothesis, alpha7* nAChR and NMDA receptor functions were studied in mice with a targeted null mutation in the gene encoding kynurenine aminotransferase II (mKat-2-/- mice), an enzyme responsible for brain KYNA synthesis. At 21 postnatal days, mKat-2-/- mice had lower hippocampal KYNA levels and higher spontaneous locomotor activity than wild-type (WT) mice. At this age, alpha7* nAChR activity induced by exogenous application of agonists to CA1 stratum radiatum interneurons was approximately 65% higher in mKat-2-/- than WT mice. Binding studies indicated that the enhanced receptor activity may not have resulted from an increase in alpha7* nAChR number. In 21-d-old mKat-2-/- mice, endogenous alpha7* nAChR activity in the hippocampus was also increased, leading to an enhancement of GABAergic activity impinging onto CA1 pyramidal neurons that could be reduced significantly by acute exposure to KYNA (100 nM). The activities of GABA(A) and NMDA receptors in the interneurons and of alpha3beta4* nAChRs regulating glutamate release onto these neurons were comparable between mKat-2-/- and WT mice. By 60 d of age, KYNA levels and GABAergic transmission in the hippocampus and locomotor activity were similar between mKat-2-/- and WT mice. Our findings that alpha7* nAChRs are major targets for KYNA in the brain may provide insights into the pathophysiology of schizophrenia and Alzheimer's disease, disorders in which brain KYNA levels are increased and alpha7* nAChR functions are impaired.

  3. Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme.

    PubMed

    Jomrit, Juntratip; Summpunn, Pijug; Meevootisom, Vithaya; Wiyakrutta, Suthep

    2011-02-25

    A sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in (2)H(2)O with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'-(2)H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The (2)H at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the (2)H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of (2)H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.

  4. Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme

    SciTech Connect

    Jomrit, Juntratip; Summpunn, Pijug; Meevootisom, Vithaya; Wiyakrutta, Suthep

    2011-02-25

    Research highlights: {yields} Stereochemical mechanism of PLP enzymes is important but difficult to determine. {yields} This new method is significantly less complicated than the previous ones. {yields} This assay is as sensitive as the radioactive based method. {yields} LC-MS/MS positively identify the analyte coenzyme. {yields} The method can be used with enzyme whose apo form is unstable. -- Abstract: A sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in {sup 2}H{sub 2}O with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'-{sup 2}H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The {sup 2}H at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the {sup 2}H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of {sup 2}H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2 mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.

  5. Un-catalyzed peptide bond formation between two monomers of glycine, alanine, serine, threonine, and aspartic acid in gas phase: a density functional theory study

    NASA Astrophysics Data System (ADS)

    Bhunia, Snehasis; Singh, Ajeet; Ojha, Animesh K.

    2016-05-01

    In the present report, un-catalyzed peptide bond formation between two monomers of glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), and aspartic acid (Asp) has been investigated in gas phase via two steps reaction mechanism and concerted mechanism at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. The peptide bond is formed through a nucleophilic reaction via transition states, TS1 and TS2 in stepwise mechanism. The TS1 reveals formation of a new C-N bond while TS2 illustrate the formation of C=O bond. In case of concerted mechanism, C-N bond is formed by a single four-centre transition state (TS3). The energy barrier is used to explain the involvement of energy at each step of the reaction. The energy barrier (20-48 kcal/mol) is required for the transformation of reactant state R1 to TS1 state and intermediate state I1 to TS2 state. The large value of energy barrier is explained in terms of distortion and interaction energies for stepwise mechanism. The energy barrier of TS3 in concerted mechanism is very close to the energy barrier of the first transition state (TS1) of the stepwise mechanism for the formation of Gly-Gly and Ala-Ala di- peptide. However, in case of Ser-Ser, Thr-Thr and Asp-Asp di-peptide, the energy barrier of TS3 is relatively high than that of the energy barrier of TS1 calculated at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. In both the mechanisms, the value of energy barrier calculated at B3LYP/6-31G(d,p) level of theory is greater than that of the value calculated at M062X/6-31G(d,p) level of theory.

  6. Crystal Structure of Human Kynurenine Aminotransferase ll*

    SciTech Connect

    Han,Q.; Robinson, H.; Li, J.

    2008-01-01

    Human kynurenine aminotransferase II (hKAT-II) efficiently catalyzes the transamination of knunrenine to kynurenic acid (KYNA). KYNA is the only known endogenous antagonist of N-methyl-d-aspartate (NMDA) receptors and is also an antagonist of 7-nicotinic acetylcholine receptors. Abnormal concentrations of brain KYNA have been implicated in the pathogenesis and development of several neurological and psychiatric diseases in humans. Consequently, enzymes involved in the production of brain KYNA have been considered potential regulatory targets. In this article, we report a 2.16 Angstroms crystal structure of hKAT-II and a 1.95 Angstroms structure of its complex with kynurenine. The protein architecture of hKAT-II reveals that it belongs to the fold-type I pyridoxal 5-phosphate (PLP)-dependent enzymes. In comparison with all subclasses of fold-type I-PLP-dependent enzymes, we propose that hKAT-II represents a novel subclass in the fold-type I enzymes because of the unique folding of its first 65 N-terminal residues. This study provides a molecular basis for future effort in maintaining physiological concentrations of KYNA through molecular and biochemical regulation of hKAT-II.

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

    PubMed

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

    2008-01-01

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

  8. Differential effect of beta-N-oxalylamino-L-alanine, the Lathyrus sativus neurotoxin, and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate on the excitatory amino acid and taurine levels in the brain of freely moving rats.

    PubMed

    La Bella, V; Piccoli, F

    2000-05-01

    We studied the effect of beta-oxalylamino-L-alanine, a glutamate analog present in Lathyrus sativus seeds and implicated in the etiopathogenesis of neurolathyrism, and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate on the extracellular levels of aspartate, glutamate and taurine in the primary motor cortex of freely moving rats. We found that while both neurotoxins increase the level of aspartate and glutamate, only (+/-)-alpha(-amino-3-hydroxy-5-methylisoxazole-4-propionate is able to modulate the level of taurine. GYKI-52466, a non-competitive non-NMDA antagonist, inhibited beta-oxalylamino-L-alanine-induced increase of aspartate, but not that of glutamate. Conversely, this antagonist proved to be very efficient in blocking the stimulating effect of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate on all three amino acids. We suggest that beta-oxalylamino-L-alanine increases the level of glutamate in vivo by a mechanism not connected to its effect on the non-NMDA receptors, which might involve the inhibition of glutamate transport. This would allow the excitatory neurotransmitter to reach a concentration sufficient to stimulate the non-NMDA receptors, which in their turn mediate the specific release of aspartate. Although the role of aspartate as a neurotransmitter is still under discussion, it might indeed amplify the excitotoxic cascade through its action on NMDA receptors. We speculate that this sequence of events might represent an important step in the molecular cascade leading to the appearance of the selective motoneuron degeneration in neurolathyrism.

  9. Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues.

    PubMed

    Karakawa, Sachise; Shimbo, Kazutaka; Yamada, Naoyuki; Mizukoshi, Toshimi; Miyano, Hiroshi; Mita, Masashi; Lindner, Wolfgang; Hamase, Kenji

    2015-11-10

    A highly sensitive and selective chiral LC-MS/MS method for D-alanine, D-aspartic acid and D-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1-500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples.

  10. Kynurenine Aminotransferase Isozyme Inhibitors: A Review

    PubMed Central

    Nematollahi, Alireza; Sun, Guanchen; Jayawickrama, Gayan S.; Church, W. Bret

    2016-01-01

    Kynurenine aminotransferase isozymes (KATs 1–4) are members of the pyridoxal-5’-phosphate (PLP)-dependent enzyme family, which catalyse the permanent conversion of l-kynurenine (l-KYN) to kynurenic acid (KYNA), a known neuroactive agent. As KATs are found in the mammalian brain and have key roles in the kynurenine pathway, involved in different categories of central nervous system (CNS) diseases, the KATs are prominent targets in the quest to treat neurodegenerative and cognitive impairment disorders. Recent studies suggest that inhibiting these enzymes would produce effects beneficial to patients with these conditions, as abnormally high levels of KYNA are observed. KAT-1 and KAT-3 share the highest sequence similarity of the isozymes in this family, and their active site pockets are also similar. Importantly, KAT-2 has the major role of kynurenic acid production (70%) in the human brain, and it is considered therefore that suitable inhibition of this isozyme would be most effective in managing major aspects of CNS diseases. Human KAT-2 inhibitors have been developed, but the most potent of them, chosen for further investigations, did not proceed in clinical studies due to the cross toxicity caused by their irreversible interaction with PLP, the required cofactor of the KAT isozymes, and any other PLP-dependent enzymes. As a consequence of the possibility of extensive undesirable adverse effects, it is also important to pursue KAT inhibitors that reversibly inhibit KATs and to include a strategy that seeks compounds likely to achieve substantial interaction with regions of the active site other than the PLP. The main purpose of this treatise is to review the recent developments with the inhibitors of KAT isozymes. This treatise also includes analyses of their crystallographic structures in complex with this enzyme family, which provides further insight for researchers in this and related studies. PMID:27314340

  11. Clinicopathological features of choledocholithiasis patients with high aminotransferase levels without cholangitis

    PubMed Central

    Huh, Cheal Wung; Jang, Sung Ill; Lim, Beom Jin; Kim, Hee Wook; Kim, Jae Keun; Park, Jun Sung; Kim, Ja Kyung; Lee, Se Joon; Lee, Dong Ki

    2016-01-01

    Abstract Common bile duct (CBD) stones are generally associated with greater elevations of alkaline phosphatase and gamma-glutamyl transpeptidase levels than aspartate aminotransferase and alanine aminotransferase levels. However, some patients with CBD stones show markedly increased aminotransferase levels, sometimes leading to the misdiagnosis of liver disease. Therefore, the aim of this study was to investigate the clinicopathologic features of patients with CBD stones and high aminotransferase levels. This prospective cohort study included 882 patients diagnosed with CBD stones using endoscopic retrograde cholangiopancreatography (ERCP). Among these patients, 38 (4.3%) exhibited aminotransferase levels above 400 IU/L without cholangitis (gallstone hepatitis [GSH] group), and 116 (13.2%) exhibited normal aminotransferase levels (control group). We compared groups in terms of clinical features, laboratory test results, radiologic images, and ERCP findings such as CBD diameter, CBD stone diameter and number, and periampullary diverticulum. Liver biopsy was performed for patients in the GSH group. GSH patients were younger and more likely to have gallbladder stones than control patients, implying a higher incidence of gallbladder stone migration. Also, GSH patients experienced more severe, short-lasting abdominal pain. ERCP showed narrower CBDs in GSH patients than in control patients. Histological analysis of liver tissue from GSH patients showed no abnormalities except for mild inflammation. Compared with control patients, GSH patients were younger and showed more severe, short-lasting abdominal pain, which could be due to a sudden increase of CBD pressure resulting from the migration of gallstones through narrower CBDs. These clinical features could be helpful not only for the differential diagnosis of liver disease but also for investigating the underlying mechanisms of liver damage in obstructive jaundice. Moreover, we propose a new definition of

  12. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    SciTech Connect

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

    2013-04-01

    The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  13. Salivary lactate dehydrogenase and aminotransferases in diabetic patients

    PubMed Central

    Malicka, Barbara; Skoskiewicz-Malinowska, Katarzyna; Kaczmarek, Urszula

    2016-01-01

    Abstract Diabetes mellitus (DM) is a group of metabolic diseases resulting from impaired insulin secretion and/or action. DM is characterized by hyperglycemia that can lead to the dysfunction or damage of organs, including the salivary glands. The aim of this study was to compare the levels of salivary lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in diabetic patients. The study was approved by the Bioethics Committee of Wroclaw Medical University (Poland). The study comprised 90 adults of both sexes, aged 21 to 57 years. The patients were divided into 3 groups: type 1 diabetics (D1), type 2 diabetics (D2), and a healthy control group (C). Each group consisted of 30 age- and sex-matched subjects. Total protein (P, by Lowry method), LDH, AST, ALT (with Alpha Diagnostics kits), and salivary flow rate were measured in unstimulated mixed saliva. The level of glycosylated hemoglobin (HbA1c) was measured with DCA 2000 Reagent Kit. The obtained data were analyzed using the Mann–Whitney U test and the Spearman rank at a significance level of P < 0.05 with the use of STATISTICA 9.0 software. In comparison with C, D1 presented a significantly higher activity of LDH (P < 0.001), AST (P < 0.001), and ALT (P < 0.01), whereas D2 indicated higher levels of LDH (P < 0.001) and ALT (P < 0.05) compared with C. Comparing D1 to D2, approximately 3-fold higher activity of AST (P < 0.01) and approximately 4.5-fold higher activity of ALT (P < 0.01) was observed. Higher levels of salivary LDH, AST, and ALT in D1 compared with D2 and C confirm that salivary glands of D1 might be attributed to autoimmunological damage associated with the pathomechanism of DM. PMID:27893660

  14. The Relationships between Respiratory Virus Infection and Aminotransferase in Children

    PubMed Central

    Oh, Jun Suk; Choi, Jun Sik; Lee, Young Hyuk; Ko, Kyung Og; Lim, Jae Woo; Cheon, Eun Jung; Lee, Gyung Min

    2016-01-01

    Purpose We sought to examine the relationship between the clinical manifestations of nonspecific reactive hepatitis and respiratory virus infection in pediatric patients. Methods Patients admitted to the pediatric unit of Konyang University Hospital for lower respiratory tract disease between January 1, 2014 and December 31, 2014 and who underwent reverse transcriptase polymerase chain reaction tests were examined. The patients were divided into those with increased levels of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) and those with normal ALT or AST levels. Further, patients with increased ALT and AST levels were individually compared with patients in the normal group, and the blood test results were compared according to the type of respiratory virus. Results Patients with increased ALT or AST levels had one more day of hospital stay, on average, compared with patients in the normal group (5.3±3.1 days vs. 4.4±3.0 days, p=0.019). Patients in the increased ALT level group were younger and had a longer mean hospital stay, compared with patients in the normal group (p=0.022 and 0.003, respectively). The incidences of increased ALT or AST were the highest in adenovirus infections (6/24, 25.0%), followed by enterovirus (2/11, 18.2%) and respiratory syncytial virus A (21/131, 16.0%) infections. Conclusion Nonspecific reactive hepatitis is more common among patients with adenovirus, enterovirus and respiratory syncytial virus infection, as well as among those infected at a younger age. Compared with AST levels, ALT levels are better indicators of the severity of nonspecific reactive hepatitis. PMID:28090469

  15. Primary structure of a key enzyme in plant tetrapyrrole synthesis: glutamate 1-semialdehyde aminotransferase.

    PubMed Central

    Grimm, B

    1990-01-01

    The formation of delta-aminolevulinate from glutamate 1-semialdehyde (GSA) is catalyzed by glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8). The active form of the barley enzyme appears to be a dimer of identical subunits with a molecular mass of 46 kDa. From the purified enzyme, amino acid sequences of the N-terminal ends of the mature protein as well as an internal peptide were determined. DNA primers deduced from these peptide sequences were used to amplify with the polymerase chain reaction a cDNA sequence encoding part of the enzyme. Screening a cDNA library with this DNA fragment identified a full-length clone encoding the 49,540-Da precursor of the GSA aminotransferase. The transit peptide for chloroplast import consists of 34 amino acids. GSA aminotransferase and a precursor form were expressed on a multicopy plasmid in Escherichia coli. Both recombinant gene products reacted with an antibody against the barley GSA aminotransferase. Active barley GSA aminotransferase expressed in E. coli was shown to be active in assays of bacterial cell extracts. As a gene symbol for barley GSA aminotransferase, Gsa is proposed. Images PMID:2349227

  16. 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.

  17. Catabolism of amino acids in livers from cafeteria-fed rats.

    PubMed

    de Castro Ghizoni, Cristiane Vizioli; Gasparin, Fabiana Rodrigues Silva; Júnior, Antonio Sueiti Maeda; Carreño, Fernando Olinto; Constantin, Rodrigo Polimeni; Bracht, Adelar; Ishii Iwamoto, Emy Luiza; Constantin, Jorgete

    2013-01-01

    Most studies using a hypercaloric diet to induce obesity have focused on the metabolism of fat and carbohydrates. Less concern has been given to the metabolism of amino acids, despite evidence of modifications in nitrogen metabolism during obesity. The aim of this study was to evaluate amino acid metabolism in livers from cafeteria diet-induced obese rats. Blood parameters were analysed, and histological sections of livers were stained with Sudan III. The enzymatic activities of some enzymes were determined in liver homogenates. Gluconeogenesis, ureagenesis, and oxygen consumption were evaluated in rat livers perfused with glutamine, alanine, or ammonium chloride. Compared to control rats, cafeteria-fed rats demonstrated higher levels of triacylglycerol and glucose in the blood and greater accumulation of fat in livers. Gluconeogenesis and urea production in livers perfused with glutamine and alanine at higher concentrations showed a substantial reduction in cafeteria-fed rats. However, no significant difference was observed among groups perfused with ammonium chloride. The activities of the enzymes alanine aminotransferase, glutaminase, and aspartate aminotransferase in the livers were reduced in cafeteria-fed rats. Taken together, these data are consistent with the hypothesis that livers from cafeteria diet-induced obese rats exhibit a limitation in their maximal capacity to metabolise glutamine and alanine to glucose, ammonia, and urea, not because of an impairment in gluconeogenesis and/or ureagenesis, but rather due to a depression in the activities of enzymes that catalyse the initial steps of amino acid metabolism.

  18. Production of D-Alanine by Corynebacterium fascians

    PubMed Central

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

    1973-01-01

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

  19. Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity.

    PubMed

    Lerchner, Alexandra; Jarasch, Alexander; Skerra, Arne

    2016-09-01

    The l-alanine dehydrogenase of Bacillus subtilis (BasAlaDH), which is strictly dependent on NADH as redox cofactor, efficiently catalyzes the reductive amination of pyruvate to l-alanine using ammonia as amino group donor. To enable application of BasAlaDH as regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases, we alterated its cofactor specificity from NADH to NADPH via protein engineering. By introducing two amino acid exchanges, D196A and L197R, high catalytic efficiency for NADPH was achieved, with kcat /KM  = 54.1 µM(-1)  Min(-1) (KM  = 32 ± 3 µM; kcat  = 1,730 ± 39 Min(-1) ), almost the same as the wild-type enzyme for NADH (kcat /KM  = 59.9 µM(-1)  Min(-1) ; KM  = 14 ± 2 µM; kcat  = 838 ± 21 Min(-1) ). Conversely, recognition of NADH was much diminished in the mutated enzyme (kcat /KM  = 3 µM(-1)  Min(-1) ). BasAlaDH(D196A/L197R) was applied in a coupled oxidation/transamination reaction of the chiral dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans ω-aminotransferase, thus allowing recycling of the two cosubstrates NADP(+) and l-Ala. An excellent cofactor regeneration with recycling factors of 33 for NADP(+) and 13 for l-Ala was observed with the engineered BasAlaDH in a small-scale biocatalysis experiment. This opens a biocatalytic route to novel building blocks for industrial high-performance polymers.

  20. Structure of the O-polysaccharide of Proteus vulgaris O44: a new O-antigen that contains an amide of D-glucuronic acid with L-alanine.

    PubMed

    Toukach, Filip V; Perepelov, Andrei V; Bartodziejska, Beata; Shashkov, Alexander S; Blaszczyk, Aleksandra; Arbatsky, Nikolay P; Rozalski, Antoni; Knirel, Yuriy A

    2003-06-23

    The O-polysaccharide of Proteus vulgaris O44, strain PrK 67/57 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H, 13C HMQC, HMQC-TOCSY and HMBC experiments. The polysaccharide was found to contain an amide of D-glucuronic acid with L-alanine [D-GlcA6(L-Ala)], and the following structure of the linear pentasaccharide repeating unit was established: [structure: see text]. The structural data of the O-polysaccharide and the results of serological studies with P. vulgaris O44 O-antiserum showed that the strain studied is unique among Proteus bacteria, which is in agreement with its classification in a separate Proteus serogroup, O44.

  1. Weaning Induced Hepatic Oxidative Stress, Apoptosis, and Aminotransferases through MAPK Signaling Pathways in Piglets

    PubMed Central

    Luo, Zhen; Zhu, Wei; Guo, Qi; Luo, Wenli; Zhang, Jing; Xu, Weina

    2016-01-01

    This study investigated the effects of weaning on the hepatic redox status, apoptosis, function, and the mitogen-activated protein kinase (MAPK) signaling pathways during the first week after weaning in piglets. A total of 12 litters of piglets were weaned at d 21 and divided into the weaning group (WG) and the control group (CG). Six piglets from each group were slaughtered at d 0 (d 20, referred to weaning), d 1, d 4, and d 7 after weaning. Results showed that weaning significantly increased the concentrations of hepatic free radicals H2O2 and NO, malondialdehyde (MDA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG), while significantly decreasing the inhibitory hydroxyl ability (IHA) and glutathione peroxidase (GSH-Px), and altered the level of superoxide dismutase (SOD). The apoptosis results showed that weaning increased the concentrations of caspase-3, caspase-8, caspase-9 and the ratio of Bax/Bcl-2. In addition, aspartate aminotransferase transaminase (AST) and alanine aminotransferase (ALT) in liver homogenates increased after weaning. The phosphorylated JNK and ERK1/2 increased, while the activated p38 initially decreased and then increased. Our results suggested that weaning increased the hepatic oxidative stress and aminotransferases and initiated apoptosis, which may be related to the activated MAPK pathways in postweaning piglets. PMID:27807471

  2. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity.

    PubMed

    Sayer, Christopher; Isupov, Michail N; Westlake, Aaron; Littlechild, Jennifer A

    2013-04-01

    The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  3. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    PubMed Central

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

    2013-01-01

    The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-­aminotransferases. PMID:23519665

  4. Genome-enabled determination of amino acid biosynthesis in Xanthomonas campestris pv. campestris and identification of biosynthetic pathways for alanine, glycine, and isoleucine by 13C-isotopologue profiling.

    PubMed

    Schatschneider, Sarah; Vorhölter, Frank-Jörg; Rückert, Christian; Becker, Anke; Eisenreich, Wolfgang; Pühler, Alfred; Niehaus, Karsten

    2011-10-01

    To elucidate the biosynthetic pathways for all proteinogenic amino acids in Xanthomonas campestris pv. campestris, this study combines results obtained by in silico genome analysis and by (13)C-NMR-based isotopologue profiling to provide a panoramic view on a substantial section of bacterial metabolism. Initially, biosynthesis pathways were reconstructed from an improved annotation of the complete genome of X. campestris pv. campestris B100. This metabolic reconstruction resulted in the unequivocal identification of biosynthesis routes for 17 amino acids in total: arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Ambiguous pathways were reconstructed from the genome data for alanine, glycine, and isoleucine biosynthesis. (13)C-NMR analyses supported the identification of the metabolically active pathways. The biosynthetic routes for these amino acids were derived from the precursor molecules pyruvate, serine, and pyruvate, respectively. By combining genome analysis and isotopologue profiling, a comprehensive set of biosynthetic pathways covering all proteinogenic amino acids was unraveled for this plant pathogenic bacterium, which plays an important role in biotechnology as a producer of the exopolysaccharide xanthan. The data obtained lay ground for subsequent functional analyses in post-genomics and biotechnology, while the innovative combination of in silico and wet lab technology described here is promising as a general approach to elucidate metabolic pathways.

  5. Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase

    PubMed Central

    Hawker, Dustin D.; Silverman, Richard B.

    2012-01-01

    Two principal neurotransmitters are involved in the regulation of mammalian neuronal activity, namely, γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, and L-glutamic acid, an excitatory neurotransmitter. Low GABA levels in the brain have been implicated in epilepsy and several other neurological diseases. Because of GABA’s poor ability to cross the blood-brain barrier (BBB), a successful strategy to raise brain GABA concentrations is the use of a compound that does cross the BBB and inhibits or inactivates GABA aminotransferase (GABA-AT), the enzyme responsible for GABA catabolism. Vigabatrin, a mechanism-based inactivator of GABA-AT, is currently a successful therapeutic for epilepsy, but has harmful side effects, leaving a need for improved GABA-AT inactivators. Here, we report the synthesis and evaluation of a series of heteroaromatic GABA analogues as substrates of GABA-AT, which will be used as the basis for the design of novel enzyme inactivators. PMID:22944334

  6. Systematic detection of BMAA (β-N-methylamino-l-alanine) and DAB (2,4-diaminobutyric acid) in mollusks collected in shellfish production areas along the French coasts.

    PubMed

    Réveillon, Damien; Séchet, Véronique; Hess, Philipp; Amzil, Zouher

    2016-02-01

    The neurotoxin β-N-methylamino-l-alanine (BMAA) is naturally present in some microalgal species in the marine environment. The accumulation of BMAA has widely been observed in filter-feeding bivalves that are known to consume primary producers constituting the base of complex aquatic food webs. This study was performed to assess the occurrence of BMAA and isomers in mollusks collected from nine representative shellfish production areas located on the three French coasts (Channel, Atlantic and Mediterranean sites). The use of a highly selective and sensitive HILIC-MS/MS method, with D5DAB as internal standard, revealed the systematic detection of BMAA and DAB, in concentrations ranging from 0.20 to 6.7 μg g(-1) dry weight of digestive gland tissues of mollusks. While we detected BMAA in four strains of diatoms in a previous study, here BMAA was only detected in one diatom species previously not investigated out of the 23 microalgal species examined (belonging to seven classes). The concentrations of BMAA and DAB in mussels and oysters were similar at different sampling locations and despite the high diversity of phytoplankton populations that mollusks feed on at these locations. Only small variations of BMAA and DAB levels were observed and these were not correlated to any of the phytoplankton species reported. Therefore, extensive research should be performed on both origin and metabolism of BMAA in shellfish. The levels observed in this study are similar to those found in other studies in France or elsewhere. A previous study had related such levels to a cluster of Amyotrophic Lateral Sclerosis in the South of France; hence the widespread occurrence of BMAA in shellfish from all coasts in France found in this study suggests the need for further epidemiological and toxicological studies to establish the levels that are relevant for a link between the consumption of BMAA-containing foodstuffs and neurodegenerative diseases.

  7. Biochemical properties and crystal structure of a β-phenylalanine aminotransferase from Variovorax paradoxus.

    PubMed

    Crismaru, Ciprian G; Wybenga, Gjalt G; Szymanski, Wiktor; Wijma, Hein J; Wu, Bian; Bartsch, Sebastian; de Wildeman, Stefaan; Poelarends, Gerrit J; Feringa, Ben L; Dijkstra, Bauke W; Janssen, Dick B

    2013-01-01

    By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg(-1) for (S)-β-phenylalanine at 30°C and 33 U mg(-1) at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.

  8. Improved detection of β-N-methylamino-L-alanine using N-hydroxysuccinimide ester of N-butylnicotinic acid for the localization of BMAA in blue mussels (Mytilus edulis).

    PubMed

    Andrýs, Rudolf; Zurita, Javier; Zguna, Nadezda; Verschueren, Klaas; De Borggraeve, Wim M; Ilag, Leopold L

    2015-05-01

    β-N-Methylamino-L-alanine (BMAA) is an important non-protein amino acid linked to neurodegenerative diseases, specifically amyotrophic lateral sclerosis (ALS). Because it can be transferred and bioaccumulated higher up the food chain, it poses significant public health concerns; thus, improved detection methods are of prime importance for the identification and management of these toxins. Here, we report the successful use of N-hydroxysuccinimide ester of N-butylnicotinic acid (C4-NA-NHS) for the efficient separation of BMAA from its isomers and higher sensitivity in detecting BMAA compared to the current method of choice using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization. Implementation of this efficient method allowed localization of BMAA in the non-visceral tissues of blue mussels, suggesting that more efficient depuration may be required to remove this toxin prior to consumption. This is a crucial method in establishing the absence or presence of the neurotoxic amino acid BMAA in food, environmental or biomedical samples.

  9. LC-MS/MS determination of the isomeric neurotoxins BMAA (beta-N-methylamino-L-alanine) and DAB (2,4-diaminobutyric acid) in cyanobacteria and seeds of Cycas revoluta and Lathyrus latifolius.

    PubMed

    Krüger, Thomas; Mönch, Bettina; Oppenhäuser, Steven; Luckas, Bernd

    2010-01-01

    Since diverse taxa of cyanobacteria has been linked to biosynthesis of BMAA, a controversy has arisen about the detection of neurotoxic amino acids in cyanobacteria. In this context, a novel LC-MS/MS method was developed for the unambiguous determination of beta-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) in cyanobacteria and selected plant seeds. Both neurotoxic and non-proteinogenic amino acids were analyzed without derivatization considering the total concentration of the free and protein-bound form. The investigation of overall 62 cyanobacterial samples of worldwide origin by application of this method revealed the absence of BMAA, whereas seeds of Cycas revoluta contained 6.96 microg g(-1) of free BMAA. In contrast, the isomer DAB was confirmed in 16 cyanobacterial samples in concentrations of 0.07-0.83 microg g(-1),whereof one sample is distributed as nutritional supplement. In addition, seeds of Lathyrus latifolius contained 4.21 microg g(-1) of free DAB. Limits of detection were for BMAA<1.0 microg g(-1) in the cyanobacterial matrix and<0.14 microg g(-1) in angiosperm seeds. DAB exhibits higher sensitivities of <0.06 microg g(-1) in cyanobacteria and <0.008 microg g(-1) in angiosperm seeds. The highly specific analysis method with increased detection sensitivity eliminates the disadvantages of derivatization-based methods to be discussed.

  10. THE EFFECT OF THE HYDROGEN ION CONCENTRATION ON THE RATE OF HYDROLYSIS OF GLYCYL GLYCINE, GLYCYL LEUCINE, GLYCYL ALANINE, GLYCYL ASPARAGINE, GLYCYL ASPARTIC ACID, AND BIURET BASE BY EREPSIN.

    PubMed

    Northrop, J H; Simms, H S

    1928-11-20

    1. The rate of hydrolysis at different pH values of glycyl glycine, glycyl leucine, glycyl alanine, glycyl asparagine, glycyl aspartic acid and biuret base has been determined. 2. The pH-activity curves obtained in this way differ for the different substrates. 3. The curves can be satisfactorily predicted by the assumption that erepsin is a weak acid or base with a dissociation constant of 10(-7.6) and that the reaction takes place between a particular ionic species of the enzyme and of the substrate. There are several possible arrangements which will predict the experimental results. 4. The rate of inactivation of erepsin at various pH values has been determined and found to agree with the assumption used above, that the enzyme is a weak acid or base with a dissociation constant of about 10(-7.6). 5. It is pointed out that if the mechanism assumed is correct, the determination of a significant value for the relative rate of hydrolysis of various peptides is a very uncertain procedure.

  11. Analytical continuation in coupling constant method; application to the calculation of resonance energies and widths for organic molecules: Glycine, alanine and valine and dimer of formic acid

    NASA Astrophysics Data System (ADS)

    Papp, P.; Matejčík, Š.; Mach, P.; Urban, J.; Paidarová, I.; Horáček, J.

    2013-06-01

    The method of analytic continuation in the coupling constant (ACCC) in combination with use of the statistical Padé approximation is applied to the determination of resonance energy and width of some amino acids and formic acid dimer. Standard quantum chemistry codes provide accurate data which can be used for analytic continuation in the coupling constant to obtain the resonance energy and width of organic molecules with a good accuracy. The obtained results are compared with the existing experimental ones.

  12. Determination of the neurotoxins BMAA (beta-N-methylamino-L-alanine) and DAB (alpha-,gamma-diaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms.

    PubMed

    Faassen, Elisabeth J; Gillissen, Frits; Zweers, Hans A J; Lürling, Miquel

    2009-01-01

    We aimed to determine concentrations of the neurotoxic amino acids beta-N-methylamino-L-alanine (BMAA) and alpha-,gamma-diaminobutyric acid (DAB) in mixed species scum material from Dutch urban waters that suffer from cyanobacterial blooms. BMAA and DAB were analysed in scum material without derivatization by LC-MSMS (liquid chromatography coupled to tandem mass spectrometry) using hydrophilic interaction chromatography (HILIC). Our method showed high selectivity, good recovery of added compounds after sample extraction (86% for BMAA and 85% for DAB), acceptable recovery after sample hydrolysation (70% for BMAA and 56% for DAB) and acceptable precision. BMAA and DAB could be detected at an injected amount of 0.34 pmol. Free BMAA was detected in nine of the 21 sampled locations with a maximum concentration of 42 microg/g DW. Free DAB was detected in two locations with a maximum concentration of 4 microg/g DW. No protein-associated forms were detected. This study is the first to detect underivatized BMAA in cyanobacterial scum material using LC-MSMS. Ubiquity of BMAA in cyanobacteria scums of Dutch urban waters could not be confirmed, where BMAA and DAB concentrations were relatively low; however, co-occurrence with other cyanobacterial neurotoxins might pose a serious health risk including chronic effects from low-level doses.

  13. Neutral penta- and hexacoordinate silicon(IV) complexes containing two bidentate ligands derived from the alpha-amino acids (S)-alanine, (S)-phenylalanine, and (S)-tert-leucine.

    PubMed

    Cota, Smaranda; Beyer, Matthias; Bertermann, Rüdiger; Burschka, Christian; Götz, Kathrin; Kaupp, Martin; Tacke, Reinhold

    2010-06-11

    The neutral hexacoordinate silicon(IV) complex 6 (SiO(2)N(4) skeleton) and the neutral pentacoordinate silicon(IV) complexes 7-11 (SiO(2)N(2)C skeletons) were synthesized from Si(NCO)(4) and RSi(NCO)(3) (R = Me, Ph), respectively. The compounds were structurally characterized by solid-state NMR spectroscopy (6-11), solution NMR spectroscopy (6 and 10), and single-crystal X-ray diffraction (8 and 11 were studied as the solvates 8 x CH(3)CN and 11 x C(5)H(12) x 0.5 CH(3)CN, respectively). The silicon(IV) complexes 6 (octahedral Si-coordination polyhedron) and 7-11 (trigonal-bipyramidal Si-coordination polyhedra) each contain two bidentate ligands derived from an alpha-amino acid: (S)-alanine, (S)-phenylalanine, or (S)-tert-leucine. The deprotonated amino acids act as monoanionic (6) or as mono- and dianionic ligands (7-11). The experimental investigations were complemented by computational studies of the stereoisomers of 6 and 7.

  14. 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.

  15. Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti.

    PubMed Central

    Alfano, J R; Kahn, M L

    1993-01-01

    Aspartate aminotransferase (AAT) is an important enzyme in aspartate catabolism and biosynthesis and, by converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carbon metabolism with nitrogen metabolism. To examine the role of AAT in symbiotic nitrogen fixation further, plasmids encoding three different aminotransferases from Rhizobium meliloti 104A14 were isolated by complementation of an Escherichia coli auxotroph that lacks three aminotransferases. pJA10 contained a gene, aatB, that coded for a previously undescribed AAT, AatB. pJA30 encoded an aromatic aminotransferase, TatA, that had significant AAT activity, and pJA20 encoded a branched-chain aminotransferase designated BatA. Genes for the latter two enzymes, tatA and batA, were previously isolated from R. meliloti. aatB is distinct from but hybridizes to aatA, which codes for AatA, a protein required for symbiotic nitrogen fixation. The DNA sequence of aatB contained an open reading frame that could encode a protein 410 amino acids long and with a monomer molecular mass of 45,100 Da. The amino acid sequence of aatB is unusual, and AatB appears to be a member of a newly described class of AATs. AatB expressed in E. coli has a Km for aspartate of 5.3 mM and a Km for 2-oxoglutarate of 0.87 mM. Its pH optimum is between 8.0 and 8.5. Mutations were constructed in aatB and tatA and transferred to the genome of R. meliloti 104A14. Both mutants were prototrophs and were able to carry out symbiotic nitrogen fixation. Images PMID:8320232

  16. Comparison of effect of cafetière and filtered coffee on serum concentrations of liver aminotransferases and lipids: six month randomised controlled trial.

    PubMed Central

    Urgert, R.; Meyboom, S.; Kuilman, M.; Rexwinkel, H.; Vissers, M. N.; Klerk, M.; Katan, M. B.

    1996-01-01

    OBJECTIVE: To study the effects of prolonged intake of cafetière coffee, which is rich in the diterpenes cafestol and kahweol, on serum aminotransferase and lipid concentrations. DESIGN: Randomised parallel controlled trial. SUBJECTS: 46 healthy men and women aged 19 to 69. INTERVENTION: Consumption of five to six strong cups (0.9 litres) a day of either cafetière (22 subjects) or filtered coffee (24 subjects) for 24 weeks. MAIN OUTCOME MEASURES: Mean changes in serum aminotransferase and lipid concentrations. RESULTS: Cafetière coffee raised alanine aminotransferase concentration by up to 80% above baseline values relative to filtered coffee. After 24 weeks the rise was still 45% (9 U/l (95% confidence interval 3 to 15 U/l), P = 0.007). Alanine aminotransferase concentration exceeded the upper limit of normal in eight of the 22 subjects drinking cafetière coffee, being twice the upper limit of normal in three of them. Cafetière coffee raised low density lipoprotein cholesterol concentrations by 9-14%. After 24 weeks the rise was 0.26 mmol/l (0.04 to 0.47 mmol/l) (P = 0.03) relative to filtered coffee. Triglyceride concentrations initially rose by 26% with cafetière coffee but returned close to baseline values within six months. All increases were reversible after the intervention was stopped. CONCLUSIONS: Daily consumption of five to six cups of strong cafetière coffee affects the integrity of liver cells as suggested by small increases in serum alanine aminotransferase concentration. The effect does not subside with prolonged intake. High intakes of coffee brews rich in cafestol and kahweol may thus be responsible for unexplained increases in this enzyme activity in apparently healthy subjects. Cafetière coffee also raises low density lipoprotein cholesterol concentration and thus the risk of coronary heart disease. PMID:8956701

  17. Histidine-40 of ribonuclease T1 acts as base catalyst when the true catalytic base, glutamic acid-58, is replaced by alanine.

    PubMed

    Steyaert, J; Hallenga, K; Wyns, L; Stanssens, P

    1990-09-25

    Mechanisms for the ribonuclease T1 (RNase T1; EC 3.1.27.3) catalyzed transesterification reaction generally include the proposal that Glu58 and His92 provide general base and general acid assistance, respectively [Heinemann, U., & Saenger, W. (1982) Nature (London) 299, 27-31]. This view was recently challenged by the observation that mutants substituted at position 58 retain high residual activity; a revised mechanism was proposed in which His40, and not Glu58, is engaged in catalysis as general base [Nishikawa, S., Morioka, H., Kim, H., Fuchimura, K., Tanaka, T., Uesugi, S., Hakoshima, T., Tomita, K., Ohtsuka, E., & Ikehara, M. (1987) Biochemistry 26, 8620-8624]. To clarify the functional roles of His40, Glu58, and His92, we analyzed the consequences of several amino acid substitutions (His40Ala, His40Lys, His40Asp, Glu58Ala, Glu58Gln, and His92Gln) on the kinetics of GpC transesterification. The dominant effect of all mutations is on Kcat, implicating His40, Glu58, and His92 in catalysis rather than in substrate binding. Plots of log (Kcat/Km) vs pH for wild-type, His40Lys, and Glu58Ala RNase T1, together with the NMR-determined pKa values of the histidines of these enzymes, strongly support the view that Glu58-His92 acts as the base-acid couple. The curves also show that His40 is required in its protonated form for optimal activity of wild-type enzyme. We propose that the charged His40 participates in electrostatic stabilization of the transition state; the magnitude of the catalytic defect (a factor of 2000) from the His40 to Ala replacement suggests that electrostatic catalysis contributes considerably to the overall rate acceleration. For Glu58Ala RNase T1, the pH dependence of the catalytic parameters suggests an altered mechanism in which His40 and His92 act as base and acid catalyst, respectively. The ability of His40 to adopt the function of general base must account for the significant activity remaining in Glu58-mutated enzymes.

  18. [Conformation of aspartate aminotransferase in crystals].

    PubMed

    Borisov, V V; Borisova, S N; Sosfenov, N I; Dikson, Kh BF

    1983-01-01

    X-ray study of chicken cytosolic aspartate aminotransferase revealed conformational changes in the protein of two kinds: (1) a shift of the small domain adjacent to substrate-binding area due to interaction of the protein with two carboxyl groups of substrate and (2) a change in inclination of the coenzyme plane due to replacement of C = N bond of the coenzyme with Lys-258 by C = N bond with a substrate. An asymmetry in subunit behaviour is observed in both cases: the domain is shifted in one subunit and the coenzyme is rotated in other. Substrate-binding properties of each subunit are strictly dependent on the protein conformation in substrate-binding area.

  19. Elucidation of matrix effects and performance of solid-phase extraction for LC-MS/MS analysis of β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) neurotoxins in cyanobacteria.

    PubMed

    Li, Aifeng; Fan, Hua; Ma, Feifei; McCarron, Pearse; Thomas, Krista; Tang, Xianghai; Quilliam, Michael A

    2012-03-07

    A liquid chromatography-mass spectrometry (LC-MS/MS) method using hydrophilic interaction liquid chromatography (HILIC) was developed for the analysis of neurotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), using multiple reaction monitoring (MRM) scan mode. Oasis-MCX and Strata-X-C polymeric cation-exchange cartridges were used to clean extracts of cyanobacterial cultures, including two strains of Microcystis aeruginosa and one strain of Nostoc sp. The performance of the solid-phase extraction (SPE) cartridges for BMAA and DAB were evaluated using mixed standards and spiked cyanobacterial extracts, which demonstrated recoveries of BMAA and DAB ranging from 66% to 91%. Matrix effects in LC-MS/MS were evaluated, and while there was no effect on BMAA quantitation, suppression of DAB was found. Full scan (Q1) and enhanced product ion (EPI) monitoring showed that the DAB suppression may be due to closely eluting compounds, including lysine, histidine, arginine and three other compounds with [M + H](+) m/z of 88, 164 and 191. The procedures developed allow the sensitive and effective analysis of trace BMAA and DAB levels in cyanobacteria. While DAB was confirmed to be present, no BMAA was found in the cyanobacterial samples tested in the present study.

  20. Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii.

    PubMed

    Astegno, Alessandra; Maresi, Elena; Bertoldi, Mariarita; La Verde, Valentina; Paiardini, Alessandro; Dominici, Paola

    2017-03-07

    Toxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine aminotransferase from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity in vitro, suggesting a role in both arginine and GABA metabolism in vivo The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention.

  1. Design and Mechanism of Tetrahydrothiophene-based GABA Aminotransferase Inactivators

    PubMed Central

    Le, Hoang V.; Hawker, Dustin D.; Wu, Rui; Doud, Emma; Widom, Julia; Sanishvili, Ruslan; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.

    2015-01-01

    Low levels of γ-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the blood-brain barrier and inhibit the activity of γ-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally-restricted, tetrahydrothiophene-based GABA analogs with a properly-positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is eight times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bond interactions with Arg-192, a π-π interaction with Phe-189, and a weak nonbonded S···O=C interaction with Glu-270, thereby inactivating the enzyme. PMID:25781189

  2. Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode.

    PubMed Central

    Blankenfeldt, W.; Nowicki, C.; Montemartini-Kalisz, M.; Kalisz, H. M.; Hecht, H. J.

    1999-01-01

    The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group. PMID:10595543

  3. Structure, expression, and function of kynurenine aminotransferases in human and rodent brains.

    PubMed

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

    2010-02-01

    Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), an endogenous antagonist of N-methyl-D: -aspartate and alpha 7-nicotinic acetylcholine receptors. Abnormal KYNA levels in human brains are implicated in the pathophysiology of schizophrenia, Alzheimer's disease, and other neurological disorders. Four KATs have been reported in mammalian brains, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase. KAT II has a striking tertiary structure in N-terminal part and forms a new subgroup in fold type I aminotransferases, which has been classified as subgroup Iepsilon. Knowledge regarding KATs is vast and complex; therefore, this review is focused on recent important progress of their gene characterization, physiological and biochemical function, and structural properties. The biochemical differences of four KATs, specific enzyme activity assays, and the structural insights into the mechanism of catalysis and inhibition of these enzymes are discussed.

  4. Structure, expression, and function of kynurenine aminotransferases in human and rodent brains

    PubMed Central

    Han, Qian; Cai, Tao; Tagle, Danilo A.

    2010-01-01

    Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), an endogenous antagonist of N-methyl-d-aspartate and alpha 7-nicotinic acetylcholine receptors. Abnormal KYNA levels in human brains are implicated in the pathophysiology of schizophrenia, Alzheimer's disease, and other neurological disorders. Four KATs have been reported in mammalian brains, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase. KAT II has a striking tertiary structure in N-terminal part and forms a new subgroup in fold type I aminotransferases, which has been classified as subgroup Iε. Knowledge regarding KATs is vast and complex; therefore, this review is focused on recent important progress of their gene characterization, physiological and biochemical function, and structural properties. The biochemical differences of four KATs, specific enzyme activity assays, and the structural insights into the mechanism of catalysis and inhibition of these enzymes are discussed. PMID:19826765

  5. Structure Expression and Function of kynurenine Aminotransferases in Human and Rodent Brains

    SciTech Connect

    Q Han; T Cai; D Tagle; J Li

    2011-12-31

    Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), an endogenous antagonist of N-methyl-D: -aspartate and alpha 7-nicotinic acetylcholine receptors. Abnormal KYNA levels in human brains are implicated in the pathophysiology of schizophrenia, Alzheimer's disease, and other neurological disorders. Four KATs have been reported in mammalian brains, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase. KAT II has a striking tertiary structure in N-terminal part and forms a new subgroup in fold type I aminotransferases, which has been classified as subgroup Iepsilon. Knowledge regarding KATs is vast and complex; therefore, this review is focused on recent important progress of their gene characterization, physiological and biochemical function, and structural properties. The biochemical differences of four KATs, specific enzyme activity assays, and the structural insights into the mechanism of catalysis and inhibition of these enzymes are discussed.

  6. The past and present of serum aminotransferases and the future of liver injury biomarkers

    PubMed Central

    McGill, Mitchell R.

    2016-01-01

    Laboratory testing is important in the diagnosis and monitoring of liver injury and disease. Current liver tests include plasma markers of injury (e.g. aminotransferases, γ-glutamyl transferase, and alkaline phosphatase), markers of function (e.g. prothrombin time, bilirubin), viral hepatitis serologies, and markers of proliferation (e.g. α-fetoprotein). Among the injury markers, the alanine and aspartate aminotransferases (ALT and AST, respectively) are the most commonly used. However, interpretation of ALT and AST plasma levels can be complicated. Furthermore, both have poor prognostic utility in acute liver injury and liver failure. New biomarkers of liver injury are rapidly being developed, and the US Food and Drug Administration the European Medicines Agency have recently expressed support for use of some of these biomarkers in drug trials. The purpose of this paper is to review the history of liver biomarkers, to summarize mechanisms and interpretation of ALT and AST elevation in plasma in liver injury (particularly acute liver injury), and to discuss emerging liver injury biomarkers that may complement or even replace ALT and AST in the future. PMID:28337112

  7. Preoperative Aspartate Aminotransferase to White Blood Cell Count Ratio Predicting Postoperative Outcomes of Hepatocellular Carcinoma.

    PubMed

    Liao, Weijia; Wang, Yongqin; Liao, Yan; He, Songqing; Jin, Junfei

    2016-04-01

    Effective biomarkers for predicting prognosis of hepatocellular carcinoma (HCC) patients after hepatectomy is urgently needed. The purpose of this study is to evaluate the value of the preoperative peripheral aspartate aminotransferase to white blood cell count ratio (AWR) for the prognostication of patients with HCC.Clinical data of 396 HCC patients who underwent radical hepatectomy were retrospectively analyzed. The patients were divided into the low-AWR group (AWR ≤5.2) and the high-AWR group (AWR >5.2); univariate analysis, Kaplan-Meier method analysis, and the multivariate analysis by Cox regression were conducted, respectively.The results showed that AWR was associated with alpha-fetoprotein (AFP), tumor size, Barcelona clinic liver cancer (BCLC) stage, portal vein tumor thrombus (PVTT), and alanine aminotransferase (ALT) in HCC. AWR > 5.2, AFP > 100 ng/mL, size of tumor >6 cm, number of multiple tumors, B-C of BCLC stage, PVTT, and distant metastasis were predictors of poorer disease-free survival (DFS) and overall survival (OS). Except for recurrence, which was an independent predictor for OS only, AWR >5.2, size of tumor >6 cm, and PVTT were independent predictors of both DFS and OS.We concluded that preoperative AWR > 5.2 was an adverse predictor of DFS and OS in HCC after hepatectomy, AWR might be a novel prognostic biomarker in HCC after curative resection.

  8. A New Octadecenoic Acid Derivative from Caesalpinia gilliesii Flowers with Potent Hepatoprotective Activity

    PubMed Central

    Osman, Samir M.; El-Haddad, Alaadin E.; El-Raey, Mohamed A.; Abd El-Khalik, Soad M.; Koheil, Mahmoud A.; Wink, Michael

    2016-01-01

    Background: Caesalpinia gilliesii Hook is an ornamental shrub with showy yellow flowers. It was used in folk medicine due to its contents of different classes of secondary metabolites. In our previous study, dichloromethane extract of C. gilliesii flowers showed a good antioxidant activity. Aim of the Study: Isolation and identification of bioactive hepatoprotective compounds from C. gilliesii flowers dichloromethane fraction. Materials and Methods: The hepatoprotective activity of dichloromethane fraction and isolated compounds were studied in CCl4-intoxicated rat liver slices by measuring liver injury markers (alanine aminotransferase, aspartate aminotransferase and glutathione [GSH]). All compounds were structurally elucidated on the basis of electron ionization-mass spectrometry, one- and two-dimensional nuclear magnetic resonance. Results: A new 12,13,16-trihydroxy-14(Z)-octadecenoic acid was identified in addition to the known β-sitosterol-3-O-butyl, daucosterol, isorhamnetin, isorhamnetin-3-O-rhamnoside, luteolin-7,4’-dimethyl ether, genistein-5-methyl ether, luteolin-7-O-rhamnoside, isovanillic acid, and p-methoxybenzoic acid. Dichloromethane fraction and isorhamnetin were able to significantly protect the liver against intoxication. Moreover, the dichloromethane fraction and the isolated phytosterols induced GSH above the normal level. Conclusion: The hepatoprotective activity of C. gilliesii may be attributed to its high content of phytosterols and phenolic compounds. SUMMARY Bioactive Hepatoprotective phytosterols and phenolics from chloroform extract of Caesalpinia gilliesii Abbreviations used: ALT: Alanine Aminotransferase; AST: Aspartate aminotransferase; GSH: Glutathione; SC50: Scavenging Capacity 50 (SC 50); COSY: Correlation spectroscopy; NMR: Nuclear Magnetic Resonance; CC: Column chromatography; EI-MS: Electron-impact mass spectrometry; HSQC: Heteronuclear single-quantum correlation. PMID:27563221

  9. [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.

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

    PubMed

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

    2016-01-01

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

  11. Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

    SciTech Connect

    Mehere, P.; Robinson, H.; Han, Q.; Lemkul, J. A.; Vavricka, C. J.; Bevan, D. R.; Li, J.

    2010-11-01

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  12. Tyrosine Aminotransferase: Biochemical and Structural Properties and Molecular Dynamics Simulations

    SciTech Connect

    P Mehere; Q Han; J Lemkul; C Vavricka; H Robinson; D Bevan; J Li

    2011-12-31

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  13. Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.

    PubMed

    Lee, Eun-Jeong; Facchini, Peter J

    2011-11-01

    Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

  14. Cloning and expression of human tyrosine aminotransferase cDNA.

    PubMed

    Séralini, G E; Luu-Thé, V; Labrie, F

    1995-01-02

    Complementary DNA clones encoding human tyrosine aminotransferase (TAT) were isolated by screening a normal adult woman liver lambda gt11 library with rat TAT cDNA. The largest isolated cDNA is 2051 bp long (EMBL accession number X55675). This cDNA was subcloned downstream of the cytomegalovirus promoter in the pCMV vector for transfection into human cervical carcinoma HeLa cells. Expression of the TAT cDNA resulted in the synthesis of a protein with a molecular mass of approximately 50 kDa, as assessed by Western analysis, a value which is in close agreement with the predicted molecular weight of 50,399, for a deduced sequence of 454 amino acids. The expressed protein catalyzed specifically the conversion of L-[14C]tyrosine into p-[14C]hydroxyphenylpyruvate. The availability of a functional TAT cDNA provides a useful tool for detailed study of the structure-function relationship of the enzyme and its mutated derivatives.

  15. 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

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

    PubMed

    Gong, Yan; Wang, Wen-qing

    2006-01-01

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

  17. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes*

    PubMed Central

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-01-01

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of the kinetic studies. We purified His6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (Km = 0.35 ± 0.03 mm for l-aspartate, Km = 0.098 ± 0 mm for d-aspartate, Km = 26 ± 2 mm for l-alanine, Km = 3.3 ± 0.2 mm for d-alanine). Competitive inhibition by various amino acids of l-aspartate or l-alanine in self-exchange reactions revealed that l-cysteine selectively inhibited l-aspartate self-exchange but only weakly inhibited l-alanine self-exchange. Additionally, l-serine selectively inhibited l-alanine self-exchange but barely inhibited l-aspartate self-exchange. The aspartate analogs l-cysteine sulfinic acid, l-cysteic acid, and d-cysteic acid competitively and strongly inhibited l-aspartate self-exchange compared with l-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of l-aspartate and l-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  18. Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

    PubMed

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-08-19

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.

  19. 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.

  20. Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris).

    PubMed

    Harr, Kendal E; Allison, Kathryn; Bonde, Robert K; Murphy, David; Harvey, John W

    2008-06-01

    Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement

  1. Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

    SciTech Connect

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

    2009-01-01

    Kynurenine aminotransferase III (KAT III) has been considered to be involved in the production of mammalian brain kynurenic acid (KYNA), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. The enzyme was identified based on its high sequence identity with mammalian KAT I, but its activity toward kynurenine and its structural characteristics have not been established. In this study, the biochemical and structural properties of mouse KAT III (mKAT III) were determined. Specifically, mKAT III cDNA was amplified from a mouse brain cDNA library, and its recombinant protein was expressed in an insect cell protein expression system. We established that mKAT III is able to efficiently catalyze the transamination of kynurenine to KYNA and has optimum activity at relatively basic conditions of around pH 9.0 and at relatively high temperatures of 50 to 60C. In addition, mKAT III is active toward a number of other amino acids. Its activity toward kynurenine is significantly decreased in the presence of methionine, histidine, glutamine, leucine, cysteine, and 3-hydroxykynurenine. Through macromolecular crystallography, we determined the mKAT III crystal structure and its structures in complex with kynurenine and glutamine. Structural analysis revealed the overall architecture of mKAT III and its cofactor binding site and active center residues. This is the first report concerning the biochemical characteristics and crystal structures of KAT III enzymes and provides a basis toward understanding the overall physiological role of mammalian KAT III in vivo and insight into regulating the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  2. Biochemical and structural properties of mouse kynurenine aminotransferase III.

    PubMed

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

    2009-02-01

    Kynurenine aminotransferase III (KAT III) has been considered to be involved in the production of mammalian brain kynurenic acid (KYNA), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. The enzyme was identified based on its high sequence identity with mammalian KAT I, but its activity toward kynurenine and its structural characteristics have not been established. In this study, the biochemical and structural properties of mouse KAT III (mKAT III) were determined. Specifically, mKAT III cDNA was amplified from a mouse brain cDNA library, and its recombinant protein was expressed in an insect cell protein expression system. We established that mKAT III is able to efficiently catalyze the transamination of kynurenine to KYNA and has optimum activity at relatively basic conditions of around pH 9.0 and at relatively high temperatures of 50 to 60 degrees C. In addition, mKAT III is active toward a number of other amino acids. Its activity toward kynurenine is significantly decreased in the presence of methionine, histidine, glutamine, leucine, cysteine, and 3-hydroxykynurenine. Through macromolecular crystallography, we determined the mKAT III crystal structure and its structures in complex with kynurenine and glutamine. Structural analysis revealed the overall architecture of mKAT III and its cofactor binding site and active center residues. This is the first report concerning the biochemical characteristics and crystal structures of KAT III enzymes and provides a basis toward understanding the overall physiological role of mammalian KAT III in vivo and insight into regulating the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  3. Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III▿

    PubMed Central

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

    2009-01-01

    Kynurenine aminotransferase III (KAT III) has been considered to be involved in the production of mammalian brain kynurenic acid (KYNA), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. The enzyme was identified based on its high sequence identity with mammalian KAT I, but its activity toward kynurenine and its structural characteristics have not been established. In this study, the biochemical and structural properties of mouse KAT III (mKAT III) were determined. Specifically, mKAT III cDNA was amplified from a mouse brain cDNA library, and its recombinant protein was expressed in an insect cell protein expression system. We established that mKAT III is able to efficiently catalyze the transamination of kynurenine to KYNA and has optimum activity at relatively basic conditions of around pH 9.0 and at relatively high temperatures of 50 to 60°C. In addition, mKAT III is active toward a number of other amino acids. Its activity toward kynurenine is significantly decreased in the presence of methionine, histidine, glutamine, leucine, cysteine, and 3-hydroxykynurenine. Through macromolecular crystallography, we determined the mKAT III crystal structure and its structures in complex with kynurenine and glutamine. Structural analysis revealed the overall architecture of mKAT III and its cofactor binding site and active center residues. This is the first report concerning the biochemical characteristics and crystal structures of KAT III enzymes and provides a basis toward understanding the overall physiological role of mammalian KAT III in vivo and insight into regulating the levels of endogenous KYNA through modulation of the enzyme in the mouse brain. PMID:19029248

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

    NASA Astrophysics Data System (ADS)

    Chatterjee, Subhojyoti; Ahmed, Marawan; Wang, Feng

    2016-02-01

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

  5. 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.

  6. Protective effect of L-ascorbic acid against oxidative damage in the liver of rats with water-immersion restraint stress.

    PubMed

    Kaida, Shingo; Ohta, Yoshiji; Imai, Yoichiro; Kawanishi, Minoru

    2010-01-01

    We examined whether L-ascorbic acid (AA) (or reduced ascorbic acid) protects against oxidative damage in the liver of rats subjected to water-immersion stress (WIRS). AA (100, 250 or 500 mg/kg) was orally administered at 0.5 h before the onset of WIRS. Rats with 6 h of WIRS had increased serum corticosterone, glucose, total ascorbic acid (T-AA), AA, lipid peroxide (LPO), and NOx concentrations and alanine aminotransferase and aspartate aminotrasferase activities. The stressed rats had increased hepatic LPO, NOx, and dehydroascorbic acid concentrations and myeloperoxidase activity, decreased hepatic T-AA, AA, reduced glutathione concentrations and superoxide dismutase activity, and unchanged hepatic vitamin E concentration. Pre-administered AA attenuated the stress-induced changes in serum LPO and NOx concentrations and alanine aminotransferase and aspartate aminotrasferase activities and hepatic LPO, NOx, and T-AA, AA, dehydroascorbic acid, and reduced glutathione concentrations and myeloperoxidase and superoxide dismutase activities dose-dependently. Pre-administered AA did not affect the stress-induced changes in serum corticosterone and glucose concentrations. These results indicate that pre-administered AA protects against oxidative damage in the liver of rats with WIRS possibly by attenuating disruption of the antioxidant defense system and increases in NO generation and neutrophil infiltration in the tissue.

  7. Deciphering the role of aspartate and prephenate aminotransferase activities in plastid nitrogen metabolism.

    PubMed

    de la Torre, Fernando; El-Azaz, Jorge; Avila, Concepción; Cánovas, Francisco M

    2014-01-01

    Chloroplasts and plastids of nonphotosynthetic plant cells contain two aspartate (Asp) aminotransferases: a eukaryotic type (Asp5) and a prokaryotic-type bifunctional enzyme displaying Asp and prephenate aminotransferase activities (PAT). We have identified the entire Asp aminotransferase gene family in Nicotiana benthamiana and isolated and cloned the genes encoding the isoenzymes with plastidic localization: NbAsp5 and NbPAT. Using a virus-induced gene silencing approach, we obtained N. benthamiana plants silenced for NbAsp5 and/or NbPAT. Phenotypic and metabolic analyses were conducted in silenced plants to investigate the specific roles of these enzymes in the biosynthesis of essential amino acids within the plastid. The NbAsp5 silenced plants had no changes in phenotype, exhibiting similar levels of free Asp and glutamate as control plants, but contained diminished levels of asparagine and much higher levels of lysine. In contrast, the suppression of NbPAT led to a severe reduction in growth and strong chlorosis symptoms. NbPAT silenced plants exhibited extremely reduced levels of asparagine and were greatly affected in their phenylalanine metabolism and lignin deposition. Furthermore, NbPAT suppression triggered a transcriptional reprogramming in plastid nitrogen metabolism. Taken together, our results indicate that NbPAT has an overlapping role with NbAsp5 in the biosynthesis of Asp and a key role in the production of phenylalanine for the biosynthesis of phenylpropanoids. The analysis of NbAsp5/NbPAT cosilenced plants highlights the central role of both plastidic aminotransferases in nitrogen metabolism; however, only NbPAT is essential for plant growth and development.

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

    SciTech Connect

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

    1988-09-01

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

  9. 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

  10. Structure of putrescine aminotransferase from Escherichia coli provides insights into the substrate specificity among class III aminotransferases.

    PubMed

    Cha, Hyung Jin; Jeong, Jae-Hee; Rojviriya, Catleya; Kim, Yeon-Gil

    2014-01-01

    YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

  11. METHANOGENS WITH PSEUDOMUREIN USE DIAMINOPIMELATE AMINOTRANSFERASE IN LYSINE BIOSYNTHESIS

    PubMed Central

    Graham, David E.; Huse, Holly K.

    2008-01-01

    Methanothermobacter thermautotrophicus uses lysine for both protein synthesis and cross-linking pseudomurein in its cell wall. A diaminopimelate aminotransferase enzyme from this methanogen (MTH0052) converts tetrahydrodipicolinate to L,L-diaminopimelate, a lysine precursor. This gene complemented an Escherichia coli diaminopimelate auxotrophy, and the purified protein catalyzed the transamination of diaminopimelate to tetrahydrodipicolinate. Phylogenetic analysis indicated this gene was recruited from anaerobic Gram-positive bacteria. These results expand the family of diaminopimelate aminotransferases to a diverse set of plant, bacterial and archaeal homologs. In contrast marine methanogens from the Methanococcales, which lack pseudomurein, appear to use a different diaminopimelate pathway for lysine biosynthesis. PMID:18371309

  12. 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.

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

    PubMed Central

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

    2013-01-01

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

  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. 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.

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

    PubMed

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

    2017-05-01

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

  17. Chlorogenic and caftaric acids in liver toxicity and oxidative stress induced by methamphetamine.

    PubMed

    Koriem, Khaled M M; Soliman, Rowan E

    2014-01-01

    Methamphetamine intoxication can cause acute hepatic failure. Chlorogenic and caftaric acids are the major dietary polyphenols present in various foods. The aim of this study was to evaluate the protective role of chlorogenic and caftaric acids in liver toxicity and oxidative stress induced by methamphetamine in rats. Thirty-two male albino rats were divided into 4 equal groups. Group 1, which was control group, was injected (i.p) with saline (1 mL/kg) twice a day over seven-day period. Groups 2, 3, and 4 were injected (i.p) with methamphetamine (10 mg/kg) twice a day over seven-day period, where groups 3 and 4 were injected (i.p) with 60 mg/kg chlorogenic acid and 40 mg/kg caftaric acid, respectively, one day before methamphetamine injections. Methamphetamine increased serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, bilirubin, cholesterol, low-density lipoprotein, and triglycerides. Also, malondialdehyde in serum, liver, and brain and plasma and liver nitric oxide levels were increased while methamphetamine induced a significant decrease in serum total protein, albumin, globulin, albumin/globulin ratio, brain serotonin, norepinephrine and dopamine, blood and liver superoxide dismutase, and glutathione peroxidase levels. Chlorogenic and caftaric acids prior to methamphetamine injections restored all the above parameters to normal values. In conclusion, chlorogenic and caftaric acids before methamphetamine injections prevented liver toxicity and oxidative stress where chlorogenic acid was more effective.

  18. Protective effect of boric acid against carbon tetrachloride-induced hepatotoxicity in mice.

    PubMed

    Ince, Sinan; Keles, Hikmet; Erdogan, Metin; Hazman, Omer; Kucukkurt, Ismail

    2012-07-01

    The protective effect of boric acid against liver damage was evaluated by its attenuation of carbon tetrachloride (CCl(4))-induced hepatotoxicity in mice. Male albino mice were treated intraperitoneally (i.p.) with boric acid (50, 100, and 200 mg/kg) or silymarin daily for 7 days and received 0.2% CCl(4) in olive oil (10 mL/kg, i.p.) on day 7. Results showed that administration of boric acid significantly reduced the elevation in serum levels of aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase, and the level of malondialdehyde in the liver that were induced by CCl(4) in mice. Boric acid treatment significantly increased glutathione content, as well as the activities of superoxide dismutase and catalase in the liver. Boric acid treatment improved the catalytic activity of cytochrome P450 2E1 and maintained activation of nuclear factor kappa light-chain enhancer of activated B cell gene expression, with no effect on inducible nitric oxide synthase gene expression in the livers of mice. Histopathologically, clear decreases in the severity of CCl(4)-induced lesions were observed, particularly at high boric acid concentrations. Results suggest that boric acid exhibits potent hepatoprotective effects on CCl(4)-induced liver damage in mice, likely the result of both the increase in antioxidant-defense system activity and the inhibition of lipid peroxidation.

  19. 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.

  20. Interactions of L-alanine with alumina as studied by vibrational spectroscopy.

    PubMed

    Garcia, Ana R; de Barros, Ricardo Brito; Fidalgo, Alexandra; Ilharco, Laura M

    2007-09-25

    The interactions of L-alanine with gamma- and alpha-alumina have been investigated by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). L-alanine/alumina samples were dried from aqueous suspensions, at 36.5 degrees C, with two amino acid concentrations (0.4 and 0.8 mmol g-1) and at different pH values (1, 6, and 13). The vibrational spectra proved that the nature of L-alanine interactions with both aluminas is the same (hydrogen bonding), although the groups involved depend on the L-alanine form and on alumina surface groups, both controlled by the pH. For samples prepared at pH 1, cationic L-alanine [CH3CH(NH3+)COOH] displaces physisorbed water from alumina, and strong hydrogen bonds are established between the carbonyl groups of alanine, as electron donors, and the surface Al-OH2+ groups of alumina. This occurs at the expense of alanine dimer dissociation and breaking of intramolecular bonds. When samples are prepared at pH 6, the interacting groups are Al-OH2+ and the carboxylate groups of zwitterionic L-alanine [CH3CH(NH3+)COO-]. The affinity of L-alanine toward alumina decreases, as the strong NH3+...-OOC intermolecular hydrogen bonds prevail over the interactions with alumina. Thus, for a load of 0.8 mmol g-1, phase segregation is observed. On alpha-alumina, crystal deposition is even observed for a load of 0.4 mmol g-1. At pH 13, the carboxylate groups of anionic L-alanine [CH3CH(NH2)COO-] are not affected by alumina. Instead, hydrogen bond interactions occur between NH2 and the Al-OH surface groups of the substrate. Complementary N2 adsorption-desorption isotherms showed that adsorption of L-alanine occurs onto the alumina pore network for samples prepared at pH 1 and 13, whereas at pH 6 the amino acid/alumina interactions are not strong enough to promote adsorption. The mesoporous structure and the high specific surface area of gamma-alumina make it a more efficient substrate for adsorption of L-alanine. For each alumina, however, it is

  1. Thermal stability, pH dependence and inhibition of four murine kynurenine aminotransferases

    PubMed Central

    2010-01-01

    Background Kynurenine aminotransferase (KAT) catalyzes the transamination of kynunrenine to kynurenic acid (KYNA). KYNA is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of N-methyl-D-aspartate receptors. Four KAT enzymes, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase, have been reported in mammalian brains. Because of the substrate overlap of the four KAT enzymes, it is difficult to assay the specific activity of each KAT in animal brains. Results This study concerns the functional expression and comparative characterization of KAT I, II, III, and IV from mice. At the applied test conditions, equimolar tryptophan with kynurenine significantly inhibited only mouse KAT I and IV, equimolar methionine inhibited only mouse KAT III and equimolar aspartate inhibited only mouse KAT IV. The activity of mouse KAT II was not significantly inhibited by any proteinogenic amino acids at equimolar concentrations. pH optima, temperature preferences of four KATs were also tested in this study. Midpoint temperatures of the protein melting, half life values at 65°C, and pKa values of mouse KAT I, II, III, and IV were 69.8, 65.9, 64.8 and 66.5°C; 69.7, 27.4, 3.9 and 6.5 min; pH 7.6, 5.7, 8.7 and 6.9, respectively. Conclusion The characteristics reported here could be used to develop specific assay methods for each of the four murine KATs. These specific assays could be used to identify which KAT is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of KAT-involved human diseases. PMID:20482848

  2. 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.

  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 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.

  5. Effect of abomasal glucose infusion on alanine metabolism and urea production in sheep.

    PubMed

    Obitsu, T; Bremner, D; Milne, E; Lobley, G E

    2000-08-01

    The effect of abomasal infusion of glucose (120 kJ/d per kg body weight (BW)0.75, 758 mmol/d) on urea production, plasma alanine-N flux rate and the conversion of alanine-N to urea was studied in sheep offered a low-N diet at limited energy intake (500 kJ/d per kg BW0.75), based on hay and grass pellets. Glucose provision reduced urinary N (P = 0.040) and urea (P = 0.009) elimination but this was offset by poorer N digestibility. Urea-N production was significantly reduced (822 v. 619 mmol/d, P = 0.024) by glucose while plasma alanine-N flux rate was elevated (295 v. 342 mmol/d, P = 0.011). The quantity of urea-N derived from alanine tended to be decreased by glucose (127 v. 95 mmol/d) but the fraction of urea production from alanine was unaltered (15%). Plasma urea and alanine concentrations (plus those of the branched chain amino acids) decreased in response to exogenous glucose, an effect probably related to enhanced anabolic usage of amino acids and lowered urea production.

  6. Histidine degradation via an aminotransferase increases the nutritional flexibility of Candida glabrata.

    PubMed

    Brunke, Sascha; Seider, Katja; Richter, Martin Ernst; Bremer-Streck, Sibylle; Ramachandra, Shruthi; Kiehntopf, Michael; Brock, Matthias; Hube, Bernhard

    2014-06-01

    The ability to acquire nutrients during infections is an important attribute in microbial pathogenesis. Amino acids are a valuable source of nitrogen if they can be degraded by the infecting organism. In this work, we analyzed histidine utilization in the fungal pathogen of humans Candida glabrata. Hemiascomycete fungi, like C. glabrata or Saccharomyces cerevisiae, possess no gene coding for a histidine ammonia-lyase, which catalyzes the first step of a major histidine degradation pathway in most other organisms. We show that C. glabrata instead initializes histidine degradation via the aromatic amino acid aminotransferase Aro8. Although ARO8 is also present in S. cerevisiae and is induced by extracellular histidine, the yeast cannot use histidine as its sole nitrogen source, possibly due to growth inhibition by a downstream degradation product. Furthermore, C. glabrata relies only on Aro8 for phenylalanine and tryptophan utilization, since ARO8, but not its homologue ARO9, was transcriptionally activated in the presence of these amino acids. Accordingly, an ARO9 deletion had no effect on growth with aromatic amino acids. In contrast, in S. cerevisiae, ARO9 is strongly induced by tryptophan and is known to support growth on aromatic amino acids. Differences in the genomic structure of the ARO9 gene between C. glabrata and S. cerevisiae indicate a possible disruption in the regulatory upstream region. Thus, we show that, in contrast to S. cerevisiae, C. glabrata has adapted to use histidine as a sole source of nitrogen and that the aromatic amino acid aminotransferase Aro8, but not Aro9, is the enzyme required for this process.

  7. Ultraviolet radiation induces stress in etiolated Landoltia punctata, as evidenced by the presence of alanine, a universal stress signal: a ¹⁵N NMR study.

    PubMed

    Monselise, E B-I; Levkovitz, A; Kost, D

    2015-01-01

    Analysis with (15) N NMR revealed that alanine, a universal cellular stress signal, accumulates in etiolated duckweed plants exposed to 15-min pulsed UV light, but not in the absence of UV irradiation. The addition of 10 mm vitamin C, a radical scavenger, reduced alanine levels to zero, indicating the involvement of free radicals. Free D-alanine was detected in (15) N NMR analysis of the chiral amino acid content, using D-tartaric acid as solvent. The accumulation of D-alanine under stress conditions presents a new perspective on the biochemical processes taking place in prokaryote and eukaryote cells.

  8. Glucose and Alanine Metabolism in Children with Maple Syrup Urine Disease

    PubMed Central

    Haymond, Morey W.; Ben-Galim, Ehud; Strobel, Karen E.

    1978-01-01

    In vitro studies have suggested that catabolism of branched chain amino acids is linked with alanine and glutamine formed in, and released from, muscle. To explore this possibility in vivo, static and kinetic studies were performed in three patients with classical, and one patient with partial, branched chain α-ketoacid decarboxylase deficiency (maple syrup urine disease, MSUD) and compared to similar studies in eight age-matched controls. The subjects underwent a 24-30-h fast, and a glucose-alanine flux study using stable isotopes. Basal plasma leucine concentrations were elevated (P <0.001) in patients with MSUD (1,140±125 μM vs. 155±18 μM in controls); and in contrast to the controls, branched chain amino acid concentrations in plasma increased during the fast in the MSUD patients. Basal plasma alanine concentrations were lower (P <0.01) in patients with classical MSUD (153±8 μM vs. 495±27 μM in controls). This discrepancy was maintained throughout the fast despite a decrease in alanine concentrations in both groups. Plasma alanine and leucine concentrations in the patient with partial MSUD were intermediate between those of the controls and the subjects with the classical form of the disease. Circulating ketone bodies and glucoregulatory hormones concentrations were similar in the MSUD and normal subjects during the fast. Alanine flux rates in two patients with classical MSUD (3.76 and 4.00 μmol/Kg per min) and the patient with partial MSUD (5.76 μmol/Kg per min) were clearly lower than those of the controls (11.72±2.53 [SD] μmol/Kg per min). After short-term starvation, glucose flux and fasting concentrations were similar in the MSUD patients and normal subjects. These data indicate that branched chain amino acid catabolism is an important rate limiting event for alanine production in vivo. PMID:670400

  9. 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.

  10. Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris)

    USGS Publications Warehouse

    Harr, K.E.; Allison, K.; Bonde, R.K.; Murphy, D.; Harvey, J.W.

    2008-01-01

    Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate amino-transferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 ??mol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement

  11. Hepatoprotective effects of pantothenic acid on carbon tetrachloride-induced toxicity in rats

    PubMed Central

    Eidi, Akram; Mortazavi, Pejman; Tehrani, Masoud Ebrahim; Rohani, Ali Haeri; Safi, Shahabaldin

    2012-01-01

    The present study was undertaken to investigate the hepatoprotective effect of pantothenic acid on CCl4-induced liver damage. Male Wistar rats were orally treated with pantothenic acid (0.005, 0.01, 0.025, 0.05 and 0.1 g/kg) daily, with administration of CCl4 (1 mL/kg, 50 % CCl4 in olive oil) twice a week for 28 days. The effect of pantothenic acid on serum markers (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyltransferase) was measured in CCl4-induced hepatotoxicity in rat. Further, the effects on enzymatic antioxidant (superoxide dismutase) were estimated in the liver samples. CCl4 challenge not only elevated the serum marker enzyme activities but also suppressed hepatic antioxidative defense system including superoxide dismutase. The biochemical observations were supplemented with histopathological examination of rat liver sections. Histopathological examination of livers showed that pantothenic acid reduced fatty degeneration, cytoplasmic vacuolization and necrosis in CCl4-treated rats. Therefore, pantothenic acid may be an effective hepatoprotective agent and viable candidate for treating hepatic disorders and other oxidative stress-related diseases. PMID:27847457

  12. 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

  13. Intrahepatic Cholestasis of Pregnancy with Severe Elevation of Bile Acids in the Setting of Acute Hepatitis C Infection

    PubMed Central

    Critchfield, Agatha S.

    2016-01-01

    Intrahepatic cholestasis of pregnancy (ICP) is a complication of pregnancy resulting in elevation of serum bile acid levels. ICP is often associated with underlying liver disease, including hepatitis C. Bile acids in relationship to the acute infection of hepatitis C virus have not yet been delineated in the literature. A 26-year-old gravida 4 para 2103 with dichorionic, diamniotic twin gestation and history of intravenous drug abuse developed ICP in the setting of acute hepatitis C infection. In addition to clinical symptoms of pruritus and right upper quadrant pain, she developed severe elevation in bile acids, 239 micromol/L, and transaminitis aspartate aminotransferase 1033 U/L, and alanine aminotransferase 448 U/L. She received ursodeoxycholic acid and antenatal testing was performed. Patient delivered vaginally at 33-week gestation following preterm rupture of membranes. Neonates were admitted to NICU and had uncomplicated neonatal courses. In the setting of ICP with significant transaminitis and severe elevation of bile acids, consideration of acute viral hepatitis is important, especially considering the worsening opioid epidemic and concurrent increase in intravenous drug use in the United States. Further study is needed regarding the acute form of HCV infection and its effect on ICP and associated bile acids. PMID:27891271

  14. Computation of energy interaction parameters as well as electric dipole intensity parameters for the absorption spectral study of the interaction of Pr(III) with L-phenylalanine, L-glycine, L-alanine and L-aspartic acid in the presence and absence of Ca 2+ in organic solvents

    NASA Astrophysics Data System (ADS)

    Moaienla, T.; Singh, Th. David; Singh, N. Rajmuhon; Devi, M. Indira

    2009-10-01

    Studying the absorption difference and comparative absorption spectra of the interaction of Pr(III) and Nd(III) with L-phenylalanine, L-glycine, L-alanine and L-aspartic acid in the presence and absence of Ca 2+ in organic solvents, various energy interaction parameters like Slater-Condon ( FK), Racah ( Ek), Lande factor ( ξ4f), nephelauxetic ratio ( β), bonding ( b1/2), percentage-covalency ( δ) have been evaluated applying partial and multiple regression analysis. The values of oscillator strength ( P) and Judd-Ofelt electric dipole intensity parameter Tλ ( λ = 2, 4, 6) for different 4f-4f transitions have been computed. On analysis of the variation of the various energy interaction parameters as well as the changes in the oscillator strength ( P) and Tλ values reveal the mode of binding with different ligands.

  15. Effects of endogenous D-alanine synthesis and autoinhibition of Bacillus anthracis germination on in vitro and in vivo infections.

    PubMed

    McKevitt, Matthew T; Bryant, Katie M; Shakir, Salika M; Larabee, Jason L; Blanke, Steven R; Lovchik, Julie; Lyons, C Rick; Ballard, Jimmy D

    2007-12-01

    Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of D-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.

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

    PubMed

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

    2016-12-16

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

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. Effects of slightly acidic electrolysed drinking water on mice.

    PubMed

    Inagaki, Hideaki; Shibata, Yoshiko; Obata, Takahiro; Kawagoe, Masami; Ikeda, Katsuhisa; Sato, Masayoshi; Toida, Kazumi; Kushima, Hidemi; Matsuda, Yukihisa

    2011-10-01

    Slightly acidic electrolysed (SAE) water is a sanitizer with strong bactericidal activity due to hypochlorous acid. We assessed the safety of SAE water as drinking water for mice at a 5 ppm total residual chlorine (TRC) concentration to examine the possibility of SAE water as a labour- and energy-saving alternative to sterile water. We provided SAE water or sterile water to mice for 12 weeks, during which time we recorded changes in body weight and weekly water and food intakes. At the end of the experiment, all of the subject animals were sacrificed to assess serum aspartate aminotransferase, alanine aminotransferase and creatinine levels and to examine the main organs histopathologically under a light microscope. In addition, we investigated the bacteria levels of both types of water. We found no difference in functional and morphological health condition indices between the groups. Compared with sterile water, SAE water had a relatively higher ability to suppress bacterial growth. We suggest that SAE water at 5 ppm TRC is a safe and useful alternative to sterile water for use as drinking water in laboratory animal facilities.

  3. Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia camphorata in a mouse model of acute hepatic injury.

    PubMed

    Huang, Guan-Jhong; Deng, Jeng-Shyan; Huang, Shyh-Shyun; Lee, Chao-Ying; Hou, Wen-Chi; Wang, Sheng-Yang; Sung, Ping-Jyun; Kuo, Yueh-Hsiung

    2013-12-01

    The hepatoprotective effects of eburicoic acid (TR1) and dehydroeburicoic acid (TR2) from Antrodia camphorata (AC) against carbon tetrachloride (CCl4)-induced liver damage were investigated in mice. TR1 and TR2 was administered intraperitoneally (i.p.) for 7 days prior to the administration of CCl4. Pretreatment with TR1 and TR2 prevented the elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and liver lipid peroxides in CCl4-treated mice. The activities of antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)], nitric oxide (NO) production, and tumour necrosis factor-alpha (TNF-α) were decreased after the treatment with TR1 and TR2 in CCl4-treated mice. Western blotting revealed that TR1 and TR2 significantly decreased inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions and increased the expression of cytochrome P4502E1 (CYP2E1) in CCl4-treated mice. Therefore, we speculate that TR1 and TR2 protect the liver from CCl4-induced hepatic damage via antioxidant and anti-inflammatory mechanisms.

  4. 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.

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

    PubMed

    Schmieden, V; Kuhse, J; Betz, H

    1993-10-08

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

  6. Isotope labeling studies on the formation of multiple addition products of alanine in the pyrolysis residue of glucose/alanine mixtures by high-resolution ESI-TOF-MS.

    PubMed

    Chu, Fong Lam; Sleno, Lekha; Yaylayan, Varoujan A

    2011-11-09

    Pyrolysis was used as a microscale sample preparation tool to generate glucose/alanine reaction products to minimize the use of expensive labeled precursors in isotope labeling studies. The residue remaining after the pyrolysis at 250 °C was analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). It was observed that a peak at m/z 199.1445 in the ESI-TOF-MS spectrum appeared only when the model system contained at least 2-fold excess alanine. The accurate mass determination indeed indicated the presence of two nitrogen atoms in the molecular formula (C(10)H(18)N(2)O(2)). To verify the origin of the carbon atoms in this unknown compound, model studies with [(13)U(6)]glucose, [(13)C-1]alanine, [(13)C-2]alanine, [(13)C-3]alanine, and [(15)N]alanine were also performed. Glucose furnished six carbon atoms, and alanine provides four carbon (2 × C-2 and 2 × C-3) and two nitrogen atoms. When commercially available fructosylalanine (N-attached to C-1) was reacted with only 1 mol of alanine, a peak at m/z 199.1445 was once again observed. In addition, when 3-deoxyglucosone (3-DG) was reacted with a 2-fold excess of alanine, a peak at m/z 199.1433 was also generated, confirming the points of attachment of the two amino acids at C-1 and C-2 atoms of 3-DG. These studies have indicated that amino acids can undergo multiple addition reactions with 1,2-dicarbonyl compounds such as 3-deoxyglucosone and eventually form a tetrahydropyrazine moiety.

  7. Biocatalytic potential of vanillin aminotransferase from Capsicum chinense

    PubMed Central

    2014-01-01

    Background The conversion of vanillin to vanillylamine is a key step in the biosynthetic route towards capsaicinoids in pungent cultivars of Capsicum sp. The reaction has previously been annotated to be catalysed by PAMT (putative aminotransferase; [GenBank: AAC78480.1, Swiss-Prot: O82521]), however, the enzyme has previously not been biochemically characterised in vitro. Results The biochemical activity of the transaminase was confirmed by direct measurement of the reaction with purified recombinant enzyme. The enzyme accepted pyruvate, and oxaloacetate but not 2-oxoglutarate as co-substrate, which is in accordance with other characterised transaminases from the plant kingdom. The enzyme was also able to convert (S)-1-phenylethylamine into acetophenone with high stereo-selectivity. Additionally, it was shown to be active at a broad pH range. Conclusions We suggest PAMT to be renamed to VAMT (vanillin aminotransferase, abbreviation used in this study) as formation of vanillin from vanillylamine could be demonstrated. Furthermore, due to high stereoselectivity and activity at physiological pH, VAMT is a suitable candidate for biocatalytic transamination in a recombinant whole-cell system. PMID:24712445

  8. Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte-neuron co-cultures.

    PubMed

    Dadsetan, Sherry; Kukolj, Eva; Bak, Lasse K; Sørensen, Michael; Ott, Peter; Vilstrup, Hendrik; Schousboe, Arne; Keiding, Susanne; Waagepetersen, Helle S

    2013-08-01

    Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of (15)NH4(+) in alanine during acute hyperammonemia. We observed a fourfold increased amount of (15)NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to (15)NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of (15)NH4 into alanine together with increased (15)N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy.

  9. The hepatoprotection of caffeic acid and rosmarinic acid, major compounds of Perilla frutescens, against t-BHP-induced oxidative liver damage.

    PubMed

    Yang, Sung-Yong; Hong, Chung-Oui; Lee, Gung Pyo; Kim, Cheong-Tae; Lee, Kwang-Won

    2013-05-01

    Perilla frutescens leaves are often used in East Asian gourmet food. In this study, we investigated the hepatoprotective effects of caffeic acid (CA), rosmarinic acid (RA), and their combination. P. frutescens contains 1.32μg CA/mg dry material (DM) and 26.84μg RA/mg DM analyzed by HPLC-DAD and HPLC-MS. CA remarkably reduced the oxidative damage than rosmarinic acid in an in vitro study. Oral intubation with CA or RA alone for five days was conducted prior to treatment with a single dose of tert-butyl hydroperoxide (0.5mmol/kg b.w., i.p.), which led to a significant reduction of indicators of hepatic toxicity, such as aspartate aminotransferase, alanine aminotransferase, oxidized glutathione, lipid peroxidation and enzyme activities related to antioxidant such as catalase, glutathione peroxidase and superoxide dismutase. Interestingly, compared to treatment with CA or RA alone, a combination of both compounds more increased the endogenous antioxidant enzymes and glutathione (GSH) and decreased lipid peroxidation in livers. These results suggest that CA from perilla leaves plays a role in the increased hepatic GSH concentration, and shows an additive hepatic protection with RA against oxidative hepatic damage.

  10. DL-canaline and 5-fluoromethylornithine. Comparison of two inactivators of ornithine aminotransferase.

    PubMed Central

    Bolkenius, F N; Knödgen, B; Seiler, N

    1990-01-01

    5-Fluoromethylornithine (5FMOrn) is an enzyme-activated irreversible inhibitor or ornithine aminotransferase (L-ornithine:2-oxo-acid 5-aminotransferase, OAT). For purified rat liver OAT, Ki(app.) was found to be 30 microM. and tau 1/2 = 4 min. Of the four stereomers of 5FMOrn only one reacts with OAT. The formation of a chromophore with an absorption maximum at 458 nm after inactivation of OAT by 5FMOrn suggests the formation of an enamine intermediate, which is slowly hydrolysed to release an unsaturated ketone. L-Canaline [(S)-2-amino-4-amino-oxybutyric acid] is a well-known irreversible inhibitor of OAT. Not only the natural L-enantiomer but also the D-enantiomer reacts by oxime formation with pyridoxal 5'-phosphate in the active site of the enzyme, although considerably more slowly. This demonstrates that the stereochemistry at C-2 of ornithine is not absolutely stringent. In vitro, canaline reacted faster than 5FMOrn with OAT. In vivo, however, only incomplete OAT inhibition was observed with canaline. Whereas intraperitoneal administration of 10 mg of 5FMOrn/kg body wt. to mice was sufficient to inactivate OAT in brain and liver by 90% for 24 h, 500 mg of DL-canaline/kg body wt. only produced a transient inhibition of 65-70%. The accumulation of ornithine in these tissues was considerably slower and the maximum concentrations lower than were achieved with 5FMOrn. It appears that DL-canaline, in contrast with 5FMOrn, is not useful as a tool in studies of biological consequences of OAT inhibition. PMID:2363680

  11. Phenylalanine-pyruvate aminotransferase activity in chicks subjected to phenylalanine imbalance or phenylalanine toxicity.

    PubMed

    Lu, J; Austic, R E

    2009-11-01

    Two experiments were done to determine the influence of Phe imbalance and excess on Phe-pyruvate aminotransferase (PAT) activity in the chick. Five replicates of 3 chicks (experiment 1) or 2 chicks (experiment 2) of a commercial brown egg layer strain were fed a semipurified diet for 1 wk and then received experimental diets for 10 d. Three diets were used in experiment 1: the basal diet contained 0.46% Phe; the imbalance diet was similar to the basal diet except that it contained a 10% mixture of indispensable amino acids lacking Phe (IAA - Phe) to create a Phe imbalance; the imbalance corrected diet was similar to the imbalance diet except that it was supplemented with 1.12% Phe to correct the imbalance. A 3 x 2 factorial arrangement of treatments in experiment 2 provided 3 dietary levels (0.46, 1.58, and 2.46%) of Phe and either no supplement or 10% supplement of IAA - Phe. Nonfasted chicks were killed and livers were sampled in experiment 1, and livers, kidneys, brains, and pectoralis major muscles were sampled in experiment 2. In experiment 1, liver PAT activity per gram of liver was 80 and 55% higher (P < 0.01) in chicks fed the imbalance and imbalance corrected diets than in chicks fed the basal diet. In experiment 2, the livers and kidneys, but not brains and muscles, of chicks that received the 10% supplement of IAA - Phe had higher activities of PAT per gram of tissue per minute and per milligram of tissue protein extract per minute than chicks that did not receive IAA - Phe (P < 0.001). No effect of dietary Phe on PAT activity was detected (P > 0.05). Phenylalanine-pyruvate aminotransferase activity appears to be regulated in response to dietary content of indispensable amino acids but not by the dietary level of Phe.

  12. 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...

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

    NASA Astrophysics Data System (ADS)

    Srinivasan, Bikshandarkoil R.

    2013-12-01

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

  14. 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.

  15. Deciphering the Role of Aspartate and Prephenate Aminotransferase Activities in Plastid Nitrogen Metabolism1[C][W][OPEN

    PubMed Central

    de la Torre, Fernando; El-Azaz, Jorge; Ávila, Concepción; Cánovas, Francisco M.

    2014-01-01

    Chloroplasts and plastids of nonphotosynthetic plant cells contain two aspartate (Asp) aminotransferases: a eukaryotic type (Asp5) and a prokaryotic-type bifunctional enzyme displaying Asp and prephenate aminotransferase activities (PAT). We have identified the entire Asp aminotransferase gene family in Nicotiana benthamiana and isolated and cloned the genes encoding the isoenzymes with plastidic localization: NbAsp5 and NbPAT. Using a virus-induced gene silencing approach, we obtained N. benthamiana plants silenced for NbAsp5 and/or NbPAT. Phenotypic and metabolic analyses were conducted in silenced plants to investigate the specific roles of these enzymes in the biosynthesis of essential amino acids within the plastid. The NbAsp5 silenced plants had no changes in phenotype, exhibiting similar levels of free Asp and glutamate as control plants, but contained diminished levels of asparagine and much higher levels of lysine. In contrast, the suppression of NbPAT led to a severe reduction in growth and strong chlorosis symptoms. NbPAT silenced plants exhibited extremely reduced levels of asparagine and were greatly affected in their phenylalanine metabolism and lignin deposition. Furthermore, NbPAT suppression triggered a transcriptional reprogramming in plastid nitrogen metabolism. Taken together, our results indicate that NbPAT has an overlapping role with NbAsp5 in the biosynthesis of Asp and a key role in the production of phenylalanine for the biosynthesis of phenylpropanoids. The analysis of NbAsp5/NbPAT cosilenced plants highlights the central role of both plastidic aminotransferases in nitrogen metabolism; however, only NbPAT is essential for plant growth and development. PMID:24296073

  16. Fragmentation of α- and β-alanine molecules by ions at Bragg-peak energies

    NASA Astrophysics Data System (ADS)

    Bari, S.; Sobocinski, P.; Postma, J.; Alvarado, F.; Hoekstra, R.; Bernigaud, V.; Manil, B.; Rangama, J.; Huber, B.; Schlathölter, T.

    2008-02-01

    The interaction of keV He+, He2+, and O5+ ions with isolated α and β isomers of the amino acid alanine was studied by means of high resolution coincidence time-of-flight mass spectrometry. We observed a strong isomer dependence of characteristic fragmentation channels which manifests in strongly altered branching ratios. Despite the ultrashort initial perturbation by the incoming ion, evidence for molecular rearrangement leading to the formation of H3+ was found. The measured kinetic energies of ionic alanine fragments can be sufficient to induce secondary damage to DNA in a biological environment.

  17. 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.

  18. Prevalence and Predictors of Elevated Aspartate Aminotransferase-to-Platelet Ratio Index in Latin American Perinatally HIV-infected Children

    PubMed Central

    Siberry, George K.; Cohen, Rachel A.; Harris, D. Robert; Cruz, Maria Leticia Santos; Oliveira, Ricardo; Peixoto, Mario F.; Cervi, Maria Celia; Hazra, Rohan; Pinto, Jorge A.

    2013-01-01

    Background Chronic liver disease has emerged as an important problem in adults with longstanding HIV infection, but data are lacking for children. We characterized elevated aspartate aminotransferase (AST)-to-platelet ratio index (APRI ), a marker of possible liver fibrosis, in perinatally HIV-infected children. Methods NISDI [NICHD (National Institute of Child Health and Human Development) International Site Development Initiative] enrolled HIV-infected children (ages 0.1-20.1 years) from five Latin American countries in an observational cohort from 2002–2009. Twice yearly visits included medical history, physical examination and laboratory evaluations. The prevalence (95% confidence interval [CI]) of APRI>1.5 was calculated and associations with demographic, HIV-related and liver-related variables were investigated in bivariate analyses. Results APRI was available for 1012 of 1032 children. APRI was >1.5 in 32 (3.2%, 95% CI: 2.2%-4.4%) including 2 of 4 participants with hepatitis B (HBV) infection. Factors significantly associated with APRI>1.5 (p<0.01 compared to APRI≤1.5) included country, younger age, past or current HBV, higher alanine aminotransferase, lower total cholesterol, higher log10 current viral load, lower current CD4 count, lower nadir CD4 count, use of hepatotoxic non-antiretroviral (ARV) medications, and no prior ARV use. Rates of APRI>1.5 varied significantly by current ARV regimen (p=0.0002), from 8.0% for no ARV to 3.2% for non-protease inhibitor (PI) regimens to 1.5% for PI-based regimens. Conclusions Elevated APRI occurred in approximately 3% of perinatally HIV-infected children. PI-based ARVs appeared protective while inadequate HIV control appeared to increase risk of elevated APRI. Additional investigations are needed to better assess potential subclinical, chronic liver disease in HIV-infected children. PMID:23799515

  19. 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.

  20. 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.

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

    PubMed Central

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

    1978-01-01

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

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

    PubMed

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

    2009-05-14

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

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

    PubMed Central

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

    2010-01-01

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

  4. Innovative effect of illite on improved microbiological conversion of L-tyrosine to 3,4 dihydroxy phenyl L-alanine (L-DOPA) by Aspergillus oryzae ME2 under acidic reaction conditions.

    PubMed

    Sikander, Ali; Ikram-ul-Haq

    2006-11-01

    In the present investigation, the previous ultraviolet irradiated mutant strain of Aspergillus oryzae UV-7 was further improved in terms of 3,4 dihydroxy phenyl L-alanine (L-DOPA) activity after chemical mutagenesis through 1-methyl 3-nitro 1-nitroso guanidine (MNNG = 250-1500 microg/ml) treatment (0-30 min). Among several mutant variants, the one that produced a larger amount of L-DOPA from L-tyrosine was designated to as ME2 and it was made 2-deoxy-D-glucose-resistant by growing it at various concentrations of 2 dg (0.01-0.025 %, w/v) in Vogel's agar medium. Relatively better production of L-DOPA (> 0.60 mg/ml) was obtained when 2.0% (w/v) glucose was used as a carbon source in the mycelium production medium and the tyrosinase activity increased constitutively (1.08 mg/ml), which resulted in a greater production of L-DOPA. At optimum pH0 (pH 6.0) and reaction time (60 min), more than 65% sugar was utilized for cell mass formation. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved 60 min after the biochemical reaction. Mould mycelium was used for microbiological conversion of L-tyrosine to L-DOPA because tyrosinases, beta-carboxylases, and tyrosine hydroxylases are intracellular enzymes. The effect of illite (1.0 x 10(6)-6.0 x 10(6) M) on biochemical conversion of L-tyrosine to L-DOPA by Aspergillus oryzae ME(2 )was also carried out. Best results of L-DOPA biosynthesis were observed when the concentration of illite was 3.5 x 10(-6) M (1.686 mg/ml L-DOPA produced with 1.525 mg/ml consumption of L-tyrosine). It was noted that the addition of illite not only increased enzyme activity but also enhanced the permeability of cell membrane to facilitate the secretion of enzymes into the reaction broth. The comparison of kinetic parameters showed the ability of mutant to yield L-DOPA (i.e., Yp/x 7.360 +/- 0.04 mg/mg). When the culture grown on various illite concentrations was monitored for Qp, Qs, and qp, there was significant enhancement (p < 0

  5. Ambient Ionization Mass Spectrometry Measurement of Aminotransferase Activity

    NASA Astrophysics Data System (ADS)

    Yan, Xin; Li, Xin; Zhang, Chengsen; Xu, Yang; Cooks, R. Graham

    2017-01-01

    A change in enzyme activity has been used as a clinical biomarker for diagnosis and is useful in evaluating patient prognosis. Current laboratory measurements of enzyme activity involve multi-step derivatization of the reaction products followed by quantitative analysis of these derivatives. This study simplified the reaction systems by using only the target enzymatic reaction and directly detecting its product. A protocol using paper spray mass spectrometry for identifying and quantifying the reaction product has been developed. Evaluation of the activity of aspartate aminotransferase (AST) was chosen as a proof-of-principle. The volume of sample needed is greatly reduced compared with the traditional method. Paper spray has a desalting effect that avoids sprayer clogging problems seen when examining serum samples by nanoESI. This very simple method does not require sample pretreatment and additional derivatization reactions, yet it gives high quality kinetic data, excellent limits of detection (60 ppb from serum), and coefficients of variation <10% in quantitation.

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  7. Dietary freshwater clam (Corbicula fluminea) extract suppresses accumulation of hepatic lipids and increases in serum cholesterol and aminotransferase activities induced by dietary chloretone in rats.

    PubMed

    Chijimatsu, Takeshi; Umeki, Miki; Kobayashi, Satoru; Kataoka, Yutaro; Yamada, Koji; Oda, Hiroaki; Mochizuki, Satoshi

    2015-01-01

    We investigated the ameliorative effect of freshwater clam extract (FCE) on fatty liver, hypercholesterolemia, and liver injury in rats exposed to chloretone. Furthermore, we examined the effects of major FCE components (fat and protein fractions) to determine the active components in FCE. Chloretone increased serum aminotransferase activities and led to hepatic lipid accumulation. Serum aminotransferase activities and hepatic lipid content were lower in rats fed total FCE or fat/protein fractions of FCE. Expression of fatty acid synthase and fatty acid desaturase genes was upregulated by chloretone. Total FCE and fat/protein fractions of FCE suppressed the increase in gene expression involved in fatty acid synthesis. Serum cholesterol levels increased twofold upon chloretone exposure. Total FCE or fat/protein fractions of FCE showed hypocholesterolemic effects in rats with hypercholesterolemia induced by chloretone. These suggest that FCE contains at least two active components against fatty liver, hypercholesterolemia, and liver injury in rats exposed to chloretone.

  8. Hydroxycitric acid does not promote inflammation or liver toxicity

    PubMed Central

    Clouatre, Dallas L; Preuss, Harry G

    2013-01-01

    Garcinia cambogia extract (GC) with its active component consisting of hydroxycitric acid (HCA) is widely utilized for weight loss. Various HCA salts are available, including calcium, magnesium, potassium and mixtures of these. Experimentally, these salts exhibit different properties with some, but not all, improving glucose tolerance and blood pressure. Recently, obesity-prone C57BL/6J mice were fed a high-fat diet (HFD, 45 kcal% fat) with or without GC (1%, w/w) for 16 wk. The active arm reduced visceral fat, adipocyte size and serum glucose, yet purportedly also exhibited hepatic collagen accumulation, lipid peroxidation and increased mRNA levels of genes related to oxidative stress. The latter findings are at odds with a large body of animal and human studies that have been conducted on the safety and efficacy of HCA. This literature shows HCA to be protective against the liver toxicity associated with ethanol and dexamethasone administration, and to maintain serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase at near normal levels. In both animal and clinical literature, elevated intakes of HCA per se have not led to signs of inflammation or hepatotoxicity. The compound has been found to reduce markers of inflammation in brain, intestines, kidney and serum. PMID:24307814

  9. Effect of glucose, independent of changes in insulin and glucagon secretion, on alanine metabolism in the conscious dog.

    PubMed Central

    Shulman, G I; Lacy, W W; Liljenquist, J E; Keller, U; Williams, P E; Cherrington, A D

    1980-01-01

    To study the effects of hyperglycemia on the metabolism of alanine and lactate independent of changes in plasma insulin and glucagon, glucose was infused into five 36-h-fasted dogs along with somatostatin and constant replacement amounts of both insulin and glucagon. Hepatic uptakes of alanine and lactate were calculated using the arteriovenous difference technique. [14C]Alanine was infused to measure the conversion of alanine and lactate into glucose. Hyperglycemia (delta 115 mg/dl) of 2 h duration caused the plasma alanine level to increase by over 50%. This change was caused by an increase in the inflow of alanine into plasma since the net hepatic uptake of the amino acid did not change. Taken together, the above findings indicate that glucose per se can significantly impair the fractional extraction of alanine by the liver. Hepatic extraction of lactate was also affected by hyperglycemia and had fallen to zero within 90 min of starting the glucose infusion. This fall was associated with a doubling of arterial lactate level. Conversion of [14C]-alanine and [14C]lactate into [14C]glucose was suppressed by 60 +/- 11% after 2 h of hyperglycemia, and because this fall could not be entirely accounted for by decreased lactate extraction an inhibitory effect of glucose on gluconeogenesis within the liver is suggested. These studies indicate that the plasma glucose level per se can be an important determinant of the level of alanine and lactate in plasma as well as the rate at which they are converted to glucose. PMID:7356691

  10. 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.

  11. NQR in Alanine and Lysine Iodates

    NASA Astrophysics Data System (ADS)

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

    2000-02-01

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

  12. Overproduction and preliminary crystallographic study of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3

    SciTech Connect

    Chon, Hyongi; Matsumura, Hiroyoshi; Shimizu, Shoko; Maeda, Nao; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2005-03-01

    A human kynurenine aminotransferase II homologue from P. horikoshii OT3 has been overproduced in E. coli, purified, and characterized. Crystals of this protein have been obtained and analyzed by X-ray diffraction. The Pyrococcus horikoshii OT3 genome contains a gene encoding a human kynurenine aminotransferase II (KAT II) homologue, which consists of 428 amino-acid residues and shows an amino-acid sequence identity of 30% to human KAT II. This gene was overexpressed in Escherichia coli and the recombinant protein (Ph-KAT II) was purified. Gel-filtration chromatography showed that Ph-KAT II exists as a homodimer. Ph-KAT II exhibited enzymatic activity that catalyzes the transamination of l-kynurenine to produce kynurenic acid. Crystals of Ph-KAT II were grown using the sitting-drop vapour-diffusion method and native X-ray diffraction data were collected to 2.2 Å resolution using synchrotron radiation from station BL44XU at SPring-8. The crystals belong to the centred orthorhombic space group C222{sub 1}, with unit-cell parameters a = 71.75, b = 86.84, c = 137.30 Å. Assuming one molecule per asymmetric unit, the V{sub M} value was 2.19 Å{sup 3} Da{sup −1} and the solvent content was 43.3%.

  13. Effects of glycine, beta-alanine and diazepam upon morphine-tolerant-dependent mice.

    PubMed

    Contreras, E; Tamayo, L

    1980-05-01

    The effects in mice of glycine, beta-alanine and diazepam on the analgesic response to morphine, on the intensity of tolerance and on the physical dependence on the analgesic have been examined. The two amino acids increased the analgesic response to morphine in a dose-related manner. However, both compounds were ineffective in the analgesic test (hot plate) when administered without morphine. Diazepam was ineffective in the analgesic test and it did not alter morphine analgesia, except when administered in a high dose which decreased and analgesic response. Glycine, either in single or repeated doses, did not modify tolerance to morphine, whereas beta-alanine induced a dose-related partial antagonism, which promptly reached a plateau. Diazepam induced a small decrease in the intensity of tolerance to the analgesic. The abstinence syndrome to morphine, induced by naloxone administration to primed mice, was reduced by single doses of glycine or beta-alanine. Diazepam behaved as a weak inhibitor of the abstinence syndrome when administered at a high dose. The potentiation of morphine analgesia and the antagonism of the abstinence syndrome induced by the amino acids may be related to their hyperpolarizing action in the c.n. system. The effects of beta-alanine on morphine tolerance cannot be explained by the same mechanism.

  14. 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.

  15. Hepatoprotective effect of Matrine salvianolic acid B salt on Carbon Tetrachloride-Induced Hepatic Fibrosis

    PubMed Central

    2012-01-01

    The aim of this study was to investigate the hepatoprotective effect of Matrine salvianolic acid B salt on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats. Salvianolic acid B and Matrine has long been used to treat liver fibrosis. Matrine salvianolic acid B salt is a new compound containing Salvianolic acid B and Matrine. Hepatic fibrosis induced by CCl4 was studied in animal models using Wistar rats. Organ coefficient, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), hexadecenoic acid (HA), laminin (LN), hydroxyproline (Hyp), and glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) in liver tissues were measured, respectively. Histopathological changes in the livers were studied by hematoxylin-eosin (H&E) staining and Masson Trichrome (MT) examination. The expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) was observed by immunohistochemical analysis. A significant reduction in serum levels of AST, ALT, HA, LN and Hyp was observed in the Matrine salvianolic acid B salt treated groups, suggesting that the salt had hepatoprotective effects. The depletion of GSH and SOD, as well as MDA accumulation in liver tissues was suppressed by Matrine salvianolic acid B salt too. The expression of TGF-β1 and α-SMA measured by immunohistology was significantly reduced by Matrine salvianolic acid B salt in a dose-dependent manner. Matrine salvianolic acid B salt treatment attenuated the necro-inflammation and fibrogenesis induced by CCl4 injection, and thus it is promising as a therapeutic anti-fibrotic agent against hepatic fibrosis. PMID:22559721

  16. Characterisation of Potential Antimicrobial Targets in Bacillus spp. I. Aminotransferases and Methionine Regeneration in Bacillus subtilis

    DTIC Science & Technology

    2002-07-01

    targets in Bacillus spp. I. Aminotransferases and methionine regeneration in Bacillus subtilis. Bradley J. Berger and Marvin H. Knodel Defence R&D...Characterisation of potential antimicrobial targets in Bacillus spp. I. Aminotransferases and methionine regeneration in Bacillus subtilis. Bradley J...examined in the gram-positive bacterium Bacillus subtilis. Homogenates of this bacterium were able to convert ketomethiobutyrate to methionine, utilising

  17. Enantiomeric Excesses of Acid Labile Amino Acid Precursors of the Murchison Meteorite

    NASA Technical Reports Server (NTRS)

    Pizzarello, Sandra

    1998-01-01

    Amino acids present in carbonaceous chondrite are extracted in water in part as free compounds and in approximately equal part as acid labile precursors. On the assumption that they would be free of contamination, the precursors of two Murchison amino acids that have terrestrial occurrence, alanine and glutamic acid, have been targeted for analysis of their enantiomeric ratios. Pyroglutamic acid, the precursor of glutamic acid, was found with an L-enantiomeric excess comparable to that of the free acid, while alanine's precursor, N-acetyl alanine, appears approximately racemic. Also alpha-imino propioacetic acid, a proposed end product of alanine synthesis in the meteorite, was analyzed and found racemic.

  18. [Association between occupational stress and aminotransferase activity in patients with metabolic syndrome].

    PubMed

    Zhao, H; Song, L; Qiang, Y; Liu, H R; Qiu, F Y; Li, X Z; Song, H

    2016-12-20

    Objective: To investigate the association between occupational stress and activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in patients with metabolic syndrome. Methods: A case-control study was performed. According to inclusion and exclusion criteria, among the staff members of enterprises and public institutions aged 20~60 years who underwent physical examination in The Affiliated Hospital of Ningxia Medical University and The People's Hospital of Wuzhong from October 2011 to October 2012, 622 patients with metabolic syndrome who did not have a blood relationship with each other were enrolled as case group, and 600 healthy staff members who also did not have a blood relationshipwith each otherwere enrolled as control group. Questionnaire investigation, chronic occupational stress investigation, physical examination, and laboratory tests were performed for all subjects. Results: Compared with the control group, the case group had significantly higher serum levels and abnormal rates of AST and ALT (t=-4.338 and-5.485, χ(2)=11.168 and 34.302, all P<0.05) . There were no significantdifferences in the serum level and abnormal rate of AST between the subgroups with different occupational stresses in both groups (F=2.192 and 2.567, χ(2)=2.694 and 5.402, all P>0.05) , but there were significant differencesbetween the subgroups in all subjects (F=5.005, χ(2)=6.398, all P<0.05) . There were no significant differences in the serum level and abnormal rate of ALT between thesubgroups with different occupational stresses in the case group, the control group, and all subjects (F=0.845, 0.450, and 1.416, χ(2)=2.564, 1.344, and 3.147, all P>0.05) . The partial correlation analysis showed that the total score of occupational stress was positively correlated withthe serum level of AST (r=0.071, P<0.05) and was not correlated with the serum level of ALT (r=-0.044, P>0.05) , and that the serum level of AST was positively correlated with that of ALT

  19. N-acetylcysteine and meso-2,3-dimercaptosuccinic acid alleviate oxidative stress and hepatic dysfunction induced by sodium arsenite in male rats

    PubMed Central

    Abu El-Saad, Ahmed M; Al-Kahtani, Mohammed A; Abdel-Moneim, Ashraf M

    2016-01-01

    Environmental exposure to arsenic represents a serious challenge to humans and other animals. The aim of the present study was to test the protective effect of antioxidant N-acetylcysteine (NAC) either individually or in combination with a chelating agent, meso-2,3-dimercaptosuccinic acid (DMSA), against sodium arsenite oral toxicity in male rats. Five groups were used: control; arsenic group (orally administrated in a concentration of 2 mg/kg body weight [b.w.]); the other three groups were orally administrated sodium arsenite in a concentration of 2 mg/kg b.w. followed by either NAC (10 mg/kg b.w., intraperitoneally [i.p.]), DMSA (50 mg/kg b.w., i.p.) or NAC plus DMSA. Arsenic toxicity caused significant rise in serum aspartate aminotransferase, alanine aminotransferase and total bilirubin, and a significant decrease in total protein (TP) and albumin levels after 3 weeks of experimental period. In addition, arsenic-treated rats showed significantly higher arsenic content in liver and significant rise in hepatic malondialdehyde level. By contrast, sharp decreases in glutathione content and catalase and glutathione reductase activities were discernible. NAC and/or DMSA counteracted most of these physiologic and biochemical defects. NAC monotherapy was more effective than DMSA in increasing TP, while DMSA was more effective in decreasing alanine aminotransferase. The combined treatment was superior over monotherapies in recovery of TP and glutathione. Biochemical data were well supported by histopathological and ultrastructural findings. In conclusion, the combination therapy of NAC and DMSA may be an ideal choice against oxidative insult induced by arsenic poisoning. PMID:27799742

  20. 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.

  1. 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.

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

    SciTech Connect

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

    1988-05-03

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

  3. Meta-analysis of the influence of TM6SF2 E167K variant on Plasma Concentration of Aminotransferases across different Populations and Diverse Liver Phenotypes

    PubMed Central

    Sookoian, Silvia; Pirola, Carlos J.

    2016-01-01

    A nonsynonymous E167K (rs58542926 C/T) variant in TM6SF2 gene was recently associated with nonalcoholic fatty liver disease (NAFLD). We explored the association between E167K and plasma concentrations of alanine (ALT) and aspartate (AST) aminotransferases through a meta-analysis. We also estimated the strength of the effect across diverse liver phenotypes, including NAFLD and chronic viral hepatitis; fourteen studies were included. We found that ALT (p = 3.2 × 10−6, n = 94,414) and AST (p = 0007, n = 93,809) levels were significantly associated with rs58542926 in NAFLD. By contrast, rs58542926 was not associated with either ALT (p = 0.24, n = 4187) or AST (p = 0.17, n = 2678) levels in four studies on chronic hepatitis. In conclusion, the results of the pooled estimates in patients with NAFLD showed that carriers of the T allele (EK + KK), when compared with homozygous subjects for the C allele (EE genotype) have increased levels of aminotransferases; however, this increase represents –2.5 (9.8%) and 1.2 (5%) IU/L of ALT and AST respectively, which is fairly small compared with the large effect of PNPLA3- rs738409-G allele that is associated with a –28% increase in serum ALT. PMID:27278285

  4. Effects of changes in cell volume on the rates of glutamine and alanine release from rat skeletal muscle in vitro.

    PubMed

    Parry-Billings, M; Bevan, S J; Opara, E; Newsholme, E A

    1991-06-01

    The effect of changes in cell volume on the rates of release of glutamine and alanine from muscle and on the concentrations of these amino acids in muscle were investigated by using an isolated preparation of rat skeletal muscle incubated in the presence of hypo- and hyper-osmotic media. Changes in cell volume were associated with changes in the rates of release of glutamine and alanine from muscle: incubation in hypo-osmotic medium decreased the rates of release of glutamine and alanine, and incubation in hyperosmotic medium increased these rates. These changes were rapidly reversed by a change in osmoticity of the medium. Despite marked changes in cell volume, the concentrations of these amino acids in muscle were maintained. It is suggested that cell volume may play a role in the regulation of amino acid metabolism in skeletal muscle.

  5. Structure-based design of a new series of D-glutamic acid based inhibitors of bacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD).

    PubMed

    Tomasić, Tihomir; Zidar, Nace; Sink, Roman; Kovac, Andreja; Blanot, Didier; Contreras-Martel, Carlos; Dessen, Andréa; Müller-Premru, Manica; Zega, Anamarija; Gobec, Stanislav; Kikelj, Danijel; Masic, Lucija Peterlin

    2011-07-14

    MurD ligase is one of the key enzymes participating in the intracellular steps of peptidoglycan biosynthesis and constitutes a viable target in the search for novel antibacterial drugs to combat bacterial drug-resistance. We have designed, synthesized, and evaluated a new series of D-glutamic acid-based Escherichia coli MurD inhibitors incorporating the 5-benzylidenethiazolidin-4-one scaffold. The crystal structure of 16 in the MurD active site has provided a good starting point for the design of structurally optimized inhibitors 73-75 endowed with improved MurD inhibitory potency (IC(50) between 3 and 7 μM). Inhibitors 74 and 75 showed weak activity against Gram-positive Staphylococcus aureus and Enterococcus faecalis. Compounds 73-75, with IC(50) values in the low micromolar range, represent the most potent D-Glu-based MurD inhibitors reported to date.

  6. Prophylactic effects of humic acid-glucan combination against experimental liver injury

    PubMed Central

    Vetvicka, Vaclav; Garcia-Mina, Jose Maria; Yvin, Jean-Claude

    2015-01-01

    Aim: Despite intensive research, liver diseases represent a significant health problem and current medicine does not offer a substance able to significantly inhibit the hepatotoxicity leading to various stages of liver disease. Based on our previously published studies showing the protective effects of a glucan-humic acid (HA) combination, we focused on the hypothesis that the combination of these two natural molecules can offer prophylactic protection against experimentally induced hepatotoxicity. Materials and Methods: Lipopolysaccharide, carbon tetrachloride, and ethanol were used to experimentally damage the liver. Levels of aspartate aminotransferase, alanine transaminase, alkaline phosphatase, glutathione, superoxide dismutase, and malondialdehyde, known to correspond to the liver damage, were assayed. Results: Using three different hepatotoxins, we found that in all cases, some samples of HA and most of all the glucan-HA combination, offer strong protection against liver damage. Conclusion: Glucan-HA combination is a promising agent for use in liver protection. PMID:26401416

  7. Effects of Omega-3 Fatty Acid in Nonalcoholic Fatty Liver Disease: A Meta-Analysis

    PubMed Central

    Lu, Wenxia; Li, Sainan; Li, Jingjing; Wang, Jianrong; Zhang, Rong; Zhou, Yuqing; Yin, Qin; Wang, Fan; Xia, Yujing; Liu, Tong; Lu, Jie; Zhou, Yingqun

    2016-01-01

    A meta-analysis was conducted to assess the effect of omega-3 fatty acid supplementation (n-3 PUFAs) in lowering liver fat, liver enzyme (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyltransferase (GGT) levels), and blood lipids (triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL), and low density lipoprotein (LDL)) in patients with nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). Methods. MEDLINE/PubMed, EMBASE, the Cochrane Central Register of Controlled Trials, CINAHL, Science Citation Index (ISI Web of Science), Chinese Biomedical Literature Database (CBM), and Chinese National Knowledge Infrastructure (CNKI) were searched for relevant randomized controlled trials on the effects of n-3 polyunsaturated fatty acids (PUFAs) in patients with NAFLD from inception to May 2015. Ten studies were included in this meta-analysis. Results. 577 cases of NAFLD/NASH in ten randomized controlled trials (RCTs) were included. The results of the meta-analysis showed that benefit changes in liver fat favored PUFA treatment, and it was also beneficial for GGT, but it was not significant on ALT, AST, TC, and LDL. Conclusions. In this meta-analysis, omega-3 PUFAs improved liver fat, GGT, TG, and HDL in patients with NAFLD/NASH. Therefore, n-3 PUFAs may be a new treatment option for NAFLD. PMID:27651787

  8. Chicoric acid regulates behavioral and biochemical alterations induced by chronic stress in experimental Swiss albino mice.

    PubMed

    Kour, Kiranjeet; Bani, Sarang

    2011-09-01

    The present study was taken up to see the effect of chicoric acid (CA) on behavioral and biochemical alterations induced by chronic restraint stress in experimental Swiss albino mice. CA at 1mg/kg dose level exhibited considerable antidepressant activity as shown by significant decrease in immobility period in the Porsolt's swim stress-induced behavioral despair test and escape failures in Learned "helplessness test". The antidepressant activity shown by CA can be attributed to its modulating effect on nor-adrenaline (NA), dopamine (DA) and 5- hydroxy tryptamine (5-HT) as shown by their quantification in CA treated chronically stressed mice. Further, a significant antioxidant effect was exhibited by CA as shown by estimation of lipid peroxidation, glutathione (GSH) and glycogen in liver of chronically stressed mice. It also normalized altered values of serum glucose, triglycerides, aspartate aminotransferase (AST) alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in a dose dependent manner. The stress busting potential of CA was further confirmed by its regulating effect on raised plasma corticosterone levels and significant attenuation of the depleted ascorbic acid, cholesterol and corticosterone levels in adrenal glands. Thus, our results suggest that CA possesses considerable stress busting potential, and that anti-oxidation may be one of the mechanisms underlying its antistress action.

  9. THE ULTRASTRUCTURAL LOCALIZATION OF THE ISOZYMES OF ASPARTATE AMINOTRANSFERASE IN MURINE TISSUES

    PubMed Central

    Papadimitriou, J. M.; Van Duijn, P.

    1970-01-01

    Two isozymes of aspartate aminotransferase have been demonstrated biochemically. One isozyme is found in the mitochondrial fraction of the cytoplasm, the other ("soluble") in the supernatant. Both isozymes can be demonstrated by the cytochemical technique of Lee and Torack, as reported in the preceding report. Aldehyde fixation rapidly inactivates both isozymes, especially the soluble one. Inactivation can be delayed by addition of ketoglutarate to the fixative. The ketoglutarate probably competes with the fixative for the active site of the enzyme, thus protecting that region of the molecule. This enables adequate tissue preservation with enough remaining enzymatic activity to be demonstrated by the precipitation of oxaloacetate as the lead salt from a medium containing α-ketoglutaric acid aspartic acid, and lead nitrate. Electron-opaque material was found not only in mitochondria but, as the result of substrate protection, on the plasma membranes of many cells including erythrocytes and bacteria, the limiting membrane of peroxisomes, and the transverse tubular system of striated muscle. Occasional centrioles, neurotubules, tubules in the tails of spermatozoa, the A-I band junction in myofibrils of striated muscle, and the ground substance between cisternae of endoplasmic reticulum in intestinal goblet cells also showed precipitate. In all cases, replacement of L-aspartic acid by D-aspartic acid in the medium resulted in unstained sections. The sensitivity of extramitochondrial sites to fixation, the need of ketoglutarate as an agent for protecting the enzymatic activity during the fixation process, and the known presence of only soluble isozyme in erythrocytes indicate that enzymatic activity at these sites can be attributed to the soluble isozyme. Localization of the soluble isozyme on the plasma membrane may be related to possible involvement in depolarization phenomena, amino acid transport, or synthesis of plasma membrane-bound mucopolysaccharides. PMID

  10. Ornithine-δ-Aminotransferase Inhibits Neurogenesis During Xenopus Embryonic Development

    PubMed Central

    Peng, Ying; Cooper, Sandra K.; Li, Yi; Mei, Jay M.; Qiu, Shuwei; Borchert, Gregory L.; Donald, Steven P.; Kung, Hsiang-fu; Phang, James M.

    2015-01-01

    Purpose. In humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development. Methods. We cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations. Results. We showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development. Conclusions. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development. PMID:25783604

  11. Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

    PubMed Central

    Moreno, Miguel Angel; Alonso, Ana; Alcolea, Pedro Jose; Abramov, Ariel; de Lacoba, Mario García; Abendroth, Jan; Zhang, Sunny; Edwards, Thomas; Lorimer, Don; Myler, Peter John; Larraga, Vicente

    2014-01-01

    Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs. PMID:25516846

  12. Cloning and molecular characterization of tick kynurenine aminotransferase (HlKAT) from Haemaphysalis longicornis (Acari: Ixodidae).

    PubMed

    Battsetseg, Badgar; Boldbaatar, Damdinsuren; Battur, Banzragch; Xuan, Xuenan; Fujisaki, Kozo

    2009-09-01

    A complementary DNA coding a novel kynurenine aminotransferase (KAT) molecule from Haemaphysalis longicornis tick embryo was cloned and characterized. The transcription of the HlKAT occurs at all stages during tick development as well as in the midgut, salivary glands, ovary, and synganglion of adult ticks, and protein expression levels increased during the blood-feeding course. The HlKAT gene without signal peptide was successfully expressed as a glutathione S-transferase fusion protein in soluble form, which is capable of catalyzing the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid, respectively. The purified recombinant HlKAT showed dose-dependent inhibition effect on the growth of equine babesial parasite, Babesia caballi, in in vitro culture. All results suggested that a specific HlKAT is present in tick and HlKAT may play an important physiological role in H. longicornis. This is the first report of a member enzyme of tryptophan pathway in Chelicerata.

  13. 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.

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

    PubMed

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

    2009-12-10

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

  15. Troglitazone acts by PPARgamma and PPARgamma-independent pathways on LLC-PK1-F+ acid-base metabolism.

    PubMed

    Welbourne, Tomas; Friday, Ellen; Fowler, Rocky; Turturro, Francesco; Nissim, Itzhak

    2004-01-01

    Troglitazone was studied in pH-sensitive LLC-PK1-F+ cells to determine the effect on pHi and glutamine metabolism as well as the role of peroxisome proliferator-activated receptor (PPARgamma)-dependent and PPARgamma-independent signaling pathways. Troglitazone induces a dose-dependent cellular acidosis that occurs within 4 min and persists over 18 h as a result of inhibiting Na+/H+ exchanger-mediated acid extrusion. Cellular acidosis was associated with glutamine-dependent augmented [15N]ammonium production and decreased [15N]alanine formation from 15N-labeled glutamine. The shift in glutamine metabolism from alanine to ammoniagenesis appears within 3 h and is associated after 18 h with both a reduction in assayable alanine aminotransferase (ALT) activity as well as cellular acidosis. The relative contribution of troglitazone-induced cellular acidosis vs. the decrease in assayable ALT activity to alanine production could be demonstrated. The PPARgamma antagonist bisphenol A diglycide ether (BADGE) reversed both the troglitazone-induced cellular acidosis and ammoniagenesis but enhanced the troglitazone reduction of assayable ALT activity; BADGE also blocked troglitazone induction of peroxisome proliferator response element-driven firefly luciferase activity. The protein kinase C (PKC) inhibitor chelerythrine mimics troglitazone effects, whereas phorbol ester reverses the effects on ammoniagenesis consistent with troglitazone negatively regulating the DAG/PKC/ERK pathway. Although functional PPARgamma signaling occurs in this cell line, the major troglitazone-induced acid-base responses appear to be mediated by pathway(s) involving PKC/ERK.

  16. Expression and processing of human ornithine-delta-aminotransferase in Saccharomyces cerevisiae.

    PubMed

    Dougherty, K M; Swanson, D A; Brody, L C; Valle, D

    1993-11-01

    Ornithine-delta-aminotransferase catalyzes the conversion of ornithine to glutamate-gamma-semialdehyde. In humans, deficiency of this mitochondrial matrix enzyme results in the progressive blinding disorder, gyrate atrophy of the choroid and retina. To explore yeast as an expression system, we introduced a cDNA encoding human ornithine-delta-aminotransferase into an ornithine aminotransferase-deficient strain of Saccharomyces cerevisiae. The human enzyme was expressed at high levels, with activity 20-fold greater than that of wild-type yeast and 10-fold higher than in human fibroblasts. Although the normal location of ornithine-delta-aminotransferase in S. cerevisiae is cytosolic, human ornithine-delta-aminotransferase expressed in S. cerevisiae was localized to the mitochondrial matrix with correct proteolytic processing of its mitochondrial leader sequence. Despite this anomalous location in yeast, human ornithine-delta-aminotransferase complemented the phenotype of the mutant strain, restoring its ability to utilize ornithine as a sole nitrogen source. We also expressed a vitamin B6-responsive missense allele of ornithine-delta-aminotransferase (V332M) and showed that the biochemical phenotype of this allele is easily demonstrated confirming the usefulness of this system for examining mutations causing gyrate atrophy.

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

    PubMed

    Lu, Yongzhi; Xu, Hongyan; Zhao, Xiaojun

    2010-08-01

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

  18. Positron and electron scattering by glycine and alanine: Shape resonances and methylation effect

    NASA Astrophysics Data System (ADS)

    Nunes, Fernanda B.; Bettega, Márcio H. F.; Sanchez, Sergio d'Almeida

    2016-12-01

    We report integral cross sections (ICSs) for both positron and electron scattering by glycine and alanine amino acids. These molecules differ only by a methyl group. We computed the scattering cross sections using the Schwinger multichannel method for both glycine and alanine in different levels of approximation for both projectiles. The alanine ICSs are greater in magnitude than the glycine ICSs for both positron and electron scattering, probably due to the larger size of the molecule. In electron scattering calculations, we found two resonances for each molecule. Glycine presents one at 1.8 eV, and another centered at around 8.5 eV, in the static-exchange plus polarization (SEP) approximation. The ICS for alanine shows one resonance at 2.5 eV and another at around 9.5 eV, also in SEP approximation. The results are in good agreement with most of the data present in the literature. The comparison of the electron scattering ICSs for both molecules indicates that the methylation of glycine destabilizes the resonances, shifting them to higher energies.

  19. Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae.

    PubMed

    Kingsbury, Joanne M; Sen, Neelam D; Cardenas, Maria E

    2015-12-01

    The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals.

  20. Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae

    PubMed Central

    Kingsbury, Joanne M.; Sen, Neelam D.; Cardenas, Maria E.

    2015-01-01

    The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals. PMID:26659116

  1. 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.

  2. 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.

  3. Effect of betaine supplementation on changes in hepatic metabolism of sulfur-containing amino acids and experimental cholestasis induced by alpha-naphthylisothiocyanate.

    PubMed

    Kim, Young C; Jung, Young S; Kim, Sang K

    2005-05-01

    Alterations in the hepatic metabolism of sulfur amino acids in experimental cholestasis induced by alpha-naphthylisothiocyanate (ANIT) (100 mg/kg, po) were monitored in male mice for 1 week. We also examined the effects of betaine supplementation (1% in drinking water) for 2 weeks on the hepatotoxicity and changes in the sulfur amino acid metabolism induced by ANIT treatment. Acute ANIT challenge elevated the serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities, and total bilirubin contents from 5 h after the treatment, reaching a peak at t = 48-72 h. Hepatic S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels were decreased significantly in a manner almost inversely proportional to the changes in serum parameters measured to determine the ANIT-induced toxicity. Hepatic glutathione and cysteine levels were elevated at t = 120 h after the treatment. Betaine supplementation blocked or significantly attenuated induction of the hepatotoxicity by ANIT. The decrease in SAM and SAH levels was also inhibited by betaine intake. The results indicate that betaine supplementation may antagonize the induction of experimental cholestasis and changes in the metabolism of sulfur amino acids associated with ANIT treatment. The underlying mechanism and pharmacological significance of its action are discussed.

  4. 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

  5. Chlorogenic acid a dietary polyphenol attenuates isoproterenol induced myocardial oxidative stress in rat myocardium: An in vivo study.

    PubMed

    Akila, Palaniyandi; Vennila, Lakshmanan

    2016-12-01

    Intent of the present study has been made to appraise the cardioprotective effect of chlorogenic acid (CGA) on isoproterenol (ISO) induced myocardial infarction (MI) in male albino Wistar rats. ISO-induced myocardial damage was indicated by the elevated levels of marker enzymes such as creatine kinase (CK), creatine kinase-MB (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and troponin T and I (cTnT, cTnI) in the serum. In addition, the levels of lipid peroxidation products such as thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD) and lipid hydroperoxides (LHPs) were significantly increased in the plasma and heart tissue. Activities of enzymic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and the non enzymic antioxidants like vitamin C, vitamin E and reduced glutathione (GSH) were decreased in the erythrocytes, plasma and heart tissue of the ISO-induced rats and myocardium infarct size as observed by staining with triphenyltetrazolium chloride (TTC). Histopathological observation corroborated with the bioochemical parameters. Oral administration of CGA at different doses (10, 20, 40mg/kg BW) for 19days prevented the above changes. The 40mg/kg BW of CGA was more pronounced than other two doses and brought back all the above parameters to near normalcy.

  6. Alleviation of alcoholic liver injury by betaine involves an enhancement of antioxidant defense via regulation of sulfur amino acid metabolism.

    PubMed

    Jung, Young Suk; Kim, Sun Ju; Kwon, Do Young; Ahn, Chul Won; Kim, Young Soon; Choi, Dal Woong; Kim, Young Chul

    2013-12-01

    Previous studies suggested that the hepatoprotective activity of betaine is associated with its effects on sulfur amino acid metabolism. We examined the mechanism by which betaine prevents the progression of alcoholic liver injury and its therapeutic potential. Rats received a liquid ethanol diet for 6 wk. Ethanol consumption elevated serum triglyceride and TNFα levels, alanine aminotransferase and aspartate aminotransferase activities, and lipid accumulation in liver. The oxyradical scavenging capacity of liver was reduced, and expression of CD14, TNFα, COX-2, and iNOS mRNAs was induced markedly. These ethanol-induced changes were all inhibited effectively by betaine supplementation. Hepatic S-adenosylmethionine, cysteine, and glutathione levels, reduced in the ethanol-fed rats, were increased by betaine supplementation. Methionine adenosyltransferase and cystathionine γ-lyase were induced, but cysteine dioxygenase was down-regulated, which appeared to account for the increment in cysteine availability for glutathione synthesis in the rats supplemented with betaine. Betaine supplementation for the final 2 wk of ethanol intake resulted in a similar degree of hepatoprotection, revealing its potential therapeutic value in alcoholic liver. It is concluded that the protective effects of betaine against alcoholic liver injury may be attributed to the fortification of antioxidant defense via improvement of impaired sulfur amino acid metabolism.

  7. The structure of alanine racemase from Acinetobacter baumannii.

    PubMed

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

    2014-09-01

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

  8. Auxin and Tryptophan Homeostasis Are Facilitated by the ISS1/VAS1 Aromatic Aminotransferase in Arabidopsis

    PubMed Central

    Pieck, Michael; Yuan, Youxi; Godfrey, Jason; Fisher, Christopher; Zolj, Sanda; Vaughan, Dylan; Thomas, Nicholas; Wu, Connie; Ramos, Julian; Lee, Norman; Normanly, Jennifer; Celenza, John L.

    2015-01-01

    Indole-3-acetic acid (IAA) plays a critical role in regulating numerous aspects of plant growth and development. While there is much genetic support for tryptophan-dependent (Trp-D) IAA synthesis pathways, there is little genetic evidence for tryptophan-independent (Trp-I) IAA synthesis pathways. Using Arabidopsis, we identified two mutant alleles of ISS1 (Indole Severe Sensitive) that display indole-dependent IAA overproduction phenotypes including leaf epinasty and adventitious rooting. Stable isotope labeling showed that iss1, but not WT, uses primarily Trp-I IAA synthesis when grown on indole-supplemented medium. In contrast, both iss1 and WT use primarily Trp-D IAA synthesis when grown on unsupplemented medium. iss1 seedlings produce 8-fold higher levels of IAA when grown on indole and surprisingly have a 174-fold increase in Trp. These findings indicate that the iss1 mutant’s increase in Trp-I IAA synthesis is due to a loss of Trp catabolism. ISS1 was identified as At1g80360, a predicted aromatic aminotransferase, and in vitro and in vivo analysis confirmed this activity. At1g80360 was previously shown to primarily carry out the conversion of indole-3-pyruvic acid to Trp as an IAA homeostatic mechanism in young seedlings. Our results suggest that in addition to this activity, in more mature plants ISS1 has a role in Trp catabolism and possibly in the metabolism of other aromatic amino acids. We postulate that this loss of Trp catabolism impacts the use of Trp-D and/or Trp-I IAA synthesis pathways. PMID:26163189

  9. Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy1[W

    PubMed Central

    Lee, Eun-Jeong; Facchini, Peter J.

    2011-01-01

    Tyrosine aminotransferase (TyrAT) catalyzes the transamination of l-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and l-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5′-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for l-Tyr and α-ketoglutarate, with apparent Km values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of l-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde. PMID:21949209

  10. Chronotypic induction of tyrosine aminotransferase by. cap alpha. -methyl-p-tyrosine. [Rat liver

    SciTech Connect

    Cahill, A.L.; Ferguson, S.M.; Ehret, C.F.

    1981-04-06

    ..cap alpha..Methyl-p-tyrosine induced hepatic tyrosine aminotransferase activity to different extents depending upon the time of day of administration of the drug. Maximal induction occurred when ..cap alpha..-methyl-p-tyrosine (100 mg/kg) was injected intraperitoneally during the first several hours of the light phase of the daily cycle, but the magnitude of the induction depended on the nutritional state of the animal. Induction was 4- to 5-fold greater in fasting rats. The effect of ..cap alpha..-methyl-ptyrosine on hepatic tyrosine aminotransferase is believed to be mediated by decreases in hypothalamic norepinephrine. This hypothesis was supported by the demonstration that decreasing levels of hypothalamic norepinephrine at times of day when hypothalamic turnover of norepinephrine was greatest resulted in the greatest induction of tyrosine aminotransferase, while lowering hypothalamic norepinephrine at times when turnover was minimal resulted in minimal induction of tyrosine aminotransferase.

  11. An alternate method for demonstration of erythrocytic aminotransferases on starch gels

    PubMed Central

    Scott, Edward M.; Wright, Rita C.

    1981-01-01

    A two-stage procedure using MTT tetrazolium for the demonstration of aminotransferases (GPT and GOT) either singly or together was developed. Identification of phenotypes was unequivocal in over 99% of the individuals studied. ImagesFig. 1 PMID:7258187

  12. Regulation of 2-oxoglutarate metabolism in rat liver by NADP-isocitrate dehydrogenase and aspartate aminotransferase.

    PubMed

    Rakhmanova, T I; Popova, T N

    2006-02-01

    Kinetic and regulatory properties of NADP-isocitrate dehydrogenase (NADP-IDH) and aspartate aminotransferase (AsAT) responsible for 2-oxoglutarate metabolism in the cytoplasm and mitochondria of rat liver were studied. Based on the subcellular location of these enzymes and their kinetic parameters (Km, Ksi) obtained with highly purified enzyme preparations, it is suggested that synthesis of 2-oxoglutarate should be mainly determined by cytoplasmic NADP-IDH (86% of the total activity in the cell), whereas its utilization should depend on cytoplasmic AsAT (78% of the total activity). AsAT from the rat liver was specified by substrate inhibition and also by changes in the enzyme affinity for the substrates under the influence of some intermediates of the tricarboxylic acid cycle: isocitrate, succinate, fumarate, and citrate. Key intermediates of nitrogen metabolism (glutamate, glutamine, and aspartate) are involved in the regulation of NADP-IDH and AsAT. These enzymes are regulated oppositely, and the catalytic activity of one enzyme can be stimulated concurrently with a decrease in the activity of the other. Obviously, carbon and nitrogen metabolism in the rat liver can be controlled through redistribution of 2-oxoglutarate between different metabolic processes via regulatory mechanisms influencing differently located forms of NADP-IDH and AsAT.

  13. Structural Basis for the Stereochemical Control of Amine Installation in Nucleotide Sugar Aminotransferases.

    PubMed

    Wang, Fengbin; Singh, Shanteri; Xu, Weijun; Helmich, Kate E; Miller, Mitchell D; Cao, Hongnan; Bingman, Craig A; Thorson, Jon S; Phillips, George N

    2015-09-18

    Sugar aminotransferases (SATs) are an important class of tailoring enzymes that catalyze the 5'-pyridoxal phosphate (PLP)-dependent stereo- and regiospecific installation of an amino group from an amino acid donor (typically L-Glu or L-Gln) to a corresponding ketosugar nucleotide acceptor. Herein we report the strategic structural study of two homologous C4 SATs (Micromonospora echinospora CalS13 and Escherichia coli WecE) that utilize identical substrates but differ in their stereochemistry of aminotransfer. This study reveals for the first time a new mode of SAT sugar nucleotide binding and, in conjunction with previously reported SAT structural studies, provides the basis from which to propose a universal model for SAT stereo- and regiochemical control of amine installation. Specifically, the universal model put forth highlights catalytic divergence to derive solely from distinctions within nucleotide sugar orientation upon binding within a relatively fixed SAT active site where the available ligand bound structures of the three out of four representative C3 and C4 SAT examples provide a basis for the overall model. Importantly, this study presents a new predictive model to support SAT functional annotation, biochemical study and rational engineering.

  14. Alanine racemase is essential for the growth and interspecies competitiveness of Streptococcus mutans

    PubMed Central

    Wei, Yuan; Qiu, Wei; Zhou, Xue-Dong; Zheng, Xin; Zhang, Ke-Ke; Wang, Shi-Da; Li, Yu-Qing; Cheng, Lei; Li, Ji-Yao; Xu, Xin; Li, Ming-Yun

    2016-01-01

    D-alanine (D-Ala) is an essential amino acid that has a key role in bacterial cell wall synthesis. Alanine racemase (Alr) is a unique enzyme that interconverts L-alanine and D-alanine in most bacteria, making this enzyme a potential target for antimicrobial drug development. Streptococcus mutans is a major causative factor of dental caries. The factors involved in the survival, virulence and interspecies interactions of S. mutans could be exploited as potential targets for caries control. The current study aimed to investigate the physiological role of Alr in S. mutans. We constructed alr mutant strain of S. mutans and evaluated its phenotypic traits and interspecies competitiveness compared with the wild-type strain. We found that alr deletion was lethal to S. mutans. A minimal supplement of D-Ala (150 μg·mL−1) was required for the optimal growth of the alr mutant. The depletion of D-alanine in the growth medium resulted in cell wall perforation and cell lysis in the alr mutant strain. We also determined the compromised competitiveness of the alr mutant strain relative to the wild-type S. mutans against other oral streptococci (S. sanguinis or S. gordonii), demonstrated using either conditioned medium assays or dual-species fluorescent in situ hybridization analysis. Given the importance and necessity of alr to the growth and competitiveness of S. mutans, Alr may represent a promising target to modulate the cariogenicity of oral biofilms and to benefit the management of dental caries. PMID:27740612

  15. A root-expressed L-phenylalanine:4-hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna.

    PubMed

    Bedewitz, Matthew A; Góngora-Castillo, Elsa; Uebler, Joseph B; Gonzales-Vigil, Eliana; Wiegert-Rininger, Krystle E; Childs, Kevin L; Hamilton, John P; Vaillancourt, Brieanne; Yeo, Yun-Soo; Chappell, Joseph; DellaPenna, Dean; Jones, A Daniel; Buell, C Robin; Barry, Cornelius S

    2014-09-01

    The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.

  16. Protective effects of caffeic acid phenethyl ester against acute radiation-induced hepatic injury in rats.

    PubMed

    Chu, JianJun; Zhang, Xiaojun; Jin, Liugen; Chen, Junliang; Du, Bin; Pang, Qingfeng

    2015-03-01

    Caffeic acid phenyl ester (CAPE) is a potent anti-inflammatory agent and it can eliminate the free radicals. The current study was intended to evaluate the protective effect of CAPE against the acute radiation-induced liver damage in rats. Male Sprague-Dawley rats were intraperitoneally administered with CAPE (30 mg/kg) for 3 consecutive days before exposing them to a single dose of 30 Gy of β-ray irradiation to upper abdomen. We found that pretreatment with CAPE significantly decreased the serum levels of alanine aminotransferase and aspartate aminotransferase and increased the activity of superoxide dismutase and glutathione. Histological evaluation further confirmed the protection of CAPE against radiation-induced hepatotoxicity. TUNEL assay showed that CAPE pretreatment inhibited hepatocyte apoptosis. Moreover, CAPE inhibited the nuclear transport of NF-κB p65 subunit, decreased the level of tumor necrosis factor-α, nitric oxide and inducible nitric oxide synthase. Taken together, these results suggest that pretreatment with CAPE offers protection against radiation-induced hepatic injury.

  17. Mechanism underlying mitochondrial protection of asiatic acid against hepatotoxicity in mice.

    PubMed

    Gao, Jing; Chen, Jin; Tang, Xinhui; Pan, Liya; Fang, Feng; Xu, Lizhi; Zhao, Xiaoning; Xu, Qiang

    2006-02-01

    Asiatic acid (AA) is one of the triterpenoid components of Terminalia catappa L., which has antioxidative, anti-inflammatory and hepatoprotective activity. This research focused on the mitochondrial protection of AA against acute liver injury induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN) in mice. It was found that pretreatment with 25, 50 or 100 mg kg(-1) AA significantly blocked the LPS + D-GalN-induced increase in both serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT) levels, which was confirmed by ultrastructural observation under an electron microscope, showing improved nuclear condensation, ameliorated mitochondrion proliferation and less lipid deposition. Meanwhile, different doses of AA could decrease both the transcription and the translation level of voltage-dependent anion channels (VDACs), the most important mitochondrial PTP component protein, and block the translocation of cytochrome c from mitochondria to cytosol. On the other hand, pre-incubation with 25, 50 and 100 microg mL(-1) AA inhibited the Ca(2+)-induced mitochondrial permeability transition (MPT), including mitochondrial swelling, membrane potential dissipation and releasing of matrix Ca(2+) in liver mitochondria separated from normal mice, indicating the direct role of AA on mitochondria. Collectively, the above data suggest that AA could protect liver from damage and the mechanism might be related to up-regulating mitochondrial VDACs and inhibiting the process of MPT.

  18. Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport

    PubMed Central

    Day, Pricilla E.; Ntani, Georgia; Crozier, Sarah R.; Mahon, Pam A.; Inskip, Hazel M.; Cooper, Cyrus; Harvey, Nicholas C.; Godfrey, Keith M.; Hanson, Mark A.; Lewis, Rohan M.; Cleal, Jane K.

    2015-01-01

    Introduction Maternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth. Methods RNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR. Results Increased placental LAT2 (p = 0.01), y+LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001). Conclusion A number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter

  19. Clinical Features of Lysosomal Acid Lipase Deficiency

    PubMed Central

    Burton, Barbara K.; Deegan, Patrick B.; Enns, Gregory M.; Guardamagna, Ornella; Horslen, Simon; Hovingh, Gerard K.; Lobritto, Steve J.; Malinova, Vera; McLin, Valerie A.; Raiman, Julian; Di Rocco, Maja; Santra, Saikat; Sharma, Reena; Sykut-Cegielska, Jolanta; Whitley, Chester B.; Eckert, Stephen; Valayannopoulos, Vassili; Quinn, Anthony G.

    2015-01-01

    Abstract Objective: The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. Methods: Investigators reviewed medical records of LAL D patients ages ≥5 years, extracted historical data, and obtained prospective laboratory and imaging data on living patients to develop a longitudinal dataset. Results: A total of 49 patients were enrolled; 48 had confirmed LAL D. Mean age at first disease-related abnormality was 9.0 years (range 0–42); mean age at diagnosis was 15.2 years (range 1–46). Twenty-nine (60%) were male patients, and 27 (56%) were <20 years of age at the time of consent/assent. Serum transaminases were elevated in most patients with 458 of 499 (92%) of alanine aminotransferase values and 265 of 448 (59%) of aspartate aminotransferase values above the upper limit of normal. Most patients had elevated low-density lipoprotein (64% patients) and total cholesterol (63%) at baseline despite most being on lipid-lowering therapies, and 44% had high-density lipoprotein levels below the lower limit of normal. More than half of the patients with liver biopsies (n = 31, mean age 13 years) had documented evidence of steatosis (87%) and/or fibrosis (52%). Imaging assessments revealed that the median liver volume was ∼1.15 multiples of normal (MN) and median spleen volume was ∼2.2 MN. Six (13%) patients had undergone a liver transplant (ages 9–43.5 years). Conclusion: This study provides the largest longitudinal case review of patients with LAL D and confirms that LAL D is predominantly a pediatric disease causing early and progressive hepatic dysfunction associated with dyslipidemia that often leads to liver failure and transplantation. PMID:26252914

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

    PubMed

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

    2002-02-15

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

  1. 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.

  2. Dietary verbascoside supplementation in donkeys: effects on milk fatty acid profile during lactation, and serum biochemical parameters and oxidative markers.

    PubMed

    D'Alessandro, A G; Vizzarri, F; Palazzo, M; Martemucci, G

    2017-03-07

    Various uses of donkeys' milk have been recently proposed for human consumption on the basis of its nutritional characteristics. Improvements in milk fatty acid profile and animal oxidative status can be induced through dietary supplementation of phenolic compounds. The study aimed to evaluate in donkeys the effects of dietary supplementation with verbascoside (VB) on: (i) the fatty acid profile and vitamins A and E contents of milk during a whole lactation, and (ii) blood biochemical parameters and markers of oxidative status of the animals. At foaling, 12 lactating jennies were subdivided into two groups (n 6): control, without VB supplement; VB, receiving a lipid-encapsulated VB supplement. Gross composition, fatty acid profile and vitamins A and E contents in milk were assessed monthly over the 6 months of lactation. Serum total cholesterol, high-density lipoproteins cholesterol and low-density lipoproteins cholesterol, tryglicerides, non-esterified fatty acid, bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase, reactive oxygen metabolites, thiobarbituric acid reactive substances (TBARs), vitamin A and vitamin E were evaluated at 8 days after foaling (D0) and then at D90, D105 and D120 of lactation. In milk, the VB supplementation decreased the saturated fatty acids (P<0.05) and increased the monounsaturated fatty acids (P<0.05), and vitamins A and E (P<0.01) values. On the serum parameters, the VB supplementation decreased total cholesterol (P<0.01), tryglicerides, bilirubin, ALT and TBARs, and increased (P<0.01) vitamin E. In conclusion, the VB dietary supplementation affects the nutritional quality of donkey's milk with a benefit on the oxidative status and serum lipidic profile of the animals.

  3. L,L-Diaminopimelate Aminotransferase from Chlamydomonas reinhardtii: A Target for Algaecide Development

    PubMed Central

    Dobson, Renwick C. J.; Girón, Irma; Hudson, André O.

    2011-01-01

    In some bacterial species and photosynthetic cohorts, including algae, the enzyme l,l-diaminopimelate aminotransferase (DapL) (E.C. 2.6.1.83) is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA) to l,l-diaminopimelate (l,l-DAP), in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL). The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and l,l-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid l-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides. PMID:21633707

  4. L,L-diaminopimelate aminotransferase from Chlamydomonas reinhardtii: a target for algaecide development.

    PubMed

    Dobson, Renwick C J; Girón, Irma; Hudson, André O

    2011-01-01

    In some bacterial species and photosynthetic cohorts, including algae, the enzyme L,L-diaminopimelate aminotransferase (DapL) (E.C. 2.6.1.83) is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA) to L,L-diaminopimelate (L,L-DAP), in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL). The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and L,L-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid L-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.

  5. Characterization of kynurenine aminotransferase III, a novel member of a phylogenetically conserved KAT family.

    PubMed

    Yu, Ping; Li, Zhengsheng; Zhang, Ling; Tagle, Danilo A; Cai, Tao

    2006-01-03

    Kynurenine aminotransferase (KAT) is an enzyme responsible for synthesis of kynurenic acid (KYNA), a well established neuroprotective and anticonvulsant agent, involved in synaptic transmission and implicated in the pathophysiology of schizophrenia, Huntington's disease and other neurological disorders. We have shown previously that kat2-/- mice had lower hippocampal KYNA levels and were more hyperactive than wild-type mice. However, these abnormalities occur early and are transitory coinciding with restoration of KYNA levels, suggesting that compensatory changes or ontogenetic expression of another unknown homolog may account for the normalization of KYNA levels in the adult kat2-/- mice brain. Here, we report the isolation of a novel KAT molecule, kat3, from mouse and human brain cDNA libraries. The encoded 454 amino acids of human KAT III share 64.8% similarity to that of KAT I and 30.1% to KAT II. Northern blot analysis demonstrated that kat3 mRNA is widely expressed but with higher expression levels in liver, kidney, heart, and neuroendocrine tissues. RT-PCR and Northern analysis showed that kat3 expression starts as early as postnatal day (PND) 7 and peaks in adult. The mRNA level of kat3 and kat1 when measured together is significantly higher at PND 60 in kat2-/- mice than those of wild-type mice indicating possible co-regulation of expression levels. RNA-interference (RNAi) directed towards transcripts for either R03A10.4 or F28H6.3 in Caenorhabditis elegans which are kat1 and kat3 orthologs, respectively, did not result in any gross abnormalities. Our results show that upregulation of kat3 and kat1 may be responsible for the phenotypic rescue on kat2-/- mice.

  6. Biochemical and phenotypic abnormalities in kynurenine aminotransferase II-deficient mice.

    PubMed

    Yu, Ping; Di Prospero, Nicholas A; Sapko, Michael T; Cai, Tao; Chen, Amy; Melendez-Ferro, Miguel; Du, Fu; Whetsell, William O; Guidetti, Paolo; Schwarcz, Robert; Tagle, Danilo A

    2004-08-01

    Kynurenic acid (KYNA) can act as an endogenous modulator of excitatory neurotransmission and has been implicated in the pathogenesis of several neurological and psychiatric diseases. To evaluate its role in the brain, we disrupted the murine gene for kynurenine aminotransferase II (KAT II), the principal enzyme responsible for the synthesis of KYNA in the rat brain. mKat-2(-/-) mice showed no detectable KAT II mRNA or protein. Total brain KAT activity and KYNA levels were reduced during the first month but returned to normal levels thereafter. In contrast, liver KAT activity and KYNA levels in mKat-2(-/-) mice were decreased by >90% throughout life, though no hepatic abnormalities were observed histologically. KYNA-associated metabolites kynurenine, 3-hydroxykynurenine, and quinolinic acid were unchanged in the brain and liver of knockout mice. mKat-2(-/-) mice began to manifest hyperactivity and abnormal motor coordination at 2 weeks of age but were indistinguishable from wild type after 1 month of age. Golgi staining of cortical and striatal neurons revealed enlarged dendritic spines and a significant increase in spine density in 3-week-old mKat-2(-/-) mice but not in 2-month-old animals. Our results show that gene targeting of mKat-2 in mice leads to early and transitory decreases in brain KAT activity and KYNA levels with commensurate behavioral and neuropathological changes and suggest that compensatory changes or ontogenic expression of another isoform may account for the normalization of KYNA levels in the adult mKat-2(-/-) brain.

  7. 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.

  8. 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.

  9. Membrane topology of the electrogenic aspartate-alanine antiporter AspT of Tetragenococcus halophilus.

    PubMed

    Nanatani, Kei; Ohonishi, Fumito; Yoneyama, Hiroshi; Nakajima, Tasuku; Abe, Keietsu

    2005-03-04

    AspT is an electrogenic aspartate:alanine exchange protein that represents the vectorial component of a proton-motive metabolic cycle found in some strains of Tetragenococcus halophilus. AspT is the sole member of a new family, the Aspartate: Alanine Exchanger (AAE) family, in secondary transporters, according to the computational classification proposed by Saier et al. (http://www.biology.ucsd.edu/~msaier/transport/). We analyzed the topology of AspT biochemically, by using fusion methods in combination with alkaline phosphatase or beta-lactamase. These results suggested that AspT has a unique topology; 8 TMS, a large cytoplasmic loop (183 amino acids) between TMS5 and TMS6, and N- and C-termini that both face the periplasm. These results demonstrated a unique 2D-structure of AspT as the novel AAE family.

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

    PubMed

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

    2000-01-01

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

  11. β-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling.

    PubMed

    Engskog, Mikael K R; Ersson, Lisa; Haglöf, Jakob; Arvidsson, Torbjörn; Pettersson, Curt; Brittebo, Eva

    2017-02-04

    β-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC-MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

  12. Hepatic serine and alanine metabolism during endotoxin-induced fever in sheep.

    PubMed Central

    Southorn, B G; Thompson, J R

    1987-01-01

    Time course changes in plasma amino acid concentrations and the hepatic metabolism of serine and alanine were measured in six mature wethers during endotoxin-induced fever. In separate trials, the animals' responses to injections of saline and endotoxin were measured. The endotoxin was from Escherichia coli serotype 055:B5 and was injected intravenously (4 micrograms/kg body weight). Liver biopsies were obtained from the sheep at 6 h postinjection during both endotoxin and saline injection trials. Rectal temperature in the endotoxin treated animals was increased (P less than 0.05, above that in control animals from 4.25 h to 9 h postinjection, with a maximum rise of 2.43 degrees C at 5.5 h postinjection. Glucose concentration in jugular plasma decreased (P less than 0.05) by 3 h postinjection and remained depressed throughout the 24 h postinjection sampling period. Plasma serine concentration was decreased (P less than 0.05) by 3 h postinjection. Plasma alanine concentration was decreased significantly (P less than 0.05) only at 24 h postinjection. Endotoxin injection increased (P less than 0.05) hepatic oxidation of 14C-serine (162%) and the net incorporation of 14C-serine carbon into hepatic protein (173%) and glycogen (275%). The net incorporation of 14C-alanine carbon into hepatic protein (172%) and glycogen (323%) were increased (P less than 0.05) by endotoxin injection, while alanine oxidation was not affected by endotoxin treatment (P greater than 0.05). The increased hepatic use of serine may explain, in part, the dramatic decrease in plasma concentrations of this amino acid following endotoxin injection into sheep. PMID:3115552

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

    PubMed

    Zahmatkesh, Saeed; Hajipour, Abdol R

    2010-04-01

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

  14. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity

    PubMed Central

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury. PMID:26208104

  15. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

    PubMed

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.

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

    PubMed

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

    2012-08-09

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

  17. 3-Hydroxykynurenine transaminase identity with alanine glyoxylate transaminase. A probable detoxification protein in Aedes aegypti.

    PubMed

    Han, Qian; Fang, Jianmin; Li, Jianyong

    2002-05-03

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

  18. Crystal and molecular structure of N-(4-nitrophenyl)-β-alanine--its vibrational spectra and theoretical calculations.

    PubMed

    Marchewka, M K; Drozd, M; Janczak, J

    2011-08-15

    The N-(4-nitrophenyl)-β-alanine in crystalline form directly by the addition of 4-nitroaniline to the acrylic acid in aqueous solution has been obtained. The title β-alanine derivative crystallizes in the P2(1)/c space group of monoclinic system with four molecules per unit cell. The X-ray geometry of β-alanine derivative molecule has been compared with those obtained by molecular orbital calculations corresponding to the gas phase. In the crystal the molecules related by an inversion center interact via symmetrically equivalent O-H···O hydrogen bonds with O···O distance of 2.656(2) Å forming a dimeric structure. The dimers of β-alanine derivative weakly interact via N-H···O hydrogen bonds between the H atom of β-amine groups and one of O atom of nitro groups. The room temperature powder vibrational (infrared and Raman) measurements are in accordance with the X-ray analysis. In aqueous solution of 4-nitroaniline and acrylic acid, the double CC bond of vinyl group of acrylic acid breaks as result of 4-nitroaniline addition.

  19. Oleanolic acid alters bile acid metabolism and produces cholestatic liver injury in mice

    SciTech Connect

    Liu, Jie; Lu, Yuan-Fu; Zhang, Youcai; Wu, Kai Connie; Fan, Fang; Klaassen, Curtis D.

    2013-11-01

    Oleanolic acid (OA) is a triterpenoids that exists widely in plants. OA is effective in protecting against hepatotoxicants. Whereas a low dose of OA is hepatoprotective, higher doses and longer-term use of OA produce liver injury. This study characterized OA-induced liver injury in mice. Adult C57BL/6 mice were given OA at doses of 0, 22.5, 45, 90, and 135 mg/kg, s.c., daily for 5 days, and liver injury was observed at doses of 90 mg/kg and above, as evidenced by increases in serum activities of alanine aminotransferase and alkaline phosphatase, increases in serum total bilirubin, as well as by liver histopathology. OA-induced cholestatic liver injury was further evidenced by marked increases of both unconjugated and conjugated bile acids (BAs) in serum. Gene and protein expression analysis suggested that livers of OA-treated mice had adaptive responses to prevent BA accumulation by suppressing BA biosynthetic enzyme genes (Cyp7a1, 8b1, 27a1, and 7b1); lowering BA uptake transporters (Ntcp and Oatp1b2); and increasing a BA efflux transporter (Ostβ). OA increased the expression of Nrf2 and its target gene, Nqo1, but decreased the expression of AhR, CAR and PPARα along with their target genes, Cyp1a2, Cyp2b10 and Cyp4a10. OA had minimal effects on PXR and Cyp3a11. Taken together, the present study characterized OA-induced liver injury, which is associated with altered BA homeostasis, and alerts its toxicity potential. - Highlights: • Oleanolic acid at higher doses and long-term use may produce liver injury. • Oleanolic acid increased serum ALT, ALP, bilirubin and bile acid concentrations. • OA produced feathery degeneration, inflammation and cell death in the liver. • OA altered bile acid homeostasis, affecting bile acid synthesis and transport.

  20. A general and practical palladium-catalyzed monoarylation of β-methyl C(sp³)-H of alanine.

    PubMed

    Chen, Kai; Zhang, Shuo-Qing; Xu, Jing-Wen; Hu, Fang; Shi, Bing-Feng

    2014-11-21

    A palladium-catalyzed monoarylation of β-methyl C(sp(3))-H of an alanine derivative with aryl iodides using an 8-aminoquinoline auxiliary is described. The reaction is highly efficient, scalable and compatible with a variety of functional groups with complete retention of chirality, providing a general and practical access to various β-aryl-α-amino acids. The synthetic potential of this protocol is further demonstrated in the sequential synthesis of diverse β-branched α-amino acids.

  1. Comparison of triglycerides and phospholipids as supplemental sources of dietary long-chain polyunsaturated fatty acids in piglets.

    PubMed

    Mathews, Susan A; Oliver, William T; Phillips, Oulayvanh T; Odle, Jack; Diersen-Schade, Deborah A; Harrell, Robert J

    2002-10-01

    Addition of arachidonic acid (AA) and docosahexaenoic acid (DHA) to infant formula promotes visual and neural development. This study was designed to determine whether the source of dietary long-chain polyunsaturated fatty acids (LCPUFA) affected overall animal health and safety. Piglets consumed ad libitum from 1 to 16 d of age a skim milk-based formula with different fat sources added to provide 50% of the metabolizable energy. Treatment groups were as follows: control (CNTL; no added LCPUFA), egg phospholipid (PL), algal/fungal triglyceride (TG) oils, TG plus PL (soy lecithin source) added to match phospholipid treatment (TG + PL) and essential fatty acid deficient (EFAD). Formulas with LCPUFA provided 0.6 and 0.3 g/100 g total fatty acids as AA and DHA, respectively. CNTL piglets had 40% longer ileal villi than PL piglets (P < 0.03), but the TG group was not different from the CNTL group. Gross liver histology did not differ among any of the formula-fed groups (P > 0.1). Apparent dry matter digestibility was 10% greater in CNTL, TG and TG + PL groups compared with PL piglets (P < 0.002). No differences in alanine aminotransferase were detected among treatments, but aspartate aminotransferase was elevated (P < 0.03) in PL piglets compared with TG + PL piglets. Total plasma AA concentration was greater in the TG group compared with CNTL piglets (P < 0.05). Total plasma DHA concentrations were greater in TG piglets compared with PL (P < 0.06) or CNTL (P < 0.02) piglets. These data demonstrate that the algal/fungal TG sources of DHA and AA may be a more appropriate supplement for infant formulas than the egg PL source based on piglet plasma fatty acid profiles and apparent dry matter digestibilities.

  2. Cloning Two Genes for Nicotianamine Aminotransferase, a Critical Enzyme in Iron Acquisition (Strategy II) in Graminaceous Plants

    PubMed Central

    Takahashi, Michiko; Yamaguchi, Hirotaka; Nakanishi, Hiromi; Shioiri, Takayuki; Nishizawa, Naoko-Kishi; Mori, Satoshi

    1999-01-01

    Nicotianamine aminotransferase (NAAT), the key enzyme involved in the biosynthesis of mugineic acid family phytosiderophores (MAs), catalyzes the amino transfer of nicotianamine (NA). MAs are found only in graminaceous plants, although NA has been detected in every plant so far investigated. Therefore, this amino transfer reaction is the first step in the unique biosynthesis of MAs that has evolved in graminaceous plants. NAAT activity is dramatically induced by Fe deficiency and suppressed by Fe resupply. Based on the protein sequence of NAAT purified from Fe-deficient barley (Hordeum vulgare) roots, two distinct cDNA clones encoding NAAT, naat-A and naat-B, were identified. Their deduced amino acid sequences were homologous to several aminotransferases, and shared consensus sequences for the pyridoxal phosphate-binding site lysine residue and its surrounding residues. The expression of both naat-A and naat-B is increased in Fe-deficient barley roots, while naat-B has a low level of constitutive expression in Fe-sufficient barley roots. No detectable mRNA from either naat-A or naat-B was present in the leaves of either Fe-deficient or Fe-sufficient barley. One genomic clone with a tandem array of naat-B and naat-A in this order was identified. naat-B and naat-A each have six introns at the same locations. The isolation of NAAT genes will pave the way to understanding the mechanism of the response to Fe in graminaceous plants, and may lead to the development of cultivars tolerant to Fe deficiency that can grow in calcareous soils. PMID:10557244

  3. Taurine-like GABA aminotransferase inhibitors prevent rabbit brain slices against oxygen-glucose deprivation-induced damage.

    PubMed

    Ricci, Lorenzo; Valoti, Massimo; Sgaragli, Giampietro; Frosini, Maria

    2012-06-01

    The activation of the GABAergic system has been shown to protect brain tissues against the damage that occurs after cerebral ischaemia. On the other hand, the taurine analogues (±)Piperidine-3-sulphonic- (PSA), 2-aminoethane phosphonic- (AEP), 2-(N-acetylamino) cyclohexane sulfonic-acids (ATAHS) and 2-aminobenzene sulfonate-acids (ANSA) have been reported to block GABA metabolism by inhibiting rabbit brain GABA aminotransferase and to increase GABA content in rabbit brain slices. The present investigation explored the neuroprotection provided by GABA, Vigabatrin (VIGA) and taurine analogues in the course of oxygen-glucose deprivation and reperfusion induced damage of rabbit brain slices. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, measured as the index of cell swelling. GABA (30-300 μM) and VIGA (30-300 μM) significantly antagonised LDH and glutamate release, as well as tissue water gain caused by oxygen-glucose deprivation and reperfusion. Lower (1-10 μM) or higher concentrations (up to 3,000 μM) were ineffective. ANSA, PSA and ATAHS significantly reduced glutamate and LDH release and tissue water gain in a range of concentrations between 30 and 300 μM. Lower (0-10 μM) or higher (up to 3,000 μM) concentrations were ineffective. Both mechanisms suggest hormetic ("U-shaped") effects. These results indicate that the GABAergic system activation performed directly by GABA or indirectly through GABA aminotransferase inhibition is a promising approach for protecting the brain against ischemia and reperfusion-induced damage.

  4. Ferulic Acid against Cyclophosphamide-Induced Heart Toxicity in Mice by Inhibiting NF-κB Pathway

    PubMed Central

    Song, Yafan; Zhang, Chunyan; Wang, Congxia; Zhao, Ling; Wang, Zheng; Dai, Zhijun; Lin, Shuai; Kang, Huafeng; Ma, Xiaobin

    2016-01-01

    The purpose of the present study was to elucidate the protective effects of ferulic acid (FA) against cyclophosphamide- (CTX-) induced changes in mice. Forty-eight male ICR mice were divided into four groups. Control group was intraperitoneally (i.p.) injected with 200 μL of phosphate buffer saline (PBS). Model group was intraperitoneally injected with a single dose of CTX (200 mg/kg). FA (50 mg/kg) and FA (100 mg/kg) groups were intraperitoneally injected with a single dose of CTX (200 mg/kg) followed by the intragastric treatment with FA (50, 100 mg/kg) for 7 consecutive days. After 12 d, the mice were sacrificed to analyze the hematological, biochemical, histological parameters and mechanism research. The results indicated that FA significantly decreased the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) in CTX-injected mice. In addition, FA effectively reduced the total numbers of white blood cells (WBCs), red blood cells, platelets, and hemoglobin content. FA also obviously attenuated the histological changes of the heart tissues caused by CTX. Moreover, Western blot demonstrated that FA inhibited the phosphorylations of NF-κB signaling pathway in CTX-stimulated cardiac tissues. In conclusion, FA might be considered as an effective agent in the amelioration of the heart toxicity resulting from CTX treatment. PMID:26881001

  5. Arsenic induced toxicity in broiler chicks and its alleviation with ascorbic acid: a toxico-patho-biochemical study

    USGS Publications Warehouse

    Khan, Ahrar; Sharaf, Rabia; Khan, Muhammad Zargham; Saleemi, Muhammad Kashif; Mahmood, Fazal

    2013-01-01

    To find out toxico-pathological effects of arsenic (As) and ameliorating effect of ascorbic acid (Vit C), broilers birds were administered 50 and 250 mg/kg arsenic and Vit C, respectively alone/in combination. As-treated birds exhibited severe signs of toxicity such as dullness, depression, increased thirst, open mouth breathing and watery diarrhea. All these signs were partially ameliorated with the treatment of Vit C. As-treated birds showed a significant decrease in serum total proteins while serum enzymes, urea and creatinine were significantly increased. Alkaline phosphatase and lactate dehydrogenase completely whereas proteins, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine were partial ameliorated in birds treated with As+Vit C as compared to As-treated and control birds. Pale and hemorrhagic liver and swollen kidneys were observed in As-treated birds. Histopathologically, liver exhibited congestion and cytoplasmic vacuolation while in kidneys, condensation of tubular epithelium nuclei, epithelial necrosis, increased urinary spaces, sloughing of tubules from basement membrane and cast deposition were observed in As-treated birds. Pathological lesions were partially ameliorated with the treatment of Vit C. It can be concluded that arsenic induces biochemical and histopathological alterations in broiler birds; however, these toxic effects can be partially attenuated by Vit C.

  6. Relationship Between Hepatic Steatosis and the Elevation of Aminotransferases in HBV-Infected Patients With HBe-Antigen Negativity and a Low Viral Load

    PubMed Central

    Enomoto, Hirayuki; Aizawa, Nobuhiro; Nishikawa, Hiroki; Ikeda, Naoto; Sakai, Yoshiyuki; Takata, Ryo; Hasegawa, Kunihiro; Nakano, Chikage; Nishimura, Takashi; Yoh, Kazunori; Ishii, Akio; Takashima, Tomoyuki; Iwata, Yoshinori; Iijima, Hiroko; Nishiguchi, Shuhei

    2016-01-01

    Abstract Nonalcoholic fatty liver disease has been suggested to be associated with alanine aminotransferase (ALT) elevation in hepatitis B virus (HBV)-infected patients with HBe antigen (HBeAg)-negativity and a low HBV-DNA level. However, few studies have evaluated the association according to histological findings of the liver. Among a total of 198 HBV-infected patients who received a percutaneous liver biopsy, we studied the histological and laboratory findings of HBeAg-negative patients without receiving nucleoside/nucleotide analogues treatment (N = 70) in order to evaluate whether hepatic steatosis and its related metabolic disorders were associated with an elevation in ALT levels in HBeAg-negative patients. In HBeAg-negative patients with a high serum HBV-DNA level (≥2000 IU/mL), the level of HBV-DNA was the only significant factor related to ALT elevation. However, in HBeAg-negative patients with a low HBV-DNA level, the serum ferritin level, and histologically observed hepatic steatosis were significantly associated factors with ALT elevation. When we evaluated 2 metabolic variables (serum ferritin and fasting insulin) that are suggested to be relevant to the presence of progressive disease in Japanese patients, we found that the rate of metabolic disorders was significantly higher among patients with a high ALT level and a low HBV-DNA level than it was among those with other conditions. The triglyceride level and the frequency of moderate or severe hepatic steatosis were significantly higher in patients with a low HBV-DNA level than in those with a high HBV-DNA level. Histologically proven hepatic steatosis and its related metabolic disorders are suggested to be involved in the elevation of aminotransferases of HBeAg-negative patients, particularly those with low HBV-DNA levels. PMID:27124068

  7. Relaxed Evolution in the Tyrosine Aminotransferase Gene Tat in Old World Fruit Bats (Chiroptera: Pteropodidae)

    PubMed Central

    Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M.; Zhang, Shuyi

    2014-01-01

    Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet. PMID:24824435

  8. Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae).

    PubMed

    Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M; Zhang, Shuyi

    2014-01-01

    Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

  9. 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.

  10. 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.

  11. 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.

  12. Taurine and beta-alanine act on both GABA and glycine receptors in Xenopus oocyte injected with mouse brain messenger RNA.

    PubMed

    Horikoshi, T; Asanuma, A; Yanagisawa, K; Anzai, K; Goto, S

    1988-09-01

    The responding pathway (process from agonist binding to channel opening) of taurine and beta-alanine was investigated in Xenopus oocytes injected with mouse brain poly(A)+ RNA. Responses to gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine were induced in oocytes injected with poly(A)+ RNA extracted from 3 regions, cerebrum, cerebellum and brainstem of the mouse brain. From comparison, responses to these 4 inhibitory amino acids in each regional poly(A)+ RNA-injected oocytes were categorized into at least 3 groups: (1) GABA, (2) glycine, and (3) taurine and beta-alanine. No cross-desensitization was observed between GABA response and glycine response, but taurine and beta-alanine responses cross-desensitized both the GABA and glycine responses. Taurine and beta-alanine responses were partially inhibited by the GABA antagonist, bicuculline, and also by the glycine antagonist, strychnine. The results suggest that the taurine or the beta-alanine response in the brain is caused through both the GABA receptor and the glycine receptor.

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

    PubMed

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

    2015-01-01

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

  14. Folic acid protects against lead acetate-induced hepatotoxicity by decreasing NF-κB, IL-1β production and lipid peroxidation mediataed cell injury.

    PubMed

    Abd Allah, Eman S H; Badary, Dalia M

    2017-03-01

    Folic acid plays an important role in cellular metabolic activities. The present study was designed to investigate the protective effect of folic acid against lead acetate-induced hepatotoxicity. Twenty four male Wistar albino rats were randomly divided into four groups, six animals each. Negative control group received the vehicle, positive control group received 1mg/kg folic acid for five consecutive days/week for 4 weeks orally, lead-exposed group received 10mg/kg lead acetate intraperitoneally (IP) for five consecutive days/week for 4 weeks, and lead-treated group received 10mg/kg lead acetate IP and 1mg/kg folic acid orally for five consecutive days/week for 4 weeks concurrently. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ- glutamyltransferase (GGT) were measured. Hepatic total peroxide and interleukin-1β (IL-1β) were also investigated. Histopathological studies using hematoxylin-eosin (H&E) and periodic acid shiff's (PAS) were carried out. The expression of nuclear factor kappa B (NF-κB) was evaluated using immunohistochemistry. Serum AST, ALT and GGT and hepatic total peroxide and IL-1β were significantly increased in lead-exposed group and were positively correlated with hepatic lead level. Moreover, lead-exposed rats showed hydropic degeneration, nuclear vesiculation, high lymphocytic infiltration, depletion of glycogen content and NF-κB expression. Concomitant folic acid administration resulted in a significant alleviation of biochemical and structural alteration-induced by lead. This was associated with reduction of hepatic total peroxide and IL-1β and reduction of NF-κB expression. In conclusion, folic acid protects against lead acetate-induced hepatotoxicity by decreasing NF-κB, IL-1β production and lipid peroxidation mediataed cell injury.

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

    PubMed

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

    2004-10-26

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

  16. 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.

  17. Thiophenyl-substituted triazolyl-thione L-alanine: asymmetric synthesis, aggregation and biological properties.

    PubMed

    Saghyan, Ashot S; Simonyan, Hayarpi M; Petrosyan, Satenik G; Geolchanyan, Arpine V; Roviello, Giovanni N; Musumeci, Domenica; Roviello, Valentina

    2014-10-01

    In this work, we report the asymmetric synthesis and characterization of an artificial amino acid based on triazolyl-thione L-alanine, which was modified with a thiophenyl-substituted moiety, as well as in vitro studies of its nucleic acid-binding ability. We found, by dynamic light scattering studies, that the synthetic amino acid was able to form supramolecular aggregates having a hydrodynamic diameter higher than 200 nm. Furthermore, we demonstrated, by UV and CD experiments, that the heteroaromatic amino acid, whose enzymatic stability was demonstrated by HPLC analysis also after 24 h of incubation in human serum, was able to bind a RNA complex, which is a feature of biomedical interest in view of innovative antiviral strategies based on modulation of RNA-RNA molecular recognition.

  18. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial

    PubMed Central

    Neuschwander-Tetri, Brent A; Loomba, Rohit; Sanyal, Arun J; Lavine, Joel E; Van Natta, Mark L; Abdelmalek, Manal F; Chalasani, Naga; Dasarathy, Srinivasan; Diehl, Anna Mae; Hameed, Bilal; Kowdley, Kris V; McCullough, Arthur; Terrault, Norah; Clark, Jeanne M; Tonascia, James; Brunt, Elizabeth M; Kleiner, David E; Doo, Edward

    2015-01-01

    Summary Background The bile acid derivative 6-ethylchenodeoxycholic acid (obeticholic acid) is a potent activator of the farnesoid X nuclear receptor that reduces liver fat and fibrosis in animal models of fatty liver disease. We assessed the efficacy of obeticholic acid in adult patients with non-alcoholic steatohepatitis. Methods We did a multicentre, double-blind, placebo-controlled, parallel group, randomised clinical trial at medical centres in the USA in patients with non-cirrhotic, non-alcoholic steatohepatitis to assess treatment with obeticholic acid given orally (25 mg daily) or placebo for 72 weeks. Patients were randomly assigned 1:1 using a computer-generated, centrally administered procedure, stratified by clinical centre and diabetes status. The primary outcome measure was improvement in centrally scored liver histology defined as a decrease in non-alcoholic fatty liver disease activity score by at least 2 points without worsening of fibrosis from baseline to the end of treatment. A planned interim analysis of change in alanine aminotransferase at 24 weeks undertaken before end-of-treatment (72 weeks) biopsies supported the decision to continue the trial (relative change in alanine aminotransferase −24%, 95% CI −45 to −3). A planned interim analysis of the primary outcome showed improved efficacy of obeticholic acid (p=0·0024) and supported a decision not to do end-of-treatment biopsies and end treatment early in 64 patients, but to continue the trial to obtain the 24-week post-treatment measures. Analyses were done by intention-to-treat. This trial was registered with ClinicalTrials.gov, number NCT01265498. Findings Between March 16, 2011, and Dec 3, 2012, 141 patients were randomly assigned to receive obeticholic acid and 142 to placebo. 50 (45%) of 110 patients in the obeticholic acid group who were meant to have biopsies at baseline and 72 weeks had improved liver histology compared with 23 (21%) of 109 such patients in the placebo group

  19. Effect of combined therapy of diabinese and nicotinic acid on liver enzymes in rabbits with dithizone-induced diabetes.

    PubMed

    Monago, Comfort C; Onwuka, Frank; Osaro, Erhabor

    2010-01-01

    The effects of diabinese, a known antidiabetic drug, and the combined effects of diabinese and nicotinic acid, a vitamin and antilipidemic drug, were studied in rabbits with dithizone-induced diabetes. Side effects of diabinese include hypoglycemia and liver toxicity. Dithizone was used to induce partial experimental diabetes and to increase blood glucose significantly (P < 0.05) by 31.3%, 23.5%, 19.5, 24.7%, and 23.9% in groups A (single therapy of diabinese 10 mg/kg body weight), B (10 mg of diabinese and nicotinic acid 150 mg/kg), C (10 mg diabinese and nicotinic acid 200 mg/kg), D (10 mg diabinese and nicotinic acid 250 mg/kg) and E control (distilled water 5 mL), respectively. Dithizone administration also increased bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels by 28.9%-35.6%, 41.2%-54.8%, 40.1%-46.1%, and 60.9%-68.4%, respectively. Diabinese monotherapy reduced bilirubin levels, while combined therapy reduced glucose, ALP, AST, and ALT levels more than single therapy. Reduction from the hyperglycemic level 48 hours after drug administration was 20.0%, 24.6%, 41.0%, and 42.0% for groups A, B, C, and D, respectively, and was concentration-dependent. Also, combined therapy produced a significant (P < 0.05) decrease in AST and ALT levels, especially at 72 hours after drug administration, but did not affect ALP levels. No significant changes in glucose, bilirubin, ALP, AST, and ALT levels were observed in Group E (control). This study shows that liver toxicity and the hypoglycemic side effects of diabinese could be managed by the concomitant administration of nicotinic acid.

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed

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

    2002-06-01

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

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

    PubMed

    Begum, N A; Datta, A G

    1992-08-18

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

  3. Hepatoprotective Effects of Schisandra sphenanthera Extract against Lithocholic Acid-Induced Cholestasis in Male Mice Are Associated with Activation of the Pregnane X Receptor Pathway and Promotion of Liver Regeneration.

    PubMed

    Zeng, Hang; Li, Dongshun; Qin, Xiaoling; Chen, Pan; Tan, Huasen; Zeng, Xuezhen; Li, Xi; Fan, Xiaomei; Jiang, Yiming; Zhou, Yawen; Chen, Yixin; Wang, Ying; Huang, Min; Bi, Huichang

    2016-03-01

    We previously reported that the ethanol extract of Schisandra sphenanthera [Wuzhi (WZ) tablet] significantly protects against acetaminophen-induced hepatoxicity. However, whether WZ exerts a protective effect against cholestasis remains unclear. In this study, the protective effect of WZ on lithocholic acid (LCA)-induced intrahepatic cholestasis in mice was characterized and the involved mechanisms were investigated. WZ pretreatment (350 mg/kg) with LCA significantly reversed liver necrosis and decreased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity. More importantly, serum total bile acids and total bilirubin were also remarkably reduced. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis showed that hepatic expression of pregnane X receptor (PXR) target genes such as CYP3A11 and UDP-glucuronosyltransferase (UGT) 1A1 were significantly increased by WZ treatment. Luciferase assays performed in LS174T cells illustrated that WZ extract and its six bioactive lignans could all activate human PXR. In addition, WZ treatment significantly promoted liver regeneration via inhibition of p53/p21 to induce cell proliferation-associated proteins such as cyclin D1 and proliferating cell nuclear antigen. In conclusion, WZ has a protective effect against LCA-induced intrahepatic cholestasis, partially owing to activation of the PXR pathway and promotion of liver regeneration.

  4. Regulation of pyruvate dehydrogenase during infusion of fatty acids of varying chain lengths in the perfused rat heart.

    PubMed

    Latipää, P M; Peuhkurinen, K J; Hiltunen, J K; Hassinen, I E

    1985-12-01

    The effects of a homologous series of fatty acids with a chain length of two to eight on the rate of pyruvate oxidation and covalent interconversions of the pyruvate dehydrogenase complex (PDH) were studied in isolated perfused rat hearts. In the Langendorff-perfused heart beating at 5 Hz against an aortic pressure of 59 mmHg (7.85 kPa), a positive linear correlation was found between the fraction of PDH existing in the active non-phosphorylated form of pyruvate dehydrogenase complex (PDHa) and the pyruvate oxidation rate until the PDHa fraction increased to 48%. This value resulted in a saturation of the citric acid cycle and further activation did not increase the metabolic flux. The PDHa content of the tissue was higher during infusion of odd carbon number fatty acids than during infusion of even carbon number fatty acids. Propionate caused an almost maximal (93%) activation of PDH. A negative correlation was found between the mitochondrial NADH/NAD+ ratio and the PDHa content. A negative correlation was also found between the acetyl-CoA/CoA ratio and the tissue PDHa content. The rate of labelled CO2 production, the specific radioactivity of tissue alanine and the metabolic balance sheet demonstrated that the alanine aminotransferase reaction in the total tissue does not reach equilibrium with the mitochondrial pyruvate pool during propionate oxidation, but the equilibrium is reached during the oxidation of even-number carbon fatty acids. This suggests that pyruvate is formed from propionate-derived metabolites also in the cytosol, although the primary metabolism of propionate occurs in the mitochondria. The results indicate that the rate of pyruvate oxidation in the myocardium is mainly regulated by covalent interconversion of PDH. During propionate oxidation the PDHa content in the tissue can increase beyond the point of saturation of the citric acid cycle and this indicates that feedback inhibition of the enzyme is rate-determining under these conditions.

  5. β-Alanine as an Ethylene Precursor. Investigations Towards Preparation, and Properties, of a Soluble Enzyme System From a Subcellular Particulate Fraction of Bean Cotyledons 1

    PubMed Central

    Stinson, Robert A.; Spencer, Mary

    1969-01-01

    A method is described for the preparation, from a subcellular particulate fraction of wax bean cotyledons, of a soluble enzyme system that is capable of converting β-alanine to ethylene. In the presence of ATP, CoA, thiamine pyrophosphate, MgSO4, and pyridoxal phosphate, ethylene production is maximum at a 0.5 mm concentration of β-alanine. The system exhibits a pH optimum at 7.0 but when the pH is raised above 8, evolution of the volatile again increases and continues to do so up to pH 12. The enzyme system is stimulated by either NADPH or NADH; the concentration of NADPH necessary to obtain maximum activity is twice that of NADH. The requirement for a reducing agent is in agreement with the proposal that malonate semialdehyde, formed by an aminotransferase reaction from β-alanine, is reduced to β-hydroxypropionate. Both malonate semialdehyde and β-hydroxypropionate are better stimulators of production of the volatile in the soluble system than is β-alanine, and β-hydroxypropionate is a better stimulator than malonate semialdehyde. This system is also able to incorporate tritium from tritiated water into ethylene; this supports the proposal that ethylene is formed by the decarboxylation of acrylate, the latter being formed from β-hydroxypropionate. Experiments with both cold and labeled malonate suggest that this compound stimulates ethylene production by acting as an end product inhibitor that prevents the loss of malonate semialdehyde from the pathway. Malonate does not appear to serve as a precursor. Addition of cytoplasmic enzymes to the `soluble system' (prepared from particulate enzymes) results in a considerable boost in ethylene production, but the specific activity (mμ1 / mg protein) is lowered from that of the particulate enzymes alone. PMID:16657194

  6. Methanococci use the diaminopimelate aminotransferase (DapL) pathway for lysine biosynthesis.

    PubMed

    Liu, Yuchen; White, Robert H; Whitman, William B

    2010-07-01

    The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to ll-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapL1 group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Deltammp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using ll-DAP and alpha-ketoglutarate as substrates was 24.3 + or - 2.0 nmol min(-1) mg of protein(-1). The gene encoding the DapL homolog in Methanocaldococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70 degrees C and pH 8.0 to 9.0. The apparent K(m)s of MJ1391 for ll-DAP and alpha-ketoglutarate were 82.8 + or - 10 microM and 0.42 + or - 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl-DAP as a substrate. Phylogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales.

  7. Methanococci Use the Diaminopimelate Aminotransferase (DapL) Pathway for Lysine Biosynthesis ▿

    PubMed Central

    Liu, Yuchen; White, Robert H.; Whitman, William B.

    2010-01-01

    The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to ll-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapL1 group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Δmmp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using ll-DAP and α-ketoglutarate as substrates was 24.3 ± 2.0 nmol min−1 mg of protein−1. The gene encoding the DapL homolog in Methanocaldococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70°C and pH 8.0 to 9.0. The apparent Kms of MJ1391 for ll-DAP and α-ketoglutarate were 82.8 ± 10 μM and 0.42 ± 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl-DAP as a substrate. Phylogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales. PMID:20418392

  8. The effect of arsenic contamination on amino acids metabolism in Spinacia oleracea L.

    PubMed

    Pavlík, Milan; Pavlíková, Daniela; Staszková, Ludmila; Neuberg, Marek; Kaliszová, Regina; Száková, Jirina; Tlustos, Pavel

    2010-09-01

    Changes of amino acid concentrations (proline, glutamate, asparagine, aspartate, alanine) and glutamate kinase activity (GKA) in plants under arsenic chronic stress reported here reveal their role in plant arsenic stress adaptation. Results of the pot experiment confirmed the toxic effect of arsenic at tested levels (As1=25 mg As kg(-1) soil, As2=50 mg As kg(-1) soil, As3=75 mg As kg(-1) soil) for spinach. Growing available arsenic contents in soil were associated with the strong inhibition of above-ground biomass and with the enhancement of As plant content. The changes of glutamate, asparagine, aspartate and proline levels in the plants showed strong linear dependences on arsenic concentration in plants (R2=0.60-0.90). Compared to the untreated control, concentrations of free proline and aspartate of As3 treatment were enhanced up to 381% and 162%, respectively. The significant changes of glutamate were observed on As2 and As3 treatments (increased level up to 188, i.e. 617%). Arsenic in plants was shown to be an inhibitor of glutamase kinase activity (R2=0.91). Inhibition of GKA resulted in an increase in the content of glutamate that is used in synthesis of phytochelatins in plant cells. Concentration of alanine did not have a confirmed linear dependence on arsenic concentration in plant (R2=0.05). The changes of its concentrations could be affected by changes of pH in plant cell or induction of alanine aminotransferase by hypoxia.

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  10. 3-Phosphono-L-alanine as pyrophosphate mimic for DNA synthesis using HIV-1 reverse transcriptase.

    PubMed

    Yang, Shiqiong; Froeyen, Mathy; Lescrinier, Eveline; Marlière, Philippe; Herdewijn, Piet

    2011-01-07

    A series of sulf(on)ate and phosph(on)ate amino acid phosphoramidate analogues of deoxynucleotides were synthesized as potential substrates for HIV-1 reverse transcriptase. Taurine, L-cysteic acid, 3-phosphono-L-alanine, O-sulfonato-L-serine, and O-phospho-L-serine were investigated as leaving groups in an enzyme catalyzed DNA synthesis protocol. Among these analogues, the phosphonate congener performed best and 3-phosphono-L-alanine can be considered as an excellent mimic of the pyrophosphate (PPi) moiety of deoxyadenosine triphosphate, to be used in enzymatic synthesis of nucleic acids. During a single nucleotide incorporation assay the use of 3-phosphono-L-Ala-dAMP as substrate resulted in 95% conversion to a P + 1 strand in 60 min at 50 μM (a concentration 10 times less than found for L-Asp-dAMP) and with improved incorporation kinetics and less stalling. For the sequences investigated, the efficiency of the incorporation is base dependent and decreases in the order (A ≥ T = G > C). In all cases, the incorporation follows Watson-Crick rules.

  11. 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.

  12. Observation on therapeutic efficacy of ursodeoxycholic acid in Chinese patients with primary biliary cirrhosis: a 2-year follow-up study.

    PubMed

    Zhu, Jiangyi; Shi, Yongquan; Zhou, Xinmin; Li, Zengshan; Huang, Xiaofeng; Han, Zheyi; Wang, Jianhong; Wang, Ruian; Ding, Jie; Wu, Kaichun; Han, Ying; Fan, Daiming

    2013-06-01

    The efficacy of ursodeoxycholic acid (UDCA) on long-term outcome of primary biliary cirrhosis (PBC) has been less documented in Chinese cohort. We aimed to assess the therapeutic effect of UDCA on Chinese patients with PBC. In the present study, 67 patients with PBC were treated with UDCA (13-15 mg·kg(-1)·day(-1)) and followed up for 2 years to evaluate the changes of symptoms, laboratory values and histological features. As the results indicated, fatigue and pruritus were obviously improved by UDCA, particularly in patients with mild or moderate symptoms. The alkaline phosphatase and γ-glutamyl transpetidase levels significantly declined at year 2 comparing to baseline values, with the most profound effects achieved in patients at stage 2. The levels of alanine aminotransferase and aspartate aminotransferase significantly decreased whereas serum bilirubin and immunoglobulin M levels exhibited no significant change. Histological feature was stable in patients at stages 1-2 but still progressed in patients at stages 3-4. The biochemical response of patients at stage 2 was much better than that of patients at stages 3-4. These data suggest that, when treated in earlier stage, patients in long-term administration of UDCA can gain favorable results not only on symptoms and biochemical responses but also on histology. It is also indicated that later histological stage, bad biochemical response and severe symptom may be indicators of poor prognosis for UDCA therapy.

  13. Synergistic ameliorative effects of sesame oil and alpha-lipoic acid against subacute diazinon toxicity in rats: hematological, biochemical, and antioxidant studies.

    PubMed

    Abdel-Daim, Mohamed M; Taha, Ramadan; Ghazy, Emad W; El-Sayed, Yasser S

    2016-01-01

    Diazinon (DZN) is a common organophosphorus insecticide extensively used for agriculture and veterinary purposes. DZN toxicity is not limited to insects; it also induces harmful effects in mammals and birds. Our experiment evaluated the protective and antioxidant potential of sesame oil (SO) and (or) alpha-lipoic acid (ALA) against DZN toxicity in male Wistar albino rats. DZN-treated animals exhibited macrocytic hypochromic anemia and significant increases in serum biochemical parameters related to liver injury, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (γGT), cholesterol, and triglycerides. They also had elevated levels of markers related to cardiac injury, such as lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), and increased biomarkers of renal injury, urea and creatinine. DZN also increased hepatic, renal, and cardiac lipid peroxidation and decreased antioxidant biomarker levels. SO and (or) ALA supplementation ameliorated the deleterious effects of DZN intoxication. Treatment improved hematology and serum parameters, enhanced endogenous antioxidant status, and reduced lipid peroxidation. Importantly, they exerted synergistic hepatoprotective, nephroprotective, and cardioprotective effects. Our findings demonstrate that SO and (or) ALA supplementation can alleviate the toxic effects of DZN via their potent antioxidant and free radical-scavenging activities.

  14. Structural Insights into a Novel Class of Aspartate Aminotransferase from Corynebacterium glutamicum

    PubMed Central

    Son, Hyeoncheol Francis; Kim, Kyung-Jin

    2016-01-01

    Aspartate aminotransferase from Corynebacterium glutamicum (CgAspAT) is a PLP-dependent enzyme that catalyzes the production of L-aspartate and α-ketoglutarate from L-glutamate and oxaloacetate in L-lysine biosynthesis. In order to understand the molecular mechanism of CgAspAT and compare it with those of other aspartate aminotransferases (AspATs) from the aminotransferase class I, we determined the crystal structure of CgAspAT. CgAspAT functions as a dimer, and the CgAspAT monomer consists of two domains, the core domain and the auxiliary domain. The PLP cofactor is found to be bound to CgAspAT and stabilized through unique residues. In our current structure, a citrate molecule is bound at the active site of one molecule and mimics binding of the glutamate substrate. The residues involved in binding of the PLP cofactor and the glutamate substrate were confirmed by site-directed mutagenesis. Interestingly, compared with other AspATs from aminotransferase subgroup Ia and Ib, CgAspAT exhibited unique binding sites for both cofactor and substrate; moreover, it was found to have unusual structural features in the auxiliary domain. Based on these structural differences, we propose that CgAspAT does not belong to either subgroup Ia or Ib, and can be categorized into a subgroup Ic. The phylogenetic tree and RMSD analysis also indicates that CgAspAT is located in an independent AspAT subgroup. PMID:27355211

  15. Gene expression profiles of murine fatty liver induced by the administration of valproic acid

    SciTech Connect

    Lee, Min-Ho; Hong, Il; Kim, Mingoo; Lee, Byung Hoon; Kim, Ju-Han; Kang, Kyung-Sun; Kim, Hyung-Lae; Yoon, Byung-Il; Chung, Heekyoung; Kong, Gu; Lee, Mi-Ock . E-mail: molee@snu.ac.kr

    2007-04-01

    Valproic acid (VPA) has been used as anticonvulsants, however, it induces hepatotoxicity such as microvesicular steatosis and necrosis in the liver. To explore the mechanisms of VPA-induced steatosis, we profiled the gene expression patterns of the mouse liver that were altered by treatment with VPA using microarray analysis. VPA was orally administered as a single dose of 100 mg/kg (low-dose) or 1000 mg/kg (high-dose) to ICR mice and the animals were killed at 6, 24, or 72 h after treatment. Serum alanine aminotransferase and aspartate aminotransferase levels were not significantly altered in the experimental animals. However, symptoms of steatosis were observed at 72 h with low-dose and at 24 h and 72 h with high-dose. After microarray data analysis, 1910 genes were selected by two-way ANOVA (P < 0.05) as VPA-responsive genes. Hierarchical clustering revealed that gene expression changes depended on the time rather than the dose of VPA treatment. Gene profiling data showed striking changes in the expression of genes associated with lipid, fatty acid, and steroid metabolism, oncogenesis, signal transduction, and development. Functional categorization of 1156 characteristically up- and down-regulated genes (cutoff > 1.5-fold) revealed that 60 genes were involved in lipid metabolism that was interconnected with biological pathways for biosynthesis of triglyceride and cholesterol, catabolism of fatty acid, and lipid transport. This gene expression profile may be associated with the known steatogenic hepatotoxicity of VPA and it may provide useful information for prediction of hepatotoxicity of unknown chemicals or new drug candidates through pattern recognition.

  16. Dietary docosahexaenoic acid and eicosapentaenoic acid influence liver triacylglycerol and insulin resistance in rats fed a high-fructose diet.

    PubMed

    de Castro, Gabriela Salim; Deminice, Rafael; Simões-Ambrosio, Livia Maria Cordeiro; Calder, Philip C; Jordão, Alceu A; Vannucchi, Helio

    2015-04-01

    This study aimed to examine the benefits of different amounts of omega-3 (n-3) polyunsaturated fatty acids from fish oil (FO) on lipid metabolism, insulin resistance and gene expression in rats fed a high-fructose diet. Male Wistar rats were separated into two groups: Control (C, n = 6) and Fructose (Fr, n = 32), the latter receiving a diet containing 63% by weight fructose for 60 days. After this period, 24 animals from Fr group were allocated to three groups: FrFO2 (n = 8) receiving 63% fructose and 2% FO plus 5% soybean oil; FrFO5 (n = 8) receiving 63% fructose and 5% FO plus 2% soybean oil; and FrFO7 (n = 8) receiving 63% fructose and 7% FO. Animals were fed these diets for 30 days. Fructose led to an increase in liver weight, hepatic and serum triacylglycerol, serum alanine aminotransferase and HOMA1-IR index. These alterations were reversed by 5% and 7% FO. FO had a dose-dependent effect on expression of genes related to hepatic β-oxidation (increased) and hepatic lipogenesis (decreased). The group receiving the highest FO amount had increased markers of oxidative stress. It is concluded that n-3 fatty acids may be able to reverse the adverse metabolic effects induced by a high fructose diet.

  17. A theoretical study of the XP and NEXAFS spectra of alanine: gas phase molecule, crystal, and adsorbate at the ZnO(10 ̅10) surface.

    PubMed

    Gao, You Kun; Traeger, Franziska; Kotsis, Konstantinos; Staemmler, Volker

    2011-06-14

    The adsorption of alanine on the mixed-terminated ZnO(10 ̅10) surface is studied by means of quantum-chemical ab initio calculations. Using a finite cluster model and the adsorption geometry as obtained both by periodic CPMD and embedded cluster calculations, the C1s, N1s and O1s X-ray photoelectron spectra (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectra are calculated for single alanine molecules on ZnO(10 ̅10). These spectra are compared with the spectra calculated for alanine in the gas phase and in its crystalline form and with experimental XPS and NEXAFS data for the isolated alanine molecule and for alanine adsorbed on ZnO(10 ̅10) at multilayer and monolayer coverage. The excellent agreement between the experimental and calculated XP and NEXAFS spectra confirms the calculated adsorption geometry: A single alanine molecule is bound to ZnO(10 ̅10) in a dissociated bidentate form with the two O atoms of the acid group bound to two Zn atoms of the surface and the proton transferred to one O atom of the surface. Other possible structures, such as adsorption of alanine in one of its neutral or zwitterionic forms in which the proton of the -COOH group remains at this group or is transferred to the amino group, can be excluded since they would give rise to quite different XP spectra. In the multilayer coverage regime, on the other hand, alanine is in its crystalline form as is also shown by the analysis of the XP spectra.

  18. 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.

  19. 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.

  20. 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.

  1. Post-translational Introduction of D-Alanine into Ribosomally Synthesized Peptides by the Dehydroalanine Reductase NpnJ.

    PubMed

    Yang, Xiao; van der Donk, Wilfred A

    2015-10-07

    Ribosomally synthesized peptides are generally limited to L-amino acid building blocks. Given the advantageous properties of peptides containing D-amino acids such as stabilization of certain turns and against proteolytic degradation, methods to introduce D-stereocenters are valuable. Here we report the first in vitro reconstitution and characterization of a dehydrogenase that carries out the asymmetric reduction of dehydroalanine. NpnJA reduces dehydroalanine to D-Ala using NAPDH as cosubstrate. The enzyme displays high substrate tolerance allowing introduction of D-Ala into a range of non-native substrates. In addition to the in vitro reactions, we describe five examples of using Escherichia coli as biosynthetic host for D-alanine introduction into ribosomal peptides. A deuterium-label-based coupled-enzyme assay was used to rapidly determine the stereochemistry of the newly installed alanine.

  2. [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.

  3. Waist circumference, body mass index, serum uric acid, blood sugar, and triglyceride levels are important risk factors for abnormal liver function tests in the Taiwanese population.

    PubMed

    Hsieh, Meng-Hsuan; Lin, Wen-Yi; Chien, Hsu-Han; Chien, Li-Ho; Huang, Chao-Kuan; Yang, Jeng-Fu; Chang, Ning-Chia; Huang, Chung-Feng; Wang, Chao-Ling; Chuang, Wan-Long; Yu, Ming-Lung; Dai, Chia-Yen; Ho, Chi-Kung

    2012-09-01

    Several studies have found that metabolic syndrome and uric acid level are related to abnormal liver function test results. The aim of this study was to explore the associations of risk factors [including blood pressure, blood sugar, total cholesterol, triglyceride, uric acid, waist circumference and body mass index (BMI) measurements] with abnormal liver function in the Taiwanese population.In total, 11,411 Taiwanese adults were enrolled in this study. Blood pressure was assessed according to the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure criteria, fasting blood sugar level according to the Bureau of Health Promotion, Department of Health, R.O.C., criteria, total cholesterol and triglyceride levels according to the Third Report of the National Cholesterol Education Program Adult Treatment Panel III criteria, BMI according to the Asia-Pacific criteria, and waist circumference according to the Revised Diagnostic Criteria of Metabolic Syndrome in Taiwan. The prevalence of a past history of hypertension and diabetes mellitus was 17.7% and 6.5%, respectively, and the rates of abnormal measurements of blood pressure, BMI, waist circumference, fasting blood sugar, triglyceride, total cholesterol, uric acid (male/female), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were 76.2%, 67.6%, 40.0%, 28.6%, 30.6%, 57.3%, 37.9%/21.9%, 14.6% and 21.3%, respectively. Multivariate analysis showed that waist circumference, BMI, serum uric acid, blood sugar, and triglyceride levels were related to abnormal AST and ALT (p<0.05), but the odds ratio for waist circumference was larger than that for BMI. In conclusion, waist circumference, BMI, serum uric acid, blood sugar, and triglyceride levels are important risk factors for abnormal AST and ALT readings in Taiwanese adults. Waist circumference might be a better indicator of risk of abnormal liver function than BMI.

  4. Effect of hypochlorous acid solution on the eradication and prevention of Pseudomonas aeruginosa infection, serum biochemical variables, and cecum microbiota in rats.

    PubMed

    Goto, Kazuo; Kuwayama, Eri; Nozu, Ryoko; Ueno, Masami; Hayashimoto, Nobuhito

    2015-01-01

    In this study, hypochlorous acid solution, a weak acid, provided as drinking water to rats, was evaluated for its ability to eradicate and prevent Pseudomonas aeruginosa infection, while monitoring its simultaneous effect on serum biochemical variables and microbiota in the rat cecum. The results suggest that the solution could not eliminate the bacteria in the experimentally infected rats; however, the administration of a 10-parts-per-million (ppm) hypochlorous acid solution as drinking water was effective in inhibiting horizontal spread of P. aeruginosa infection among cage mates. Additionally, exposure to hypochlorous solution did not have any effect on serum biochemical variables of the rat including levels of total cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, total bilirubin, lipase, amylase, urea nitrogen, total protein, calcium (Ca), phosphorus (P), sodium (Na), chlorine (Cl), except for potassium (K) levels. The most frequently isolated bacteria in the rat cecum included species belonging to Bacteroidales, Lactobacillus, Clostridiales, Erysipelotrichaceae, Akkermansia, Coriobacteriales, and Firmicutes. The ratio of the terminal restriction fragment length polymorphism (T-RFLP) peaks did not differ across rats administered with 5 and 10 ppm weak acid solution as compared to the control group for any of the bacteria, except for Erysipelotrichaceae and Firmicutes, where the ratio of T-RFLP peaks was higher in the 5 ppm group for Erysipelotrichaceae and in the 10 ppm group for Firmicutes than that in the control group (P<0.01). The results suggest that the weak acid hypochlorous solution could not eradicate P. aeruginosa completely from rats. The solution was effective in preventing infection without affecting serum biochemical variables; however, some of bacterial microbiota may have changed due to administration of the solution.

  5. 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).

  6. A theoretical study of alanine dipeptide and analogs

    SciTech Connect

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

    1989-01-01

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

  7. 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.

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

    PubMed

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

    2010-08-01

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

  9. The influence of secretion elicitors and external pH on the kinetics of D-alanine uptake by the trap lobes of Dionaea muscipula Ellis (Venus's Flytrap).

    PubMed

    Rea, P A; Whatley, F R

    1983-08-01

    Simple kinetic techniques were used to examine the mechanism of D-alanine uptake by the adaxial surfaces of the trap lobes of Dionaea muscipula Ellis (Venus's Flytrap.) On the basis of these analyses, the uptake of D-alanine was found to depend on the time during which the trap lobes were inoculated with elicitors of secretion before excision and measurement of uptake. Disks taken from traps that had not been subjected to a preceding period of inoculation with secretion elicitors showed a low basal rate of uptake which was neither pH-dependent nor exhibited saturation with respect to external D-alanine concentration. Disks from preinoculated traps, on the other hand, displayed an enhanced rate of uptake which showed both pH-dependence and saturation with respect to external D-alanine concentration. The capacity for enhanced uptake was lost upon prolonged inoculation or when inoculation was stopped. Of the compounds tested, only elicitors of secretion caused an enhancement of uptake. The enhanced rate of D-alanine uptake is temperature-sensitive with a Q10 characteristic of a mediated process. Uncouplers cause an instantaneous abolition of uptake whereas the effects of terminal-oxidase inhibitors are time-dependent. The pH-dependence of uptake is inferred to result from an increased affinity of the carrier system for D-alanine at low pH values. Although the ionic state of D-alanine is relatively unaffected over the pH range examined, a decrease in the external pH from 6.0 to 3.8 decreases the apparent K m for uptake by four-fold but increases V max by only 30%. It is concluded that the acid secreted by the digestive glands of Dionaea plays a direct role in facilitating the uptake of amino acids from the trap cavity.

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

    PubMed Central

    Levin, Alfred P.; Hartman, Philip E.

    1963-01-01

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

  11. Alanine substitutions of noncysteine residues in the cysteine-stabilized αβ motif

    PubMed Central

    Yang, Ying-Fang; Cheng, Kuo-Chang; Tsai, Ping-Hsing; Liu, Chung-Cheng; Lee, Tian-Ren; Ping-Chiang Lyu

    2009-01-01

    The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized αβ motif (CSαβ) consists of an α-helix and an antiparallel triple-stranded β-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering. PMID:19533758

  12. In situ detection of myocardial infarction in pig by measurements of aspartate aminotransferase (ASAT) activity in the interstitial fluid.

    PubMed

    Kennergren, C; Nyström, B; Nyström, U; Berglin, E; Larsson, G; Mantovani, V; Lönnroth, P; Hamberger, A

    1997-01-01

    Microdialysis probes permeable to large molecules (m.w. cut-off > 200 kD) were introduced into the myocardium of anaesthetized pigs in order to evaluate their potential for early detection of myocardial ischaemia and enzyme markers for infarction. The left anterior descending coronary artery was occluded for 30 min and the myocardium was reperfused for 3 h. The concentrations of aspartate aminotransferase (ASAT), lactate, glucose and selected free amino acids were measured. The levels in the interstitium of ischaemic and non-ischaemic myocardium were compared with those in plasma from the coronary sinus as well as from a peripheral vein. Twelve probes were inserted in six pigs and withdrawn after 8-72 hours of sampling. No complications occurred. Simultaneous 100% increase of ASAT and lactate was found in myocardial dialysates after 30 min of ischaemia. ASAT activity remained at that level until the end of reperfusion. The plasma peak ASAT level was not attained until after 3 h. Glutamate was the only amino acid which increased significantly in the myocardial interstitium during ischaemia, peaking after 30 min of reperfusion. Dialysates from the unaffected myocardium showed no effects on lactate, ASAT or glutamate. The use of myocardial microdialysis for pre- and postoperative recordings in man is discussed.

  13. The aspartate aminotransferase family in conifers: biochemical analysis of a prokaryotic-type enzyme from maritime pine.

    PubMed

    de la Torre, Fernando; Suárez, María Fernanda; Santis, Laura de; Cánovas, Francisco M

    2007-09-01

    Plant aspartate aminotransferase (AAT, EC 2.6.1.1) plays a key role in primary nitrogen assimilation, the transfer of reducing equivalents and the interchanges of carbon and nitrogen pools between subcellular compartments. We investigated the AAT family in conifers using maritime pine as the experimental model. Genes for cytosolic, mitochondrial and two plastidic isoenzymes (eukaryotic- and prokaryotic-types) were identified and their deduced amino acid sequences compared. The primary structure of the eukaryotic-type enzymes is quite well conserved, whereas the prokaryotic-type AAT is highly divergent (15% of identity). These molecular data were confirmed by the absence of immunological cross-reactivity between the two types of native AATs. The mature prokaryotic-type polypeptide was overexpressed in Escherichia coli, and the native enzyme was purified to apparent homogeneity and its molecular properties determined. The fully active recombinant holoenzyme showed highest catalytic activity at 50-60 degrees C and was moderately thermostable, retaining about 50% of its activity after incubation at 70 degrees C for 5-10 min. The presence of pyridoxal 5'-phosphate significantly increased the thermostability of the enzyme. These molecular characteristics were exploited to develop a rapid protocol for the purification of this prokaryotic-type enzyme from pine cotyledons. The results will be useful for studying aspartate and amino acid metabolism in trees.

  14. 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.

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

    PubMed

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

    2014-03-01

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

  16. An epoxysuccinic acid derivative(loxistatin)-induced hepatic injury in rats and hamsters

    SciTech Connect

    Fukushima, K.; Arai, M.; Kohno, Y.; Suwa, T.; Satoh, T. )

    1990-08-01

    Loxistatin is a possible therapeutic agent of muscular dystrophy. A single oral administration of loxistatin to male rats caused focal necrosis of the liver with inflammatory cell infiltration. The severity of the lesions was dose-dependent up to 200 mg/kg and also manifest by an increase in serum alanine aminotransferase and aspartate aminotransferase activities. Hepatic glutathione (GSH) levels decreased with a maximum 20% depletion within 5 hr after the oral administration of loxistatin. Pretreatment with diethyl maleate did not potentiate the loxistatin-induced hepatic injury. On the other hand, the hepatoprotective effect of cysteamine was observed when cysteamine was administered 24 hr before loxistatin dosing, but the effect was not observed when the antidote was administered concomitantly with loxistatin. Pretreatment of rats with phenobarbital or trans-stilbene oxide provided partial protection against the hepatotoxic effect of loxistatin. Pretreatment with SKF-525A resulted in increased hepatic injury, while pretreatment with piperonyl butoxide, cimetidine, or 3-methylcholanthrene had no effect on hepatic damage by loxistatin. Five hours after (14C)loxistatin administration to rats, the covalent binding of the radioactivity to proteins was greatest in the liver, followed by the kidney, then muscle and blood to a lesser extent. (14C)Loxistatin acid, the pharmacologically active form of loxistatin, irreversibly bound to rat liver microsomal proteins; more binding occurred when the NADPH-generating system was omitted and when the microsomes were boiled first. GSH did not alter the extent of irreversible binding, whereas N-ethylmaleimide decreased the binding of (14C)loxistatin acid to rat liver microsomal proteins by 75%. Unlike the rat, administration of loxistatin to hamsters caused neither hepatic injury nor hepatic GSH depletion.

  17. L,L-diaminopimelate aminotransferase (DapL): a putative target for the development of narrow-spectrum antibacterial compounds.

    PubMed

    Triassi, Alexander J; Wheatley, Matthew S; Savka, Michael A; Gan, Han Ming; Dobson, Renwick C J; Hudson, André O

    2014-01-01

    Despite the urgent need for sustained development of novel antibacterial compounds to combat the drastic rise in antibiotic resistant and emerging bacterial infections, only a few clinically relevant antibacterial drugs have been recently developed. One of the bottlenecks impeding the development of novel antibacterial compounds is the identification of new enzymatic targets. The nutritionally essential amino acid anabolic pathways, for example lysine biosynthesis, provide an opportunity to explore the development of antibacterial compounds, since human genomes do not possess the genes necessary to synthesize these amino acids de novo. The diaminopimelate (DAP)/lysine (lys) anabolic pathways are attractive targets for antibacterial development since the penultimate lys precursor meso-DAP (m-DAP) is a cross-linking amino acid in the peptidoglycan (PG) cell wall of most Gram-negative bacteria and lys plays a similar role in the PG of most Gram-positive bacteria, in addition to its role as one of the 20 proteogenic amino acids. The L,L-diaminopimelate aminotransferase (DapL) pathway was recently identified as a novel variant of the DAP/lys anabolic pathways. The DapL pathway has been identified in the pathogenic bacteria belonging to the genus; Chlamydia, Leptospira, and Treponema. The dapL gene has been identified in the genomes of 381 or approximately 13% of the 2771 bacteria that have been sequenced, annotated and reposited in the NCBI database, as of May 23, 2014. The narrow distribution of the DapL pathway in the bacterial domain provides an opportunity for the development and or discovery of narrow spectrum antibacterial compounds.

  18. L,L-diaminopimelate aminotransferase (DapL): a putative target for the development of narrow-spectrum antibacterial compounds

    PubMed Central

    Triassi, Alexander J.; Wheatley, Matthew S.; Savka, Michael A.; Gan, Han Ming; Dobson, Renwick C. J.; Hudson, André O.

    2014-01-01

    Despite the urgent need for sustained development of novel antibacterial compounds to combat the drastic rise in antibiotic resistant and emerging bacterial infections, only a few clinically relevant antibacterial drugs have been recently developed. One of the bottlenecks impeding the development of novel antibacterial compounds is the identification of new enzymatic targets. The nutritionally essential amino acid anabolic pathways, for example lysine biosynthesis, provide an opportunity to explore the development of antibacterial compounds, since human genomes do not possess the genes necessary to synthesize these amino acids de novo. The diaminopimelate (DAP)/lysine (lys) anabolic pathways are attractive targets for antibacterial development since the penultimate lys precursor meso-DAP (m-DAP) is a cross-linking amino acid in the peptidoglycan (PG) cell wall of most Gram-negative bacteria and lys plays a similar role in the PG of most Gram-positive bacteria, in addition to its role as one of the 20 proteogenic amino acids. The L,L-diaminopimelate aminotransferase (DapL) pathway was recently identified as a novel variant of the DAP/lys anabolic pathways. The DapL pathway has been identified in the pathogenic bacteria belonging to the genus; Chlamydia, Leptospira, and Treponema. The dapL gene has been identified in the genomes of 381 or approximately 13% of the 2771 bacteria that have been sequenced, annotated and reposited in the NCBI database, as of May 23, 2014. The narrow distribution of the DapL pathway in the bacterial domain provides an opportunity for the development and or discovery of narrow spectrum antibacterial compounds. PMID:25309529

  19. Participation of cysteine and cystine in inactivation of tyrosine aminotransferase in rat liver homogenates.

    PubMed Central

    Buckley, W T; Milligan, L P

    1978-01-01

    1. Inactivation of tyrosine aminotransferase was studied in rat liver homogenates. Under an O2 atmosphere with cysteine added, inactivation was rapid after a lag period of approx. 1h, whereas a N2 atmosphere extended the lag period to approx. 3h. 2. Replacement of cysteine with cystine resulted in rapid inactivation both aerobically and anaerobically. 3. Removal of the particulate fraction by centrifuging rat liver homogenates at 13,000g for 9min resulted in an aerobic lag period of 0.5h in the presence of cystine and approx. 3h in the presence of cysteine. 4. It is proposed that the stimulatory effect of cysteine on tyrosine aminotransferase inactivation occurs largely as a result of oxidation to cystine, which appears to be a more directly effective agent. PMID:33669

  20. 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.

  1. 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.

  2. 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.

  3. The influence of various cations on the catalytic properties of clays. [polymerization of alanine adenylate

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The polymerization of alanine adenylate in the presence of the sodium form of various clays was studied, and hectorite was found to cause more polymerization than nontronite and montmorillonite (in that order) although the differences were not great. The effect on polymerization of presaturating montmorillonite with different cations was determined. Hectorite, with increased basicity of the interspatial planes, allows polymerization of lysine, which montmorillonite does not. The general trend is that, for the same amino acid, higher degrees of polymerization are obtained when the cation in the octahedral lattice of the clay is divalent rather than trivalent. With the exchangeable cations the order is reversed, for a reason that is explained. The main role of clays in the polymerization mechanism of amino acids is concentration and neutralization of charges.

  4. Structural Analysis of QdtB, an Aminotransferase Required for the Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-[alpha]-D-glucose

    SciTech Connect

    Thoden, James B.; Schaffer, Christina; Messner, Paul; Holden, Hazel M.

    2009-05-21

    3-Acetamido-3,6-dideoxy-{alpha}-D-glucose or Quip3NAc is an unusual deoxyamino sugar found in the O-antigens of some Gram-negative bacteria and in the S-layers of Gram-positive bacteria. It is synthesized in these organisms as a dTDP-linked sugar via the action of five enzymes. The focus of this investigation is on QdtB from Thermoanaerobacterium thermosaccharolyticum E207-71, a PLP-dependent aminotransferase that catalyzes the penultimate step in the production of dTDP-Quip3NAc. For this analysis, the enzyme was crystallized in the presence of its product, dTDP-Quip3N, and the structure was solved and refined to 2.15 {angstrom} resolution. QdtB is a dimer, and its overall fold places it into the well-characterized aspartate aminotransferase superfamily. Electron density corresponding to the bound product reveals the presence of a Schiff base between C-4' of the PLP cofactor and the amino nitrogen of the sugar. Those amino acid side chains involved in binding the dTDP-sugar into the active site include Tyr 183, His 309, and Tyr 310 from subunit 1 and Lys 219 from subunit 2. Notably there is a decided lack of interactions between the pyranosyl C-4' hydroxyl of the dTDP-sugar and the protein. In keeping with this observation, we show that QdtB can also turn over dTDP-3-acetamido-3,6-dideoxy-{alpha}-D-galactose. This investigation represents the first structural analysis of a sugar-modifying aminotransferase with a bound product in its active site that functions at the C-3' rather than the C-4' position of the hexose.

  5. Effects of ursodeoxycholic acid treatment on nutrition and liver function in patients with cystic fibrosis and longstanding cholestasis.

    PubMed Central

    Cotting, J; Lentze, M J; Reichen, J

    1990-01-01

    The prevalence of biliary and hepatic diseases is increasing in patients with cystic fibrosis as more of them reach adult life. There is no effective treatment or method of preventing cholestasis in cystic fibrosis, although beneficial effects have been ascribed to the tertiary bile acid, ursodeoxycholate, in other forms of chronic cholestasis. We evaluated prospectively the effects of a six month course of ursodeoxycholate (15-20 mg/kg per day) in eight, mostly adult, patients with cystic fibrosis and chronic cholestasis. Bile acid treatment improved inflammatory activity (average decrease in alanine aminotransferase, 60%, p less than 0.005) and cholestasis (alkaline phosphatase, 47%; p less than 0.01) in all patients. Quantitative liver function, measured by 45 minute sulphobromophthalein retention and by the 14C-aminopyrine breath test, improved in all patients while galactose elimination capacity showed a slight decrease. Patients' nutritional state improved as evidenced by a 1.8 kg weight gain and an increase in muscle mass suggested by a 26% increase in 24 hour urinary creatinine excretion. Steatorrhea was not affected by bile acid treatment. Ursodeoxycholic acid may be beneficial in the treatment of chronic cholestasis in cystic fibrosis by improving liver function and also the patient's nutritional state. PMID:2387518

  6. Effects of ursodeoxycholic acid treatment on nutrition and liver function in patients with cystic fibrosis and longstanding cholestasis.

    PubMed

    Cotting, J; Lentze, M J; Reichen, J

    1990-08-01

    The prevalence of biliary and hepatic diseases is increasing in patients with cystic fibrosis as more of them reach adult life. There is no effective treatment or method of preventing cholestasis in cystic fibrosis, although beneficial effects have been ascribed to the tertiary bile acid, ursodeoxycholate, in other forms of chronic cholestasis. We evaluated prospectively the effects of a six month course of ursodeoxycholate (15-20 mg/kg per day) in eight, mostly adult, patients with cystic fibrosis and chronic cholestasis. Bile acid treatment improved inflammatory activity (average decrease in alanine aminotransferase, 60%, p less than 0.005) and cholestasis (alkaline phosphatase, 47%; p less than 0.01) in all patients. Quantitative liver function, measured by 45 minute sulphobromophthalein retention and by the 14C-aminopyrine breath test, improved in all patients while galactose elimination capacity showed a slight decrease. Patients' nutritional state improved as evidenced by a 1.8 kg weight gain and an increase in muscle mass suggested by a 26% increase in 24 hour urinary creatinine excretion. Steatorrhea was not affected by bile acid treatment. Ursodeoxycholic acid may be beneficial in the treatment of chronic cholestasis in cystic fibrosis by improving liver function and also the patient's nutritional state.

  7. 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.

  8. 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).

  9. Carrot Juice Administration Decreases Liver Stearoyl-CoA Desaturase 1 and Improves Docosahexaenoic Acid Levels, but Not Steatosis in High Fructose Diet-Fed Weanling Wistar Rats

    PubMed Central

    Mahesh, Malleswarapu; Bharathi, Munugala; Reddy, Mooli Raja Gopal; Kumar, Manchiryala Sravan; Putcha, Uday Kumar; Vajreswari, Ayyalasomayajula; Jeyakumar, Shanmugam M.

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases associated with an altered lifestyle, besides genetic factors. The control and management of NAFLD mostly depend on lifestyle modifications, due to the lack of a specific therapeutic approach. In this context, we assessed the effect of carrot juice on the development of high fructose-induced hepatic steatosis. For this purpose, male weanling Wistar rats were divided into 4 groups, fed either a control (Con) or high fructose (HFr) diet of AIN93G composition, with or without carrot juice (CJ) for 8 weeks. At the end of the experimental period, plasma biochemical markers, such as triglycerides, alanine aminotransferase, and β-hydroxy butyrate levels were comparable among the 4 groups. Although, the liver injury marker, aspartate aminotransferase, levels in plasma showed a reduction, hepatic triglycerides levels were not significantly reduced by carrot juice ingestion in the HFr diet-fed rats (HFr-CJ). On the other hand, the key triglyceride synthesis pathway enzyme, hepatic stearoyl-CoA desaturase 1 (SCD1), expression at mRNA level was augmented by carrot juice ingestion, while their protein levels showed a significant reduction, which corroborated with decreased monounsaturated fatty acids (MUFA), particularly palmitoleic (C16:1) and oleic (C18:1) acids. Notably, it also improved the long chain n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA; C22:6) content of the liver in HFr-CJ. In conclusion, carrot juice ingestion decreased the SCD1-mediated production of MUFA and improved DHA levels in liver, under high fructose diet-fed conditions. However, these changes did not significantly lower the hepatic triglyceride levels. PMID:27752492

  10. Evaluation of oxidative stress via total antioxidant status, sialic acid, malondialdehyde and RT-PCR findings in sheep affected with bluetongue

    PubMed Central

    Aytekin, I.; Aksit, H.; Sait, A.; Kaya, F.; Aksit, D.; Gokmen, M.; Baca, A. Unsal

    2015-01-01

    Introduction Bluetongue (BT) is a non-contagious infectious disease of ruminants. The disease agent bluetongue virus (BTV) is classified in the Reoviridae family Orbivirus. Aims and objectives The aim of this study was to determine serum malondialdehyde (MDA), total antioxidative stres (TAS), total sialic acid (TSA), ceruloplasmin, triglyceride, alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), cholesterol, creatinine, albumin, and total protein levels in sheep with and without bluetongue (BT). Materials and Methods The study included 13 Sakiz crossbreed sheep, aged 1–4 years and usually in the last stage of pregnancy, as the BT group and a control group consisting of 10 healthy sheep. All sheep were clinically examined before collecting blood samples. Serum ALT, AST, cholesterol, triglyceride, albumin, GGT, total protein, creatinine and TAS levels were measured using commercially available kits as per manufacturer's recommendations using a Biochemistry Auto Analyzer (Sinnowa D280, China). Serum lipid peroxidation was estimated through a previously described method in which MDA reacts with thiobarbituric acid (TBA) to form a coloured complex at a maximum absorbance of 535 nm. The TSA value was measured at 549 nm using the method described by Warren (1959): sialic acid was oxidised to formyl-pyruvic acid, which reacts with TBA to form a pink product. The ceruloplasmin concentration was measured according to Sunderman and Nomoto (1970): ceruloplasmin and p-phenylenediamine formed a coloured oxidation product that was proportional to the concentration of serum ceruloplasmin. Real time RT-PCR and conventional RT-PCR were performed as described by Shaw and others (2007). Results Biochemistry analysis of serum showed that in the BT group, TSA, MDA, triglyceride and ALT and AST were higher and that ceruloplasmin and TAS were lower than in the control group. Serum albumin, cholesterol, creatinine, total protein and GGT did

  11. Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-α in LPS-stimulated monocytes in human whole-blood.

    PubMed

    Raspé, C; Czeslick, E; Weimann, A; Schinke, C; Leimert, A; Kellner, P; Simm, A; Bucher, M; Sablotzki, A

    2013-04-01

    To investigate the effects of the commonly-used immunomodulators l-glutamine, l-alanine, and the combination of both l-alanyl-l-glutamine (Dipeptamin(®)) on intracellular expression of IL-6, IL-8, and TNF-α during endotoxemia, lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system were investigated by flow cytometry. Whole blood of twenty-seven healthy volunteers was stimulated with LPS and incubated with three different amino acid solutions (1. l-glutamine, 2. l-alanine, 3. l-alanyl-l-glutamine, each concentration 2 mM, 5 mM, incubation time 3 h). CD14(+) monocytes were phenotyped in whole-blood and intracellular expression of cytokines was assessed by flow cytometry. Our investigations showed for the first time in whole blood probes, imitating best physiologically present cellular interactions, that l-glutamine caused a dose-independent inhibitory effect on IL-6 and TNF-α production in human monocytes stimulated with LPS. However, l-alanine had contrary effects on IL-6 expression, significantly upregulating expression of IL-6 in LPS-treated monocytes. The impact of l-alanine on the expression of TNF-α was comparable with glutamine. Neither amino acid was able to affect IL-8 production in LPS-stimulated monocytes. The combination of both did not influence significantly IL-6 and IL-8 expression in monocytes during endotoxemia, however strongly reduced TNF-α production. For the regulation of TNF-α, l-glutamine, l-alanine and the combination of both show a congruent and exponentiated downregulating effect during endotoxemia, for the modulation of IL-6, l-glutamine and l-alanine featured opposite regulation leading to a canceling impact of each other when recombining both amino acids.

  12. Combined supplementation of carbohydrate, alanine, and proline is effective in maintaining blood glucose and increasing endurance performance during long-term exercise in mice.

    PubMed

    Nogusa, Yoshihito; Mizugaki, Ami; Hirabayashi-Osada, Yuri; Furuta, Chie; Ohyama, Kana; Suzuki, Katsuya; Kobayashi, Hisamine

    2014-01-01

    Carbohydrate supplementation is extremely important during prolonged exercise because it maintains blood glucose levels during later stages of exercise. In this study, we examined whether maintaining blood glucose levels by carbohydrate supplementation could be enhanced during long-term exercise by combining this supplementation with alanine and proline, which are gluconeogenic amino acids, and whether such a combination would affect exercise endurance performance. Male C57BL/6J mice were orally administered either maltodextrin (1.25 g/kg) or maltodextrin (1.0 g/kg) with alanine (0.225 g/kg) and proline (0.025 g/kg) 15 min before running for 170 min. Combined supplementation of maltodextrin, alanine, and proline induced higher blood glucose levels than isocaloric maltodextrin alone during the late exercise phase (100-170 min). The hepatic glycogen content of mice administered maltodextrin, alanine, and proline was higher than that of mice ingesting maltodextrin alone 60 min after beginning exercise, but the glycogen content of the gastrocnemius muscle showed no difference. We conducted a treadmill running test to determine the effect of alanine and proline on endurance performance. The test showed that running time to exhaustion of mice that were supplemented with maltodextrin (2.0 g/kg) was longer than that of mice that were supplemented with water alone. Maltodextrin supplementation (1.0 g/kg) with alanine (0.9 g/kg) and proline (0.1 g/kg) further increased running time to exhaustion compared to maltodextrin alone (2.0 g/kg). These results indicate that combined supplementation of carbohydrate, alanine, and proline is effective for maintaining blood glucose and hepatic glycogen levels and increasing endurance performance during long-term exercise in mice.

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

    PubMed

    Baffa, Oswaldo; Kinoshita, Angela

    2014-05-01

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

  14. GMXPBSA 2.1: A GROMACS tool to perform MM/PBSA and computational alanine scanning

    NASA Astrophysics Data System (ADS)

    Paissoni, C.; Spiliotopoulos, D.; Musco, G.; Spitaleri, A.

    2015-01-01