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Sample records for pyruvic transaminase serum

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

    Science.gov (United States)

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

    2010-01-01

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

  2. Chemical protection against radiation effects on Serum transaminase and the levels of glutamic and pyruvic acids following gamma irradiation of rats

    International Nuclear Information System (INIS)

    Mahdy, A.M.; EL-Kashef, H.S.

    1988-01-01

    The present study been carried out to evaluate the radioprotective efficiency of urea and vitamin E for protecting certain enzymatic systems from deleterious radiation effects. The activities of serum transaminase; aspartate aminotransferase (A S T) and alanine aminotransferase (A L T); as well as their relative substrates; glutamic and pyruvic acid levels; were selected for this study. The results indicated that whole body gamma irradiation at the dose of 7 Gy caused an evident elevation in the activities of both A S T and A L T and in the level of pyruvic acid at the experiment period (first,third,seventh and tenth days post irradiation). On the other hand the free glutamic acid level decreased at all post irradiation days. The variation in both enzymatic activities, pyruvic and glutamic acid levels became less pronounced in rats treated with either urea or vitamin E as chemical radioprotectors before whole body gamma irradiation. The results showed that the two agents are good radioprotectors, with respect to these parameters under investigation

  3. A comparative study of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in the saliva of diabetic and normal patients.

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    Verma, M; Metgud, R; Madhusudan, A S; Verma, N; Saxena, M; Soni, A

    2014-10-01

    Diabetes has been reported to affect salivary glands adversely in humans and experimental models. Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) are salivary enzymes that also are widely distributed in animal tissues. We determined GOT and GPT levels in saliva samples of 100 type 1 and 30 type 2 diabetic patients using reflectance spectrophotometry and compared them to 30 age and sex matched healthy controls. Statistically significant differences were observed in the mean values of GOT and GPT in type 1 diabetics compared to type 2 and control groups. Significantly higher GOT levels were found in the 1-20 year age group of type 1 diabetics. Our findings suggest that salivary gland damage is due to the same immunological attack that affects pancreatic β cells and results in type 1 diabetes.

  4. omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.

    Science.gov (United States)

    Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

    2004-04-01

    Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-ABA, the apparent K(m) and V(max) for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.

  5. ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

    Science.gov (United States)

    Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

    2004-01-01

    Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for l-β-amino-n-butyric acid (l-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and Vmax for l-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM l-β-ABA, the apparent Km and Vmax for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM d,l-β-ABA, producing optically pure d-β-ABA (99% enantiomeric excess) with 53% conversion. PMID:15066855

  6. Insulin-induced inhibition of gluconeogenesis genes, including glutamic pyruvic transaminase 2, is associated with reduced histone acetylation in a human liver cell line.

    Science.gov (United States)

    Honma, Kazue; Kamikubo, Michiko; Mochizuki, Kazuki; Goda, Toshinao

    2017-06-01

    Hepatic glutamic pyruvic transaminase (GPT; also known as alanine aminotransferase) is a gluconeogenesis enzyme that catalyzes conversions between alanine and pyruvic acid. It is also used as a blood biomarker for hepatic damage. In this study, we investigated whether insulin regulates GPT expression, as it does for other gluconeogenesis genes, and if this involves the epigenetic modification of histone acetylation. Human liver-derived HepG2 cells were cultured with 0.5-100nM insulin for 8h, and the mRNA expression of GPT, glutamic-oxaloacetic transaminase (GOT), γ-glutamyltransferase (GGT), PCK1, G6PC and FBP1 was measured. We also investigated the extent of histone acetylation around these genes. Insulin suppressed the mRNA expression of gluconeogenesis genes (GPT2, GOT1, GOT2, GGT1, GGT2, G6PC, and PCK1) in HepG2 cells in a dose-dependent manner. mRNA levels of GPT2, but not GPT1, were decreased by insulin. Histone acetylation was also reduced around GPT2, G6PC, and PCK1 in response to insulin. The expression of GPT2 and other gluconeogenesis genes such as G6PC and PCK1 was suppressed by insulin, in association with decreases in histone H3 and H4 acetylation surrounding these genes. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Protection from radiation induced changes in liver and serum transaminase of whole body gamma irradiated rats

    International Nuclear Information System (INIS)

    Elkashef, H.S.; Roushdy, H.M.; Saada, H.N.; Abdelsamie, M.

    1986-01-01

    Whole body gamma irradiation of rats with a dose of 5.5 Gy induced significant changes in the activity of liver and serum transaminase. The results indicated that this radiation dose caused a significant increase in the activity of serum Got and GPT on the third and seventh days after irradiation. This was followed by significant decreases on the fourteenth post-irradiation day. The activity of Got returned to is control activity, while the activity of GPT was significantly above the control on the twenty ones post-irradiation day. The activity of Got, in the liver of irradiated rats was elevated during the post-irradiation days, but on the twenty one day activity was about the normal value. The activity of liver GPT firstly decreased and then increased very much but attained the control level on the fourteenth after irradiation. The intraperitoneal injection of testosterone-vitamin E mixture 10 days before whole body gamma irradiation caused complete recovery for the activity of liver and serum Got. No indication of remarkable recovery in the case of GPT activity was recorded either in liver or in serum of irradiated rats. The applied mixture could protect against radiation induced changes in Got activity of liver and serum but could not protect or ameliorate the changes which occurred in the activity of GPT of the two tissues. 2 tab

  8. Binding of ethyl pyruvate to bovine serum albumin: Calorimetric, spectroscopic and molecular docking studies

    Energy Technology Data Exchange (ETDEWEB)

    Pathak, Mallika [Department of Chemistry, Miranda House, University of Delhi, Delhi 11007 (India); Mishra, Rashmi; Agarwala, Paban K. [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Ojha, Himanshu, E-mail: himanshu.drdo@gmail.com [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Singh, Bhawna [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Singh, Anju; Kukreti, Shrikant [Nucleic Acid Research Laboratory, Department of Chemistry, University of Delhi, Delhi 11007 (India)

    2016-06-10

    Highlights: • ITC study showed binding of ethyl pyruvate with BSA with high binding affinity. • Ethyl pyruvate binding caused conformation alteration of BSA. • Fluorescence quenching mechanism is static in nature. • Electrostatic, hydrogen bonding and hydrophobic forces involved in binding. • Docking confirmed role of electrostatic, hydrogen bonding and hydrophobic forces. - Abstract: Various in vitro and in vivo studies have shown the anti-inflammatory and anticancer potential role of ethyl pyruvate. Bio-distribution of drugs is significantly influenced by the drug-serum protein binding. Therefore, the binding mechanism of the ethyl pyruvate with bovine serum albumin was investigated using UV–vis absorption, fluorescence, circular dichroism, isothermal titration calorimetry and molecular docking techniques. Absorption and fluorescence quenching studies indicated the binding of ethyl pyruvate with protein. Circular dichroism spectra of bovine serum albumin confirmed significant change in the conformation of protein upon binding. Thermodynamic data confirmed that ethyl pyruvate binds to bovine serum albumin at the two different sites with high affinity. Binding of ethyl pyruvate to bovine serum albumin involves hydrogen bonding, van der Waal and hydrophobic interactions. Further, docking studies indicated that ethyl pyruvate could bind significantly at the three binding sites. The results will definitely contribute to the development of ethyl pyruvate as drug.

  9. Pengaruh Ekstrak Daun Sambiloto (Andrographis paniculata terhadap Struktur Mikroanatomi Hepar dan Kadar Glutamat Piruvat Transaminase Serum Mencit (Mus musculus yang Terpapar Diazinon

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    TRI WULANDARI

    2007-11-01

    Full Text Available Diazinon is a pesticide which is often using by farmer to kill insect as theenemy of the plant. The over using of pesticide may result in the remaining of diazinon residue in farming product. This residue can cause the damage of body tissue, especially liver. The aim of research were to find out the effect of leaves sambiloto (Andrographis paniculata Ness. extract on microanatomic structure of liver and serum glutamate pyruvate transaminase (GPT level of mice (Mus musculus L. exposed to diazinon. The research used Compelete Random Design with five treatments. The treatment of each group were using CMC 1% (placebo control, diazinon solution 40 mg/Kg BW (negative ontrol and the leaves sambiloto extract 12,6; 25,2 and 37,8 mg /kg BW. Diazinon solution was given within 10 days and continued with extract of sambiloto leaves also within 10 days. Parameter observed was the microanatomic structure of liver and serum GPT level. The data was analyzed of Analysis of Varians (Anova and continued with DMRT at significance 5%. The result of the research showed that the giving of the extract of sambiloto leaves in some dose variation degree is significantly influential to repair the microanatomic structure of liver and to decrease the serum GPT level was 37,8 mg/Kg BW.

  10. NÍVEIS SÉRICOS DE TRANSAMINASE GLUTÂMICA-OXALACÉTICA (GOT E TRANSAMINASE GLUTÂMICA-PIRÚVICA (GPT EM BOVINOS DO ESTADO DE GOIÁS SERUM LEVELS OF GOT AND GPT IN BOVINES IN THE GOIÁS STATE

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    Eduardo Cavalheiro Jardim

    2007-09-01

    Full Text Available

    Foram analisados 162 hemossoros de bovinos mestiços zebu com a finalidade de obtenção dos níveis séricos da transaminase glutâmica-oxalacética e da transaminase glutâmica-pirúvica, de 18 bezerros(as, 31 novilhos(as e 83 vacas, procedentes de municípios do Estado de Goiás. Os valores encontrados foram os seguintes (URF: TGO, 77,5 +/- 27,3 (bezerros(as, 72,9 +/- 19,6 (novilhos(as e 81,8 +/- 28,4 (vacas; TGP, 29,5 +/- 8,1 (novilhos(as, 31,1 +/- 8.3 (novilhos(as e 34,1 +/- 10,6 (vacas. É muito difícil estandadizar condições de manejo, e os dados encontrados no presente trabalho confirmam que as transaminases são enzimas, dinâmicas, capazes de responderem rapidamente a um grande número de trocas fisiológicas. Posteriores trabalhos devem ser desenvolvidos buscando uniformizar os grupos de trabalho sob os diferentes pontos considerados no presente estudo.

    With the objective of studying the serum levels of GPT and GOT, 162 blood serum from crossbred zebu were analyzed. The blood was taken from 48 calves, 31 steers and 83 cows from farms of counties in the State of Goiás. GOT serum levels (URF were as fo1lows: calves, 72.5 +/- 27.3, steers, 72.9 +/- 19.6 and cows 81.8 +/-28.4. For GPT serum levels (URF was found: calves, 29.5 +/- 8.1, steers, 31.1 +/- 8.3 and cows 34.1 +/- 10.6. Some differences were found in GOT and GPT, in comparison with other foreign reports in mature cows.

  11. Alanine transaminase (ALT) blood test

    Science.gov (United States)

    ... gov/ency/article/003473.htm Alanine transaminase (ALT) blood test To use the sharing features on this page, please enable JavaScript. The alanine transaminase (ALT) blood test measures the level of the enzyme ALT in ...

  12. EFFECT OF CIGARETTE SMOKING ON SERUM TRANSAMINASES ...

    African Journals Online (AJOL)

    Global Journal of Medical Sciences. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue · Archives · Journal Home > Vol 1, No 1 (2002) >. Log in or Register to get access to full text downloads.

  13. Identification of branched-chain amino acid aminotransferases active towards (R)-(+)-1-phenylethylamine among PLP fold type IV transaminases.

    Science.gov (United States)

    Bezsudnova, Ekaterina Yu; Dibrova, Daria V; Nikolaeva, Alena Yu; Rakitina, Tatiana V; Popov, Vladimir O

    2018-04-10

    New class IV transaminases with activity towards L-Leu, which is typical of branched-chain amino acid aminotransferases (BCAT), and with activity towards (R)-(+)-1-phenylethylamine ((R)-PEA), which is typical of (R)-selective (R)-amine:pyruvate transaminases, were identified by bioinformatics analysis, obtained in recombinant form, and analyzed. The values of catalytic activities in the reaction with L-Leu and (R)-PEA are comparable to those measured for characteristic transaminases with the corresponding specificity. Earlier, (R)-selective class IV transaminases were found to be active, apart from (R)-PEA, only with some other (R)-primary amines and D-amino acids. Sequences encoding new transaminases with mixed type of activity were found by searching for changes in the conserved motifs of sequences of BCAT by different bioinformatics tools. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Serum Reactivity Against Bacterial Pyruvate Dehydrogenase: Increasing the Specificity of Anti-Mitochondrial Antibodies for the Diagnosis of Primary Biliary Cirrhosis

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    Hiroshi Miyakawa

    2006-01-01

    Full Text Available Antimitochondrial antibodies (AMA are the serum hallmark of primary biliary cirrhosis (PBC. However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2 which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH, 10 with primary sclerosing cholangitis (PSC, and 27 healthy individuals for their reactivities at serial dilutions (1:10, 1:20 and 1:40 against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB. A murine anti-human PDC-E2 monoclonal antibody (mAB was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38% of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.

  15. Genetics Home Reference: GABA-transaminase deficiency

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    ... Description GABA-transaminase deficiency is a brain disease (encephalopathy) that begins in infancy. Babies with this disorder ... genetic testing? What is precision medicine? What is newborn screening? New Pages LMNA-related congenital muscular dystrophy ...

  16. Pyruvate kinase blood test

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003357.htm Pyruvate kinase blood test To use the sharing features on this page, ... energy when oxygen levels are low. How the Test is Performed A blood sample is needed. In the laboratory, white blood ...

  17. Study on the effect of food irradiation on some blood serum enzymes in rats

    International Nuclear Information System (INIS)

    Metwalli, O.M.

    1977-01-01

    Rats were fed irradiated diets as part of a study of screening tests for the safety of irradiated food. The diet consisted (g/100 g) of casein, 8.5; skim-milk, 9.4; potato starch, 50.00; wheat flour, 16.50; sucrose, 5.00; sunflower oil, 6.00; choline chloride, 0.10; salt mixture, 3.50; and vitamin mix.ure, 1.00. Diets were irradiated at 2.5 or 4.5 Mrad and were fed ad lib. for 4 months. Levels of serum (i) glutanic-pyruvic transaminase, (ii) glutamic-oxalacetic transaminase, and (iii) lactic dehydrogenase were determined. No significant changes were observed in (i) or (iii) on ieeding irradiated diets, or in (ii) for male rats. Significant decreases (P [de

  18. Radioprotective effect against gamma-irradiation of methylene blue in the rat with reference to serum enzymes and pancreatic protein fractions examined by isoelectric focussing

    Energy Technology Data Exchange (ETDEWEB)

    Chung, S O; Nam, S Y [Kyung Hee Univ., Seoul (Korea). Dept. of Biology

    1975-12-01

    The Sprague-Dawley male rats were given 360 rads of single whole-body gamma-irradiation following an intraperitoneal injection of methylene blue (40 mg/kg). Serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum amylase, serum lipase, and serum lecithinase A activities, and isoelectric focussing pattern of pancreatic juice proteins were determined at various time intervals after exposure. Methylene blue reduced generally the rise of SGOT, sGPT, amylase, and lipase activities. Methylene blue delayed also the serum lecithinase A fall after exposure. Mean number of protein bands as revealed by isoelectric focussing of pancreatic juice were significantly altered in both the control and the methylene blue-treated group after exposure. Especially methylene blue-treated group showed a marked delay in the decrease in number of protein bands after exposure. The possibility of using the SGOT, the SGPT, the serum amylase, the serum lipase, and the serum lecithinase A levels, and the number of protein bands in isoelectric focussing pattern of pancreatic juice as an early index of radiation injury is suggested.

  19. Cerebrospinal fluid lactate and pyruvate concentrations and their ratio.

    Science.gov (United States)

    Zhang, Wan-Ming; Natowicz, Marvin R

    2013-05-01

    Determinations of cerebrospinal fluid (CSF) lactate and pyruvate concentrations and CSF lactate:pyruvate (L/P) ratios are important in several clinical settings, yet published normative data have significant limitations. We sought to determine a large dataset of stringently-defined normative data for CSF lactate and pyruvate concentrations and CSF L/P ratios. We evaluated data from 627 patients who had determinations of CSF lactate and/or CSF pyruvate from 2001 to 2011 at the Cleveland Clinic. Inclusion in the normal reference population required normal CSF cell counts, glucose and protein and routine serum chemistries and absence of progressive brain disorder, epilepsy, or seizure within 24h. Brain MRI, if done, showed no evidence of tumor, acute changes or basal ganglia abnormality. CSF cytology, CSF alanine and immunoglobulin levels, and oligoclonal band analysis were required to be normal, if done. Various inclusion/exclusion criteria were compared. 92 patients fulfilled inclusion/exclusion criteria for a reference population. The 95% central intervals (2.5%-97.5%) for CSF lactate and pyruvate levels were 1.01-2.09mM and 0.03-0.15mM, respectively, and 9.05-26.37 for CSF L/P. There were no significant gender-related differences of CSF lactate or pyruvate concentrations or of CSF L/P. Weak positive correlations between the concentration of CSF lactate or pyruvate and age were noted. Using stringent inclusion/exclusion criteria, we determined normative data for CSF lactate and pyruvate concentrations and CSF L/P ratios in a large, well-characterized reference population. Normalcy of routine CSF and blood analytes are the most important parameters in determining reference intervals for CSF lactate and pyruvate. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  20. SIGNIFICANCE OF LACTATE DEHYDROGENASE AND ASPARTATE TRANSAMINASE AS BIOCHEMICAL MARKERS AND AS PREDICTORS OF SEVERITY OF PREGNANCY-INDUCED HYPERTENSION AND ITS COMPLICATIONS

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    Ramesh Sonowal

    2017-03-01

    Full Text Available BACKGROUND To compare serum Lactate Dehydrogenase (LDH and serum Aspartate Transaminase (AST of normotensive pregnant women with those of preeclamptic and eclamptic women. To determine the relationship of levels of serum lactate dehydrogenase and serum aspartate transaminase with severity of pregnancy-induced hypertension and its complications. MATERIALSAND METHODS The study was carried out on pregnant hypertensive patients attending outpatient department of Obstetrics and Gynaecology department, AMCH, Dibrugarh, Assam from 1 st July 2013 to 30 th June 2014. Normotensive pregnant women were taken as controls. Each serum sample from both the control group as well as study group was estimated for lactate dehydrogenase and aspartate transaminase using standard methods and a comparison is drawn and analysed using t-test and Chi-square test. RESULTS Serum lactate dehydrogenase and serum aspartate transaminase levels were higher in the study group in comparison to the study groups. The mean serum LDH was 198±30.03U/L in control group, whereas in preeclampsia and eclampsia, mean serum levels of LDH were 817±114U/L and 927±108U/L, respectively. The levels of the serum AST were found to be less than 600U/L in normotensive and preeclampsia patients and more than 600 U/L in eclampsia and other complications of PIH. CONCLUSION Serum lactate dehydrogenase and serum aspartate transaminase levels in patients suffering from preeclampsia and its complications are consistently higher compared to the normotensive pregnant patients. To determine the usefulness of inclusion of these enzymes along with other cardiac enzymes in the panel of investigations of pregnant women universally needs further large scale comparative studies.

  1. Effect of subchronic zinc toxicity on rat salivary glands and serum composition.

    Science.gov (United States)

    Mizari, Nazer; Hirbod-Mobarakeh, Armin; Shahinpour, Shervin; Ghalichi-Tabriz, Mostafa; Beigy, Maani; Yamini, Ali; Dehpour, Ahmad Reza

    2012-11-01

    Zinc plays an important role in a wide variety of metabolic processes in animal systems. The role of zinc in preservative treatment, fungicidal action and medicine, and addition of supplementary zinc have increased the probability of zinc toxicity, specially the chronic type. It is known that the composition and quantity of saliva influence the oral health. Regarding people's exposure to zinc in routine life and the importance of saliva, our purpose was to investigate the effects of oral zinc intoxication on secretory function in rat salivary glands and also on serum composition. In this study, there were five groups of female rats. Four groups received zinc acetate dehydrate through their drinking water. After 3 months of experiment, the chemical characteristics and flow rate of saliva and weight of salivary glands were determined. The effects of zinc on hematological and chemical factors of plasma were assessed too. Flow rate of submandibular glands was significantly lower in experimental groups and there were significant changes in Na(+), Ca(2+) and K(+) concentration both in saliva and in plasma. The serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, glucose levels in the plasma and urine creatinine levels were also altered in experimental groups in comparison with the control group. Our results show that zinc toxicity will affect the quantity and quality of saliva probably through changes in the various neurologic pathways to the salivary glands or effects on acinar cells of the salivary glands. Furthermore, our results showed that zinc toxicity will affect the liver and renal function.

  2. GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria.

    Science.gov (United States)

    Cao, Juxiang; Barbosa, Jose M; Singh, Narendra; Locy, Robert D

    2013-07-01

    GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.

  3. Porcine alanine transaminase after liver allo-and xenotransplantation

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

    2012-01-01

    Aspartate transaminase (AST) and alanine transaminase (ALT) are measured following liver transplantation as indicators of hepatocellular injury. During a series of orthotopic liver allo-and xenotransplants, we observed that there was an increase in AST in all cases. The anticipated concomitant rise in ALT did not occur when a wild-type (WT) pig was the source of the liver graft, but did occur when a baboon or a genetically engineered (α1,3-galactosyltransferase gene-knockout [GTKO]) pig was t...

  4. Termoativação da transaminase glutâmico oxaloacética

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    Hélion Póvoa Júnior

    1973-01-01

    Full Text Available Estudou-se a atividade da transaminase glutâmico oxaloacética (TGO em diferentes tecidos (fígado, músculo, rim, pulmão, baço e soro sanguíneo de ratos e de soro humano. verificou-se que a atividade da enzima proveniente de qualquer um destes tecidos é aumentada cerca de tr~es vezes quando a incubação se faz a 60ºC, ao invés de 37ºC. São feitas considerações acerca da importância deste fato.Glutamic Oxalacetic transaminase is thermoativated. Its optimum of catalytical activity is at 60ºC. At this temperature, colour is approximately three times more intense than at 37ºC, temperature usually utilized for determination of enzyme activity. This phenomenon is observed in human blood serum and several rat tissues (liver, heart, striated muscle, spleen, lung, kidney and blood serum.

  5. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

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    Baggetto, L G; Lehninger, A L

    1987-05-29

    Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

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

    Science.gov (United States)

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

    2015-07-01

    A Pseudomonas species [Pseudomonas sp. strain amino alkanoate catabolism (AAC)] was identified that has the capacity to use 12-aminododecanoic acid, the constituent building block of homo-nylon-12, as a sole nitrogen source. Growth of Pseudomonas sp. strain AAC could also be supported using a range of additional ω-amino alkanoates. This metabolic function was shown to be most probably dependent upon one or more transaminases (TAs). Fourteen genes encoding putative TAs were identified from the genome of Pseudomonas sp. AAC. Each of the 14 genes was cloned, 11 of which were successfully expressed in Escherichia coli and tested for activity against 12-aminododecanoic acid. In addition, physiological functions were proposed for 9 of the 14 TAs. Of the 14 proteins, activity was demonstrated in 9, and of note, 3 TAs were shown to be able to catalyse the transfer of the ω-amine from 12-aminododecanoic acid to pyruvate. Based on this study, three enzymes have been identified that are promising biocatalysts for the production of nylon and related polymers. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  7. A rational approach for ω-transaminase-catalyzed process design

    DEFF Research Database (Denmark)

    T. Gundersen, Maria; Lloyd, Richard; Tufvesson, Pär

    Herein we describe a novel rational approach to the design of a ω-transaminase process such that it will fulfill criteria necessary for industrial use. By first determining the fundamental properties of the reaction system, it is possible to suggest appropriate process strategies that may be used...

  8. beta-Chloro-L-alanine inhibition of the Escherichia coli alanine-valine transaminase.

    OpenAIRE

    Whalen, W A; Wang, M D; Berg, C M

    1985-01-01

    beta-Chloro-L-alanine, an amino acid analog which inhibits a number of enzymes, reversibly inhibited the Escherichia coli K-12 alanine-valine transaminase, transaminase C. This inhibition, along with the inhibition of transaminase B, accounted for the isoleucine-plus-valine requirement of E. coli in the presence of beta-chloro-L-alanine.

  9. Pyruvate reduces 4-aminophenol in vitro toxicity

    International Nuclear Information System (INIS)

    Harmon, R. Christopher; Kiningham, Kinsley K.; Valentovic, Monica A.

    2006-01-01

    Pyruvate has been observed to reduce the nephrotoxicity of some agents by maintaining glutathione status and preventing lipid peroxidation. This study examined the mechanism for pyruvate protection of p-aminophenol (PAP) nephrotoxicity. Renal cortical slices from male Fischer 344 rats were incubated for 30-120 min with 0, 0.1, 0.25 or 0.5 mM PAP in oxygenated Krebs buffer containing 0 or 10 mM pyruvate or glucose (1.28 or 5.5 mM). LDH leakage was increased above control by 0.25 and 0.5 mM PAP beginning at 60 min and by 0.1 mM PAP at 120 min. Pyruvate prevented an increase in LDH leakage at 60- and 120-min exposure to 0.1 and 0.25 mM PAP. Pyruvate also prevented a decline in ATP levels. Glucose (1.28 and 5.5 mM) provided less protection than pyruvate from PAP toxicity. Total glutathione levels were diminished by 0.1 and 0.25 mM PAP within 60 and 30 min, respectively. Pyruvate prevented the decline in glutathione by 0.1 mM PAP at both time periods and at 30 min for 0.25 mM PAP. Pyruvate reduced the magnitude of glutathione depletion by 0.25 mM PAP following a 60-min incubation. Glutathione disulfide (GSSG) levels in renal slices were increased at 60 min by exposure to 0.25 mM PAP, while pyruvate prevented increased GSSG levels by PAP. Pyruvate also reduced the extent of 4-hydroxynonenal (4-HNE)-adducted proteins present after a 90-min incubation with PAP. These results indicate that pyruvate provided protection for PAP toxicity by providing an energy substrate and reducing oxidative stress

  10. Applying Enzymatic Cascades for ISCPR in ω-transaminase Systems

    DEFF Research Database (Denmark)

    Janes, Kresimir; Woodley, John; Tufvesson, Pär

    , esterases, ketoreductases and proteases and many more emerging biocatalysts such are monoamine oxidases, transaminases and P450 monooxygenases to name a few. The focus of this thesis is the biocatalytic synthesis of small molecule pharmaceuticals (Mw... in an industrial process context have thus not been considered properly. In this research lactate dehydrogenase (LDH) (E.C. 1.1.1.27), alanine dehydrogenase (E.C. 1.4.1.1) (AlaDH) and yeast alcohol dehydrogenase (E.C. 1.1.1.1) (YADH) have been researched as co-product degrading enzymes and glucose dehydrogenase...

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

    Science.gov (United States)

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

    2012-01-01

    Aspartate transaminase (AST) and alanine transaminase (ALT) are measured following liver transplantation as indicators of hepatocellular injury. During a series of orthotopic liver allo-and xenotransplants, we observed that there was an increase in AST in all cases. The anticipated concomitant rise in ALT did not occur when a wild-type (WT) pig was the source of the liver graft, but did occur when a baboon or a genetically engineered (α1,3-galactosyltransferase gene-knockout [GTKO]) pig was the source of the graft. We hypothesized that the cience of Galα1,3Gal in GTKO pig livers may render pig hepatocytes similar to human and baboon hepatocytes in their response to hepatocellular injury. Reviewing the literature, after WT pig liver allotransplantation or xenotransplantation, in the majority of reports, although changes in AST were reported, no mention was made of changes in ALT, suggesting that there was no change in ALT. However, Ramirez et al. reported two cases of liver xenotransplants from hCD55 pigs, following which there were increases in both AST and ALT, suggesting that it is not simply the cience of expression of Galα1,3Gal that is the cause. We acknowledge that our observation is based on a small number of experiments, but we believe it is worth recording. © 2012 John Wiley & Sons A/S.

  12. Pediatric FAST and elevated liver transaminases: An effective screening tool in blunt abdominal trauma.

    Science.gov (United States)

    Sola, Juan E; Cheung, Michael C; Yang, Relin; Koslow, Starr; Lanuti, Emma; Seaver, Chris; Neville, Holly L; Schulman, Carl I

    2009-11-01

    The current standard for the evaluation of children with blunt abdominal trauma (BAT) consists of physical examination, screening lab values, and computed tomography (CT) scan. We sought to determine if the focused assessment with sonography for trauma (FAST) combined with elevated liver transaminases (AST/ALT) could be used as a screening tool for intra-abdominal injury (IAI) in pediatric patients with BAT. Registry data at a level 1 trauma center was retrospectively reviewed from 1991-2007. Data collected on BAT patients under the age of 16 y included demographics, injury mechanism, ISS, GCS, imaging studies, serum ALT and AST levels, and disposition. AST and ALT were considered positive if either one was >100 IU/L. Overall, 3171 cases were identified. A total of 1008 (31.8%) patients received CT scan, 1148 (36.2%) had FAST, and 497 (15.7%) patients received both. Of the 497 patients, 400 (87.1%) also had AST and ALT measured. FAST was 50% sensitive, 91% specific, with a positive predictive value (PPV) of 68%, negative predictive value (NPV) of 83%, and accuracy of 80%. Combining FAST with elevated AST or ALT resulted in a statistically significant increase in all measures (sensitivity 88%, specificity 98%, PPV 94%, NPV 96%, accuracy 96%). FAST combined with AST or ALT > 100 IU/L is an effective screening tool for IAI in children following BAT. Pediatric patients with a negative FAST and liver transaminases < 100 IU/L should be observed rather than subjected to the radiation risk of CT.

  13. Adult Kawasaki's disease with myocarditis, splenomegaly, and highly elevated serum ferritin levels.

    Science.gov (United States)

    Cunha, Burke A; Pherez, Francisco M; Alexiadis, Varvara; Gagos, Marios; Strollo, Stephanie

    2010-01-01

    Kawasaki's disease is a disease of unknown cause. The characteristic clinical features of Kawasaki's disease are fever> or =102 degrees F for> or =5 days accompanied by a bilateral bulbar conjunctivitis/conjunctival suffusion, erythematous rash, cervical adenopathy, pharyngeal erythema, and swelling of the dorsum of the hands/feet. Kawasaki's disease primarily affects children and is rare in adults. In children, Kawasaki's disease is more likely to be associated with aseptic meningitis, coronary artery aneurysms, and thrombocytosis. In adult Kawasaki's disease, unilateral cervical adenopathy, arthritis, conjunctival suffusion/conjunctivitis, and elevated serum transaminases (serum glutamic oxaloacetic transaminase [SGOT]/serum glutamate pyruvate transaminase [SGPT]) are more likely. Kawasaki's disease in adults may be mimicked by other acute infections with fever and rash, that is, group A streptococcal scarlet fever, toxic shock syndrome (TSS), and Rocky Mountain Spotted Fever (RMSF). Because there are no specific tests for Kawasaki's disease, diagnosis is based on clinical criteria and the syndromic approach. In addition to rash and fever, scarlet fever is characterized by circumoral pallor, oropharyngeal edema, Pastia's lines, and peripheral eosinophilia, but not conjunctival suffusion, splenomegaly, swelling of the dorsum of the hands/feet, thrombocytosis, or an elevated SGOT/SGPT. In TSS, in addition to rash and fever, there is conjunctival suffusion, oropharyngeal erythema, and edema of the dorsum of the hands/feet, an elevated SGOT/SGPT, and thrombocytopenia. Patients with TSS do not have cervical adenopathy or splenomegaly. RMSF presents with fever and a maculopapular rash that becomes petechial, first appearing on the wrists/ankles after 3 to 5 days. RMSF is accompanied by a prominent headache, periorbital edema, conjunctival suffusion, splenomegaly, thrombocytopenia, an elevated SGOT/SGPT, swelling of the dorsum of the hands/feet, but not oropharyngeal

  14. Dynamics of pyruvate metabolism in Lactococcus lactis

    DEFF Research Database (Denmark)

    Melchiorsen, Claus Rix; Jensen, Niels B.S.; Christensen, Bjarke

    2001-01-01

    The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end...... product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acid-product formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis...

  15. Cui et al., Afr J Tradit Complement Altern Med. (2016) 13(5):114-122 ...

    African Journals Online (AJOL)

    oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in serum, .... [2, 2′-azino-bis (3-ethylbenzothiazoline)-6-sulphonic acid] diamonium salt ..... Chaenomeles sinensis fruit extract in streptozotocin-induced diabetic rats.

  16. Cultivation of parasitic leptospires: effect of pyruvate.

    Science.gov (United States)

    Johnson, R C; Walby, J; Henry, R A; Auran, N E

    1973-07-01

    Sodium pyruvate (100 mug/ml) is a useful addition to the Tween 80-albumin medium for the cultivation of parasitic serotypes. It is most effective in promoting growth from small inocula and growth of the nutritionally fastidious serotypes.

  17. Pyruvate transport by thermogenic-tissue mitochondria.

    OpenAIRE

    Proudlove, M O; Beechey, R B; Moore, A L

    1987-01-01

    1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced...

  18. Phosphorylation site on yeast pyruvate dehydrogenase complex

    International Nuclear Information System (INIS)

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the 32 P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation

  19. Ethyl Pyruvate Ameliorates Hepatic Ischemia-Reperfusion Injury by Inhibiting Intrinsic Pathway of Apoptosis and Autophagy

    Directory of Open Access Journals (Sweden)

    Miao Shen

    2013-01-01

    Full Text Available Background. Hepatic ischemia-reperfusion (I/R injury is a pivotal clinical problem occurring in many clinical conditions such as transplantation, trauma, and hepatic failure after hemorrhagic shock. Apoptosis and autophagy have been shown to contribute to cell death in hepatic I/R injury. Ethyl pyruvate, a stable and simple lipophilic ester, has been shown to have anti-inflammatory properties. In this study, the purpose is to explore both the effect of ethyl pyruvate on hepatic I/R injury and regulation of intrinsic pathway of apoptosis and autophagy. Methods. Three doses of ethyl pyruvate (20 mg/kg, 40 mg/kg, and 80 mg/kg were administered 1 h before a model of segmental (70% hepatic warm ischemia was established in Balb/c mice. All serum and liver tissues were obtained at three different time points (4 h, 8 h, and 16 h. Results. Alanine aminotransferase (ALT, aspartate aminotransferase (AST, and pathological features were significantly ameliorated by ethyl pyruvate (80 mg/kg. The expression of Bcl-2, Bax, Beclin-1, and LC3, which play an important role in the regulation of intrinsic pathway of apoptosis and autophagy, was also obviously decreased by ethyl pyruvate (80 mg/kg. Furthermore, ethyl pyruvate inhibited the HMGB1/TLR4/ NF-κb axis and the release of cytokines (TNF-α and IL-6. Conclusion. Our results showed that ethyl pyruvate might attenuate to hepatic I/R injury by inhibiting intrinsic pathway of apoptosis and autophagy, mediated partly through downregulation of HMGB1/TLR4/ NF-κb axis and the competitive interaction with Beclin-1 of HMGB1.

  20. Study on the protective effect of ethyl pyruvate on mouse models of sepsis-induced lung injury

    International Nuclear Information System (INIS)

    Ti Dongdong; Deng Zihui; Xue Hui; Wang Luhuan; Lin Ji; Yan Guangtao

    2008-01-01

    Objective: To investigate the protective role of ethyl pyruvate on mouse models of lung injury from sepsis. Methods: Mouse sepsis models were established by cecal ligation-perforation. Four enzyme parameters related to synthesis of free radicals in lung homogenized fluids namely malonaldehyde (MDA), pyruvate acid, lactic acid and total anti-oxidative capacity (TAOC) were determined with spectrophotometry, and serum leptin levels were detected with radioimmunoassay at 3, 6, 9, 12h after operation in these models. Half of the models were treated with intraperitoneal injection of ethyl pyruvate (EP) (75mg/kg). Results: In the models treated with ethyl pyruvate injection, the activity of malonaldehyde, pyruvate acid, lactic acid and total anti-oxidative capacity were affected to certain extent, at some time frames but the results were not unanimously inhibitive or promotive. Serum leptin levels in EP injection models at 6h and 12h after sepsis were significantly higher than those in non-treated models. Conclusion: Ethyl pyruvate perhaps exerted its protective effect on sepsis-induced lung injury through increase of leptin levels in the models. (authors)

  1. The progression from a lower to a higher invasive stage of bladder cancer is associated with severe alterations in glucose and pyruvate metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Conde, Vanessa R. [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal); Oliveira, Pedro F. [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal); Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine, Abel Salazar Institute of Biomedical Sciences, University of Porto – UMIB/ICBAS/UP (Portugal); Nunes, Ana R.; Rocha, Cátia S. [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal); Ramalhosa, Elsa; Pereira, José A. [Mountain Research Centre (CIMO), School of Agriculture, Polytechnic Institute of Bragança (Portugal); Alves, Marco G., E-mail: alvesmarc@gmail.com [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal); Silva, Branca M., E-mail: bmcms@ubi.pt [CICS-UBI–Health Sciences Research Centre, University of Beira Interior, Covilhã (Portugal)

    2015-07-01

    Cancer cells present a particular metabolic behavior. We hypothesized that the progression of bladder cancer could be accompanied by changes in cells glycolytic profile. We studied two human bladder cancer cells, RT4 and TCCSUP, in which the latter represents a more invasive stage. The levels of glucose, pyruvate, alanine and lactate in the extracellular media were measured by Proton Nuclear Magnetic Resonance. The protein expression levels of glucose transporters 1 (GLUT1) and 3 (GLUT3), monocarboxylate transporter 4 (MCT4), phosphofructokinase-1 (PFK1), glutamic-pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) were determined. Our data showed that glucose consumption and GLUT3 levels were similar in both cell lines, but TCCSUP cells displayed lower levels of GLUT1 and PFK expression. An increase in pyruvate consumption, concordant with the higher levels of lactate and alanine production, was also detected in TCCSUP cells. Moreover, TCCSUP cells presented lower protein expression levels of GPT and LDH. These results illustrate that bladder cancer progression is associated with alterations in cells glycolytic profile, namely the switch from glucose to pyruvate consumption in the more aggressive stage. This may be useful to develop new therapies and to identify biomarkers for cancer progression. - Highlights: • Metabolic phenotype of less and high invasive bladder cancer cells was studied. • Bladder cancer progression involves alterations in cells glycolytic profile. • More invasive bladder cancer cells switch from glucose to pyruvate consumption. • Our results may help to identify metabolic biomarkers of bladder cancer progression.

  2. Crystal structure of Cryptosporidium parvum pyruvate kinase.

    Directory of Open Access Journals (Sweden)

    William J Cook

    Full Text Available Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303-320 of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.

  3. Microorganisms and methods for producing pyruvate, ethanol, and other compounds

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Jennifer L.; Zhang, Xiaolin

    2017-12-26

    Microorganisms comprising modifications for producing pyruvate, ethanol, and other compounds. The microorganisms comprise modifications that reduce or ablate activity of one or more of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, phosphate acetyltransferase, acetate kinase, pyruvate oxidase, lactate dehydrogenase, cytochrome terminal oxidase, succinate dehydrogenase, 6-phosphogluconate dehydrogenase, glutamate dehydrogenase, pyruvate formate lyase, pyruvate formate lyase activating enzyme, and isocitrate lyase. The microorganisms optionally comprise modifications that enhance expression or activity of pyruvate decarboxylase and alcohol dehydrogenase. The microorganisms are optionally evolved in defined media to enhance specific production of one or more compounds. Methods of producing compounds with the microorganisms are provided.

  4. Cultivation of Parasitic Leptospires: Effect of Pyruvate

    Science.gov (United States)

    Johnson, R. C.; Walby, J.; Henry, R. A.; Auran, N. E.

    1973-01-01

    Sodium pyruvate (100 μg/ml) is a useful addition to the Tween 80-albumin medium for the cultivation of parasitic serotypes. It is most effective in promoting growth from small inocula and growth of the nutritionally fastidious serotypes. Images PMID:4580191

  5. Compartmented pyruvate in perfused working heart

    International Nuclear Information System (INIS)

    Buenger, R.

    1985-01-01

    Pyruvate compartmentation and lactate dehydrogenase (LDH) were studied in isolated perfused working guinea pig hearts. The mean intracellular pyruvate (Pyr) contents increased with perfusate Pyr (0-2 mM) but varied only slightly with glucose (0-10 mM) and additional insulin (0.04-5 U/l), respectively. With 5-10 mM glucose plus 5 U/l insulin, but not with Pyr or lactate (Lac) as substrates, a near equilibrium between the LDH and the glycerol-3-phosphate dehydrogenase seemed to exist. Evidence for an inhibitory effect of Pyr on the activity of the LDH system of the perfused hearts was not obtained. With [U- 14 C]glucose as sole substrate, the specific activity of coronary venous Lac was near half that of precursor glucose. 14 CO 2 production was thus in quantitative agreement with rates of pyruvate oxidation that were determined as glucose uptake minus (Pyr + Lac) release. In contrast, with 0.2 mM [1- 14 C]Pyr plus 5 mM glucose, the ratio of 14 CO 2 production to specific activity of Lac overestimated Pyr oxidation judged from myocardial substrate balances and O 2 uptake, respectively; here, at least three pools of [ 14 C]HCO-3 and [ 14 C]lac, respectively, were kinetically demonstrable during washout of trace amounts of 14 C-labeled Pyr. Evidently, the specific activity of Lac was equivalent to that of mitochondrial oxidized Pyr provided [ 14 C]glucose was the sole or major precursor of cellular pyruvate. However, exogenously applied [1- 14 C]Pyr of high specific activity seemed to induce intracellular formation of both a highly and lowly labeled Pyr; the latter Pyr compartment did not seem in ready equilibrium with the cell physiologically prevailing highly labeled Pyr pool

  6. Effects of sub-chronic exposure to zinc nanoparticles on tissue accumulation, serum biochemistry and histopathological changes in tilapia (Oreochromis niloticus)

    Science.gov (United States)

    Kaya, Hasan; Duysak, Müge; Akbulut, Mehmet; Yılmaz, Sevdan; Gürkan, Mert; Arslan, Zikri; Demir, Veysel; Ateş, Mehmet

    2016-01-01

    Zinc nanoparticles (ZnNPs) are among the least investigated NPs and thus their toxicological effects are not known. In this study, tilapia (Oreochromis niloticus) were exposed to 1 and 10 mg/L suspensions of small size (SS, 40–60 nm) and large size (LS, 80–100 nm) ZnNPs for 14 days under semi-static conditions. Total Zn levels in the intestine, liver, kidney, gill, muscle tissue and brain were measured. Blood serum glucose (GLU), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) were examined to elucidate the physiological disturbances induced by ZnNPs. Organ pathologies were examined for the gills, liver and kidney to identify injuries associated with exposure. Significant accumulation was observed in the order of intestine, liver, kidney and gills. Zn levels exhibited time- and concentration-dependent increase in the organs. Accumulation in kidney was also dependent on particle size; NPs SS-ZnNPs were trapped more effectively than LS-ZnNPs. No significant accumulation occurred in the brain (p>0.05) while Zn levels in muscle tissue increased only marginally (p≥0.05). Significant disturbances were noted in serum GOT and LDH (p0.05). Histopathological tubular deformations and mononuclear cell infiltrations were observed in kidney sections. In addition, an increase in melano-macrophage aggregation intensity was identified on the 7th day in treatments exposed to LS-ZnNPs. Mononuclear cell infiltrations were identified in liver sections for all treatments. Both ZnNPs caused basal hyperplasia in gill sections. Fusions appeared in the gills after the 7th day in fish treated with 10 mg/L suspensions of SS-ZnNPs. In addition, separations in the secondary lamella epithelia were observed. The results indicated that exposure to ZnNPs could lead to disturbances in blood biochemistry and cause histopathological injuries in the tissues of O. niloticus. PMID:27464841

  7. Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy

    Directory of Open Access Journals (Sweden)

    Ximena Escalera-Fanjul

    2017-06-01

    Full Text Available Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s. Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64

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

    Science.gov (United States)

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

    2014-01-01

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

  9. Loss of Mitochondrial Pyruvate Carrier 2 in Liver Leads to Defects in Gluconeogenesis and Compensation via Pyruvate-Alanine Cycling

    Science.gov (United States)

    McCommis, Kyle S.; Chen, Zhouji; Fu, Xiaorong; McDonald, William G.; Colca, Jerry R.; Kletzien, Rolf F.; Burgess, Shawn C.; Finck, Brian N.

    2015-01-01

    SUMMARY Pyruvate transport across the inner mitochondrial membrane is believed to be a prerequisite step for gluconeogenesis in hepatocytes, which is important for maintenance of normoglycemia during prolonged food deprivation, but also contributes to hyperglycemia in diabetes. To determine the requirement for mitochondrial pyruvate import in gluconeogenesis, mice with liver-specific deletion of mitochondrial pyruvate carrier 2 (LS-Mpc2−/−) were generated. Loss of MPC2 impaired, but did not completely abolish, hepatocyte pyruvate metabolism, labelled pyruvate conversion to TCA cycle intermediates and glucose, and glucose production from pyruvate. Unbiased metabolomic analyses of livers from fasted LS-Mpc2−/− mice suggested that alterations in amino acid metabolism, including pyruvate-alanine cycling, might compensate for loss of MPC2. Indeed, inhibition of pyruvate-alanine transamination further reduced mitochondrial pyruvate metabolism and glucose production by LS-Mpc2−/− hepatocytes. These data demonstrate an important role for MPC2 in controlling hepatic gluconeogenesis and illuminate a compensatory mechanism for circumventing a block in mitochondrial pyruvate import. PMID:26344101

  10. Ethyl pyruvate protects colonic anastomosis from ischemia-reperfusion injury.

    Science.gov (United States)

    Unal, B; Karabeyoglu, M; Huner, T; Canbay, E; Eroglu, A; Yildirim, O; Dolapci, M; Bilgihan, A; Cengiz, O

    2009-03-01

    Ethyl pyruvate is a simple derivative in Ca(+2)- and K(+)-containing balanced salt solution of pyruvate to avoid the problems associated with the instability of pyruvate in solution. It has been shown to ameliorate the effects of ischemia-reperfusion (I/R) injury in many organs. It has also been shown that I/R injury delays the healing of colonic anastomosis. In this study, the effect of ethyl pyruvate on the healing of colon anastomosis and anastomotic strength after I/R injury was investigated. Anastomosis of the colon was performed in 32 adult male Wistar albino rats divided into 4 groups of 8 individuals: (1) sham-operated control group (group 1); (2) 30 minutes of intestinal I/R by superior mesenteric artery occlusion (group 2); (3) I/R+ ethyl pyruvate (group 3), ethyl pyruvate was administered as a 50-mg/kg/d single dose; and (4) I/R+ ethyl pyruvate (group 4), ethyl pyruvate administration was repeatedly (every 6 hours) at the same dose (50 mg/kg). On the fifth postoperative day, animals were killed. Perianastomotic tissue hydroxyproline contents and anastomotic bursting pressures were measured in all groups. When the anastomotic bursting pressures and tissue hydroxyproline contents were compared, it was found that they were decreased in group 2 when compared with groups 1, 3, and 4 (P .05). Ethyl pyruvate significantly prevents the delaying effect of I/R injury on anastomotic strength and healing independent from doses of administration.

  11. High Serum Adiponectin Level Is a Risk Factor for Anemia in Japanese Men: A Prospective Observational Study of 1,029 Japanese Subjects.

    Directory of Open Access Journals (Sweden)

    Kei Kohno

    Full Text Available Erythroid abnormalities including anemia and polycythemia are often observed in the general clinical setting. Because recent studies reported that adiponectin negatively affects hematopoiesis, we performed a prospective observational study to assess the relationship between anemia and adiponectin, as well as other parameters, in 1029 Japanese subjects (477 men and 552 women 40 years of age and older. Body measurements, blood tests, and nutrition intake studies were performed at baseline, and 5 to 7 years later (follow-up. Hemoglobin (Hb and hematocrit (Hct levels in men with high serum adiponectin levels were lower at follow-up than at baseline. Multiple regression analysis showed that age, body mass index, adiponectin, and glutamic-pyruvic transaminase were significantly associated with erythroid-related variables (red blood cells, Hb, and Hct in both men and women (P <0.05. In a logistic regression analysis, adiponectin, fasting blood glucose, and β-natriuretic peptide were significant risk factors for anemia in men, and blood urea nitrogen and amylase were significant risk factors in women. Physical features and nutrient intake were not risk factors for anemia. Our study demonstrates, both clinically and epidemiologically, that a high serum adiponectin level decreases the amounts of erythroid-related variables and is a risk factor for anemia in Japanese men.

  12. A Practical and Fast Method To Predict the Thermodynamic Preference of omega-Transaminase-Based Transformations

    DEFF Research Database (Denmark)

    Meier, Robert J.; Gundersen Deslauriers, Maria; Woodley, John

    2015-01-01

    A simple, easy-to-use, and fast approach method is proposed and validated that can predict whether a transaminase reaction is thermodynamically unfavourable. This allowed us to de-select, in the present case, at least 50% of the reactions because they were thermodynamically unfavourable as confir...

  13. Selections of minimal conditions for a simple intensification and scale up of w-transaminase reactions

    DEFF Research Database (Denmark)

    Gundersen, Maria T.; Lloyd, Richard C; Woodley, John

    A step wise decision matrix is presented to quickly evaluate w - transaminase for a ‘simple scale up’ in the synthetic direction . Here a ‘simple scale up’ is defined as a system without specialized equipment or process development, thus a rapid implementation . The three step method consists...

  14. Effects of aqueous extract of Basella alba leaves on haematological ...

    African Journals Online (AJOL)

    Jane

    2010-09-02

    Sep 2, 2010 ... It is high in vitamin A, vitamin C, vitamin B9 (folic acid), calcium ..... noids in serum of control and insulin dependent diabetic Spanish subjects. Clin. Chem. ... of serum glutamic oxaloacetic and glutamic pyruvic transaminases.

  15. Pyruvate carboxylase is expressed in human skeletal muscle

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2010-01-01

    Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable...

  16. Production and Recovery of Pyruvic Acid: Recent Advances

    Science.gov (United States)

    Pal, Dharm; Keshav, Amit; Mazumdar, Bidyut; Kumar, Awanish; Uslu, Hasan

    2017-12-01

    Pyruvic acid is an important keto-carboxylic acid and can be manufactured by both chemical synthesis and biotechnological routes. In the present paper an overview of recent developments and challenges in various existing technique for the production and recovery of pyruvic acid from fermentation broth or from waste streams has been presented. The main obstacle in biotechnological production of pyruvic acid is development of suitable microorganism which can provide high yield and selectivity. On the other hand, technical limitation in recovery of pyruvic acid from fermentation broth is that, it could not be separated as other carboxylic acid in the form of salts by addition of alkali. Besides, pyruvic acid cannot be crystallized. Commercial separation by distillation is very expensive because pyruvic acid decomposes at higher temperature. It is also chemically reactive due to its peculiar molecular structure and has tendency to polymerize. Thus, at high concentration the various type of reaction leads to lower yield of the product, and hence, conventional methods are not favorable. Alternate separation technologies viable to both synthetic and biological routes are the current research areas. Latest techniques such as reactive extraction is new to the field of recovery of pyruvic acid. Recent development and future prospects in downstream processing of biochemically produced pyruvic acids has been discussed in this review article.

  17. The Role of Pyruvate Dehydrogenase Kinase in Diabetes and Obesity

    Directory of Open Access Journals (Sweden)

    In-Kyu Lee

    2014-06-01

    Full Text Available The pyruvate dehydrogenase complex (PDC is an emerging target for the treatment of metabolic syndrome. To maintain a steady-state concentration of adenosine triphosphate during the feed-fast cycle, cells require efficient utilization of fatty acid and glucose, which is controlled by the PDC. The PDC converts pyruvate, coenzyme A (CoA, and oxidized nicotinamide adenine dinucleotide (NAD+ into acetyl-CoA, reduced form of nicotinamide adenine dinucleotide (NADH, and carbon dioxide. The activity of the PDC is up- and down-regulated by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase, respectively. In addition, pyruvate is a key intermediate of glucose oxidation and an important precursor for the synthesis of glucose, glycerol, fatty acids, and nonessential amino acids.

  18. An improved method for the assay of platelet pyruvate dehydrogenase

    International Nuclear Information System (INIS)

    Schofield, P.J.; Griffiths, L.R.; Rogers, S.H.

    1980-01-01

    An improved method for the assay of human platelet pyruvate dehydrogenase is described. By generating the substrate [1- 14 C]pyruvate in situ from [1- 14 C]lactate plus L-lactate dehydrogenase, the rate of spontaneous decarboxylation is dramatically reduced, allowing far greater sensitivity in the assay of low activities of pyruvate dehydrogenase. In addition, no special precautions are required for the storage and use of [1- 14 C]lactate, in contrast to those for [1- 14 C]pyruvate. These factors allow a 5-10-fold increase in sensitivity compared with current methods. The pyruvate dehydrogenase activity of normal subjects as determined by the [1- 14 C]lactate system was 215+-55 pmol min -1 mg -1 protein (n=18). The advantages of this assay system are discussed. (Auth.)

  19. Inborn Errors of Metabolism with Acidosis: Organic Acidemias and Defects of Pyruvate and Ketone Body Metabolism.

    Science.gov (United States)

    Schillaci, Lori-Anne P; DeBrosse, Suzanne D; McCandless, Shawn E

    2018-04-01

    When a child presents with high-anion gap metabolic acidosis, the pediatrician can proceed with confidence by recalling some basic principles. Defects of organic acid, pyruvate, and ketone body metabolism that present with acute acidosis are reviewed. Flowcharts for identifying the underlying cause and initiating life-saving therapy are provided. By evaluating electrolytes, blood sugar, lactate, ammonia, and urine ketones, the provider can determine the likelihood of an inborn error of metabolism. Freezing serum, plasma, and urine samples during the acute presentation for definitive diagnostic testing at the provider's convenience aids in the differential diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Effect of methanolic extract of Hibiscus sabdariffa in ethanol-induced ...

    African Journals Online (AJOL)

    The objective of this study was to evaluate the activity of Hibiscus sabdariffa on the liver of rats following repeated administration of ethanol. Hepatotoxicity was induced on the rats using ethanol and the levels of serum enzymes such as serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase ...

  1. Process engineering tools to guide implementation and scale-up of transaminase cascades

    DEFF Research Database (Denmark)

    Tufvesson, Pär; Janes, Kresimir; Lima Ramos, Joana

    Biocatalysis is gaining ground in the pharmaceutical and fine chemical industry as a selective and potentially green technology to help synthesize industrially interesting products. In particular in the last decade the application of transaminases (E.C. 2.6.1.X ) has gained particular attention...... properties and values. A major challenge in the transaminase catalysed synthesis of chiral amines is the unfavourable equilibrium position [1]. There are several solutions to such equilibrium problems, including the use of in-situ product removal (ISPR) and cascade reactions to degrade or recycle the co......-product formed. Such techniques, especially those using cascades can be a great tool to overcome the thermodynamic hurdle, but also present some new challenges with respect to compatibility of reaction conditions, recycling of co-factors and last but not least, the added cost of the cascade system components [2...

  2. and alanine (EC. 2.6.1.2) transaminases, and alkaline phosphatase

    African Journals Online (AJOL)

    The activities of aspartate (E.C. 2.6.1.1) and alanine (E.C. 2.6.1.2) transaminases (AST and ALT, respectively), and alkaline phosphatase (ALP) (E.C. 3.1.3.1) were determined in erythrocytes obtained from 20 HbAA, 15 HbAS and 12 HbSS human subjects. The results showed that the three enzymes had different levels of ...

  3. Ursodeoxycholic Acid Can Improve Liver Transaminase Quantities in Children with Anticonvulsant Drugs Hepatotoxicity: a Pilot Study.

    Directory of Open Access Journals (Sweden)

    Masoumeh Asgarshirazi

    2015-06-01

    Full Text Available The present study has been directed to investigate Ursodeoxycholic Acid (UDCA effect in children, to reduce the high Liver transaminases induced by Anticonvulsant drugs (drug induced hepatitis. This idea has been driven from Cytoprotective and antioxidant properties of UDCA to be used in drug induced inflammation in Liver. Twenty two epileptic patients aged between 4 mo - 3 yr whom were under anticonvulsant therapy with drugs such as valperoic acid, primidone, levetiracetam, Phenobarbital or any combination of them and had shown Liver transaminases rise , after rule out of Viral-Autoimmune, Metabolic and Anatomic causes, have been prescribed UDCA in dose of 10-15 mg/kg/day, at least for 6 months. Any patient who have shown confusing factors such as genetic disorders with liver involvement or spontaneous decline in enzymes or had not treatment compliance has been excluded from the study. Transaminases range changes as well as Probable side effects of the drug have been monitored. The results indicated that UDCA is effective and well tolerable in the children with drug induced hyper transaminasemia. No side effect has been seen and recorded in this study. Based on this study and its results, we recommend UDCA as a safe and effective choice in drug induced hepatotoxicities.

  4. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Jaouani Mouna

    2015-09-26

    Sep 26, 2015 ... to several mutations at the Pyruvate Kinase gene (PKLR) located on chromosome .... Tunisians (Fig. 2) [21]. The screening of whole PKLR gene revealed the presence of ..... newborns: the pitfalls of diagnosis. J Pediatr 2007 ...

  5. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Phenotypic and molecular genetic analysis of Pyruvate Kinase deficiency in a Tunisian family. Jaouani Mouna, Hamdi Nadia, Chaouch Leila, Kalai Miniar, Mellouli Fethi, Darragi Imen, Boudriga Imen, Chaouachi Dorra, Bejaoui Mohamed, Abbes Salem ...

  6. Prognostic impact of the pretreatment aspartate transaminase/alanine transaminase ratio in patients treated with first-line systemic tyrosine kinase inhibitor therapy for metastatic renal cell carcinoma.

    Science.gov (United States)

    Kang, Minyong; Yu, Jiwoong; Sung, Hyun Hwan; Jeon, Hwang Gyun; Jeong, Byong Chang; Park, Se Hoon; Jeon, Seong Soo; Lee, Hyun Moo; Choi, Han Yong; Seo, Seong Il

    2018-05-13

    To examine the prognostic role of the pretreatment aspartate transaminase/alanine transaminase or De Ritis ratio in patients with metastatic renal cell carcinoma receiving first-line systemic tyrosine kinase inhibitor therapy. We retrospectively searched the medical records of 579 patients with metastatic renal cell carcinoma who visited Samsung Medical Center, Seoul, Korea, from January 2001 through August 2016. After excluding 210 patients, we analyzed 360 patients who received first-line tyrosine kinase inhibitor therapy. Cancer-specific survival and overall survival were defined as the primary and secondary end-points, respectively. A multivariate Cox proportional hazards regression model was used to identify independent prognosticators of survival outcomes. The overall population was divided into two groups according to the pretreatment De Ritis ratio as an optimal cut-off value of 1.2, which was determined by a time-dependent receiver operating characteristic curve analysis. Patients with a higher pretreatment De Ritis ratio (≥1.2) had worse cancer-specific survival and overall survival outcomes, compared with those with a lower De Ritis ratio (<1.2). Notably, a higher De Ritis ratio (≥1.2) was found to be an independent predictor of both cancer-specific survival (hazard ratio 1.61, 95% confidence interval 1.13-2.30) and overall survival outcomes (hazard ratio 1.69, 95% confidence interval 1.19-2.39), along with male sex, multiple metastasis (≥2), non-clear cell histology, advanced pT stage (≥3), previous metastasectomy and the Memorial Sloan Kettering Cancer Center risk classification. Our findings show that the pretreatment De Ritis ratio can provide valuable information about the survival outcomes of metastatic renal cell carcinoma patients receiving first-line tyrosine kinase inhibitor therapy. © 2018 The Japanese Urological Association.

  7. The effect of antioxidant supplementation on hepatitis C viral load, transaminases and oxidative status: a randomized trial among chronic hepatitis C virus-infected patients

    DEFF Research Database (Denmark)

    Groenbaek, K.; Friis, H.; Hansen, Max

    2006-01-01

    Objective To assess the effect of antioxidant supplementation on hepatitis C viral load, transaminases and oxidative status. Methods We performed a randomized, placebo-controlled, double-blind trial to assess the effect of antioxidant supplementation on serum alanine aminotransferase, plasma...... hepatitis C viral load as well as oxidative and antioxidant markers in patients with hepatitis C virus infection. The participants received a daily dose of ascorbic acid (500 mg), D-alpha-tocopherol (9451 U) and selenium (200 mu g) or placebo tablets for 6 months. Results Twenty-three patients were included...... aminotransferase and logo-transformed plasma hepatitis C virus-RNA between the groups or changes from the baseline at any time. No consistent differences between groups or changes from the baseline with respect to erythrocyte activities of antioxidative enzymes (glutathione reductase, superoxide dismutase...

  8. Can serum free fatty acids assessment predict severe preeclampsia ...

    African Journals Online (AJOL)

    Evaluation of serum fasting FFAs, uric acid, liver transaminases (AST, ALT) during delivery were done. Results: The mean level of FFAs was significantly elevated in preeclampsia cases compared to women with normal blood pressure (2.12 ± 2.64, 0.43± 0.29 respectively, p= 0.003). Also, cases with high FFAs levels had ...

  9. Pyruvate Dehydrogenase and Pyruvate Dehydrogenase Kinase Expression in Non Small Cell Lung Cancer and Tumor-Associated Stroma

    Directory of Open Access Journals (Sweden)

    Michael I. Koukourakis

    2005-01-01

    Full Text Available Pyruvate dehydrogenase (PDH catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters into the Krebs cycle, providing adenosine triphosphate (ATP to the cell. PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs. Under hypoxic conditions, conversion of pyruvate to lactate occurs, a reaction catalyzed by lactate dehydrogenase 5 (LDH5. In cancer cells, however, pyruvate is transformed to lactate occurs, regardless of the presence of oxygen (aerobic glycolysis/Warburg effect. Although hypoxic intratumoral conditions account for HIFia stabilization and induction of anaerobic metabolism, recent data suggest that high pyruvate concentrations also result in HIFia stabilization independently of hypoxia. In the present immunohistochemical study, we provide evidence that the PDH/PDK pathway is repressed in 73% of non small cell lung carcinomas, which may be a key reason for HIFia stabilization and “aerobic glycolysis.” However, about half of PDHdeficient carcinomas are not able to switch on the HIF pathway, and patients harboring these tumors have an excellent postoperative outcome. A small subgroup of clinically aggressive tumors maintains a coherent PDH and HIF/LDH5 expression. In contrast to cancer cells, fibroblasts in the tumor-supporting stroma exhibit an intense PDH but reduced PDK1 expression favoring maximum PDH activity. This means that stroma may use lactic acid produced by tumor cells, preventing the creation of an intolerable intratumoral acidic environment at the same time.

  10. Towards a standardized way of reporting physicochemical data and process metrics for transaminase reactions

    DEFF Research Database (Denmark)

    Tufvesson, Pär; Woodley, John

    information about the system and the reaction performance is lacking in the otherwise very useful scientific reports at laboratory scale. For instance, although K equilibrium is one of the key determining factors for the design and scale-up any transaminase process, it is very rarely reported [3]. In order...... to build a broader understanding of the correlation between the underlying physicochemical properties of the system (e.g. substrate volatility) and the process performance (e.g. gram of product per gram of biocatalyst), it would be highly beneficial if these data were reported, and ideally in a consistent...

  11. Immobilization of Escherichia coli containing ω‐transaminase activity in LentiKats®

    DEFF Research Database (Denmark)

    Cárdenas‐Fernández, Max; Lima Afonso Neto, Watson; López, Carmen

    2012-01-01

    Whole Escherichia coli cells overexpressing ω‐transaminase (ω‐TA) and immobilized cells entrapped in LentiKats® were used as biocatalysts in the asymmetric synthesis of the aromatic chiral amines 1‐phenylethylamine (PEA) and 3‐amino‐1‐phenylbutane (APB). Whole cells were permeabilized...... whole cell biocatalysis, the reaction with IPA was one order of magnitude faster than with Ala. No reaction was detected when permeabilized E. coli cells containing ω‐TA were employed using Ala as the amino donor. Additionally, the synthesis of APB from 4‐phenyl‐2‐butanone and IPA was studied. Whole...

  12. Evaluation of cytotoxicity and oxidative stress induced by alcoholic ...

    African Journals Online (AJOL)

    Maqsood

    (Duke et al., 1992), sinapic acid and sinapin (Schultz and. Gmelin, 1952), and ... serum glutamic pyruvate transaminase (SGPT) were within normal levels ..... like asthma, skin disease and diabetes (Gill and Macleod, 1980; Maier et al., 1998).

  13. Correlation between hematologic profile and transaminase enzymes with hospitalization duration dengue

    Science.gov (United States)

    Tinambunan, E.; Suryani; Katu, S.; Halim, R.; Mubin, A. H.; Sahyuddin

    2018-03-01

    Dengue is an infectious disease that can be found from mild to severe andaffected the clinical spectrum of the disease. Various hematologic profiles and transaminase enzymes are thought to reflect the severity of the disease thus affecting the hospitalization duration. For determining the correlation between hematological profile and transaminase enzyme to the hospitalization duration in dengue patients, an observational design study with the cross-sectional approach on dengue subjects was from 2 hospitals in Makassar. Hemoglobin, leukocyte, thrombocyte, AST, ALT, PT, and APTT were examined for hospitalization duration. There were 65 samples (34 men, 31 women) with the length of stay dengue patients. There was no correlation between the elevated of hematocrit value (p = 0.429), thrombocytopenia (p = 1.000), elevated of AST (p = 0.456) and ALT (p = 0.285) on hospitalization duration. In conclusion, low leukocyte values and APTT prolongation correlate with hospitalization duration but did not correlate significantly with hospitalization duration for elevated hematocrit, thrombocytopenia, elevated AST, and ALT.

  14. Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids

    DEFF Research Database (Denmark)

    Mourtzakis, Marina; Saltin, B.; Graham, T.

    2006-01-01

    During prolonged exercise, carbohydrate oxidation may result from decreased pyruvate production and increased fatty acid supply and ultimately lead to reduced pyruvate dehydrogenase (PDH) activity. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate, whereby the decline...... amino acid taken up during exercise and recovery. Alanine and glutamine were also associated...... with pyruvate metabolism, and they comprised 68% of total amino-acid release during exercise and recovery. Thus reduced pyruvate production was primarily associated with reduced carbohydrate oxidation, whereas the greatest production of pyruvate was related to glutamate, glutamine, and alanine metabolism...

  15. The effectiveness of fermented turmeric powder in subjects with elevated alanine transaminase levels: a randomised controlled study

    Science.gov (United States)

    2013-01-01

    Background Previous animal studies have shown that Curcuma longa (turmeric) improves liver function. Turmeric may thus be a promising ingredient in functional foods aimed at improving liver function. The purpose of the study is to investigate the hepatoprotective effect of fermented turmeric powder (FTP) on liver function in subjects with elevated alanine transaminase (ALT) levels. Methods A randomised, double-blind, placebo-controlled trial was conducted between November 2010 and April 2012 at the clinical trial center for functional foods of the Chonbuk National University Hospital. The trial included 60 subjects, 20 years old and above, who were diagnosed mild to moderate elevated ALT levels between 40 IU/L and 200 IU/L. Sixty subjects were randomised to receive FTP 3.0 g per day or placebo 3.0 g per day for 12 weeks. The treatment group received two capsules of FTP three times a day after meals, for 12 weeks. The primary efficacy endpoint was change in the ALT levels in the two groups. The secondary efficacy endpoints included its effect on aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), total bilirubin (TB), and lipid profiles. Safety was assessed throughout the study using ongoing laboratory tests. Adverse events (AEs) were also recorded. Results Sixty subjects were randomised in the study (30 into the FTP group, 30 into the placebo group), and among them, twelve subjects were excluded from the analysis for protocol violation, adverse events or consent withdrawal. The two groups did not differ in baseline characteristics. After 12 weeks of treatment, 48 subjects were evaluated. Of the 48 subjects, 26 randomly received FTP capsules and 22 received placebo. The FTP group showed a significant reduction in ALT levels after 12 weeks of treatment compared with the placebo group (p = 0.019). There was also observed that the serum AST levels were significantly reduce in the FTP group than placebo group (p = 0.02). The GGT levels

  16. Ethyl Pyruvate Emerges as a Safe and Fast Acting Agent against Trypanosoma brucei by Targeting Pyruvate Kinase Activity.

    Directory of Open Access Journals (Sweden)

    Netsanet Worku

    Full Text Available Human African Trypanosomiasis (HAT also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties.The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM. The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions.Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross

  17. Anaplerotic roles of pyruvate carboxylase in mammalian tissues.

    Science.gov (United States)

    Jitrapakdee, S; Vidal-Puig, A; Wallace, J C

    2006-04-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC serves an anaplerotic role for the tricarboxylic acid cycle, when intermediates are removed for different biosynthetic purposes. In liver and kidney, PC provides oxaloacetate for gluconeogenesis. In adipocytes PC is involved in de novo fatty acid synthesis and glyceroneogenesis, and is regulated by the peroxisome proliferator-activated receptor-gamma, suggesting that PC is involved in the metabolic switch controlling fuel partitioning toward lipogenesis. In islets, PC is necessary for glucose-induced insulin secretion by providing oxaloacetate to form malate that participates in the 'pyruvate/malate cycle' to shuttle 3C or 4C between mitochondria and cytoplasm. Hyperglycemia and hyperlipidemia impair this cycle and affect glucose-stimulated insulin release. In astrocytes, PC is important for de novo synthesis of glutamate, an important excitatory neurotransmitter supplied to neurons. Transcriptional studies of the PC gene pinpoint some transcription factors that determine tissue-specific expression.

  18. Exogenous pyruvate facilitates cancer cell adaptation to hypoxia by serving as an oxygen surrogate.

    Science.gov (United States)

    Yin, Chengqian; He, Dan; Chen, Shuyang; Tan, Xiaoling; Sang, Nianli

    2016-07-26

    Molecular oxygen is the final electron acceptor in cellular metabolism but cancer cells often become adaptive to hypoxia, which promotes resistance to chemotherapy and radiation. The reduction of endogenous glycolytic pyruvate to lactate is known as an adaptive strategy for hypoxic cells. Whether exogenous pyruvate is required for hypoxic cell proliferation by either serving as an electron acceptor or a biosynthetic substrate remains unclear. By using both hypoxic and ρ0 cells defective in electron transfer chain, we show that exogenous pyruvate is required to sustain proliferation of both cancer and non-cancer cells that cannot utilize oxygen. Particularly, we show that absence of pyruvate led to glycolysis inhibition and AMPK activation along with decreased NAD+ levels in ρ0 cells; and exogenous pyruvate increases lactate yield, elevates NAD+/NADH ratio and suppresses AMPK activation. Knockdown of lactate dehydrogenase significantly inhibits the rescuing effects of exogenous pyruvate. In contrast, none of pyruvate-derived metabolites tested (including acetyl-CoA, α-ketoglutarate, succinate and alanine) can replace pyruvate in supporting ρ0 cell proliferation. Knockdown of pyruvate carboxylase, pyruvate dehydrogenase and citrate synthase do not impair exogenous pyruvate to rescue ρ0 cells. Importantly, we show that exogenous pyruvate relieves ATP insufficiency and mTOR inhibition and promotes proliferation of hypoxic cells, and that well-oxygenated cells release pyruvate, providing a potential in vivo source of pyruvate. Taken together, our data support a novel pyruvate cycle model in which oxygenated cells release pyruvate for hypoxic cells as an oxygen surrogate. The pyruvate cycle may be targeted as a new therapy of hypoxic cancers.

  19. Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri

    DEFF Research Database (Denmark)

    Møller, Kasper; Langkjær, Rikke Breinhold; Nielsen, Jens

    2004-01-01

    was controlled by variations in the amount of mRNA. The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S. cerevisiae. This indicates that the difference in ethanol formation between these two yeasts...... is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery. However, the PDC genes of Saccharomyces/Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin. While S. cerevisiae...

  20. Protective effect of ethyl pyruvate on mice sperm parameters in phenylhydrazine induced hemolytic anemia.

    Science.gov (United States)

    Mozafari, Ali Akbar; Shahrooz, Rasoul; Ahmadi, Abbas; Malekinjad, Hassan; Mardani, Karim

    2016-01-01

    The aim of the present study was to assess the protective effect of ethyl pyruvate (EP) on sperm quality parameters, testosterone level and malondialdehyde (MDA) in phenylhydrazine (PHZ) treated mice. For this purpose, 32 NMRI mice with the age range of 8 to 10 weeks, weight average 26.0 ± 2.0 g, were randomly divided into four equal groups. The control group (1) received normal saline (0. 1 mL per day) by intraperitoneal injection (IP). Group 2 (PHZ group) was treated with initial dose of PHZ (8 mg 100 g(-1), IP) followed by 6 mg 100 g(-1) , IP every 48 hr. Group 3, (Group PHZ+EP) received PHZ (according to the previous prescription) with EP (40 mg kg(-1), daily, IP). Ethyl pyruvate group (4) received only EP (40 mg kg(-1), daily, IP). Treatment period was 35 days. After euthanasia, sperms from caudal region of epididymis were collected and the total mean sperm count, sperm viability, motility and morphology were determined. Testis tissue MDA and serum testosterone levels of all experimental groups were also evaluated. A considerable reduction in mean percentage of number, natural morphology of sperm, sperm motility and viability and serum testosterone concentration besides DNA injury increment among mice treating with PHZ in comparison with control group were observed. However, in PHZ+EP group the above mentioned parameters were improved. This study showed that PHZ caused induction of toxicity on sperm parameters and reduction of testosterone as well as the increment of MDA level and EP as an antioxidant could reduce destructive effects of PHZ on sperm parameters, testosterone level and lipid peroxidation.

  1. Process Considerations for the Asymmetric Synthesis of Chiral Amines using ω-Transaminase

    DEFF Research Database (Denmark)

    Lima Afonso Neto, Watson

    in order to eliminate infeasible routes. This work illustrates the Laboratory scale characterization of different process options for the asymmetric synthesis of chiral amines catalysed by ω-transaminase (ω –TAm). The studied process options include: (i) the immobilization of the biocatalyst to improve its...... focused on development of comprehensive screening methodologies and guidelines to aid (i) the selection and characterization of suitable biocatalysts for the process; (ii) the selection and characterization of suitable carriers for immobilization of (S)- and (R)-selective ω-TAm; and (iii) the selection...... of suitable polymeric resins for product removal. The work has been performed in collaboration with c-LEcta GmbH (Leipzig, Germany) and DSM Innovative Synthesis (Geleen, The Netherlands) who supplied the enzymes for the case study, making possible the successful demonstration of the screening methodologies...

  2. A Rapid Selection Procedure for Simple Commercial Implementation of omega-Transaminase Reactions

    DEFF Research Database (Denmark)

    Gundersen Deslauriers, Maria; Tufvesson, Pär; Rackham, Emma J.

    2016-01-01

    A stepwise selection procedure is presented to quickly evaluate whether a given omega-transaminase reaction is suitable for a so-called "simple" scale-up for fast industrial implementation. Here "simple" is defined as a system without the need for extensive process development or specialized......, and (3) determination of product inhibition. The method is exemplified with experimental work focused on two products: 1-(4-bromophenyl)ethylamine and (S)-(+)3-amino-1-Boc-piperidine, synthesized from their corresponding pro-chiral ketones each with two alternative amine donors, propan-2-amine, and 1......-phenylethylamine. Each step of the method has a threshold value, which must be surpassed to allow "simple" implementation, helping select suitable combinations of substrates, enzymes, and donors. One reaction pair, 1-Boc-3-piperidone with propan-2-amine, met the criteria of the three-step selection procedure...

  3. A case study on robust optimal experimental design for model calibration of ω-Transaminase

    DEFF Research Database (Denmark)

    Daele, Timothy, Van; Van Hauwermeiren, Daan; Ringborg, Rolf Hoffmeyer

    the experimental space. However, it is expected that more informative experiments can be designed to increase the confidence of the parameter estimates. Therefore, we apply Optimal Experimental Design (OED) to the calibrated model of Shin and Kim (1998). The total number of samples was retained to allow fair......” parameter values are not known before finishing the model calibration. However, it is important that the chosen parameter values are close to the real parameter values, otherwise the OED can possibly yield non-informative experiments. To counter this problem, one can use robust OED. The idea of robust OED......Proper calibration of models describing enzyme kinetics can be quite challenging. This is especially the case for more complex models like transaminase models (Shin and Kim, 1998). The latter fitted model parameters, but the confidence on the parameter estimation was not derived. Hence...

  4. Glycogenic hepatopathy is an under-recognised cause of hepatomegaly and elevated liver transaminases in type 1 diabetes mellitus.

    Science.gov (United States)

    Irani, N R; Venugopal, K; Kontorinis, N; Lee, M; Sinniah, R; Bates, T R

    2015-07-01

    Glycogenic hepatopathy (GH) is an under-recognised complication of type 1 diabetes mellitus (T1DM) not controlled to target resulting in hepatomegaly and elevated liver transaminases. We report the case of a 19-year-old man with T1DM not controlled to target who presented with abdominal pain, hepatomegaly and deranged liver transaminases. He was subsequently diagnosed with GH on liver biopsy, with the mainstay of treatment being reduction in caloric intake and insulin. © 2015 Royal Australasian College of Physicians.

  5. Room-temperature solid phase ionic liquid (RTSPIL) coated Ω-transaminases: Development and application in organic solvents

    DEFF Research Database (Denmark)

    Grabner, B.; Nazario, M. A.; Gundersen, M. T.

    2018-01-01

    ω-Transaminases ATA-40, ATA-47 and ATA-82P were coated with room-temperature solid phase ionic liquids (RTSPILs) by means of three methods, melt coating, precipitation coating, and co‐lyophilization, and showed increased stability in all of the five tested organic solvents. Co‐lyophilization and ......ω-Transaminases ATA-40, ATA-47 and ATA-82P were coated with room-temperature solid phase ionic liquids (RTSPILs) by means of three methods, melt coating, precipitation coating, and co‐lyophilization, and showed increased stability in all of the five tested organic solvents. Co...

  6. Functional loss of GABA transaminase (GABA-T) expressed early leaf senescence under various stress conditions in Arabidopsis thaliana

    OpenAIRE

    Jalil, Syed Uzma; Ahmad, Iqbal; Ansari, Mohammad Israil

    2017-01-01

    GABA-transaminase (GABA-T) involved in carbon and nitrogen metabolism during the plant development process via GABA shunt and GABA-T mutant, which is defective in GABA catabolism, is ideal model to examine the role of GABA-T in plant development and leaf senescence of plant. We have characterized GABA transaminase knock out mutant pop2-1 that is transition and pop2-3 which is T-DNA insertion mutant of Arabidopsis thaliana during various stress conditions.The GABA-T knockout mutant plants disp...

  7. Novel mutations associated with pyruvate kinase deficiency in Brazil

    Directory of Open Access Journals (Sweden)

    Maria Carolina Costa Melo Svidnicki

    2018-01-01

    Full Text Available Background: Pyruvate kinase deficiency is a hereditary disease that affects the glycolytic pathway of the red blood cell, causing nonspherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait and shows a marked variability in clinical expression. This study reports on the molecular characterization of ten Brazilian pyruvate kinase-deficient patients and the genotype–phenotype correlations. Method: Sanger sequencing and in silico analysis were carried out to identify and characterize the genetic mutations. A non-affected group of Brazilian individuals were also screened for the most commonly reported variants (c.1456C>T and c.1529G>A. Results: Ten different variants were identified in the PKLR gene, of which three are reported here for the first time: p.Leu61Gln, p.Ala137Val and p.Ala428Thr. All the three missense variants involve conserved amino acids, providing a rationale for the observed enzyme deficiency. The allelic frequency of c.1456C>T was 0.1% and the 1529G>A variant was not found. Conclusion: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency from South America. The results allowed us to correlate the severity of the clinical phenotype with the identified variants. Keywords: Red cell disorder, Pyruvate kinase, Mutation, Hemolytic anemia, PKLR gene

  8. Characterization of a C 4 maize pyruvate orthophosphate dikinase ...

    African Journals Online (AJOL)

    Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in plants that utilize the C4 photosynthetic pathway to fix CO2. The enzymatic reaction catalyzed by PPDK is critically controlled by light and is one of the rate-limiting steps of the C4 pathway. The intact maize (Zea mays) C4-PPDK gene, containing its own promoter, ...

  9. Amperometric pyruvate sensor based on a pyruvate dehydrogenase-immobilized carbon paste electrode containing vitamin K3 as a mediator

    Energy Technology Data Exchange (ETDEWEB)

    Miki, K. [Nara National College of Technology, Nara (Japan); Kinoshita, H. [Kawassui Women`s College, Nagasaki (Japan); Yamamoto, Y. [Kyoto Municipal Junior College of Nursing, Kyoto (Japan); Taniguchi, N. [Kyoto Research Center for Hygiene, Kyoto (Japan); Ikeda, T. [Kyoto University, Kyoto (Japan). Faculty of Agriculture

    1995-12-05

    Pyruvate dehydrogenase (PDH) was immobilized on the surface of a carbon paste electrode containing vitamin K3 (2-Methyl-1,4-naphthoquinone, VK), and the electrode surface was covered with a dialysis membrane. The enzyme electrode produced an anodic current starting from -0.2 V to reach a limiting current at +0.1 V vs. Ag/AgCl due to the enzyme-catalyzed oxidation of pyruvate in a phosphate buffer solution of pH 7.0. The current response to pyruvate depended on the amounts of both the immobilized-PDH and VK mixed in the carbon paste electrode at low amount of the enzyme and VK, and became independent at above 0.15 mg PDH and 0.65% (w/w) VK. The electrode with 0.15mg PDH and 0.65% (w/w) VK could be used as a pyruvate sensor to measure in the range of 2 ,{mu}M to 3mM. The response time was about 60 sec, and the current was independent of pH in the range of 5.7 - 7.2. The presence of L-ascorbic acid didn`t interfere with this measurement. Phosphate ion could also be determined with this electrode in a citrate buffer solution. 14 refs., 6 figs., 1 tab.

  10. Enzymatic synthesis of 11C-pyruvic acid and 11C-L-lactic acid

    International Nuclear Information System (INIS)

    Cohen, M.B.; Spolter, L.; Chang, C.C.; Cook, J.S.; Macdonald, N.S.

    1980-01-01

    L-Lactic acid is formed as the end product of glycolysis under anaerobic conditions in all cells, but this reaction is of special significance in the myocardium. L-Lactic acid is reversibly formed from and is in equilibrium with myocardial pyruvic acid, which is its sole metabolic pathway. 11 C-Pyruvic acid is synthesized from 11 C carbon dioxide using pyruvate-ferredoxin oxidoreductase and coenzymes. The 11 C-pyruvic acid is then converted to 11 -L-lactic acid by lactic acid dehydrogenase. The availability of 11 C-pyruvic acid and 11 C-L-lactic acid will permit the in vivo investigation of lactate metabolism. (author)

  11. Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

    DEFF Research Database (Denmark)

    Knudsen, Jenny Dahl; Hägglöf, Cecilia; Weber, Nora

    2016-01-01

    yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated. RESULTS: A series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol...

  12. Explaining Operational Instability of Amine Transaminases : Substrate-Induced Inactivation Mechanism and Influence of Quaternary Structure on Enzyme-Cofactor Intermediate Stability

    NARCIS (Netherlands)

    Borner, Tim; Ramisch, Sebastian; Reddem, Eswar R.; Bartsch, Sebastian; Vogel, Andreas; Thunnissen, Andy-Mark W. H.; Adlercreutz, Patrick; Grey, Carl

    The insufficient operational stability of amine transaminases (ATA) constitutes a limiting factor for high productivity in chiral amine synthesis. In this work, we investigated the operational stability of a tetrameric ATA with 92% sequence identity to a Pseudomonas sp. transaminase and compared it

  13. Increased superoxide accumulation in pyruvate dehydrogenase complex deficient fibroblasts.

    Science.gov (United States)

    Glushakova, Lyudmyla G; Judge, Sharon; Cruz, Alex; Pourang, Deena; Mathews, Clayton E; Stacpoole, Peter W

    2011-11-01

    The pyruvate dehydrogenase complex (PDC) oxidizes pyruvate to acetyl CoA and is critically important in maintaining normal cellular energy homeostasis. Loss-of-function mutations in PDC give rise to congenital lactic acidosis and to progressive cellular energy failure. However, the subsequent biochemical consequences of PDC deficiency that may contribute to the clinical manifestations of the disorder are poorly understood. We postulated that altered flux through PDC would disrupt mitochondrial electron transport, resulting in oxidative stress. Compared to cells from 4 healthy subjects, primary cultures of skin fibroblasts from 9 patients with variable mutations in the gene encoding the alpha subunit (E1α) of pyruvate dehydrogenase (PDA1) demonstrated reduced growth and viability. Superoxide (O(2)(.-)) from the Qo site of complex III of the electron transport chain accumulated in these cells and was associated with decreased activity of manganese superoxide dismutase. The expression of uncoupling protein 2 was also decreased in patient cells, but there were no significant changes in the expression of cellular markers of protein or DNA oxidative damage. The expression of hypoxia transcription factor 1 alpha (HIF1α) also increased in PDC deficient fibroblasts. We conclude that PDC deficiency is associated with an increase in O(2)(.-) accumulation coupled to a decrease in mechanisms responsible for its removal. Increased HIF1α expression may contribute to the increase in glycolytic flux and lactate production in PDC deficiency and, by trans-activating pyruvate dehydrogenase kinase, may further suppress residual PDC activity through phosphorylation of the E1α subunit. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Small, Juan E. [Massachusetts General Hospital and Harvard Medical School, Department of Radiology, Boston, MA (United States); Gonzalez, Guido E. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Radiology, Boston, MA (United States); Clinica Alemana de Santiago, Departmento de Imagenes, Santiago (Chile); Nagao, Karina E.; Walton, David S. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Ophthalmology, Boston, MA (United States); Caruso, Paul A. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Radiology, Boston, MA (United States)

    2009-10-15

    Pyruvate dehydrogenase (PDH) deficiency is a genetic disorder of mitochondrial metabolism. The clinical manifestations range from severe neonatal lactic acidosis to chronic neurodegeneration. Optic neuropathy is an uncommon clinical sequela and the imaging findings of optic neuropathy in these patients have not previously been described. We present a patient with PDH deficiency with bilateral decreased vision in whom MRI demonstrated bilateral optic neuropathy and chiasmopathy. (orig.)

  15. Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

    International Nuclear Information System (INIS)

    Small, Juan E.; Gonzalez, Guido E.; Nagao, Karina E.; Walton, David S.; Caruso, Paul A.

    2009-01-01

    Pyruvate dehydrogenase (PDH) deficiency is a genetic disorder of mitochondrial metabolism. The clinical manifestations range from severe neonatal lactic acidosis to chronic neurodegeneration. Optic neuropathy is an uncommon clinical sequela and the imaging findings of optic neuropathy in these patients have not previously been described. We present a patient with PDH deficiency with bilateral decreased vision in whom MRI demonstrated bilateral optic neuropathy and chiasmopathy. (orig.)

  16. Occult hepatitis B virus among the patients with abnormal alanine transaminase.

    Science.gov (United States)

    Makvandi, Manoochehr; Neisi, Niloofar; Khalafkhany, Davod; Makvandi, Kamyar; Hajiani, Eskandar; Shayesteh, Ali Akbar; Masjedi Zadeh, Abdolrahim; Sina, Amir Hosein; Hamidifard, Mojtaba; Rasti, Mojtaba; Aryan, Ehsan; Ahmadi, Kambiz; Yad Yad, Mohammad Jafar

    2014-08-01

    The occult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HBV) DNA in the sera or in the liver biopsy and the absence of hepatitis B surface antigen (HBsAg) by serological test. The current study aimed to evaluate the occult HBV infection by polymerase chain reaction (PCR) and determine HBV genotyping among the patients with abnormal alanine transaminase (ALT) in Ahvaz city, Iran. The sera of 120 patients, 54 (45%) females and 66 (55%) males, with abnormal ALT 40-152 IU were collected. All the patients were negative for HBsAg for more than one year. The patients` sera were tested by PCR using primers specified for the S region of HBV. Then the positive PCR products were sequenced to determine HBV genotyping and phylogenic tree. Of these 120 subjects, 12 (10%) patients including 6 (5%) males and 6 (5%) females were found positive for HBV DNA by PCR, which indicated the presence of occult HBV infection among these patients. The sequencing results revealed that genotype D was predominant with sub-genotyping D1 among OBI patients. Occult hepatitis B infection is remarkably prevalent in Ahvaz, Iran, and should be considered as a potential risk factor for the transmission of Hepatitis B Virus throughout the community by the carriers.

  17. Amine donor and acceptor influence on the thermodynamics of ω-transaminase reactions

    DEFF Research Database (Denmark)

    Gundersen, Maria T.; Abu, Rohana; Schürmann, Martin

    2015-01-01

    In recent years biocatalytic transamination using ω-transaminase has become established as one of the most interesting routes to synthesize chiral amines with a high enantiomeric purity, especially in the pharmaceutical sector where the demand for such compounds is high. Nevertheless, one limitat...... of such reactions because it may be used to help select suitable donor/acceptor combinations. The results presented here give guidance, with respect to thermodynamics, in order to further extend the application of biocatalytic transamination....... limitation for successful implementation and scale-up is that the thermodynamics of such conversions are frequently found unfavourable. Herein we report experimental measurements of apparent equilibrium constants for several industrially relevant transamination reactions in a systematic manner to better...... understand the effect of amine acceptor and donor choice. For example, we have found that ortho-substitution of acetophenone like molecules, had a significant impact on the thermodynamic equilibrium. Likewise, the effect of cyclic amine acceptors was evaluated and compared to similar non-cyclic structures...

  18. Synchronization by food access modifies the daily variations in expression and activity of liver GABA transaminase.

    Science.gov (United States)

    De Ita-Pérez, Dalia; Méndez, Isabel; Vázquez-Martínez, Olivia; Villalobos-Leal, Mónica; Díaz-Muñoz, Mauricio

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Dalia De Ita-Pérez

    2014-01-01

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

  20. Occult Hepatitis B Virus Among the Patients With Abnormal Alanine Transaminase

    Science.gov (United States)

    Makvandi, Manoochehr; Neisi, Niloofar; Khalafkhany, Davod; Makvandi, Kamyar; Hajiani, Eskandar; Shayesteh, Ali Akbar; Masjedi Zadeh, Abdolrahim; Sina, Amir Hosein; Hamidifard, Mojtaba; Rasti, Mojtaba; Aryan, Ehsan; Ahmadi, Kambiz; Yad Yad, Mohammad Jafar

    2014-01-01

    Background: The occult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HBV) DNA in the sera or in the liver biopsy and the absence of hepatitis B surface antigen (HBsAg) by serological test. Objectives: The current study aimed to evaluate the occult HBV infection by polymerase chain reaction (PCR) and determine HBV genotyping among the patients with abnormal alanine transaminase (ALT) in Ahvaz city, Iran. Patients and Methods: The sera of 120 patients, 54 (45%) females and 66 (55%) males, with abnormal ALT 40-152 IU were collected. All the patients were negative for HBsAg for more than one year. The patients` sera were tested by PCR using primers specified for the S region of HBV. Then the positive PCR products were sequenced to determine HBV genotyping and phylogenic tree. Results: Of these 120 subjects, 12 (10%) patients including 6 (5%) males and 6 (5%) females were found positive for HBV DNA by PCR, which indicated the presence of occult HBV infection among these patients. The sequencing results revealed that genotype D was predominant with sub-genotyping D1 among OBI patients. Conclusions: Occult hepatitis B infection is remarkably prevalent in Ahvaz, Iran, and should be considered as a potential risk factor for the transmission of Hepatitis B Virus throughout the community by the carriers. PMID:25485052

  1. [The role of transaminases and mitochondrial anion porters in the energetics of animals of different ages].

    Science.gov (United States)

    Akhmerov, R N; Sultanov, Sh; Allamuratov, Sh I

    1995-01-01

    Aminohydroxyacetate, an inhibitor of aminotransferases, decreases the rate of oxygen consumption by 1-day-old young rats by 35% 10 min after intraperitoneal injection, whereas for 20-day-old rats, the inhibitory effect is 56%, and for adult mice, it is 83%. More prolonged exposure to aminohydroxyacetate leads to death of the animal. One-day-old rats die 90 min after the injection, 20-day-old rats die 30 min after the injection, and adult mice die 15 min after the injection. Butylmalonate (an inhibitor of mitochondrial dicarboxylate translocator) decreases the rate of energy metabolism to a lower extent, by 38% in adult mice and by 18% in 1-day-old rats. The animals generally remain alive after the exposure to this compound. 1,2,3-benzyltricarboxylate, an inhibitor of tricarboxylate transport, shows only a weak effect on energy metabolism of animals of any age. These results provide evidence that the role of the transaminase system in energy metabolism increases with age. Mechanisms underlying weak sensitivity of newborn animals to these inhibitors are discussed.

  2. Identification, expression and characterization of an R-ω-transaminase from Capronia semiimmersa.

    Science.gov (United States)

    Iglesias, César; Panizza, Paola; Rodriguez Giordano, Sonia

    2017-07-01

    Chiral amines are essential precursors in the production of biologically active compounds, including several important drugs. Among the biocatalytic strategies that have been developed for their synthesis, the use of ω-transaminases (ω-TA) appears as an attractive alternative allowing the stereoselective amination of prochiral ketones. However, the problems associated with narrow substrate specificity, unfavourable reaction equilibrium and expensive amine donors still hamper its industrial application. The search for novel enzymes from nature can contribute to expand the catalytic repertoire of ω-TA and help to circumvent some of these problems. A genome mining approach, based on the work described by Höhne et al., was applied for selection of potential R-ω-TA. Additional criteria were used to select an enzyme that differs from previously described ones. A candidate R-ω-TA from Capronia semiimmersa was selected, cloned and expressed in Escherichia coli. Interestingly, alignment of this enzyme with previously reported TA sequences revealed the presence of two additional amino acid residues in a loop close to the active site. The impact of this change was analysed with a structural model based on crystallized R-ω-TAs. Analysis of the substrate specificity of R-ω-TA from C. semiimmersa indicates that it accepts a diversity of ketones as substrates yielding the corresponding amine with good yields and excellent enantioselectivity. The expressed enzyme accepts isopropylamine as amine donor what makes it suitable for industrial processes.

  3. Ethnicity and elevated liver transaminases among newly diagnosed children with type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Hudson Omar D

    2012-11-01

    Full Text Available Abstract Background To examine the influence of ethnicity on liver transaminases among adolescents with type 2 diabetes mellitus (T2DM. Methods A retrospective medical chart review of 57 (30 males and 27 females newly diagnosed patients with T2DM. Ethnicity was determined by self-report and height, weight, body mass index (BMI and glycosylated hemoglobin (HbA1c were obtained using standard clinical procedures. Fasting levels of alanine aminotransaminase (ALT and aspartate aminotransferase (AST were collected prior to the initiation of any therapy. Results Age, gender, height, weight, BMI, and HbA1c did not differ between ethnic groups. Compared to African-Americans, Hispanics had significantly higher ALT (23.9 ± 3.4 vs. 107.8 ± 20.3, p=0.002 and AST (17.7 ± 2.5 vs. 71.1 ± 15.7, p Conclusions These preliminary findings suggest that Hispanic children with T2DM may be at higher risk for developing non-alcoholic fatty liver disease and indicate that a comprehensive hepatic evaluation is warranted in this population. Future studies that incorporate more precise and proximal measures of liver health are warranted in this population.

  4. PDK4 Inhibits Cardiac Pyruvate Oxidation in Late Pregnancy.

    Science.gov (United States)

    Liu, Laura X; Rowe, Glenn C; Yang, Steven; Li, Jian; Damilano, Federico; Chan, Mun Chun; Lu, Wenyun; Jang, Cholsoon; Wada, Shogo; Morley, Michael; Hesse, Michael; Fleischmann, Bernd K; Rabinowitz, Joshua D; Das, Saumya; Rosenzweig, Anthony; Arany, Zoltan

    2017-12-08

    Pregnancy profoundly alters maternal physiology. The heart hypertrophies during pregnancy, but its metabolic adaptations, are not well understood. To determine the mechanisms underlying cardiac substrate use during pregnancy. We use here 13 C glucose, 13 C lactate, and 13 C fatty acid tracing analyses to show that hearts in late pregnant mice increase fatty acid uptake and oxidation into the tricarboxylic acid cycle, while reducing glucose and lactate oxidation. Mitochondrial quantity, morphology, and function do not seem altered. Insulin signaling seems intact, and the abundance and localization of the major fatty acid and glucose transporters, CD36 (cluster of differentiation 36) and GLUT4 (glucose transporter type 4), are also unchanged. Rather, we find that the pregnancy hormone progesterone induces PDK4 (pyruvate dehydrogenase kinase 4) in cardiomyocytes and that elevated PDK4 levels in late pregnancy lead to inhibition of PDH (pyruvate dehydrogenase) and pyruvate flux into the tricarboxylic acid cycle. Blocking PDK4 reverses the metabolic changes seen in hearts in late pregnancy. Taken together, these data indicate that the hormonal environment of late pregnancy promotes metabolic remodeling in the heart at the level of PDH, rather than at the level of insulin signaling. © 2017 American Heart Association, Inc.

  5. Immobilisation of ω-transaminase for industrial application: Screening and characterisation of commercial ready to use enzyme carriers

    DEFF Research Database (Denmark)

    Lima Afonso Neto, Watson; Schürmann, Martin; Panella, Lavinia

    2015-01-01

    Despite of the advantages that enzyme immobilisation can bring to industrial biocatalysis, its utilisation is still limited to a small number of enzymes and processes. Transaminase catalysed processes are a good example where immobilisation can be of major importance and even decisive for economi...... and possibility to store the biocatalyst for more than 70 days (at room temperature) were obtained as result of the immobilisation on the selected supports.......)-selective ω-transaminases. These carriers allowed the re-use of the immobilised enzyme for 8 cycles of 24 h each, under relevant process conditions, corresponding to approximately 250 h of operation, with more than 50% of the initial activity retained. Likewise the stability towards higher temperatures...

  6. PEDIATRIC VISCERAL ADIPOSITY INDEX ADAPTATION CORRELATES WITH HOMA-IR, MATSUDA, AND TRANSAMINASES.

    Science.gov (United States)

    Hernández, María José Garcés; Klünder, Miguel; Nieto, Nayely Garibay; Alvarenga, Juan Carlos López; Gil, Jenny Vilchis; Huerta, Samuel Flores; Siccha, Rosa Quispe; Hernandez, Joselin

    2018-03-01

    Visceral adiposity index (VAI) is a mathematical model associated with cardiometabolic risk in adults, but studies on children failed to support this association. Our group has proposed a pediatric VAI model using pediatric ranges, but it has not yet been evaluated and needs further adjustments. The objective of this study was to further adjust the proposed pediatric VAI by age, creating a new pediatric metabolic index (PMI), and assess the correlation of the PMI with insulin resistance indexes and hepatic enzymes. A cross-sectional design with data from 396 children (age 5 to 17 years) was analyzed with a generalized linear model to find the coefficients for triglycerides, high-density-lipoprotein cholesterol, and waist circumference-body mass index quotient. The model was constructed according to sex and age and designated PMI. A cross-validation analysis was performed and a receiver operating characteristic curve was used to determine cut-off points. Significant moderate correlation was found between PMI and homeostatic model assessment of insulin resistance (HOMA-IR) ( r = 0.452; P = .003), Matsuda ( r = -0.366; P = .019), alanine aminotransferase ( r = 0.315, P = .045), and γ-glutamyltransferase ( r = 0.397; P = .010). A PMI score >1.7 was considered as risk. PMI correlates with HOMA-IR, Matsuda, and hepatic enzymes. It could be helpful for identifying children at risk for cardiometabolic diseases. ALT = alanine transaminase BMI = body mass index GGT = γ-glutamyltransferase HDL-C = high-density-lipoprotein cholesterol HOMA-IR = homeostatic model assessment of insulin resistance hs-CRP = high sensitivity C-reactive protein ISI = insulin sensitivity index NAFLD = nonalcoholic fatty liver disease PMI = pediatric metabolic index QUICKI = quantitative insulin sensitivity check index ROC = receiver operating characteristic TG = triglyceride TNF-α = tumor necrosis factor-alpha VAI = visceral adiposity index VAT = visceral adipose tissue WC = waist circumference.

  7. Functional loss of GABA transaminase (GABA-T expressed early leaf senescence under various stress conditions in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Syed Uzma Jalil

    2017-06-01

    Full Text Available GABA-transaminase (GABA-T involved in carbon and nitrogen metabolism during the plant development process via GABA shunt and GABA-T mutant, which is defective in GABA catabolism, is ideal model to examine the role of GABA-T in plant development and leaf senescence of plant. We have characterized GABA transaminase knock out mutant pop2-1 that is transition and pop2-3 which is T-DNA insertion mutant of Arabidopsis thaliana during various stress conditions.The GABA-T knockout mutant plants displayed precocious leaf senescence, which was accompanied by the assays of physiological parameters of leaf senescence during various stress conditions. Furthermore, our physiological evidence indicates that pop2-1 and pop2-3 mutations rapidly decreased the efficiency of leaf photosynthesis, chlorophyll content, GABA content, GABA-T, and glutamate decarboxylase (GAD activity and on the other hand increases membrane ion leakage, malondialdehyde (MDA level in stress induced leaves. However, cell viability assay by trypan blue and insitu Hydrogen peroxidation assay by 3,3-diaminobenzidine (DAB in stress induced leaves also display that pop2-1 and pop2-3 mutant leaves show oversensitivity in response to different stress conditions as compared to wild type. These results strongly indicate that the loss-of-function of GABA transaminase gene induces early leaf senescence in Arabidopsis thaliana during various stress conditions.

  8. Crystallization and preliminary X-ray crystallographic studies of β-transaminase from Mesorhizobium sp. strain LUK

    International Nuclear Information System (INIS)

    Kim, Bokyung; Park, Ok Kyeung; Bae, Ju Young; Jang, Tae-ho; Yoon, Jong Hwan; Do, Kyoung Hun; Kim, Byung-Gee; Yun, Hyungdon; Park, Hyun Ho

    2011-01-01

    β-Transaminase from Mesorhizobium sp. strain LUK was crystallized. The crystals were found to belong to the orthorhombic space group C222 1 , with unit-cell parameters a = 90.91, b = 192.17, c = 52.75 Å. The crystals were obtained at 293 K and diffracted to a resolution of 2.5 Å. β-Transaminase (β-TA) catalyzes the transamination reaction between β-aminocarboxylic acids and keto acids. This enzyme is a particularly suitable candidate for use as a biocatalyst for the asymmetric synthesis of enantiochemically pure β-amino acids for pharmaceutical purposes. The β-TA from Mesorhizobium sp. strain LUK (β-TAMs) belongs to a novel class in that it shows β-transaminase activity with a broad and unique substrate specificity. In this study, β-TAMs was overexpressed in Escherichia coli with an engineered C-terminal His tag. β-TAMs was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.5 Å from a crystal that belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 90.91, b = 192.17, c = 52.75 Å

  9. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

    OpenAIRE

    Vanderperre, Beno?t; Herzig, S?bastien; Krznar, Petra; H?rl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-01-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic p...

  10. An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion

    OpenAIRE

    McCommis, Kyle S.; Hodges, Wesley T.; Bricker, Daniel K.; Wisidagama, Dona R.; Compan, Vincent; Remedi, Maria S.; Thummel, Carl S.; Finck, Brian N.

    2016-01-01

    Objective: Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC) is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion. Methods: To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, we used genetically-engineered Drosophila and mice with loss of MPC activity in insulin-prod...

  11. The mitochondrial pyruvate carrier mediates high fat diet-induced increases in hepatic TCA cycle capacity

    OpenAIRE

    Rauckhorst, Adam J.; Gray, Lawrence R.; Sheldon, Ryan D.; Fu, Xiaorong; Pewa, Alvin D.; Feddersen, Charlotte R.; Dupuy, Adam J.; Gibson-Corley, Katherine N.; Cox, James E.; Burgess, Shawn C.; Taylor, Eric B.

    2017-01-01

    Objective: Excessive hepatic gluconeogenesis is a defining feature of type 2 diabetes (T2D). Most gluconeogenic flux is routed through mitochondria. The mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytosol into the mitochondrial matrix, thereby gating pyruvate-driven gluconeogenesis. Disruption of the hepatocyte MPC attenuates hyperglycemia in mice during high fat diet (HFD)-induced obesity but exerts minimal effects on glycemia in normal chow diet (NCD)-fed conditions. T...

  12. Pyruvate Dehydrogenase Kinase as a Novel Therapeutic Target in Oncology

    Directory of Open Access Journals (Sweden)

    Gopinath eSutendra

    2013-03-01

    Full Text Available Current drug development in oncology is non-selective as it typically focuses on pathways essential for the survival of all dividing cells. The unique metabolic profile of cancer, which is characterized by increased glycolysis and suppressed mitochondrial glucose oxidation provides cancer cells with a proliferative advantage, conducive with apoptosis resistance and even increased angiogenesis. Recent evidence suggests that targeting the cancer-specific metabolic and mitochondrial remodeling may offer selectivity in cancer treatment. Pyruvate dehydrogenase kinase (PDK is a mitochondrial enzyme that is activated in a variety of cancers and results in the selective inhibition of pyruvate dehydrogenase (PDH, a complex of enzymes that converts cytosolic pyruvate to mitochondrial acetyl-CoA, the substrate for the Krebs’ cycle. Inhibition of PDK with either small interfering RNAs or the orphan drug dichloroacetate (DCA shifts the metabolism of cancer cells from glycolysis to glucose oxidation and reverses the suppression of mitochondria-dependent apoptosis. In addition, this therapeutic strategy increases the production of diffusible Krebs’ cycle intermediates and mitochondria-derived reactive oxygen species (mROS, activating p53 or inhibiting pro-proliferative and pro-angiogenic transcription factors like nuclear factor of activated T-cells (NFAT and hypoxia-inducible factor 1α (HIF1α. These effects result in decreased tumor growth and angiogenesis in a variety of cancers with high selectivity. In a small but mechanistic clinical trial in patients with glioblastoma, a highly aggressive and vascular form of brain cancer, DCA decreased tumor angiogenesis and tumor growth, suggesting that metabolic targeting therapies can be translated directly to patients. Therefore, reversing the mitochondrial suppression with metabolic-modulating drugs, like PDK inhibitors holds promise in the rapidly expanding field of metabolic oncology.

  13. A pharmacologic increase in activity of plasma transaminase derived from small intestine in animals receiving an acyl CoA: diacylglycerol transferase (DGAT) 1 inhibitor.

    Science.gov (United States)

    Yokoyama, Hideaki; Kobayashi, Akio; Kondo, Kazuma; Oshida, Shin-Ichi; Takahashi, Tadakazu; Masuyama, Taku; Shoda, Toshiyuki; Sugai, Shoichiro

    2018-01-01

    Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the re-synthesis of triglycerides (TG) from free fatty acids and diacylglycerol. JTT-553 is a DGAT1 inhibitor and exhibits its pharmacological action (inhibition of re-synthesis of TG) in the enterocytes of the small intestine leading to suppression of a postprandial elevation of plasma lipids. After repeated oral dosing JTT-553 in rats and monkeys, plasma transaminase levels were increased but there were neither changes in other hepatic function parameters nor histopathological findings suggestive of hepatotoxicity. Based on the results of exploratory studies for investigation of the mechanism of the increase in transaminase levels, plasma transaminase levels were increased after dosing JTT-553 only when animals were fed after dosing and a main factor in the diet contributing to the increase in plasma transaminase levels was lipids. After dosing JTT-553, transaminase levels were increased in the small intestine but not in the liver, indicating that the origin of transaminase increased in the plasma was not the liver but the small intestine where JTT-553 exhibits its pharmacological action. The increase in small intestinal transaminase levels was due to increased enzyme protein synthesis and was suppressed by inhibiting fatty acid-transport to the enterocytes. In conclusion, the JTT-553-related increase in plasma transaminase levels is considered not to be due to release of the enzymes from injured cells into the circulation but to be phenomena resulting from enhancement of enzyme protein synthesis in the small intestine due to the pharmacological action of JTT-553 in this organ.

  14. Pyruvate carboxylase deficiency: An underestimated cause of lactic acidosis

    Directory of Open Access Journals (Sweden)

    F. Habarou

    2015-03-01

    Full Text Available Pyruvate carboxylase (PC is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A. Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1 and type B (patient 2. Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

  15. Pyruvate kinase M2: a potential target for regulating inflammation

    Directory of Open Access Journals (Sweden)

    Jose Carlos eAlves-Filho

    2016-04-01

    Full Text Available Pyruvate kinase (PK is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signalling pathways, affecting both the enzymatic activity of PKM2 as a pyruvate kinase and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for PKM2 as a therapeutic target in inflammatory and metabolic disorders.

  16. Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast

    NARCIS (Netherlands)

    A.J. van Maris; J.M. Geertman; A. Vermeulen; M.K. Groothuizen; A.A. Winkler; M.D. Piper; J.P. van Dijken; J.T. Pronk

    2004-01-01

    textabstractThe absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects:

  17. Field dependence of T1 for hyperpolarized [1-13C]pyruvate

    DEFF Research Database (Denmark)

    Chattergoon, N.; Martnez-Santiesteban, F.; Handler, W. B.

    2013-01-01

    conformation and properties of the dissolution media such as buffer composition, solution pH, temperature and magnetic field. We have measured the magnetic field dependence of the spin–lattice relaxation time of hyperpolarized [1-13C]pyruvate using field-cycled relaxometry. [1-13C]pyruvate was hyperpolarized...

  18. Relations between fatty acid synthesis, pyruvate concentration and cell concentration of suspensions of isolated rat hepatocytes

    NARCIS (Netherlands)

    Beynen, A.C.; Geelen, M.J.H.

    1984-01-01

    1. 1. The cell concentration of suspensions of isolated rat hepatocytes affects both the rate of pyruvate accumulation in the incubation medium and the rate of fatty acid synthesis. 2. 2. At low cell concentrations pyruvate accumulation is directly related to the cell concentration but levels off

  19. Apparent rate constant mapping using hyperpolarized [1-(13) C]pyruvate

    DEFF Research Database (Denmark)

    Khegai, O.; Schulte, R. F.; Janich, M. A.

    2014-01-01

    Hyperpolarization of [1-13C]pyruvate in solution allows real-time measurement of uptake and metabolism using MR spectroscopic methods. After injection and perfusion, pyruvate is taken up by the cells and enzymatically metabolized into downstream metabolites such as lactate, alanine, and bicarbona...

  20. A Patient With Pyruvate Carboxylase Deficiency and Nemaline Rods on Muscle Biopsy

    DEFF Research Database (Denmark)

    Unal, Ozlem; Orhan, Diclehan; Ostergaard, Elsebet

    2013-01-01

    Nemaline rods are the pathologic hallmark of nemaline myopathy, but they have also been described as a secondary phenomenon in a variety of other disorders. Nemaline rods have not been reported in pyruvate carboxylase deficiency before. Here we present a patient with pyruvate carboxylase deficiency...

  1. The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel.

    NARCIS (Netherlands)

    Huberts, D.H.; Venselaar, H.; Vriend, G.; Veenhuis, M.; Klei, I.J. van der

    2010-01-01

    Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenulapolymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional

  2. Detection of myocardial ischemia before infarction, based on accumulation of labeled pyruvate

    International Nuclear Information System (INIS)

    Goldstein, R.A.; Klein, M.S.; Sobel, B.E.

    1980-01-01

    To determine whether ischemic, but not irreversibly injured myocardium, can be differentiated from normal tissue based on accumulation of labeled pyruvate, isolated hearts were perfused with buffer containing [ 14 C]pyruvate under conditions of normal or low flow. Fifteen minutes after the hearts were exposed to labeled material, myocardial radioactivity was fourfold greater in ischemic compared to control hearts, due to accumulation of label in sequestered lactate produced from the pyruvate. Open-chest rabbits subjected to coronary occlusion exhibited a 1.73:1 ratio of radioactivity in ischemic compared with normal myocardium 15 min after systemic injection of [ 14 C]pyruvate. The results obtained suggest that zones of myocardial ischemia should be detectable in vivo by positron tomography after systemic administration of [ 11 C]pyruvate as well

  3. The crystal structure of Toxoplasma gondii pyruvate kinase 1.

    Directory of Open Access Journals (Sweden)

    Rebecca Bakszt

    2010-09-01

    Full Text Available Pyruvate kinase (PK, which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population.We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers.We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  4. The crystal structure of Toxoplasma gondii pyruvate kinase 1.

    Science.gov (United States)

    Bakszt, Rebecca; Wernimont, Amy; Allali-Hassani, Abdellah; Mok, Man Wai; Hills, Tanya; Hui, Raymond; Pizarro, Juan C

    2010-09-14

    Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  5. The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

    Energy Technology Data Exchange (ETDEWEB)

    Bakszt, R.; Wernimont, A; Allali-Hassani, A; Mok, M; Hills, T; Hui, R; Pizarro, J

    2010-01-01

    Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two {alpha}-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  6. Pyruvate metabolism: A therapeutic opportunity in radiation-induced skin injury

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Hyun; Kang, Jeong Wook [Department of Radiation Oncology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Lee, Dong Won [Department of Plastic Surgery, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Oh, Sang Ho [Department of Dermatology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Lee, Yun-Sil [College of Pharmacy & Division of Life and Pharmaceutical Sciences, Ewah Womans University, Seoul 120-750 (Korea, Republic of); Lee, Eun-Jung [Department of Radiation Oncology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Cho, Jaeho, E-mail: jjhmd@yuhs.ac [Department of Radiation Oncology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

    2015-05-08

    Ionizing radiation is used to treat a range of cancers. Despite recent technological progress, radiation therapy can damage the skin at the administration site. The specific molecular mechanisms involved in this effect have not been fully characterized. In this study, the effects of pyruvate, on radiation-induced skin injury were investigated, including the role of the pyruvate dehydrogenase kinase 2 (PDK2) signaling pathway. Next generation sequencing (NGS) identified a wide range of gene expression differences between the control and irradiated mice, including reduced expression of PDK2. This was confirmed using Q-PCR. Cell culture studies demonstrated that PDK2 overexpression and a high cellular pyruvate concentration inhibited radiation-induced cytokine expression. Immunohistochemical studies demonstrated radiation-induced skin thickening and gene expression changes. Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness and inflammatory cytokine expression. These findings indicated that regulation of the pyruvate metabolic pathway could provide an effective approach to the control of radiation-induced skin damage. - Highlights: • The effects of radiation on skin thickness in mice. • Next generation sequencing revealed that radiation inhibited pyruvate dehydrogenase kinase 2 expression. • PDK2 inhibited irradiation-induced cytokine gene expression. • Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness.

  7. Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation.

    Science.gov (United States)

    Agrimi, Gennaro; Mena, Maria C; Izumi, Kazuki; Pisano, Isabella; Germinario, Lucrezia; Fukuzaki, Hisashi; Palmieri, Luigi; Blank, Lars M; Kitagaki, Hiroshi

    2014-03-01

    Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  8. Mitochondrial Pyruvate Carrier 2 Hypomorphism in Mice Leads to Defects in Glucose-Stimulated Insulin Secretion

    Directory of Open Access Journals (Sweden)

    Patrick A. Vigueira

    2014-06-01

    Full Text Available Carrier-facilitated pyruvate transport across the inner mitochondrial membrane plays an essential role in anabolic and catabolic intermediary metabolism. Mitochondrial pyruvate carrier 2 (Mpc2 is believed to be a component of the complex that facilitates mitochondrial pyruvate import. Complete MPC2 deficiency resulted in embryonic lethality in mice. However, a second mouse line expressing an N-terminal truncated MPC2 protein (Mpc2Δ16 was viable but exhibited a reduced capacity for mitochondrial pyruvate oxidation. Metabolic studies demonstrated exaggerated blood lactate concentrations after pyruvate, glucose, or insulin challenge in Mpc2Δ16 mice. Additionally, compared with wild-type controls, Mpc2Δ16 mice exhibited normal insulin sensitivity but elevated blood glucose after bolus pyruvate or glucose injection. This was attributable to reduced glucose-stimulated insulin secretion and was corrected by sulfonylurea KATP channel inhibitor administration. Collectively, these data are consistent with a role for MPC2 in mitochondrial pyruvate import and suggest that Mpc2 deficiency results in defective pancreatic β cell glucose sensing.

  9. Elevated transaminases as a predictor of coma in a patient with anorexia nervosa: a case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Yoshida Shuhei

    2010-09-01

    Full Text Available Abstract Introduction Liver injury is a frequent complication associated with anorexia nervosa, and steatosis of the liver is thought to be the major underlying pathology. However, acute hepatic failure with transaminase levels over 1000 IU/mL and deep coma are very rare complications and the mechanism of pathogenesis is largely unknown. Case presentation A 37-year-old Japanese woman showed features of acute liver failure and hepatic coma which were not associated with hypoglycemia or hyper-ammonemia. Our patient's consciousness was significantly improved with the recovery of liver function and normalization of transaminase levels after administration of nutritional support. Conclusions Our case report demonstrates that transaminase levels had an inverse relationship with the consciousness of our patient, although the pathogenesis of coma remains largely unknown. This indicates that transaminase levels can be one of the key predictors of impending coma in patients with anorexia nervosa. Therefore, frequent monitoring of transaminase levels combined with rigorous treatment of the underlying nutritional deficiency and psychiatric disorder are necessary to prevent this severe complication.

  10. Single pyruvate intake induces blood alkalization and modification of resting metabolism in humans.

    Science.gov (United States)

    Olek, Robert A; Luszczyk, Marcin; Kujach, Sylwester; Ziemann, Ewa; Pieszko, Magdalena; Pischel, Ivo; Laskowski, Radoslaw

    2015-03-01

    Three separate studies were performed with the aim to 1) determine the effect of a single sodium pyruvate intake on the blood acid-base status in males and females; 2) compare the effect of sodium and calcium pyruvate salts and establish their role in the lipolysis rate; and 3) quantify the effect of single pyruvate intake on the resting energy metabolism. In all, 48 individuals completed three separate studies. In all the studies, participants consumed a single dose of pyruvate 0.1 g/kg 60 min before commencing the measurements. The whole blood pH, bicarbonate concentration, base excess or plasma glycerol, free fatty acids, glucose concentrations, or resting energy expenditure and calculated respiratory exchange ratio were determined. The analysis of variance for repeated measurements was performed to examine the interaction between treatment and time. The single dose of sodium pyruvate induced blood alkalization, which was more marked in the male than in the female participants. Following the ingestion of sodium or calcium pyruvate, the blood acid-base parameters were higher than in the placebo trial. Furthermore, 3-h postingestion glycerol was lower in both pyruvate trials than in placebo. Resting energy expenditure did not differ between the trials; however, carbohydrate oxidation was increased after sodium pyruvate ingestion. Pyruvate intake induced mild alkalization in a sex-dependent fashion. Moreover, it accelerated carbohydrate metabolism and delayed the rate of glycerol appearance in the blood, but had no effect on the resting energy expenditure. Furthermore, sodium salt seems to have had a greater effect on the blood buffering level than calcium salt. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. The coupling of ω-transaminase and Oppenauer oxidation reactions via intra-membrane multicomponent diffusion – A process model for the synthesis of chiral amines

    DEFF Research Database (Denmark)

    Esparza-Isunza, T.; González-Brambila, M.; Gani, Rafiqul

    2015-01-01

    amine product. Using 2-propylamine as the amine donor of the ω-transaminase reaction, gives acetone as a by-product, which in turn allows the coupling of the ω-transaminase reaction with the Oppenauer oxidation. The Oppenauer reaction converts secondary alcohols into ketones, and these can subsequently......In this study we consider the theoretical coupling of an otherwise thermodynamically limited ω-transaminase reaction to an Oppenauer oxidation, in order to shift the equilibria of both reactions, with the aim of achieving a significant (and important) increase in the yield of the desired chiral...... of this paper is to report the development of a mathematical model as a tool for the simulation and potential design of such a process for the production of a range of chiral amines. The mathematical model developed considers that each reaction is performed in a single ideally mixed isothermal reactor operating...

  12. Chiral Amine Synthesis Using ω-Transaminases: An Amine Donor that Displaces Equilibria and Enables High-Throughput Screening**

    Science.gov (United States)

    Green, Anthony P; Turner, Nicholas J; O'Reilly, Elaine

    2014-01-01

    The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by-product removal using only one equivalent of a diamine donor (ortho-xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone). Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity. PMID:25138082

  13. Effects of pyruvate dose on in vivo metabolism and quantification of hyperpolarized 13C spectra

    DEFF Research Database (Denmark)

    Janich, M. A.; Menzel, M. I.; Wiesinger, F.

    2012-01-01

    Real‐time in vivo measurements of metabolites are performed by signal enhancement of [1‐13C]pyruvate using dynamic nuclear polarization, rapid dissolution and intravenous injection, acquisition of free induction decay signals and subsequent quantification of spectra. The commonly injected dose...... uptake and metabolic conversion. The goal of this study was to examine the effects of a [1‐13C]pyruvate bolus on metabolic conversion in vivo. Spectra were quantified by three different methods: frequency‐domain fitting with LCModel, time‐domain fitting with AMARES and simple linear least‐squares fitting...... in the time domain. Since the simple linear least‐squares approach showed bleeding artifacts and LCModel produced noisier time signals. AMARES performed best in the quantification of in vivo hyperpolarized pyruvate spectra. We examined pyruvate doses of 0.1–0.4 mmol/kg (body mass) in male Wistar rats...

  14. A pyruvate-buffered dialysis fluid induces less peritoneal angiogenesis and fibrosis than a conventional solution

    NARCIS (Netherlands)

    van Westrhenen, Roos; Zweers, Machteld M.; Kunne, Cindy; de Waart, Dirk R.; van der Wal, Allard C.; Krediet, Raymond T.

    2008-01-01

    BACKGROUND: Conventional lactate-buffered peritoneal dialysis (PD) fluids containing glucose and glucose degradation products are believed to contribute to the development of fibrosis and angiogenesis in the dialyzed peritoneum. To reduce potential negative effects of lactate, pyruvate was

  15. Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

    Science.gov (United States)

    Vogel, O; Hoehn, B; Henning, U

    1972-06-01

    The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.

  16. Effect of hexoses on the levels of pyruvate decarboxylase in Mucor rouxii.

    OpenAIRE

    Barrera, C R; Corral, J

    1980-01-01

    Pyruvate decarboxylase activity in the dimorphic fungus Mucor rouxii increased 25- to 35-fold in yeastlike and mycelial cells grown in the presence of glucose as compared to the activity observed in mycelial cultures grown in the absence of glucose.

  17. Changes in myocardial lactate, pyruvate and lactate-pyruvate ratio during cardiopulmonary bypass for elective adult cardiac surgery: Early indicator of morbidity

    Directory of Open Access Journals (Sweden)

    P M Kapoor

    2011-01-01

    Full Text Available Background: Myocardial lactate assays have been established as a standard method to compare various myocardial protection strategies. This study was designed to test whether coronary sinus (CS lactates, pyruvate and lactate-pyruvate (LP ratio correlates with myocardial dysfunction and predict postoperative outcomes. Materials and Methods: This prospective observational study was conducted on 40 adult patients undergoing elective cardiac surgery with the aid of cardiopulmonary bypass (CPB. CS blood sampling was done for estimation of myocardial lactate (ML, pyruvate (MP and lactate-pyruvate ratio (MLPR namely: pre-CPB (T 1 , after removal of aortic cross clamp (T 2 and 30 minutes post-CPB (T 3 . Results: Baseline myocardial LPR strongly correlated with Troponin-I at T1 (s: 0.6. Patients were sub grouped according to the median value of myocardial lactate (2.9 at baseline T1 into low myocardial lactate (LML group, mean (2.39±0.4 mmol/l, n=19 and a high myocardial lactate (HML group, mean (3.65±0.9 mmol/l, n=21. A significant increase in PL, ML, MLPR and TropI occurred in both groups as compared to baseline. Patients in HML group had significant longer period of ICU stay. Patients with higher inotrope score had significantly higher ML (T2, T3. ML with a baseline value of 2.9 mmol/l had 70.83% sensitivity and 62.5% specificity (ROC area: 0.7109 Std error: 0.09 while myocardial pyruvate with a baseline value of 0.07 mmol/l has 79.17% sensitivity and 68.75% specificity (ROC area: 0.7852, Std error: 0.0765 for predicting inotrope requirement after CPB. Conclusion: CS lactate, pyruvate and LP ratio correlate with myocardial function and can predict postoperative outcome.

  18. Detection of Hepatitis C virus RNA in peripheral blood mononuclear cells of patients with abnormal alanine transaminase in Ahvaz

    Directory of Open Access Journals (Sweden)

    M Makvandi

    2014-01-01

    Full Text Available Purpose: Hepatitis C virus (HCV is an important agent for chronic and acute hepatitis. Occult hepatitis C remains a major health problem worldwide. Patients with chronic occult HCV may progress to cirrhosis and hepatocellular carcinoma. The aim of this study was to determine prevalence of occult hepatitis C by IS-PCR-ISH (in situ PCR in situ hybridisation in the patients with abnormal ALT. Materials and Methods: The blood samples were taken from 53 patients including 17 females (32.1% and 36 (67.9% males who had abnormal alanine transaminase (ALT for more than 1 year. The mean ALT and aspartate transaminase (AST level were 41.02 ± 9.3 and 24.17 ± 7.3, respectively. The patients′ age were between 4 and 70-years old with mean age 38 ± 13. All the patients were negative for HCV antibody, HCV RNA and HBs Ag. The peripheral blood mononuclear cells (PBMC were separated with ficoll gradient from each blood sample, then the cells were fixed on slides by cold acetone and followed by IS-PCR-ISH for HCV RNA detection. Results: Seventeen (32% patients including 6 (11.3% females and 11 (20.7% males showed positive results for HCV RNA by in situ-PCR in situ hybridisation. Ten (18.8% positive cases were between 20 and 40-years old and 6 (11.3% positive patients were between 40 and 60 years old. Ten (19.6% patients who were positive for IS-PCR-ISH also had positive anti-HBc IgG and 7 (13.2% patients were negative for HBc-IgG. Conclusion: In the present study high rate of 32% occult hepatitis C were found among the patients with elevated ALT.

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

    Science.gov (United States)

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

    2015-01-01

    Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products. Copyright © 2014 John Wiley & Sons, Ltd.

  20. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

    OpenAIRE

    Fabian V Filipp

    2013-01-01

    Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2) is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor acti...

  1. Pyruvate incubation enhances glycogen stores and sustains neuronal function during subsequent glucose deprivation.

    Science.gov (United States)

    Shetty, Pavan K; Sadgrove, Matthew P; Galeffi, Francesca; Turner, Dennis A

    2012-01-01

    The use of energy substrates, such as lactate and pyruvate, has been shown to improve synaptic function when administered during glucose deprivation. In the present study, we investigated whether prolonged incubation with monocarboxylate (pyruvate or lactate) prior rather than during glucose deprivation can also sustain synaptic and metabolic function. Pyruvate pre-incubation(3-4h) significantly prolonged (>25 min) the tolerance of rat hippocampal slices to delayed glucose deprivation compared to control and lactate pre-incubated slices, as revealed by field excitatory post synaptic potentials (fEPSPs); pre-incubation with pyruvate also reduced the marked decrease in NAD(P)H fluorescence resulting from glucose deprivation. Moreover, pyruvate exposure led to the enhancement of glycogen stores with time, compared to glucose alone (12 μmol/g tissue at 4h vs. 3.5 μmol/g tissue). Prolonged resistance to glucose deprivation following exogenous pyruvate incubation was prevented by glycogenolysis inhibitors, suggesting that enhanced glycogen mediates the delay in synaptic activity failure. The application of an adenosine A1 receptor antagonist enhanced glycogen utilization and prolonged the time to synaptic failure, further confirming this hypothesis of the importance of glycogen. Moreover, tissue levels of ATP were also significantly maintained during glucose deprivation in pyruvate pretreated slices compared to control and lactate. In summary, these experiments indicate that pyruvate exposure prior to glucose deprivation significantly increased the energy buffering capacity of hippocampal slices, particularly by enhancing internal glycogen stores, delaying synaptic failure during glucose deprivation by maintaining ATP levels, and minimizing the decrease in the levels of NAD(P)H. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Physical exercise reduces pyruvate carboxylase (PCB) and contributes to hyperglycemia reduction in obese mice.

    Science.gov (United States)

    Muñoz, Vitor Rosetto; Gaspar, Rafael Calais; Crisol, Barbara Moreira; Formigari, Guilherme Pedron; Sant'Ana, Marcella Ramos; Botezelli, José Diego; Gaspar, Rodrigo Stellzer; da Silva, Adelino S R; Cintra, Dennys Esper; de Moura, Leandro Pereira; Ropelle, Eduardo Rochete; Pauli, José Rodrigo

    2018-07-01

    The present study evaluated the effects of exercise training on pyruvate carboxylase protein (PCB) levels in hepatic tissue and glucose homeostasis control in obese mice. Swiss mice were distributed into three groups: control mice (CTL), fed a standard rodent chow; diet-induced obesity (DIO), fed an obesity-inducing diet; and a third group, which also received an obesity-inducing diet, but was subjected to an exercise training protocol (DIO + EXE). Protocol training was carried out for 1 h/d, 5 d/wk, for 8 weeks, performed at an intensity of 60% of exhaustion velocity. An insulin tolerance test (ITT) was performed in the last experimental week. Twenty-four hours after the last physical exercise session, the animals were euthanized and the liver was harvested for molecular analysis. Firstly, DIO mice showed increased epididymal fat and serum glucose and these results were accompanied by increased PCB and decreased p-Akt in hepatic tissue. On the other hand, physical exercise was able to increase the performance of the mice and attenuate PCB levels and hyperglycemia in DIO + EXE mice. The above findings show that physical exercise seems to be able to regulate hyperglycemia in obese mice, suggesting the participation of PCB, which was enhanced in the obese condition and attenuated after a treadmill running protocol. This is the first study to be aimed at the role of exercise training in hepatic PCB levels, which may be a novel mechanism that can collaborate to reduce the development of hyperglycemia and type 2 diabetes in DIO mice.

  3. Pyruvate Decarboxylase Activity Assay in situ of Different Industrial Yeast Strains

    Directory of Open Access Journals (Sweden)

    Dorota Kręgiel

    2009-01-01

    Full Text Available Cytoplasmic pyruvate decarboxylase (PDC, EC 4.1.1.1 is one of the key enzymes of yeast fermentative metabolism. PDC is the first enzyme which, under anaerobic conditions, leads to decarboxylation of pyruvate with acetaldehyde as the end product. The aim of this study is to develop a suitable method for PDC activity assay in situ for different industrial yeast strains. Saccharomyces sp. and Debaryomyces sp. yeast strains grew in fermentative medium with 12 % of glucose. Enzymatic assay was conducted in cell suspension treated with digitonin as permeabilisation agent, and with sodium pyruvate as a substrate, at temperature of 30 °C. Metabolites of PDC pathway were detected using gas chromatographic (GC technique. Various parameters like type and molar concentration of the substrate, minimal effective mass fraction of digitonin, cell concentration, reaction time and effect of pyrazole (alcohol dehydrogenase inhibitor were monitored to optimize PDC enzymatic assay in situ. In the concentration range of yeast cells from 1⋅10^7 to 1⋅10^8 per mL, linear correlation between the produced acetaldehyde and cell density was noticed. Only pyruvate was the specific substrate for pyruvate decarboxylase. In the presence of 0.05 M sodium pyruvate and 0.05 % digitonin, the enzymatic reaction was linear up to 20 min of the assay. During incubation, there was no formation of ethanol and, therefore, pyrazole was not necessary for the assay.

  4. Pyruvate dehydrogenase kinase inhibition: Reversing the Warburg effect in cancer therapy

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    Hayden Bell

    2016-06-01

    Full Text Available The poor efficacy of many cancer chemotherapeutics, which are often non-selective and highly toxic, is attributable to the remarkable heterogeneity and adaptability of cancer cells. The Warburg effect describes the up regulation of glycolysis as the main source of adenosine 5’-triphosphate in cancer cells, even under normoxic conditions, and is a unique metabolic phenotype of cancer cells. Mitochondrial suppression is also observed which may be implicated in apoptotic suppression and increased funneling of respiratory substrates to anabolic processes, conferring a survival advantage. The mitochondrial pyruvate dehydrogenase complex is subject to meticulous regulation, chiefly by pyruvate dehydrogenase kinase. At the interface between glycolysis and the tricarboxylic acid cycle, the pyruvate dehydrogenase complex functions as a metabolic gatekeeper in determining the fate of glucose, making pyruvate dehydrogenase kinase an attractive candidate in a bid to reverse the Warburg effect in cancer cells. The small pyruvate dehydrogenase kinase inhibitor dichloroacetate has, historically, been used in conditions associated with lactic acidosis but has since gained substantial interest as a potential cancer chemotherapeutic. This review considers the Warburg effect as a unique phenotype of cancer cells in-line with the history of and current approaches to cancer therapies based on pyruvate dehydrogenase kinase inhibition with particular reference to dichloroacetate and its derivatives.

  5. Decarboxylation of oxalacetate to pyruvate by purified avian liver phosphoenolpyruvate carboxykinase

    Energy Technology Data Exchange (ETDEWEB)

    Noce, P S; Utter, M F

    1975-01-01

    Phosphoenolpyruvate carboxykinase, which has been isolated from chicken liver mitochondria in essentially homogenous form, carries out the irreversible decarboxylation of oxalacetate to pyruvate in the presence of catalytic amounts of GDP or IDP, as well as the reversible decarboxylation of oxalacetate to phosphoenolpyruvate in the presence of substrate amounts of GTP or ITP. The pyruvate- and phosphoenolpyruvate-forming reactions are similar in their nucleoside specificity and appear to be carried out by the same protein. However, the two activities vary markedly in their response to added metal ions and sulfhydryl reagents. Phosphoenolpyruvate formation is completely dependent on the presence of a divalent metal ion, with Mn/sup 2 +/ the most effective species. This reaction is also stimulated by sulfhydryl reagents such as 2-mercaptoethanol. In contrast, the pyruvate-forming reaction is strongly inhibited by divalent metal ions, including Mn/sup 2 +/, and also by moderate concentrations of sulfhydryl reagents. These observations and the demonstration that pyruvate kinase-like activity is very low or absent make it unlikely that pyruvate formation proceeds via phosphoenolpyruvate as an intermediate. Although the pyruvate-forming reaction is inhibited by added metal ions, the reaction is also inhibited by metal-chelating agents such as 8-hydroxyquinoline and o-phenanthroline, suggesting that the reaction is dependent on the presence of a metal ion. It has not been possible, however, to demonstrate that the enzyme is a metalloprotein.

  6. Supplementation of pyruvate prevents palmitate-induced impairment of glucose uptake in C2 myotubes.

    Science.gov (United States)

    Jung, Jong Gab; Choi, Sung-E; Hwang, Yoon-Jung; Lee, Sang-A; Kim, Eun Kyoung; Lee, Min-Seok; Han, Seung Jin; Kim, Hae Jin; Kim, Dae Jung; Kang, Yup; Lee, Kwan-Woo

    2011-10-15

    Elevated fatty acid levels have been thought to contribute to insulin resistance. Repression of the glucose transporter 4 (GLUT4) gene as well as impaired GLUT4 translocation may be a mediator for fatty acid-induced insulin resistance. This study was initiated to determine whether palmitate treatment repressed GLUT4 expression, whether glucose/fatty acid metabolism influenced palmitate-induced GLUT4 gene repression (PIGR), and whether attempts to prevent PIGR restored palmitate-induced impairment of glucose uptake (PIIGU) in C2 myotubes. Not only stimulators of fatty acid oxidation, such as bezafibrate, AICAR, and TOFA, but also TCA cycle substrates, such as pyruvate, leucine/glutamine, and α-ketoisocaproate/monomethyl succinate, significantly prevented PIGR. In particular, supplementing with pyruvate through methyl pyruvate resulted in nearly complete prevention of PIIGU, whereas palmitate treatment reduced the intracellular pyruvate level. These results suggest that pyruvate depletion plays a critical role in PIGR and PIIGU; thus, pyruvate supplementation may help prevent obesity-induced insulin resistance in muscle cells. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Tai-Long Chien

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

  8. Serum sickness

    Science.gov (United States)

    ... the problem should be stopped. Avoid using that medicine or antiserum in the future. ... Call your provider if you received medicine or antiserum in the last 4 weeks and have symptoms of serum sickness.

  9. Assessing the transport rate of hyperpolarized pyruvate and lactate from the intra- to the extracellular space.

    Science.gov (United States)

    Reineri, Francesca; Daniele, Valeria; Cavallari, Eleonora; Aime, Silvio

    2016-08-01

    The use of [1-(13) C]pyruvate hyperpolarized by means of dynamic nuclear polarization provides a direct way to track the metabolic transformations of this metabolite in vivo and in cell cultures. The identification of the intra- and extracellular contributions to the (13) C NMR resonances is not straightforward. In order to obtain information about the rate of pyruvate and lactate transport through the cellular membrane, we set up a method that relies on the sudden 'quenching' of the extracellular metabolites' signal. The paramagnetic Gd-tetraazacyclododecane triacetic acid (Gd-DO3A) complex was used to dramatically decrease the longitudinal relaxation time constants of the (13) C-carboxylate resonances of both pyruvate and lactate. When Gd-DO3A was added to an MCF-7 cellular culture, which had previously received a dose of hyperpolarized [1-(13) C]pyruvate, the contributions of the extracellular pyruvate and lactate signals were deleted. From the analysis of the decay curves of the (13) C-carboxylate resonances of pyruvate and lactate it was possible to extract information about the exchange rate of the two metabolites across the cellular membrane. In particular, it was found that, in the reported experimental conditions, the lactate transport from the intra- to the extracellular space is not much lower than the rate of lactate formation. The method reported herein is non-destructive and it could be translated to in vivo studies. It opens a route for the use of hyperpolarized pyruvate to assess altered activity of carboxylate transporter proteins that may occur in pathological conditions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. [Diagnostic value of detection of blood levels of lactate, pyruvate and 2,3-diphosphoglycerate in children with diabetes mellitus].

    Science.gov (United States)

    Marchenko, L F; Baturin, A A; Terent'eva, E A

    1991-01-01

    Measurements were made of lactate, pyruvate and 2,3-diphosphoglycerate in 69 children admitted to the hospital in a state of diabetic ketoacidosis of different intensity. Depending on the intensity of metabolic abnormalities, the content of lactate and pyruvate was found to be increased, whereas that of 2,3-diphosphoglycerate to be lowered. Measurements of the content of lactate and the lactate/pyruvate ratio enables carrying out differential diagnosis between the ketoacidotic and lactacidotic varieties of diabetic coma.

  11. Chronic pyruvate supplementation increases exploratory activity and brain energy reserves in young and middle-aged mice

    DEFF Research Database (Denmark)

    Koivisto, Hennariikka; Leinonen, Henri; Puurula, Mari

    2016-01-01

    to brain and thereby attenuate aging- or AD-related cognitive impairment. Mice received ~800 mg/kg/day Na-pyruvate in their chow for 2-6 months. In middle-aged wild-type mice and in 6.5-month-old APP/PS1 mice, pyruvate facilitated spatial learning and increased exploration of a novel odor. However......, in passive avoidance task for fear memory, the treatment group was clearly impaired. Independent of age, long-term pyruvate increased explorative behavior, which likely explains the paradoxical impairment in passive avoidance. We also assessed pyruvate effects on body weight, muscle force, and endurance...

  12. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Wittmann Christoph

    2008-03-01

    Full Text Available Abstract Background Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS and phosphoenolpyruvate carboxylase (PEPC, whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood. Results In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose. Conclusion The metabolic

  13. Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

    Science.gov (United States)

    Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

    2010-08-01

    Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

  14. An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion

    Directory of Open Access Journals (Sweden)

    Kyle S. McCommis

    2016-08-01

    Full Text Available Objective: Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion. Methods: To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, we used genetically-engineered Drosophila and mice with loss of MPC activity in insulin-producing cells. Results: In both species, MPC deficiency results in elevated blood sugar concentrations and glucose intolerance accompanied by impaired glucose-stimulated insulin secretion. In mouse islets, β-cell MPC-deficiency resulted in decreased respiration with glucose, ATP-sensitive potassium (KATP channel hyperactivity, and impaired insulin release. Moreover, treatment of pancreas-specific MPC knockout mice with glibenclamide, a sulfonylurea KATP channel inhibitor, improved defects in islet insulin secretion and abnormalities in glucose homeostasis in vivo. Finally, using a recently-developed biosensor for MPC activity, we show that the MPC is rapidly stimulated by glucose treatment in INS-1 insulinoma cells suggesting that glucose sensing is coupled to mitochondrial pyruvate carrier activity. Conclusions: Altogether, these studies suggest that the MPC plays an important and ancestral role in insulin-secreting cells in mediating glucose sensing, regulating insulin secretion, and controlling systemic glycemia. Keywords: Stimulus-coupled secretion, Insulin, β-Cell, Diabetes, Pyruvate, Mitochondria, Drosophila

  15. The mitochondrial pyruvate carrier mediates high fat diet-induced increases in hepatic TCA cycle capacity.

    Science.gov (United States)

    Rauckhorst, Adam J; Gray, Lawrence R; Sheldon, Ryan D; Fu, Xiaorong; Pewa, Alvin D; Feddersen, Charlotte R; Dupuy, Adam J; Gibson-Corley, Katherine N; Cox, James E; Burgess, Shawn C; Taylor, Eric B

    2017-11-01

    Excessive hepatic gluconeogenesis is a defining feature of type 2 diabetes (T2D). Most gluconeogenic flux is routed through mitochondria. The mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytosol into the mitochondrial matrix, thereby gating pyruvate-driven gluconeogenesis. Disruption of the hepatocyte MPC attenuates hyperglycemia in mice during high fat diet (HFD)-induced obesity but exerts minimal effects on glycemia in normal chow diet (NCD)-fed conditions. The goal of this investigation was to test whether hepatocyte MPC disruption provides sustained protection from hyperglycemia during long-term HFD and the differential effects of hepatocyte MPC disruption on TCA cycle metabolism in NCD versus HFD conditions. We utilized long-term high fat feeding, serial measurements of postabsorptive blood glucose and metabolomic profiling and 13 C-lactate/ 13 C-pyruvate tracing to investigate the contribution of the MPC to hyperglycemia and altered hepatic TCA cycle metabolism during HFD-induced obesity. Hepatocyte MPC disruption resulted in long-term attenuation of hyperglycemia induced by HFD. HFD increased hepatic mitochondrial pyruvate utilization and TCA cycle capacity in an MPC-dependent manner. Furthermore, MPC disruption decreased progression of fibrosis and levels of transcript markers of inflammation. By contributing to chronic hyperglycemia, fibrosis, and TCA cycle expansion, the hepatocyte MPC is a key mediator of the pathophysiology induced in the HFD model of T2D. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  16. Effects of Vigabatrin, an Irreversible GABA Transaminase Inhibitor, on Ethanol Reinforcement and Ethanol Discriminative Stimuli in Mice

    Science.gov (United States)

    Griffin, William C.; Nguyen, Shaun A.; Deleon, Christopher P.; Middaugh, Lawrence D.

    2012-01-01

    We tested the hypothesis that the irreversible gamma-amino butyric acid (GABA) transaminase inhibitor, γ-vinyl GABA (Vigabatrin; VGB) would reduce ethanol reinforcement and enhance the discriminative stimulus effect of ethanol, effectively reducing ethanol intake. The present studies used adult C57BL/6J (B6) mice in well-established operant, two-bottle choice consumption, locomotor activity and ethanol discrimination procedures, to examine comprehensively the effects of VGB on ethanol-supported behaviors. VGB dose-dependently reduced operant responding for ethanol as well as ethanol consumption for long periods of time. Importantly, a low dose (200 mg/kg) of VGB was selective for reducing ethanol responding without altering intake of food or water reinforcement. Higher VGB doses (>200 mg/kg) still reduced ethanol intake, but also significantly increased water consumption and, more modestly, increased food consumption. While not affecting locomotor activity on its own, VGB interacted with ethanol to reduce the stimulatory effects of ethanol on locomotion. Finally, VGB (200 mg/kg) significantly enhanced the discriminative stimulus effects of ethanol as evidenced by significant left-ward and up-ward shifts in ethanol generalization curves. Interestingly, VGB treatment was associated with slight increases in blood ethanol concentrations. The reduction in ethanol intake by VGB appears to be related to the ability of VGB to potentiate the pharmacological effects of ethanol. PMID:22336593

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  18. Chiral amine synthesis using ω-transaminases: an amine donor that displaces equilibria and enables high-throughput screening.

    Science.gov (United States)

    Green, Anthony P; Turner, Nicholas J; O'Reilly, Elaine

    2014-09-26

    The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by-product removal using only one equivalent of a diamine donor (ortho-xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone). Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity. © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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

    Energy Technology Data Exchange (ETDEWEB)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. (Mount Sinai School of Medicine, New York, NY (USA)); Wada, H.; Horio, Y. (Univ. of Osaka (Japan))

    1990-05-01

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

  20. A new synthesis of [3-11C]pyruvic acid using alanine racemase

    International Nuclear Information System (INIS)

    Ikemoto, M.; Okamoto, E.; Sasaki, M.; Haradahira, T.; Omura, H.; Furuya, Y.; Suzuki, K.; Watanabe, Y.

    1998-01-01

    The synthesis of [3- 11 C]pyruvic acid was attempted by two reaction systems (A: alanine racemase and D-amino acid oxidase, B: alanine racemase and L-alanine dehydrogenase) utilizing a new thermostable enzyme, alanine racemase. Conversion rates from D,L-[3- 11 C]alanine to [3- 11 C]pyruvic acid were almost 100% in both methods. Similar results were obtained with immobilized enzymes packed in a single column. Furthermore, the same column could be used repeatedly without a remarkable decrease of the [3- 11 C]pyruvic acid yield. Various matrices were tested for the immobilizing enzyme, and Aminopropyl-CPG was concluded to be the most suitable since the loss of the enzyme activity was the least in the studied matrices

  1. Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

    Science.gov (United States)

    Hurd, Thomas R; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T; Baranowski, Bartlomiej; Fearnley, Ian M; Prime, Tracy A; Murphy, Michael P; James, Andrew M

    2012-10-12

    Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism.

  2. Metabolic Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-13C]Pyruvate

    Science.gov (United States)

    Nelson, Sarah J.; Kurhanewicz, John; Vigneron, Daniel B.; Larson, Peder E. Z.; Harzstark, Andrea L.; Ferrone, Marcus; van Criekinge, Mark; Chang, Jose W.; Bok, Robert; Park, Ilwoo; Reed, Galen; Carvajal, Lucas; Small, Eric J.; Munster, Pamela; Weinberg, Vivian K.; Ardenkjaer-Larsen, Jan Henrik; Chen, Albert P.; Hurd, Ralph E.; Odegardstuen, Liv-Ingrid; Robb, Fraser J.; Tropp, James; Murray, Jonathan A.

    2014-01-01

    This first-in-man imaging study evaluated the safety and feasibility of hyperpolarized [1-13C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Imaging living systems with hyperpolarized agents can result in more than 10,000-fold enhancement in signal relative to conventional magnetic resonance (MR) imaging. When combined with the rapid acquisition of in vivo 13C MR data, it is possible to evaluate the distribution of agents such as [1-13C]pyruvate and its metabolic products lactate, alanine, and bicarbonate in a matter of seconds. Preclinical studies in cancer models have detected elevated levels of hyperpolarized [1-13C]lactate in tumor, with the ratio of [1-13C]lactate/[1-13C]pyruvate being increased in high-grade tumors and decreased after successful treatment. Translation of this technology into humans was achieved by modifying the instrument that generates the hyperpolarized agent, constructing specialized radio frequency coils to detect 13C nuclei, and developing new pulse sequences to efficiently capture the signal. The study population comprised patients with biopsy-proven prostate cancer, with 31 subjects being injected with hyperpolarized [1-13C]pyruvate. The median time to deliver the agent was 66 s, and uptake was observed about 20 s after injection. No dose-limiting toxicities were observed, and the highest dose (0.43 ml/kg of 230 mM agent) gave the best signal-to-noise ratio for hyperpolarized [1-13C]pyruvate. The results were extremely promising in not only confirming the safety of the agent but also showing elevated [1-13C]lactate/[1-13C]pyruvate in regions of biopsy-proven cancer. These findings will be valuable for noninvasive cancer diagnosis and treatment monitoring in future clinical trials. PMID:23946197

  3. Pyruvate administration reduces recurrent/moderate hypoglycemia-induced cortical neuron death in diabetic rats.

    Directory of Open Access Journals (Sweden)

    Bo Young Choi

    Full Text Available Recurrent/moderate (R/M hypoglycemia is common in type 1 diabetes patients. Moderate hypoglycemia is not life-threatening, but if experienced recurrently it may present several clinical complications. Activated PARP-1 consumes cytosolic NAD, and because NAD is required for glycolysis, hypoglycemia-induced PARP-1 activation may render cells unable to use glucose even when glucose availability is restored. Pyruvate, however, can be metabolized in the absence of cytosolic NAD. We therefore hypothesized that pyruvate may be able to improve the outcome in diabetic rats subjected to insulin-induced R/M hypoglycemia by terminating hypoglycemia with glucose plus pyruvate, as compared with delivering just glucose alone. In an effort to mimic juvenile type 1 diabetes the experiments were conducted in one-month-old young rats that were rendered diabetic by streptozotocin (STZ, 50mg/kg, i.p. injection. One week after STZ injection, rats were subjected to moderate hypoglycemia by insulin injection (10 U/kg, i.p. without anesthesia for five consecutive days. Pyruvate (500 mg/kg was given by intraperitoneal injection after each R/M hypoglycemia. Three hours after last R/M hypoglycemia, zinc accumulation was evaluated. Three days after R/M hypoglycemia, neuronal death, oxidative stress, microglial activation and GSH concentrations in the cerebral cortex were analyzed. Sparse neuronal death was observed in the cortex. Zinc accumulation, oxidative injury, microglial activation and GSH loss in the cortex after R/M hypoglycemia were all reduced by pyruvate injection. These findings suggest that when delivered alongside glucose, pyruvate may significantly improve the outcome after R/M hypoglycemia by circumventing a sustained impairment in neuronal glucose utilization resulting from PARP-1 activation.

  4. Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

    Science.gov (United States)

    Plaga, W; Lottspeich, F; Oesterhelt, D

    1992-04-01

    An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

  5. Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

    Science.gov (United States)

    Wojtkowiak, Jonathan W; Cornnell, Heather C; Matsumoto, Shingo; Saito, Keita; Takakusagi, Yoichi; Dutta, Prasanta; Kim, Munju; Zhang, Xiaomeng; Leos, Rafael; Bailey, Kate M; Martinez, Gary; Lloyd, Mark C; Weber, Craig; Mitchell, James B; Lynch, Ronald M; Baker, Amanda F; Gatenby, Robert A; Rejniak, Katarzyna A; Hart, Charles; Krishna, Murali C; Gillies, Robert J

    2015-01-01

    Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to

  6. Crystal structure and thermochemical properties of a novel coordination compound sodium pyruvate C3H3O3Na(s)

    International Nuclear Information System (INIS)

    Gao, Zhen-Fei; Di, You-Ying; Liu, Su-Zhou; Lu, Dong-Fei; Dou, Jian-Min

    2014-01-01

    Graphical abstract: A novel coordination compound sodium pyruvate C 3 H 3 O 3 Na(s) is synthesised. Elemental analysis and X-ray crystallography are used to characterise the composition and crystal structure of the compound. The lattice potential energy and ionic volume of the anion are obtained from crystallographic data. The standard molar enthalpy of formation of the compound is calculated by an isoperibol solution-reaction calorimeter. Molar enthalpies of dissolution of the compound at various molalities are measured at T = 298.15 K. According to Pitzer’s theory, molar enthalpy of dissolution of the title compound at infinite dilution is calculated. The values of relative apparent molar enthalpies and relative partial molar enthalpies of the solvent and the compound at different concentrations m/(mol · kg −1 ) are derived. - Highlights: • The sodium pyruvate was synthesised and crystal structure was determined. • The enthalpy change of the synthesis reaction was obtained. • Standard molar enthalpy of formation was obtained. • Molar enthalpy of dissolution at infinite dilution was calculated. - Abstract: A novel coordination compound sodium pyruvate C 3 H 3 O 3 Na(s) is synthesised by a liquid phase reaction. The compound has an obvious bioactivity and can be used as the biological carbon source and the chemical identification of primary and secondary alcohols. It can be also used to determinate transaminase. Elemental analysis and X-ray crystallography are used to characterise the composition and crystal structure of the compound. Single crystal X-ray analysis reveals that the compound is formed by one CH 3 COCOO − anion and one Na + cation. An obvious feature of the crystal structure is the formation of the five-membered chelate ring by the coordination of O1 of carboxylate and O3 of keto form with Na + cation, and it is good for the stability of the compound in structure. The lattice potential energy and ionic volume of the anion are obtained

  7. The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel

    NARCIS (Netherlands)

    Huberts, Daphne H. E. W.; Venselaar, Hanka; Vriend, Gert; Veenhuis, Marten; van der Klei, Ida J.

    Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenula polymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional

  8. Chronic pyruvate supplementation increases exploratory activity and brain energy reserves in young and middle-aged mice

    Directory of Open Access Journals (Sweden)

    Hennariikka eKoivisto

    2016-03-01

    Full Text Available Numerous studies have reported neuroprotective effects of pyruvate when given in systemic injections. Impaired glucose uptake and metabolism are found in Alzheimer's disease (AD and in AD mouse models. We tested whether dietary pyruvate supplementation is able to provide added energy supply to brain and thereby attenuate aging- or AD-related cognitive impairment. Mice received ~ 800 mg/kg/day Na-pyruvate in their chow for 2- 6 months. In middle-aged wild-type mice and in 6.5-month-old APP/PS1 mice, pyruvate facilitated spatial learning and increased exploration of a novel odor. However, in passive avoidance task for fear memory, the treatment group was clearly impaired. Independent of age, long-term pyruvate increased explorative behavior, which likely explains the paradoxical impairment in passive avoidance. We also assessed pyruvate effects on body weight, muscle force and endurance, and found no effects. Metabolic post-mortem assays revealed increased energy compounds in nuclear magnetic resonance spectroscopy as well as increased brain glycogen storages in the pyruvate group. Pyruvate supplementation may counteract aging-related behavioral impairment but its beneficial effect seems related to increased explorative activity rather than direct memory enhancement.

  9. Mutants of GABA transaminase (POP2 suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh mutants in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Frank Ludewig

    Full Text Available BACKGROUND: The gamma-aminubutyrate (GABA shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD, the mitochondrial enzymes GABA transaminase (GABA-T; POP2 and succinic semialdehyde dehydrogenase (SSADH. We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported. PRINCIPAL FINDINGS: To elucidate the role of succinic semialdehyde (SSA, gamma-hydroxybutyrate (GHB and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants. SIGNIFICANCE: We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.

  10. Growth hormone-induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity

    DEFF Research Database (Denmark)

    Nellemann, B.; Vendelbo, M.H.; Nielsen, Thomas Svava

    2014-01-01

    Insulin resistance induced by growth hormone (GH) is linked to promotion of lipolysis by unknown mechanisms. We hypothesized that suppression of the activity of pyruvate dehydrogenase in the active form (PDHa) underlies GH-induced insulin resistance similar to what is observed during fasting....

  11. Studies to enhance the hyperpolarization level in PHIP-SAH-produced C13-pyruvate

    Science.gov (United States)

    Cavallari, Eleonora; Carrera, Carla; Aime, Silvio; Reineri, Francesca

    2018-04-01

    The use of [1-13C]pyruvate, hyperpolarized by dissolution-Dynamic Nuclear Polarization (d-DNP), in in vivo metabolic studies has developed quickly, thanks to the imaging probe's diagnostic relevance. Nevertheless, the cost of a d-DNP polarizer is quite high and the speed of hyperpolarization process is relatively slow, meaning that its use is limited to few research laboratories. ParaHydrogen Induced Polarization Side Arm Hydrogenation (PHIP-SAH) (Reineri et al., 2015) is a cost effective and easy-to-handle method that produces 13C-MR hyperpolarization in [1-13C]pyruvate and other metabolites. This work aims to identify the main determinants of the hyperpolarization levels observed in C13-pyruvate using this method. By dissecting the various steps of the PHIP-SAH procedure, it has been possible to assess the role of several experimental parameters whose optimization must be pursued if this method is to be made suitable for future translational steps. The search for possible solutions has led to improvements in the polarization of sodium [1-13C]pyruvate from 2% to 5%. Moreover, these results suggest that observed polarization levels could be increased considerably by an automatized procedure which would reduce the time required for the work-up passages that are currently carried out manually. The results reported herein mean that the attainment of polarization levels suitable for the metabolic imaging applications of these hyperpolarized substrates show significant promise.

  12. Multi site Kinetic Modeling of 13C Metabolic MR Using [1-13C]Pyruvate

    International Nuclear Information System (INIS)

    Damian, P.A.G.; Sperl, J.I.; Janich, M.A.; Wiesinger, F.; Schulte, R.F.; Menzel, M.I.; Damian, P.A.G.; Damian, P.A.G.; Haase, A.; Janich, M.A.; Schwaiger, M.; Janich, M.A.; Khegai, O.; Glaser, S.J.

    2014-01-01

    Hyperpolarized 13 C imaging allows real-time in vivo measurements of metabolite levels. Quantification of metabolite conversion between [1- 13 C]pyruvate and downstream metabolites [1- 13 C]alanine, [1- 13 C]lactate, and [ 13 C] bicarbonate can be achieved through kinetic modeling. Since pyruvate interacts dynamically and simultaneously with its downstream metabolites, the purpose of this work is the determination of parameter values through a multi site, dynamic model involving possible biochemical pathways present in MR spectroscopy. Kinetic modeling parameters were determined by fitting the multi site model to time-domain dynamic metabolite data. The results for different pyruvate doses were compared with those of different two-site models to evaluate the hypothesis that for identical data the uncertainty of a model and the signal-to-noise ratio determine the sensitivity in detecting small physiological differences in the target metabolism. In comparison to the two-site exchange models, the multi site model yielded metabolic conversion rates with smaller bias and smaller standard deviation, as demonstrated in simulations with different signal-to-noise ratio. Pyruvate dose effects observed previously were confirmed and quantified through metabolic conversion rate values. Parameter interdependency allowed an accurate quantification and can therefore be useful for monitoring metabolic activity in different tissues

  13. Dissociative electron attachment and anion-induced dimerization in pyruvic acid

    Czech Academy of Sciences Publication Activity Database

    Zawadzki, Mateusz; Ranković, Miloš; Kočišek, Jaroslav; Fedor, Juraj

    2018-01-01

    Roč. 20, č. 10 (2018), s. 6838-6844 ISSN 1463-9076 R&D Projects: GA ČR GA17-04844S; GA ČR GJ16-10995Y Institutional support: RVO:61388955 Keywords : pyruvic acid * electron attachment * dimerization Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 4.123, year: 2016

  14. Mitochondrial metabolism of pyruvate is essential for regulating glucose-stimulated insulin secretion.

    Science.gov (United States)

    Patterson, Jessica N; Cousteils, Katelyn; Lou, Jennifer W; Manning Fox, Jocelyn E; MacDonald, Patrick E; Joseph, Jamie W

    2014-05-09

    It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.

  15. Magnetic resonance and fluorescence studies on pyruvate dehydrogenase complexes and their small molecular weight constituents

    NARCIS (Netherlands)

    Grande, H.J.

    1976-01-01

    The articles presented in this thesis do not describe at first glance one well-defined subject. They are, however, in fact connected by one central theme: the study of large enzyme aggregates by molecular physical methods. Chosen was the pyruvate dehydrogenase complex (PDC) because of its

  16. Pyruvate Oxidase Influences the Sugar Utilization Pattern and Capsule Production in Streptococcus pneumoniae

    NARCIS (Netherlands)

    Carvalho, Sandra M.; Farshchi Andisi, Vahid; Gradstedt, Henrik; Neef, Jolanda; Kuipers, Oscar P.; Neves, Ana R.; Bijlsma, Jetta J. E.

    2013-01-01

    Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence,

  17. Multisite Kinetic Modeling of 13C Metabolic MR Using [1-13C]Pyruvate

    Directory of Open Access Journals (Sweden)

    Pedro A. Gómez Damián

    2014-01-01

    Full Text Available Hyperpolarized 13C imaging allows real-time in vivo measurements of metabolite levels. Quantification of metabolite conversion between [1-13C]pyruvate and downstream metabolites [1-13C]alanine, [1-13C]lactate, and [13C]bicarbonate can be achieved through kinetic modeling. Since pyruvate interacts dynamically and simultaneously with its downstream metabolites, the purpose of this work is the determination of parameter values through a multisite, dynamic model involving possible biochemical pathways present in MR spectroscopy. Kinetic modeling parameters were determined by fitting the multisite model to time-domain dynamic metabolite data. The results for different pyruvate doses were compared with those of different two-site models to evaluate the hypothesis that for identical data the uncertainty of a model and the signal-to-noise ratio determine the sensitivity in detecting small physiological differences in the target metabolism. In comparison to the two-site exchange models, the multisite model yielded metabolic conversion rates with smaller bias and smaller standard deviation, as demonstrated in simulations with different signal-to-noise ratio. Pyruvate dose effects observed previously were confirmed and quantified through metabolic conversion rate values. Parameter interdependency allowed an accurate quantification and can therefore be useful for monitoring metabolic activity in different tissues.

  18. NH4+ triggers the release of astrocytic lactate via mitochondrial pyruvate shunting

    Science.gov (United States)

    Lerchundi, Rodrigo; Fernández-Moncada, Ignacio; Contreras-Baeza, Yasna; Sotelo-Hitschfeld, Tamara; Mächler, Philipp; Wyss, Matthias T.; Stobart, Jillian; Baeza-Lehnert, Felipe; Alegría, Karin; Weber, Bruno; Barros, L. Felipe

    2015-01-01

    Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K+ as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4+, a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4+ with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4+ and in the somatosensory cortex of anesthetized mice in response to i.v. NH4+. Unexpectedly, NH4+ had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4+ diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4+ is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4+ behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes. PMID:26286989

  19. NH4(+) triggers the release of astrocytic lactate via mitochondrial pyruvate shunting.

    Science.gov (United States)

    Lerchundi, Rodrigo; Fernández-Moncada, Ignacio; Contreras-Baeza, Yasna; Sotelo-Hitschfeld, Tamara; Mächler, Philipp; Wyss, Matthias T; Stobart, Jillian; Baeza-Lehnert, Felipe; Alegría, Karin; Weber, Bruno; Barros, L Felipe

    2015-09-01

    Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K(+) as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4(+), a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4(+) with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4(+) and in the somatosensory cortex of anesthetized mice in response to i.v. NH4(+). Unexpectedly, NH4(+) had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4(+) diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4(+) is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4(+) behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes.

  20. Metabolic imaging of patients with prostate cancer using hyperpolarized [1-¹³C]pyruvate

    DEFF Research Database (Denmark)

    Nelson, Sarah J; Kurhanewicz, John; Vigneron, Daniel B

    2013-01-01

    This first-in-man imaging study evaluated the safety and feasibility of hyperpolarized [1-¹³C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Imaging living systems with hyperpolarized agents can result in more than 10,000-f...

  1. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

    Science.gov (United States)

    Krznar, Petra; Hörl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-01-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival. PMID:27176894

  2. Pyruvate Kinase Triggers a Metabolic Feedback Loop that Controls Redox Metabolism in Respiring Cells

    NARCIS (Netherlands)

    Grüning, N.M.; Rinnerthaler, M.; Bluemlein, K.; Mulleder, M.; Wamelink, M.M.C.; Lehrach, H.; Jakobs, C.A.J.M.; Breitenbach, M.; Ralser, M.

    2011-01-01

    In proliferating cells, a transition from aerobic to anaerobic metabolism is known as the Warburg effect, whose reversal inhibits cancer cell proliferation. Studying its regulator pyruvate kinase (PYK) in yeast, we discovered that central metabolism is self-adapting to synchronize redox metabolism

  3. Hyperpolarized 1-13C Pyruvate Imaging of Porcine Cardiac Metabolism shift by GIK Intervention

    DEFF Research Database (Denmark)

    Søvsø Szocska Hansen, Esben; Tougaard, Rasmus Stilling; Mikkelsen, Emmeli

    to evaluate the general feasibility to detect an imposed shift in metabolic substrate utilization during metabolic modulation with glucose, insulin and potassium (GIK) infusion. This study demonstrates that hyperpolarized 13C-pyruvate, in a large animal, is a feasible method for cardiac studies, and...

  4. Exercise-induced pyruvate dehydrogenase activation is not affected by 7 days of bed rest

    DEFF Research Database (Denmark)

    Kiilerich, Kristian; Jørgensen, Stine Ringholm; Biensø, Rasmus Sjørup

    2011-01-01

    To test the hypothesis that physical inactivity impairs the exercise-induced modulation of pyruvate dehydrogenase (PDH), 6 healthy normally physically active male subjects completed 7 days of bed rest. Before and immediately after the bed rest, the subjects completed an OGTT and a one-legged knee...

  5. Molecular and Physiological Logics of the Pyruvate-Induced Response of a Novel Transporter in Bacillus subtilis.

    Science.gov (United States)

    Charbonnier, Teddy; Le Coq, Dominique; McGovern, Stephen; Calabre, Magali; Delumeau, Olivier; Aymerich, Stéphane; Jules, Matthieu

    2017-10-03

    At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB ), the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a hetero-oligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB We show that both glucose and malate, the preferred carbon sources for B. subtilis , trigger the binding of CcpA upstream of pftAB , which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB , which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis IMPORTANCE Pyruvate is a small-molecule metabolite ubiquitous in living cells. Several species also use it as a carbon source as well as excrete it into the environment. The bacterial systems for pyruvate import/export have yet to be discovered. Here, we identified in the model bacterium Bacillus subtilis the first import

  6. Neuron-astrocyte interactions, pyruvate carboxylation and the pentose phosphate pathway in the neonatal rat brain.

    Science.gov (United States)

    Morken, Tora Sund; Brekke, Eva; Håberg, Asta; Widerøe, Marius; Brubakk, Ann-Mari; Sonnewald, Ursula

    2014-01-01

    Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-(13)C]glucose and [1,2-(13)C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-(13)C]glucose and were sacrificed 30 min later. Brain extracts were analysed using (1)H- and (13)C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-(13)C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-(13)C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-(13)C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.

  7. Protective effect of pyruvate against ethanol-induced apoptotic neurodegeneration in the developing rat brain.

    Science.gov (United States)

    Ullah, Najeeb; Naseer, Muhammad Imran; Ullah, Ikram; Lee, Hae Young; Koh, Phil Ok; Kim, Myeong Ok

    2011-12-01

    Exposure to alcohol during the early stages of brain development can lead to neurological disorders in the CNS. Apoptotic neurodegeneration due to ethanol exposure is a main feature of alcoholism. Exposure of developing animals to alcohol (during the growth spurt period in particular) elicits apoptotic neuronal death and causes fetal alcohol effects (FAE) or fetal alcohol syndrome (FAS). A single episode of ethanol intoxication (at 5 g/kg) in a seven-day-old developing rat can activate the apoptotic cascade, leading to widespread neuronal death in the brain. In the present study, we investigated the potential protective effect of pyruvate against ethanol-induced neuroapoptosis. After 4h, a single dose of ethanol induced upregulation of Bax, release of mitochondrial cytochrome-c into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1), all of which promote apoptosis. These effects were all reversed by co-treatment with pyruvate at a well-tolerated dosage (1000 mg/kg). Histopathology performed at 24 and 48 h with Fluoro-Jade-B and cresyl violet stains showed that pyruvate significantly reduced the number of dead cells in the cerebral cortex, hippocampus and thalamus. Immunohistochemical analysis at 24h confirmed that ethanol-induced cell death is both apoptotic and inhibited by pyruvate. These findings suggest that pyruvate treatment attenuates ethanol-induced neuronal cell loss in the developing rat brain and holds promise as a safe therapeutic and neuroprotective agent in the treatment of neurodegenerative disorders in newborns and infants. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. 13C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and of the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

    International Nuclear Information System (INIS)

    Cohen, S.M.

    1987-01-01

    13 C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13 C enrichments at the individual carbons of glutamate when [3- 13 C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13 C label into fatty acids was monitored in this liver preparation. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1- 13 C]acetyl-CoA (from [2- 13 C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3- 13 C]alanine plus [2- 13 C]ethanol, which are converted to [2- 13 C]acetyl-CoA. Thus, measurement of 13 C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids. In addition to obtaining the rate of lipogenesis, it was possible to distinguish the contributions of chain elongation from those of the de novo synthesis pathway and to estimate the average chain length of the 13 C-labeled fatty acids produced

  9. Effect of thiamine deficiency, pyrithiamine and oxythiamine on pyruvate metabolism in rat liver and brain in vivo

    International Nuclear Information System (INIS)

    Meghal, S.K.; O'Neal, R.M.; Koeppe, R.E.

    1977-01-01

    Rats were fed either a thiamine-deficient diet or diets containing pyrithiamine or oxythiamine. When symptoms of thiamine deficiency appeared, the animals were injected intraperitoneally with [2- 14 C] pyruvate six to twelve minutes prior to sacrifice. Free glutamic and aspartic acids were isolated from liver and brain and degraded. The results indicate that, in thiamine-deficient or oxythiamine-treated rats, pyruvate metabolism in liver and brain is similar to that in normal animals. In contrast, pyrithinamine drastically decreases the oxidative decarboxylation of pyruvate by rat liver. (auth.)

  10. Serum ferritin

    International Nuclear Information System (INIS)

    Rochna Viola, E.M.; Diaz de Domingo, N.B.; Lazarowski, A.

    1981-01-01

    Serum ferritin (SF) concentration as determined by the immunoradiometric method allows the direct measurement of a fraction of the body ferritin pool. In normal subjects, SF is an excellent index of body iron stores. In certain conditions associated with increased ferritin synthesis (such as liver disease, inflammation, malignancy, chronic disorders, ineffective erythropoiesis, or during ferrotherapy), SF may not accurately reflect body iron stores. In hyposideremic anemias SF concentration permits to differentiate those due to iron deficiency from those due to chronic disorders. With a good assay quality, subnormal SF levels are incontrovertible in the diagnosis of iron deficiency. SF determination has been investigated as possible tumor marker. When performed in combination with the alpha-fetoprotein assay, SF enhances the specificity of serodiagnosis of hepatoma. SF results must be interpreted bearing in mind the possible participation of circumstances that i) modify the body iron stores and ii) lead to increased ferritin synthesis. (author) [es

  11. Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: requirement for ADP.

    Science.gov (United States)

    Bulos, B A; Thomas, B J; Shukla, S P; Sacktor, B

    1984-11-01

    Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate ( + proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (Km = 8.0 microM) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled alpha-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (greater than 99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 microM), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 +/- 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent KI was 0.8 mM. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, alpha-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the

  12. Reprint of "How do components of real cloud water affect aqueous pyruvate oxidation?"

    Science.gov (United States)

    Boris, Alexandra J.; Desyaterik, Yury; Collett, Jeffrey L.

    2015-01-01

    Chemical oxidation of dissolved volatile or semi-volatile organic compounds within fog and cloud droplets in the atmosphere could be a major pathway for secondary organic aerosol (SOA) formation. This proposed pathway consists of: (1) dissolution of organic chemicals from the gas phase into a droplet; (2) reaction with an aqueous phase oxidant to yield low volatility products; and (3) formation of particle phase organic matter as the droplet evaporates. The common approach to simulating aqueous SOA (aqSOA) reactions is photo-oxidation of laboratory standards in pure water. Reactions leading to aqSOA formation should be studied within real cloud and fog water to determine whether additional competing processes might alter apparent rates of reaction as indicated by rates of reactant loss or product formation. To evaluate and identify the origin of any cloud water matrix effects on one example of observed aqSOA production, pyruvate oxidation experiments simulating aqSOA formation were monitored within pure water, real cloud water samples, and an aqueous solution of inorganic salts. Two analysis methods were used: online electrospray ionization high-resolution time-of-flight mass spectrometry (ESI-HR-ToF-MS), and offline anion exchange chromatography (IC) with quantitative conductivity and qualitative ESI-HR-ToF-MS detection. The apparent rate of oxidation of pyruvate was slowed in cloud water matrices: overall measured degradation rates of pyruvate were lower than in pure water. This can be at least partially accounted for by the observed formation of pyruvate from reactions of other cloud water components. Organic constituents of cloud water also compete for oxidants and/or UV light, contributing to the observed slowed degradation rates of pyruvate. The oxidation of pyruvate was not significantly affected by the presence of inorganic anions (nitrate and sulfate) at cloud-relevant concentrations. Future bulk studies of aqSOA formation reactions using simplified

  13. Saccharomyces cerevisiae Differential Functionalization of Presumed ScALT1 and ScALT2 Alanine Transaminases Has Been Driven by Diversification of Pyridoxal Phosphate Interactions

    Directory of Open Access Journals (Sweden)

    Erendira Rojas-Ortega

    2018-05-01

    Full Text Available Saccharomyces cerevisiae arose from an interspecies hybridization (allopolyploidiza-tion, followed by Whole Genome Duplication. Diversification analysis of ScAlt1/ScAlt2 indicated that while ScAlt1 is an alanine transaminase, ScAlt2 lost this activity, constituting an example in which one of the members of the gene pair lacks the apparent ancestral physiological role. This paper analyzes structural organization and pyridoxal phosphate (PLP binding properties of ScAlt1 and ScAlt2 indicating functional diversification could have determined loss of ScAlt2 alanine transaminase activity and thus its role in alanine metabolism. It was found that ScAlt1 and ScAlt2 are dimeric enzymes harboring 67% identity and intact conservation of the catalytic residues, with very similar structures. However, tertiary structure analysis indicated that ScAlt2 has a more open conformation than that of ScAlt1 so that under physiological conditions, while PLP interaction with ScAlt1 allows the formation of two tautomeric PLP isomers (enolimine and ketoenamine ScAlt2 preferentially forms the ketoenamine PLP tautomer, indicating a modified polarity of the active sites which affect the interaction of PLP with these proteins, that could result in lack of alanine transaminase activity in ScAlt2. The fact that ScAlt2 forms a catalytically active Schiff base with PLP and its position in an independent clade in “sensu strictu” yeasts suggests this protein has a yet undiscovered physiological function.

  14. Saccharomyces cerevisiae Differential Functionalization of Presumed ScALT1 and ScALT2 Alanine Transaminases Has Been Driven by Diversification of Pyridoxal Phosphate Interactions

    Science.gov (United States)

    Rojas-Ortega, Erendira; Aguirre-López, Beatriz; Reyes-Vivas, Horacio; González-Andrade, Martín; Campero-Basaldúa, Jose C.; Pardo, Juan P.; González, Alicia

    2018-01-01

    Saccharomyces cerevisiae arose from an interspecies hybridization (allopolyploidiza-tion), followed by Whole Genome Duplication. Diversification analysis of ScAlt1/ScAlt2 indicated that while ScAlt1 is an alanine transaminase, ScAlt2 lost this activity, constituting an example in which one of the members of the gene pair lacks the apparent ancestral physiological role. This paper analyzes structural organization and pyridoxal phosphate (PLP) binding properties of ScAlt1 and ScAlt2 indicating functional diversification could have determined loss of ScAlt2 alanine transaminase activity and thus its role in alanine metabolism. It was found that ScAlt1 and ScAlt2 are dimeric enzymes harboring 67% identity and intact conservation of the catalytic residues, with very similar structures. However, tertiary structure analysis indicated that ScAlt2 has a more open conformation than that of ScAlt1 so that under physiological conditions, while PLP interaction with ScAlt1 allows the formation of two tautomeric PLP isomers (enolimine and ketoenamine) ScAlt2 preferentially forms the ketoenamine PLP tautomer, indicating a modified polarity of the active sites which affect the interaction of PLP with these proteins, that could result in lack of alanine transaminase activity in ScAlt2. The fact that ScAlt2 forms a catalytically active Schiff base with PLP and its position in an independent clade in “sensu strictu” yeasts suggests this protein has a yet undiscovered physiological function. PMID:29867852

  15. Purification of a protein from serum of cattle with hepatic lipidosis, and identification of the protein as haptoglobin.

    Science.gov (United States)

    Yoshino, K; Katoh, N; Takahashi, K; Yuasa, A

    1992-06-01

    A protein that has 2 subunits with molecular weight of 35,000 and 23,000 was detected in serum of cattle with hepatic lipidosis (fatty liver). The protein was purified from serum obtained from a cow with fatty liver, and was identified as haptoglobin, which is known to have hemoglobin-binding capacity and to be an acute-phase protein. To assess the relevance of haptoglobin in fatty liver, cattle were classified in 3 groups (healthy control, haptoglobin-positive, and haptoglobin-negative); liver triglyceride content and several serum biochemical variables were evaluated for the 3 groups. Compared with the control and haptoglobin-negative cattle, haptoglobin-positive cattle had significantly (P less than 0.01) higher liver triglyceride content, serum bilirubin concentration, and aspartate transaminase activity. Serum haptoglobin concentration was high in slaughter cattle (27 of 40 cattle tested), particularly in cows (20/28).

  16. Carbon-14 tracer studies in rat-liver perfusion experiments under conditions of gluconeogenesis from lactate and pyruvate

    International Nuclear Information System (INIS)

    Muellhofer, G.; Schwab, A.; Mueller, C.; Stetten, C. von; Gruber, E.

    1977-01-01

    The intracellular events in the metabolic pathway of gluconeogenesis from lactate and pyruvate in liver tissue were assumed to be understood. Nevertheless the results of several 14 C-tracer experiments gave rise to the postulation of still unknown intracellular interactions under this condition. A contribution was made to the solution of this problem by using different 14 C labelled tracers such as [1- 14 C]lactate or pyruvate and [2- 14 C]lactate or pyruvate. [ 14 C]bicarbonate and [1- 14 C]-octanoate in perfusion experiments with livers from rats under conditions of gluconeogenesis from lactate and pyruvate. The 14 C labelling patterns of intracellular metabolities such as malate, citrate, phosphoenolpyruvate, phosphoglycerate and newly synthesized glucose were analysed under different conditions. A comparison with values calculated by using metabolic models based on the generally accepted concepts of intracellular interactions showed some fundamental discrepancies which justify the postulation. (orig./MG) [de

  17. Investigating tumor perfusion and metabolism using multiple hyperpolarized 13C compounds: HP001, pyruvate and urea

    DEFF Research Database (Denmark)

    von Morze, Cornelius; Larson, Peder E.Z.; Hu, Simon

    2012-01-01

    The metabolically inactive hyperpolarized agents HP001 (bis-1,1-(hydroxymethyl)-[1-13C]cyclopropane-d8) and urea enable a new type of perfusion magnetic resonance imaging based on a direct signal source that is background-free. The addition of perfusion information to metabolic information obtained...... (T1=95 s ex vivo, 32 s in vivo at 3 T) using a pulse sequence with balanced steady-state free precession and ramped flip angle over time for efficient utilization of the hyperpolarized magnetization and three-dimensional echo-planar spectroscopic imaging of urea copolarized with [1-13C...... of separate dynamic HP001 imaging and copolarized pyruvate/urea imaging were compared. A strong and significant correlation (R=0.73, P=.02) detected between the urea and HP001 data confirmed the value of copolarizing urea with pyruvate for simultaneous assessment of perfusion and metabolism....

  18. Propionate Increases Hepatic Pyruvate Cycling and Anaplerosis and Alters Mitochondrial Metabolism

    DEFF Research Database (Denmark)

    Perry, Rachel J; Borders, Candace B; Cline, Gary W

    2016-01-01

    /tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-(13)C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously...... at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20-30-fold, increased hepatic TCA metabolite concentrations 2-3-fold, and increased endogenous glucose production rates by 20-100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only...... tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo....

  19. Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

    International Nuclear Information System (INIS)

    Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

    2009-01-01

    Potassium tellurite (K 2 TeO 3 ) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

  20. Enzyme mechanisms for pyruvate-to-lactate flux attenuation: a study of Sherpas, Quechuas, and hummingbirds.

    Science.gov (United States)

    Hochachka, P W; Stanley, C; McKenzie, D C; Villena, A; Monge, C

    1992-10-01

    During incremental exercise to fatigue under hypobaric hypoxia, Andean Quechua natives form and accumulate less plasma lactate than do lowlanders under similar conditions. This phenomenon of low lactate accumulation despite hypobaric hypoxia, first discovered some half century ago, is known in Quechuas to be largely unaffected by acute exposure to hypoxia or by acclimatization to sea level conditions. Earlier Nuclear Magnetic Resonance (NMR) spectroscopy and metabolic biochemistry studies suggest that closer coupling of energy demand and energy supply in Quechuas allows given changes in work rate with relatively modest changes in muscle adenylate and phosphagen concentrations, thus tempering the activation of glycolytic flux to pyruvate--a coarse control mechanism operating at the level of overall pathway flux. Later studies of enzyme activities in skeletal muscles of Quechuas and of Sherpas have identified a finely-tuned control mechanism which by adaptive modifications of a few key enzymes apparently serves to specifically attenuate pyruvate flux to lactate.

  1. MCA Vmean and the arterial lactate-to-pyruvate ratio correlate during rhythmic handgrip

    DEFF Research Database (Denmark)

    Rasmussen, Peter; Plomgaard, Peter; Krogh-Madsen, Rikke

    2006-01-01

    /P ratio at two plasma lactate levels. MCA Vmean was determined by ultrasound Doppler sonography at rest, during 10 min of rhythmic handgrip exercise at approximately 65% of maximal voluntary contraction force, and during 20 min of recovery in seven healthy male volunteers during control...... and a approximately 15 mmol/l hyperglycemic clamp. Cerebral arteriovenous differences for metabolites were obtained by brachial artery and retrograde jugular venous catheterization. Control resting arterial lactate was 0.78 +/- 0.09 mmol/l (mean +/- SE) and pyruvate 55.7 +/- 12.0 micromol/l (L/P ratio 16.4 +/- 1......Regulation of cerebral blood flow during physiological activation including exercise remains unknown but may be related to the arterial lactate-to-pyruvate (L/P) ratio. We evaluated whether an exercise-induced increase in middle cerebral artery mean velocity (MCA Vmean) relates to the arterial L...

  2. Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain

    DEFF Research Database (Denmark)

    Zhang, Yiming; Liu, Guodong; Engqvist, Martin K. M.

    2015-01-01

    Background: A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole...... DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase...... further increased the maximum specific growth rate to 0.069 h-1. Conclusions: In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing...

  3. Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein

    DEFF Research Database (Denmark)

    Schreiber, K; Boes, N; Escbach, M

    2006-01-01

    the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress...... proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the PPA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression...... was also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter...

  4. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    International Nuclear Information System (INIS)

    Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P.

    2005-01-01

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression

  5. Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

    Energy Technology Data Exchange (ETDEWEB)

    Chenoufi, N.; Loreal, O.; Cariou, S.; Hubert, N.; Lescoat, G. [Univ. Hospital Pontchaillou, Unite de Recherches Hepatologiques, INSERM U 49, Rennes (France); Drenou, B. [Univ. Hospital Pontchaillou, Lab. d`Hematologie et d`Immunologie, Rennes (France); Leroyer, P.; Brissot, P. [Univ. Hospital Pontchaillou, Clinique des Maladies du Foie, Rennes (France)

    1997-03-01

    Background/Aims: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. Methods: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. Results: In EGF/pyruvate-stimulated cultures, the level of [{sup 3}H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [{sup 3}H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However,the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [{sup 3}H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. Conclusions: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma. (au) 61 refs.

  6. Sodium Pyruvate Reduced Hypoxic-Ischemic Injury to Neonatal Rat Brain

    OpenAIRE

    Pan, Rui; Rong, Zhihui; She, Yun; Cao, Yuan; Chang, Li-Wen; Lee, Wei-Hua

    2012-01-01

    Background Neonatal hypoxia-ischemia (HI) remains a major cause of severe brain damage and is often associated with high mortality and lifelong disability. Immature brains are extremely sensitive to hypoxia-ischemia, shown as prolonged mitochondrial neuronal death. Sodium pyruvate (SP), a substrate of the tricarboxylic acid cycle and an extracellular antioxidant, has been considered as a potential treatment for hypoxic-ischemic encephalopathy (HIE), but its effects have not been evaluated in ...

  7. Erythrocyte pyruvate kinase deficiency in the Ohio Amish: origin and characterization of the mutant enzyme.

    OpenAIRE

    Muir, W A; Beutler, E; Wasson, C

    1984-01-01

    We have identified eight individuals in an Amish population in Geauga County, Ohio, who have a congenital hemolytic anemia and red cell pyruvate kinase (PK) deficiency. The mutant enzyme is a low Km phosphoenolpyruvate (PEP) variant associated with a slower (77.5% of normal) electrophoretic mobility in starch gel. Because of the high consanguinity in this population, we assume the affected individuals are homozygous for the mutant gene. Genealogical records allow us to trace all eight cases b...

  8. Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

    Science.gov (United States)

    Li, Xiaoli; Li, Yaqing; Han, Gaoyang; Li, Xiaoran; Ji, Yasai; Fan, Zhirui; Zhong, Yali; Cao, Jing; Zhao, Jing; Mariusz, Goscinski; Zhang, Mingzhi; Wen, Jianguo; Nesland, Jahn M.; Suo, Zhenhe

    2016-01-01

    Pyruvate plays a critical role in the mitochondrial tricarboxylic acid (TCA) cycle, and it is the center product for the synthesis of amino acids, carbohydrates and fatty acids. Pyruvate transported across the inner mitochondrial membrane appears to be essential in anabolic and catabolic intermediary metabolism. The mitochondrial pyruvate carrier (MPC) mounted in the inner membrane of mitochondria serves as the channel to facilitate pyruvate permeating. In mammals, the MPC is formed by two paralogous subunits, MPC1 and MPC2. It is known that complete ablation of MPC2 in mice causes death on the 11th or 12th day of the embryonic period. However, MPC1 deletion and the knowledge of gene function in vivo are lacking. Using the new technology of gene manipulation known as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) systems, we gained stable MPC1 gene heterozygous mutation mice models, and the heterozygous mutations could be stably maintained in their offsprings. Only one line with homozygous 27 bases deletion in the first exon was established, but no offsprings could be obtained after four months of mating experiments, indicating infertility of the mice with such homozygous deletion. The other line of MPC1 knockout (KO) mice was only heterozygous, which mutated in the first exon with a terminator shortly afterwards. These two lines of MPC1 KO mice showed lower fertility and significantly higher bodyweight in the females. We concluded that heterozygous MPC1 KO weakens fertility and influences the metabolism of glucose and fatty acid and bodyweight in mice. PMID:27835892

  9. Beneficial effect of pyruvate therapy on Leigh syndrome due to a novel mutation in PDH E1α gene.

    Science.gov (United States)

    Koga, Yasutoshi; Povalko, Nataliya; Katayama, Koujyu; Kakimoto, Noriko; Matsuishi, Toyojiro; Naito, Etsuo; Tanaka, Masashi

    2012-02-01

    Leigh syndrome (LS) is a progressive untreatable degenerating mitochondrial disorder caused by either mitochondrial or nuclear DNA mutations. A patient was a second child of unconsanguineous parents. On the third day of birth, he was transferred to neonatal intensive care units because of severe lactic acidosis. Since he was showing continuous lactic acidosis, the oral supplementation of dichloroacetate (DCA) was introduced on 31st day of birth at initial dose of 50 mg/kg, followed by maintenance dose of 25 mg/kg/every 12 h. The patient was diagnosed with LS due to a point mutation of an A-C at nucleotide 599 in exon 6 in the pyruvate dehydrogenase E1α gene, resulting in the substitution of aspartate for threonine at position 200 (N200T). Although the concentrations of lactate and pyruvate in blood were slightly decreased, his clinical conditions were deteriorating progressively. In order to overcome the mitochondrial or cytosolic energy crisis indicated by lactic acidosis as well as clinical symptoms, we terminated the DCA and administered 0.5 g/kg/day TID of sodium pyruvate orally. We analyzed the therapeutic effects of DCA or sodium pyruvate in the patient, and found that pyruvate therapy significantly decreased lactate, pyruvate and alanine levels, showed no adverse effects such as severe neuropathy seen in DCA, and had better clinical response on development and epilepsy. Though the efficacy of pyruvate on LS will be evaluated by randomized double-blind placebo-controlled study design in future, pyruvate therapy is a possible candidate for therapeutic choice for currently incurable mitochondrial disorders such as LS. Copyright © 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  10. Single Sodium Pyruvate Ingestion Modifies Blood Acid-Base Status and Post-Exercise Lactate Concentration in Humans

    Directory of Open Access Journals (Sweden)

    Robert A. Olek

    2014-05-01

    Full Text Available This study examined the effect of a single sodium pyruvate ingestion on a blood acid-base status and exercise metabolism markers. Nine active, but non-specifically trained, male subjects participated in the double-blind, placebo-controlled, crossover study. One hour prior to the exercise, subjects ingested either 0.1 g·kg−1 of body mass of a sodium pyruvate or placebo. The capillary blood samples were obtained at rest, 60 min after ingestion, and then three and 15 min after completing the workout protocol to analyze acid-base status and lactate, pyruvate, alanine, glucose concentrations. The pulmonary gas exchange, minute ventilation and the heart rate were measured during the exercise at a constant power output, corresponding to ~90% O2max. The blood pH, bicarbonate and the base excess were significantly higher after sodium pyruvate ingestion than in the placebo trial. The blood lactate concentration was not different after the ingestion, but the post-exercise was significantly higher in the pyruvate trial (12.9 ± 0.9 mM than in the placebo trial (10.6 ± 0.3 mM, p < 0.05 and remained elevated (nonsignificant after 15 min of recovery. The blood pyruvate, alanine and glucose concentrations, as well as the overall pulmonary gas exchange during the exercise were not affected by the pyruvate ingestion. In conclusion, the sodium pyruvate ingestion one hour before workout modified the blood acid-base status and the lactate production during the exercise.

  11. MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4

    OpenAIRE

    Hong, Candice Sun; Graham, Nicholas A.; Gu, Wen; Espindola Camacho, Carolina; Mah, Vei; Maresh, Erin L.; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A.L.; Gardner, Brian K.; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K.; Hurvitz, Sara A.

    2016-01-01

    Monocarboxylate Transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but ...

  12. Molecular and Physiological Logics of the Pyruvate-Induced Response of a Novel Transporter in Bacillus subtilis

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    Teddy Charbonnier

    2017-10-01

    Full Text Available At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB, the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a hetero-oligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB. We show that both glucose and malate, the preferred carbon sources for B. subtilis, trigger the binding of CcpA upstream of pftAB, which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB, which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis.

  13. Volumetric spiral chemical shift imaging of hyperpolarized [2-(13) c]pyruvate in a rat c6 glioma model.

    Science.gov (United States)

    Park, Jae Mo; Josan, Sonal; Jang, Taichang; Merchant, Milton; Watkins, Ron; Hurd, Ralph E; Recht, Lawrence D; Mayer, Dirk; Spielman, Daniel M

    2016-03-01

    MRS of hyperpolarized [2-(13)C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the (13)C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-(13)C]pyruvate in glioma-bearing brain. Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-(13)C]pyruvate, [2-(13)C]lactate, and [5-(13)C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-(13)C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-(13)C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity. The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-(13)C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain. Robust volumetric imaging with hyperpolarized [2-(13)C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate. © 2015 Wiley Periodicals, Inc.

  14. Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

    OpenAIRE

    Compan V; Pierredon S; Vanderperre B; Krznar P; Marchiq I; Zamboni N; Pouyssegur J; Martinou JC

    2015-01-01

    The transport of pyruvate into mitochondria requires a specific carrier the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism and its activity is likely to play a key role in bioenergetics. Until now investigation of the MPC activity has been limited. However the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell popu...

  15. MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells

    OpenAIRE

    Vanderperre, Benoît; Cermakova, Kristina; Escoffier Breancon, Jessica; Kaba, Mayis; Bender, Tom; Nef, Serge; Martinou, Jean-Claude

    2016-01-01

    Selective transport of pyruvate across the inner mitochondrial membrane by the mitochondrial pyruvate carrier (MPC) is a fundamental step that couples cytosolic and mitochondrial metabolism. The recent molecular identification of the MPC complex has revealed two interacting subunits, MPC1 and MPC2. Although in yeast, an additional subunit, MPC3, can functionally replace MPC2, no alternative MPC subunits have been described in higher eukaryotes. Here, we report for the first time the existence...

  16. Breast Cancer-Derived Lung Metastases Show Increased Pyruvate Carboxylase-Dependent Anaplerosis

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    Stefan Christen

    2016-10-01

    Full Text Available Cellular proliferation depends on refilling the tricarboxylic acid (TCA cycle to support biomass production (anaplerosis. The two major anaplerotic pathways in cells are pyruvate conversion to oxaloacetate via pyruvate carboxylase (PC and glutamine conversion to α-ketoglutarate. Cancers often show an organ-specific reliance on either pathway. However, it remains unknown whether they adapt their mode of anaplerosis when metastasizing to a distant organ. We measured PC-dependent anaplerosis in breast-cancer-derived lung metastases compared to their primary cancers using in vivo 13C tracer analysis. We discovered that lung metastases have higher PC-dependent anaplerosis compared to primary breast cancers. Based on in vitro analysis and a mathematical model for the determination of compartment-specific metabolite concentrations, we found that mitochondrial pyruvate concentrations can promote PC-dependent anaplerosis via enzyme kinetics. In conclusion, we show that breast cancer cells proliferating as lung metastases activate PC-dependent anaplerosis in response to the lung microenvironment.

  17. Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase.

    Science.gov (United States)

    Westerhold, Lauren E; Bridges, Lance C; Shaikh, Saame Raza; Zeczycki, Tonya N

    2017-07-11

    Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.

  18. Stem Cell Metabolism in Cancer and Healthy Tissues: Pyruvate in the Limelight

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    Cyril Corbet

    2018-01-01

    Full Text Available Normal and cancer stem cells (CSCs share the remarkable potential to self-renew and differentiate into many distinct cell types. Although most of the stem cells remain under quiescence to maintain their undifferentiated state, they can also undergo cell divisions as required to regulate tissue homeostasis. There is now a growing evidence that cell fate determination from stem cells implies a fine-tuned regulation of their energy balance and metabolic status. Stem cells can shift their metabolic substrate utilization, between glycolysis and mitochondrial oxidative metabolism, during specification and/or differentiation, as well as in order to adapt their microenvironmental niche. Pyruvate appears as a key metabolite since it is at the crossroads of cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. This Review describes how metabolic reprogramming, focusing on pyruvate utilization, drives the fate of normal and CSCs by modulating their capacity for self-renewal, clonal expansion/differentiation, as well as metastatic potential and treatment resistance in cancer. This Review also explores potential therapeutic strategies to restore or manipulate stem cell function through the use of small molecules targeting the pyruvate metabolism.

  19. Increased production of pyruvic acid by Escherichia coli RNase G mutants in combination with cra mutations.

    Science.gov (United States)

    Sakai, Taro; Nakamura, Naoko; Umitsuki, Genryou; Nagai, Kazuo; Wachi, Masaaki

    2007-08-01

    The Escherichia coli RNase G is known as an endoribonuclease responsible for the 5'-end maturation of 16S rRNA and degradation of several specific mRNAs such as adhE and eno mRNAs. In this study, we found that an RNase G mutant derived from the MC1061 strain did not grow on a glucose minimal medium. Genetic analysis revealed that simultaneous defects of cra and ilvIH, encoding a transcriptional regulator of glycolysis/gluconeogenesis and one of isozymes of acetohydroxy acid synthase, respectively, were required for this phenomenon to occur. The results of additional experiments presented here indicate that the RNase G mutation, in combination with cra mutation, caused the increased production of pyruvic acid from glucose, which was then preferentially converted to valine due to the ilvIH mutation, resulting in depletion of isoleucine. In fact, the rng cra double mutant produced increased amount of pyruvate in the medium. These results suggest that the RNase G mutation could be applied in the breeding of producer strains of pyruvate and its derivatives such as valine.

  20. Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria.

    Science.gov (United States)

    Su, Yu-Bin; Peng, Bo; Li, Hui; Cheng, Zhi-Xue; Zhang, Tian-Tuo; Zhu, Jia-Xin; Li, Dan; Li, Min-Yi; Ye, Jin-Zhou; Du, Chao-Chao; Zhang, Song; Zhao, Xian-Liang; Yang, Man-Jun; Peng, Xuan-Xian

    2018-02-13

    The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

  1. Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency.

    Science.gov (United States)

    Tomoda, A; Lachant, N A; Noble, N A; Tanaka, K R

    1983-07-01

    Pentose phosphate shunt activity was studied by the release of 14CO2 from 14C-1-glucose and 14C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7.1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate (Ki=1.05 mM) and a noncompetitive inhibitor with NADP (Ki=3.3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their Kms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.

  2. Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high-saturated fat diet

    Science.gov (United States)

    Hwang, Byounghoon; Wu, Pengfei; Harris, Robert A.

    2012-01-01

    SUMMARY Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) might prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it might induce detrimental effects by inhibiting fatty acid oxidation. PPARα agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment with a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild type and PDK4 knockout mice fed a high fat diet. As expected, treatment of wild type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, lowered blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid and a reduction in the capacity for fatty acid synthesis by PDK4 deficiency. PMID:22429297

  3. Effect of two different hypocaloric diets in transaminases and insulin resistance in nonalcoholic fatty liver disease and obese patients.

    Science.gov (United States)

    de Luis, D A; Aller, R; Izaola, O; Gonzalez Sagrado, M; Conde, R

    2010-01-01

    The aim of our study was to examine the changes in hypertransaminasemia after weight reduction in obese patients with and without NAFLD and the relation with insulin resistance. A population of 162 obese patients was randomly allocated to two groups: a) diet I (low fat) and b) diet II (low carbohydrate), dieting along 3 months. Patients were classified as group I (n=112) when serum ALT activity was normal or group II (NAFLD, n=30) when serum ALT activity was (>or=43 UI/L). In control group with diet I, BMI, weight, fat mass, waist to hip ratio, waist circumference, systolic pressure, total cholesterol, LDL cholesterol, HOMA and insulin levels decreased. In NAFLD group with diet I improved the same parameters and glucose, triglycerides, ALT, AST, gamaglutamine transferase levels, too. In control group with diet II, BMI, weight, fat mass, waist to hip ratio, waist circumference, systolic pressure, total cholesterol, LDL cholesterol, HOMA and insulin levels decreased. In NAFLD group with diet II improved the same parameters and glucose, triglycerides, ALT and gamaglutamine transferase levels, without statistical changes in AST. We showed that weight reduction secondary to two hypocaloric diets was associated with improvement in hipertransaminasemia and insulin resistance in NAFLD patients.

  4. Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

    Science.gov (United States)

    Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

    2014-10-24

    The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex*

    Science.gov (United States)

    Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S.; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

    2014-01-01

    The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. PMID:25210042

  6. Hyperpolarized [1-(13) C]pyruvate MRI for noninvasive examination of placental metabolism and nutrient transport: A feasibility study in pregnant guinea pigs.

    Science.gov (United States)

    Friesen-Waldner, Lanette J; Sinclair, Kevin J; Wade, Trevor P; Michael, Banoub; Chen, Albert P; de Vrijer, Barbra; Regnault, Timothy R H; McKenzie, Charles A

    2016-03-01

    To test the feasibility of hyperpolarized [1-(13) C]pyruvate magnetic resonance imaging (MRI) for noninvasive examination of guinea pig fetoplacental metabolism and nutrient transport. Seven pregnant guinea pigs with a total of 30 placentae and fetuses were anesthetized and scanned at 3T. T1 -weighted (1) H images were obtained from the maternal abdomen. An 80 mM solution of hyperpolarized [1-(13) C]pyruvate (hereafter referred to as pyruvate) was injected into a vein in the maternal foot. Time-resolved 3D (13) C images were acquired starting 10 seconds after the beginning of bolus injection and every 10 seconds after to 50 seconds. The pregnant guinea pigs were recovered after imaging. Regions of interest (ROIs) were drawn around the maternal heart and each placenta and fetal liver in all slices in the (1) H images. These ROIs were copied to the (13) C images and were used to calculate the sum of the pyruvate and lactate signal intensities for each organ. The signal intensities were normalized by the volume of the organ and the maximum signal in the maternal heart. No adverse events were observed in the pregnant guinea pigs and natural pupping occurred at term (∼68 days). Pyruvate signal was observed in all 30 placentae, and lactate, a by-product of pyruvate metabolism, was also observed in all placentae. The maximum pyruvate and lactate signals in placentae occurred at 20 seconds. In addition to the observation of pyruvate and lactate signals in the placentae, both pyruvate and lactate signals were observed in all fetal livers. The maximum pyruvate and lactate signals in the fetal livers occurred at 10 seconds and 20 seconds, respectively. This work demonstrates the feasibility of using hyperpolarized [1-(13) C]pyruvate MRI to noninvasively examine fetoplacental metabolism and transport of pyruvate in guinea pigs. Hyperpolarized (13) C MRI may provide a novel method for longitudinal studies of fetoplacental abnormalities. © 2015 Wiley Periodicals, Inc.

  7. Combined Hyperpolarized 13C-pyruvate MRS and 18F-FDG PET (HyperPET) Estimates of Glycolysis in Canine Cancer Patients

    DEFF Research Database (Denmark)

    Hansen, Adam E.; Gutte, Henrik; Holst, Pernille

    2018-01-01

    13C Magnetic Resonance Spectroscopy (MRS) using hyperpolarized 13C-labeled pyruvate as a substrate offers a measure of pyruvate-lactate interconversion and is thereby a marker of the elevated aerobic glycolysis (Warburg effect) generally exhibited by cancer cells. Here, we aim to compare hyperpol......13C Magnetic Resonance Spectroscopy (MRS) using hyperpolarized 13C-labeled pyruvate as a substrate offers a measure of pyruvate-lactate interconversion and is thereby a marker of the elevated aerobic glycolysis (Warburg effect) generally exhibited by cancer cells. Here, we aim to compare...

  8. Comparison the effectiveness of pyruvic acid 50% and salicylic acid 30% in the treatment of acne

    Directory of Open Access Journals (Sweden)

    Fariba Jaffary

    2016-01-01

    Full Text Available Background: Acne vulgaris is a chronic inflammatory disease of the pilosebaceous follicles and one of the most common skin diseases. The peeling method has been recently found to be effective for acne treatment. This study aimed to compare the efficacy of pyruvic acid 50% and salicylic acid 30% peeling in the treatment of mild to moderate acne. Materials and Methods: In a prospective single-blinded clinical trial, 86 patients with acne were randomly assigned into two groups. In both groups, the routine treatment of acne (topical solution of erythromycin 4%, triclorocarban soap, and sunscreen were used twice a day for 8 weeks. In addition, salicylic acid 30% for the control group and pyruvic acid 50% for the case group were used. In both groups, acne severity index (ASI was calculated before and at week 2, 4, 6, and 8 of the treatment. Patient satisfaction was assessed at the end of the treatment. Side effects were recorded using a checklist. Results: In both groups, the reduction in the number of comedones, papules, and ASI were statistically significant (P < 0.001 in the course of treatment. However, it was not significant regarding the number of pustules (P = 0.09. None of the number of comedone, papules, pustules, and ASI was statistically different between study groups. Both treatment groups had similar side effects except for scaling in the fifth session, which was significantly lower in salicylic acid - treated patients (P = 0.015. Conclusion: Both pyruvic acid 50% and salicylic acid 30% are effective in the improvement of mild to moderate acne with no significant difference in efficacy and side effects.

  9. Comparison the effectiveness of pyruvic acid 50% and salicylic acid 30% in the treatment of acne.

    Science.gov (United States)

    Jaffary, Fariba; Faghihi, Gita; Saraeian, Sara; Hosseini, Sayed Mohsen

    2016-01-01

    Acne vulgaris is a chronic inflammatory disease of the pilosebaceous follicles and one of the most common skin diseases. The peeling method has been recently found to be effective for acne treatment. This study aimed to compare the efficacy of pyruvic acid 50% and salicylic acid 30% peeling in the treatment of mild to moderate acne. In a prospective single-blinded clinical trial, 86 patients with acne were randomly assigned into two groups. In both groups, the routine treatment of acne (topical solution of erythromycin 4%, triclorocarban soap, and sunscreen) were used twice a day for 8 weeks. In addition, salicylic acid 30% for the control group and pyruvic acid 50% for the case group were used. In both groups, acne severity index (ASI) was calculated before and at week 2, 4, 6, and 8 of the treatment. Patient satisfaction was assessed at the end of the treatment. Side effects were recorded using a checklist. In both groups, the reduction in the number of comedones, papules, and ASI were statistically significant ( P < 0.001) in the course of treatment. However, it was not significant regarding the number of pustules ( P = 0.09). None of the number of comedone, papules, pustules, and ASI was statistically different between study groups. Both treatment groups had similar side effects except for scaling in the fifth session, which was significantly lower in salicylic acid - treated patients ( P = 0.015). Both pyruvic acid 50% and salicylic acid 30% are effective in the improvement of mild to moderate acne with no significant difference in efficacy and side effects.

  10. Maturation of pig oocytes in vitro in a medium with pyruvate

    Directory of Open Access Journals (Sweden)

    H. Gonzales-Figueroa

    2005-06-01

    Full Text Available The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouseand transported to the laboratory within 2 h at 35-39ºC,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF and in SM + pFF + gonadotropins (eCG and hCG for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9% and SM + pFF (42.9% showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6% showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 ± 3.6% and SM + pFF (42.9 ± 3.7%. These results indicate that pig oocytes can be successfully matured in a chemically definedmedium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment.

  11. Protective Effect of Pyruvate Against Radiation-Induced Damage in Collagenized Tissues

    Science.gov (United States)

    Griko, Y. V.; Yan, Xiaoli

    2016-01-01

    Exposure to high doses of ionizing radiation produces both acute and late effects on the collagenized tissues and have profound effects on wound healing. Because of the crucial practical importance for new radioprotective agents, our study has been focused on evaluation of the efficacy of non-toxic naturally occurring compounds to protect tissue integrity against high-dose gamma radiation. Here, we demonstrate that molecular integrity of collagen may serve as a sensitive biological marker for quantitative evaluation of molecular damage to collagenized tissue and efficacy of radioprotective agents. Increasing doses of gamma radiation (0-50kGy) result in progressive destruction of the native collagen fibrils, which provide a structural framework, strength, and proper milieu for the regenerating tissue. The strategy used in this study involved the thermodynamic specification of all structural changes in collagenized matrix of skin, aortic heart valve, and bone tissue induced by different doses and conditions of g-irradiation. This study describes a simple biophysical approach utilizing the Differential Scanning Calorimetry (DSC) to characterize the structural resistance of the aortic valve matrix exposed to different doses of g-irradiation. It allows us to identify the specific response of each constituent as well as to determine the influence of the different treatments on the characteristic parameters of protein structure. We found that pyruvate, a substance that naturally occurs in the body, provide significant protection (up to 80%) from biochemical and biomechanical damage to the collagenized tissue through the effective targeting of reactive oxygen species. The recently discovered role of pyruvate in the cell antioxidant defense to O2 oxidation, and its essential constituency in the daily human diet, indicate that the administration of pyruvate-based radioprotective formulations may provide safe and effective protection from deleterious effects of ionizing

  12. A comparison of quantitative methods for clinical imaging with hyperpolarized (13)C-pyruvate.

    Science.gov (United States)

    Daniels, Charlie J; McLean, Mary A; Schulte, Rolf F; Robb, Fraser J; Gill, Andrew B; McGlashan, Nicholas; Graves, Martin J; Schwaiger, Markus; Lomas, David J; Brindle, Kevin M; Gallagher, Ferdia A

    2016-04-01

    Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

  13. Domain interaction in rabbit muscle pyruvate kinase. II. Small angle neutron scattering and computer simulation.

    Science.gov (United States)

    Consler, T G; Uberbacher, E C; Bunick, G J; Liebman, M N; Lee, J C

    1988-02-25

    The effects of ligands on the structure of rabbit muscle pyruvate kinase were studied by small angle neutron scattering. The radius of gyration, RG, decreases by about 1 A in the presence of the substrate phosphoenolpyruvate, but increases by about the same magnitude in the presence of the allosteric inhibitor phenylalanine. With increasing pH or in the absence of Mg2+ and K+, the RG of pyruvate kinase increases. Hence, there is a 2-A difference in RG between two alternative conformations. Length distribution analysis indicates that, under all experimental conditions which increase the radius of gyration, there is a pronounced increase observed in the probability for interatomic distance between 80 and 110 A. These small angle neutron scattering results indicate a "contraction" and "expansion" of the enzyme when it transforms between its active and inactive forms. Using the alpha-carbon coordinates of crystalline cat muscle pyruvate kinase, a length distribution profile was calculated, and it matches the scattering profile of the inactive form. These observations are expected since the crystals were grown in the absence of divalent cations (Stuart, D. I., Levine, M., Muirhead, H., and Stammers, D. K. (1979) J. Mol. Biol. 134, 109-142). Hence, results from neutron scattering, x-ray crystallographic, and sedimentation studies (Oberfelder, R. W., Lee, L. L.-Y., and Lee, J.C. (1984) Biochemistry 23, 3813-3821) are totally consistent with each other. With the aid of computer modeling, the crystal structure has been manipulated in order to effect changes that are consistent with the conformational change described by the solution scattering data. The structural manipulation involves the rotation of the B domain relative to the A domain, leading to the closure of the cleft between these domains. These manipulations resulted in the generation of new sets of atomic (C-alpha) coordinates, which were utilized in calculations, the result of which compared favorably with the

  14. Effect of gamma irradiation on the activity of alanine and aspartate transaminases in subcellular fractions of the brain and heart in white rats

    Energy Technology Data Exchange (ETDEWEB)

    Plenin, A E

    1973-01-01

    In experiments on rats, the activity of alanine (I) and aspartate transaminases (II) was studied in homogenates and subcellular fractions of the brain and myocardium under normal conditions and for 30 days after ..gamma.. irradiation at 40 rads. The activity of II in brain homogenates increased 1 hour after irradiation but decreased by 20 percent on day 3; it decreased again on days 7 and 15. The activity of brain I increased after 1 hour and 3 days but then returned to normal. The activity of I in heart homogenates increased in all the periods after irradiation. The subcellular fractions exhibited phase changes in the activity of the enzymes. These changes were different in nature from those observed after X and ..gamma.. irradiation at the same dose.

  15. Creatine and creatine pyruvate reduce hypoxia-induced effects on phrenic nerve activity in the juvenile mouse respiratory system.

    Science.gov (United States)

    Scheer, Monika; Bischoff, Anna M; Kruzliak, Peter; Opatrilova, Radka; Bovell, Douglas; Büsselberg, Dietrich

    2016-08-01

    Adequate concentrations of ATP are required to preserve physiological cell functions and protect tissue from hypoxic damage. Decreased oxygen concentration results in ATP synthesis relying increasingly on the presence of phosphocreatine. The lack of ATP through hypoxic insult to neurons that generate or regulate respiratory function, would lead to the cessation of breathing (apnea). It is not clear whether creatine plays a role in maintaining respiratory phrenic nerve (PN) activity during hypoxic challenge. The aim of the study was to test the effects of exogenously applied creatine or creatine pyruvate in maintaining PN induced respiratory rhythm against the deleterious effects of severe hypoxic insult using Working Heart-Brainstem (WHB) preparations of juvenile Swiss type mice. WHB's were perfused with control perfusate or perfusate containing either creatine [100μM] or creatine pyruvate [100μM] prior to hypoxic challenge and PN activity recorded throughout. Results showed that severe hypoxic challenge resulted in an initial transient increase in PN activity, followed by a reduction in that activity leading to respiratory apnea. The results demonstrated that perfusing the WHB preparation with creatine or creatine pyruvate, significantly reduced the onset of apnea compared to control conditions, with creatine pyruvate being the more effective substance. Overall, creatine and creatine pyruvate each produced time-dependent degrees of protection against severe hypoxic-induced disturbances of PN activity. The underlying protective mechanisms are unknown and need further investigations. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Modeling of the pyruvate production with Escherichia coli: comparison of mechanistic and neural networks-based models.

    Science.gov (United States)

    Zelić, B; Bolf, N; Vasić-Racki, D

    2006-06-01

    Three different models: the unstructured mechanistic black-box model, the input-output neural network-based model and the externally recurrent neural network model were used to describe the pyruvate production process from glucose and acetate using the genetically modified Escherichia coli YYC202 ldhA::Kan strain. The experimental data were used from the recently described batch and fed-batch experiments [ Zelić B, Study of the process development for Escherichia coli-based pyruvate production. PhD Thesis, University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia, July 2003. (In English); Zelić et al. Bioproc Biosyst Eng 26:249-258 (2004); Zelić et al. Eng Life Sci 3:299-305 (2003); Zelić et al Biotechnol Bioeng 85:638-646 (2004)]. The neural networks were built out of the experimental data obtained in the fed-batch pyruvate production experiments with the constant glucose feed rate. The model validation was performed using the experimental results obtained from the batch and fed-batch pyruvate production experiments with the constant acetate feed rate. Dynamics of the substrate and product concentration changes was estimated using two neural network-based models for biomass and pyruvate. It was shown that neural networks could be used for the modeling of complex microbial fermentation processes, even in conditions in which mechanistic unstructured models cannot be applied.

  17. Neuron-astrocyte interactions, pyruvate carboxylation and the pentose phosphate pathway in the neonatal rat brain

    OpenAIRE

    Morken, Tora Sund; Brekke, Eva Mari Førland; Håberg, Asta; Widerøe, Marius; Brubakk, Ann-Mari; Sonnewald, Ursula

    2014-01-01

    Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-(13)C]glucose and [1,2-(13)C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity duri...

  18. Dietary modulation of erythrocyte insulin receptor interaction and the regulation of adipose tissue pyruvate dehydrogenase enzyme activity in growing rats; a mechanism of action of dietary fiber in metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Ogunwole, J.O.A.

    1984-01-01

    The metabolic effects of graded cellulose (a dietary fiber) intake were studied at minimal (10%) and maximal (20%) protein levels in male weanling Sprague Dawley rats. The hypothesis was tested that the hypoglycemic effect of high fiber diets is partly mediated through increased tissue sensitivity to insulin at the cell receptor level. Erythrocyte insulin receptor interaction (IRI) and percent insulin stimulation of adipose tissue pyruvate dehydrogenase (PDH) activity (PDS) were used as indices of tissue sensitivity to insulin. IRI was determined by a standardized radioceptor assay PDS by the rate of oxidation of 1-/sup 14/C-pyruvate to /sup 14/CO/sub 2/ in epidymal fat pads and serum insulin levels by radioimmunoassay. In both protein groups, the addition of fiber in the diet resulted in a significant (P < 0.05) increase in food intake (FI) for calorie compensation. Fiber and protein intake had a significant (P < 0.01) effect on IRI and both basal (PDB) and PDS activities of PDH. At all fiber levels, specific percent /sup 125/I-insulin binding (SIB) was higher in the 20% protein groups while in the fiber-free group, a higher SIB was observed in the 10% protein group.

  19. Effect of gamma radiation on the concentration of pyruvate and lactate in erythrocytes of healthy men after submaximal physical exercise

    International Nuclear Information System (INIS)

    Zagorski, T.; Dudek, I.; Berkan, L.; Chmielewski, H.; Kedziora, J.

    1993-01-01

    The aim of this work was to study the effect of gamma radiation and submaximal physical exercise on the concentration of final products of anaerobic glycolytic pathway in erythrocytes of healthy men. Twenty one men aged 20-22 were examined. They underwent physical exercise at doses of 2 w/kg body weight for 15 min. Erythrocytes were taken in the rest and after physical exercise and were exposed to gamma radiation (500 Gy doses) from 60 Co source. The concentration of pyruvate was estimated by Fermognost tests and the concentration of lactate by Boehringer Mannheim tests. The submaximal physical exercise was found to cause a significantly increased concentration of pyruvate and lactate in the non-radiated and irradiated erythrocytes. Gamma radiation at 500 Gy dose was found to increase concentration of pyruvate in erythrocytes (in the rest and after physical exercise) with simultaneous decrease of lactate concentration. (author). 17 refs, 1 tab

  20. Recovery of Pyruvic Acid using Tri-n-butylamine Dissolved in Non-Toxic Diluent (Rice Bran Oil)

    Science.gov (United States)

    Pal, Dharm; Keshav, Amit

    2016-04-01

    An attempt has been made to investigate the effectiveness of the vegetable oil based biocompatible solvent for the separation of pyruvic acid from fermentation broth, by using rice bran oil as natural, non-toxic diluent. Reactive extraction of pyruvic acid (0.1-0.5 k mol/m3) from aqueous solutions has been studied using tri-n-butylamine (TBA; 10-70 %) as an extractant dissolved in non toxic rice bran oil at T = 30 ± 1 °C. Results were presented in terms of distribution coefficient (Kd), extraction efficiency (E %), loading ratio (Z), and complexation constant (\\varphi_{α β }). Extraction equilibrium was interpreted using mass action modeling approach. Based on the extent of loading (Z < 0.5) only (1:1), pyruvic acid: TBA complex was proposed. Equilibrium complexation constant was evaluated to 1.22 m3/k mol. Results obtained are useful in understanding the extraction mechanism.

  1. Pyruvate oxidase influences the sugar utilization pattern and capsule production in Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Sandra M Carvalho

    Full Text Available Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

  2. Characterization of cDNAs encoding human pyruvate dehydrogenase α subunit

    International Nuclear Information System (INIS)

    Ho, Lap; Wexler, I.D.; Liu, Techung; Thekkumkara, T.J.; Patel, M.S.

    1989-01-01

    A cDNA clone (1,423 base pairs) comprising the entire coding region of the precursor form of the α subunit of pyruvate dehydrogenase (E 1 α) has been isolated from a human liver cDNA library in phage λgt11. The first 29 amino acids deduced from the open reading frame correspond to a typical mitochondrial targeting leader sequence. The remaining 361 amino acids, starting at the N terminus with phenylalanine, represent the mature mitochondrial E 1 α peptide. The cDNA has 43 base pairs in the 5' untranslated region and 210 base pairs in the 3' untranslated region, including a polyadenylylation signal and a short poly(A) tract. The nucleotide sequence of human liver E 1 α cDNA was confirmed by the nucleotide sequences of three overlapping fragments generated from human liver and fibroblast RNA by reverse transcription and DNA amplification by the polymerase chain reaction. This consensus nucleotide sequence of human liver E 1 α cDNA resolves existing discrepancies among three previously reported human E 1 α cDNAs and provides the unambiguous reference sequence needed for the characterization of genetic mutations in pyruvate dehydrogenase-deficient patients

  3. Pyruvate decarboxylase provides growing pollen tubes with a competitive advantage in petunia.

    Science.gov (United States)

    Gass, Nathalie; Glagotskaia, Tatiana; Mellema, Stefan; Stuurman, Jeroen; Barone, Mario; Mandel, Therese; Roessner-Tunali, Ute; Kuhlemeier, Cris

    2005-08-01

    Rapid pollen tube growth places unique demands on energy production and biosynthetic capacity. The aim of this work is to understand how primary metabolism meets the demands of such rapid growth. Aerobically grown pollen produce ethanol in large quantities. The ethanolic fermentation pathway consists of two committed enzymes: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Because adh mutations do not affect male gametophyte function, the obvious question is why pollen synthesize an abundant enzyme if they could do just as well without. Using transposon tagging in Petunia hybrida, we isolated a null mutant in pollen-specific Pdc2. Growth of the mutant pollen tubes through the style is reduced, and the mutant allele shows reduced transmission through the male, when in competition with wild-type pollen. We propose that not ADH but rather PDC is the critical enzyme in a novel, pollen-specific pathway. This pathway serves to bypass pyruvate dehydrogenase enzymes and thereby maintain biosynthetic capacity and energy production under the unique conditions prevailing during pollen-pistil interaction.

  4. Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR Variants in a Thai Population.

    Directory of Open Access Journals (Sweden)

    Rebekah van Bruggen

    Full Text Available Pyruvate kinase (PKLR is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41 is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.

  5. Heavy-atom isotope effects on binding of reactants to lactate dehydrogenase and pyruvate kinase

    International Nuclear Information System (INIS)

    Gawlita, E.

    1993-04-01

    18 O and 13 C kinetic isotope effects have been measured on the reaction of pyruvate kinase with phospho-enol-pyruvate and ADP using a remote label technique. The magnitude of both investigated isotope effects showed a dependence on the concentration of ADP. However, while the carbon effect was simply 'washed out' to unity at high ATP concentration, the oxygen effect becomes inverse and reached 0.9928 at the highest used concentration of ADP. Such a result testifies that the assumption of the negligible effect of isotopic substitution on enzyme-substrate associations remains correct only for carbon effects. An equilibrium 18 O isotope effect on association of oxalate with lactate dehydrogenase in the presence of NADHP has been evaluated by both experimental and theoretical means. Experimental methods, which involved equilibrium dialysis and gas chromatographic/mass spectrometric measurement of isotopic ration, yielded an inverse value of 0.9840. Semiempirical methods involved vibrational analysis of oxalate in two different environments. The comparison of calculated values with the experimentally determined isotope effect indicated that the AM 1 Hamiltonian proved superior to its PM 3 counterpart in this modelling. 160 refs, 8 figs, 18 tabs

  6. n-Octyl gallate as inhibitor of pyruvate carboxylation and lactate gluconeogenesis.

    Science.gov (United States)

    Eler, Gabrielle Jacklin; Santos, Israel Souza; de Moraes, Amarilis Giaretta; Comar, Jurandir Fernando; Peralta, Rosane Marina; Bracht, Adelar

    2015-04-01

    The alkyl gallates are found in several natural and industrial products. In the latter products, these compounds are added mainly for preventing oxidation. In the present work, the potencies of methyl gallate, n-propyl gallate, n-pentyl gallate, and n-octyl gallate as inhibitors of pyruvate carboxylation and lactate gluconeogenesis were evaluated. Experiments were done with isolated mitochondria and the isolated perfused rat liver. The potency of the gallic acid esters as inhibitors of pyruvate carboxylation in isolated mitochondria obeyed the following decreasing sequence: n-octyl gallate > n-pentyl gallate > n-propyl gallate > methyl gallate. A similar sequence of decreasing potency for lactate gluconeogenesis inhibition in the perfused liver was found in terms of the portal venous concentration. Both actions correlate with the lipophilicity of the compounds. The effects are harmful at high concentrations. At appropriate concentrations, however, octyl gallate should act therapeutically because its inhibitory action on gluconeogenesis will contribute further to its proposed antihyperglycemic effects. © 2014 Wiley Periodicals, Inc.

  7. Gluconeogenesis in Leishmania mexicana: contribution of glycerol kinase, phosphoenolpyruvate carboxykinase, and pyruvate phosphate dikinase.

    Science.gov (United States)

    Rodriguez-Contreras, Dayana; Hamilton, Nicklas

    2014-11-21

    Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells

    Science.gov (United States)

    Cheng, Tzuling; Sudderth, Jessica; Yang, Chendong; Mullen, Andrew R.; Jin, Eunsook S.; Matés, José M.; DeBerardinis, Ralph J.

    2011-01-01

    Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how “glutamine-addicted” cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis. PMID:21555572

  9. Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide.

    Science.gov (United States)

    Verhoef, René; de Waard, Pieter; Schols, Henk A; Siika-aho, Matti; Voragen, Alphons G J

    2003-09-01

    The slime-forming bacterium Methylobacterium sp. was isolated from a Finnish paper machine and its exopolysaccharide (EPS) was produced on laboratory scale. Sugar compositional analysis revealed a 100% galactan (EPS). However, FT-IR showed a very strong peak at 1611 cm(-1) showing the presence of pyruvate. Analysis of the pyruvate content revealed that, based on the sugar composition, the EPS consists of a trisaccharide repeating unit consisting of D-galactopyranose and [4,6-O-(1-carboxyethylidene)]-D-galactopyranose with a molar ratio of 1:2, respectively. Both linkage analysis and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy revealed the following repeating unit: -->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1-->. By enrichment cultures from various ground and compost heap samples a polysaccharide-degrading culture was obtained that produced an endo acting enzyme able to degrade the EPS described. The enzyme hydrolysed the EPS to a large extent, releasing oligomers that mainly consisted out of two repeating units.

  10. Pyruvate oxidase influences the sugar utilization pattern and capsule production in Streptococcus pneumoniae.

    Science.gov (United States)

    Carvalho, Sandra M; Farshchi Andisi, Vahid; Gradstedt, Henrik; Neef, Jolanda; Kuipers, Oscar P; Neves, Ana R; Bijlsma, Jetta J E

    2013-01-01

    Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps) transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

  11. Ketogenic diet in pyruvate dehydrogenase complex deficiency: short- and long-term outcomes.

    Science.gov (United States)

    Sofou, Kalliopi; Dahlin, Maria; Hallböök, Tove; Lindefeldt, Marie; Viggedal, Gerd; Darin, Niklas

    2017-03-01

    Our aime was to study the short- and long-term effects of ketogenic diet on the disease course and disease-related outcomes in patients with pyruvate dehydrogenase complex deficiency, the metabolic factors implicated in treatment outcomes, and potential safety and compliance issues. Pediatric patients diagnosed with pyruvate dehydrogenase complex deficiency in Sweden and treated with ketogenic diet were evaluated. Study assessments at specific time points included developmental and neurocognitive testing, patient log books, and investigator and parental questionnaires. A systematic literature review was also performed. Nineteen patients were assessed, the majority having prenatal disease onset. Patients were treated with ketogenic diet for a median of 2.9 years. All patients alive at the time of data registration at a median age of 6 years. The treatment had a positive effect mainly in the areas of epilepsy, ataxia, sleep disturbance, speech/language development, social functioning, and frequency of hospitalizations. It was also safe-except in one patient who discontinued because of acute pancreatitis. The median plasma concentration of ketone bodies (3-hydroxybutyric acid) was 3.3 mmol/l. Poor dietary compliance was associated with relapsing ataxia and stagnation of motor and neurocognitive development. Results of neurocognitive testing are reported for 12 of 19 patients. Ketogenic diet was an effective and safe treatment for the majority of patients. Treatment effect was mainly determined by disease phenotype and attainment and maintenance of ketosis.

  12. Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

    Directory of Open Access Journals (Sweden)

    Alba Timón-Gómez

    Full Text Available Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

  13. Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

    Science.gov (United States)

    Timón-Gómez, Alba; Proft, Markus; Pascual-Ahuir, Amparo

    2013-01-01

    Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

  14. 3-Bromopyruvate antagonizes effects of lactate and pyruvate, synergizes with citrate and exerts novel anti-glioma effects.

    Science.gov (United States)

    El Sayed, S M; El-Magd, R M Abou; Shishido, Y; Chung, S P; Diem, T H; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

    2012-02-01

    Oxidative stress-energy depletion therapy using oxidative stress induced by D-amino acid oxidase (DAO) and energy depletion induced by 3-bromopyruvate (3BP) was reported recently (El Sayed et al., Cancer Gene Ther., 19, 1-18, 2012). Even in the presence of oxygen, cancer cells oxidize glucose preferentially to produce lactate (Warburg effect) which seems vital for cancer microenvironment and progression. 3BP is a closely related structure to lactate and pyruvate and may antagonize their effects as a novel mechanism of its action. Pyruvate exerted a potent H(2)O(2) scavenging effect to exogenous H(2)O(2), while lactate had no scavenging effect. 3BP induced H(2)O(2) production. Pyruvate protected against H(2)O(2)-induced C6 glioma cell death, 3BP-induced C6 glioma cell death but not against DAO/D-serine-induced cell death, while lactate had no protecting effect. Lactate and pyruvate protected against 3BP-induced C6 glioma cell death and energy depletion which were overcome with higher doses of 3BP. Lactate and pyruvate enhanced migratory power of C6 glioma which was blocked by 3BP. Pyruvate and lactate did not protect against C6 glioma cell death induced by other glycolytic inhibitors e.g. citrate (inhibitor of phosphofructokinase) and sodium fluoride (inhibitor of enolase). Serial doses of 3BP were synergistic with citrate in decreasing viability of C6 glioma cells and spheroids. Glycolysis subjected to double inhibition using 3BP with citrate depleted ATP, clonogenic power and migratory power of C6 glioma cells. 3BP induced a caspase-dependent cell death in C6 glioma. 3BP was powerful in decreasing viability of human glioblastoma multiforme cells (U373MG) and C6 glioma in a dose- and time-dependent manner.

  15. Differences between magnesium-activated and manganese-activated pyruvate kinase from the muscle of Concholepas concholepas.

    Science.gov (United States)

    González, R; Carvajal, N; Morán, A

    1984-01-01

    In contrast to the Mg2+-activated enzyme, in the presence of Mn2+ pyruvate kinase exhibits hyperbolic kinetics with respect to the substrate phosphoenolpyruvate and is insensitive to fructose 1,6-biphosphate, phenylalanine and alanine. However, with both metal activated species inhibition by excess ADP is observed. In contrast with Mg2+, which affords significant protection against inactivation caused by 5,5'-dithiobis (2-nitrobenzoic acid), the rate of inactivation by this reagent is increased in the presence of Mn2+. Differences in conformational changes induced by combination of pyruvate kinase with Mg2+ or Mn2+ were indicated by u.v. difference spectra.

  16. Preparation of C-II labeled pyruvic acid for use in assessment of hypoxia in tumors. Project 4

    International Nuclear Information System (INIS)

    Anon.

    1986-01-01

    Of the three methods of synthesis of C-II-labeled pyruvic acid that we had proposed to investigate in order to determine the best and most appropriate synthesis of C-II-labeled pyruvate, the cold chemistry of Method A, via an isocyanide intermediate, has been verified. Similarly, the cold chemistry of Method B, via the 1,3-dithiane derivative, has been verified up to the deprotection and last step of the synthesis. The difficulties which have been encountered with the biochemistry of Method C from ribulose 1,5-diphosphate, have yet to be resolved. 12 refs., 6 figs

  17. Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1‐13C]pyruvate MRS studies by infusion of glucose, insulin and potassium

    DEFF Research Database (Denmark)

    Lauritzen, Mette Hauge; Laustsen, Christoffer; Butt, Sadia Asghar

    2013-01-01

    A change in myocardial metabolism is a known effect of several diseases. MRS with hyperpolarized 13C‐labelled pyruvate is a technique capable of detecting changes in myocardial pyruvate metabolism, and has proven to be useful for the evaluation of myocardial ischaemia in vivo. However, during fas...

  18. Ethyl pyruvate inhibits proliferation and induces apoptosis of hepatocellular carcinoma via regulation of the HMGB1–RAGE and AKT pathways

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Ping; Dai, Weiqi; Wang, Fan; Lu, Jie; Shen, Miao; Chen, Kan; Li, Jingjing; Zhang, Yan; Wang, Chengfen; Yang, Jing; Zhu, Rong; Zhang, Huawei; Zheng, Yuanyuan; Guo, Chuan-Yong, E-mail: guochuanyong@hotmail.com; Xu, Ling, E-mail: xuling606@sina.com

    2014-01-24

    Highlights: • Ethyl pyruvate inhibits liver cancer. • Promotes apoptosis. • Decreased the expression of HMGB1, p-Akt. - Abstract: Ethyl pyruvate (EP) was recently identified as a stable lipophilic derivative of pyruvic acid with significant antineoplastic activities. The high mobility group box-B1 (HMGB1)–receptor for advanced glycation end-products (RAGE) and the protein kinase B (Akt) pathways play a crucial role in tumorigenesis and development of many malignant tumors. We tried to observe the effects of ethyl pyruvate on liver cancer growth and explored its effects in hepatocellular carcinoma model. In this study, three hepatocellular carcinoma cell lines were treated with ethyl pyruvate. An MTT colorimetric assay was used to assess the effects of EP on cell proliferation. Flow cytometry and TUNEL assays were used to analyze apoptosis. Real-time PCR, Western blotting and immunofluorescence demonstrated ethyl pyruvate reduced the HMGB1–RAGE and AKT pathways. The results of hepatoma orthotopic tumor model verified the antitumor effects of ethyl pyruvate in vivo. EP could induce apoptosis and slow the growth of liver cancer. Moreover, EP decreased the expression of HMGB1, RAGE, p-AKT and matrix metallopeptidase-9 (MMP9) and increased the Bax/Bcl-2 ratio. In conclusion, this study demonstrates that ethyl pyruvate induces apoptosis and cell-cycle arrest in G phase in hepatocellular carcinoma cells, plays a critical role in the treatment of cancer.

  19. Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions

    NARCIS (Netherlands)

    Vaartjes, W.J.; Breejen, J.N. den; Geelen, M.J.H.; Bergh, S.G. van den

    1980-01-01

    1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite

  20. A novel mechanism for the pyruvate protection against zinc-induced cytotoxicity: mediation by the chelating effect of citrate and isocitrate.

    Science.gov (United States)

    Sul, Jee-Won; Kim, Tae-Youn; Yoo, Hyun Ju; Kim, Jean; Suh, Young-Ah; Hwang, Jung Jin; Koh, Jae-Young

    2016-08-01

    Intracellular accumulation of free zinc contributes to neuronal death in brain injuries such as ischemia and epilepsy. Pyruvate, a glucose metabolite, has been shown to block zinc neurotoxicity. However, it is largely unknown how pyruvate shows such a selective and remarkable protective effect. In this study, we sought to find a plausible mechanism of pyruvate protection against zinc toxicity. Pyruvate almost completely blocked cortical neuronal death induced by zinc, yet showed no protective effects against death induced by calcium (ionomycin, NMDA) or ferrous iron. Of the TCA cycle intermediates, citrate, isocitrate, and to a lesser extent oxaloacetate, protected against zinc toxicity. We then noted with LC-MS/MS assay that exposure to pyruvate, and to a lesser degree oxaloacetate, increased levels of citrate and isocitrate, which are known zinc chelators. While pyruvate added only during zinc exposure did not reduce zinc toxicity, citrate and isocitrate added only during zinc exposure, as did extracellular zinc chelator CaEDTA, completely blocked it. Furthermore, addition of pyruvate after zinc exposure substantially reduced intracellular zinc levels. Our results suggest that the remarkable protective effect of pyruvate against zinc cytotoxicity may be mediated indirectly by the accumulation of intracellular citrate and isocitrate, which act as intracellular zinc chelators.

  1. Protein electrophoresis - serum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003540.htm Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  2. Serum iron test

    Science.gov (United States)

    Fe+2; Ferric ion; Fe++; Ferrous ion; Iron - serum; Anemia - serum iron; Hemochromatosis - serum iron ... A blood sample is needed. Iron levels are highest in the morning. Your health care provider will likely have you do this test in the morning.

  3. Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high saturated fat diet.

    Science.gov (United States)

    Hwang, Byounghoon; Wu, Pengfei; Harris, Robert A

    2012-05-01

    Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it may have detrimental effects by inhibiting fatty acid oxidation. Peroxisome proliferator-activated receptor α (PPARα) agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment using a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild-type and PDK4 knockout mice fed a high-fat diet. As expected, treatment of wild-type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, reduced blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid, and a reduction in the capacity for fatty acid synthesis as a result of PDK4 deficiency. Journal compilation © 2012 FEBS. No claim to original US government works.

  4. Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

    Directory of Open Access Journals (Sweden)

    Yoichi Takakusagi

    Full Text Available BACKGROUND: TH-302 is a hypoxia-activated prodrug (HAP of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. METHODOLOGY/RESULTS: The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2, with minimal effect under aerobic conditions (21% O2. Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3, significantly delayed tumor growth. CONCLUSIONS/SIGNIFICANCE: Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the

  5. Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

    Science.gov (United States)

    Takakusagi, Yoichi; Matsumoto, Shingo; Saito, Keita; Matsuo, Masayuki; Kishimoto, Shun; Wojtkowiak, Jonathan W; DeGraff, William; Kesarwala, Aparna H; Choudhuri, Rajani; Devasahayam, Nallathamby; Subramanian, Sankaran; Munasinghe, Jeeva P; Gillies, Robert J; Mitchell, James B; Hart, Charles P; Krishna, Murali C

    2014-01-01

    TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3). Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3)), significantly delayed tumor growth. Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

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

    Science.gov (United States)

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

    2014-11-07

    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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Efeito do Exercício com 1 e 3 Minutos de Intervalo de Descanso Entre as Séries na Atividade Sérica das Transaminases

    Directory of Open Access Journals (Sweden)

    Ramon Martins Alves

    2011-12-01

    Full Text Available O treinamento de força (TF intenso ocasiona danos na estrutura muscular, que podem ser detectados por meio de marcadores indiretos. OBJETIVO. Verificar se há alterações na atividade da creatina quinase (CK, da aspartato transaminase (AST e alanina transaminase (ALT com o TF em diferentes intervalos entre séries e exercícios. MÉTODOS. Treze voluntários (± 21,8 anos; ±173,6 cm; ±68,3 kg realizaram o teste de uma repetição máxima (1RM nos exercícios: supino reto, puxada (pulley, desenvolvimento sentado, tríceps (pulley e rosca bíceps. Após sete dias, todos foram submetidos a uma coleta de sangue (PRE e realizaram 4 séries máximas até a falha concêntrica com intensidade de 85% de 1RM e intervalos de 1 ou 3 minutos entre as séries e exercícios. Todos retornaram ao mesmo local 24, 48 e 72 horas após a sessão para mais uma coleta de sangue. Repetindo os procedimentos após uma semana, porém os voluntários que realizaram o teste com intervalo de 1 minuto na semana seguinte manteriam intervalos de 3 minutos e vice-versa. Realizou-se ANOVA mantendo como significância p<0,05. RESULTADOS. Houve redução significativa do número de repetições a cada série de todos os exercícios com 1 e 3 minutos de intervalo. Observou-se um aumento significativo da atividade sérica de CK e de AST 24, 48 e 72 horas utilizando ambos os intervalos. ALT não se alterou. CONCLUSÃO. O TF causou aumento na atividade de CK e AST, mas sem apresentar diferenças com os intervalos utilizados. Porém, pequenos intervalos reduzem o volume total de repetições.

  8. Simultaneous hyperpolarized 13C-pyruvate MRI and 18F-FDG-PET in cancer (hyperPET)

    DEFF Research Database (Denmark)

    Gutte, Henrik; Hansen, Adam E.; Henriksen, Sarah T.

    2015-01-01

    named this concept hyper PET. Intravenous injection of the hyperpolarized 13C-pyruvate results in an increase of 13C-lactate, 13C-alanine and 13CCO2 (13C-HCO3) resonance peaks relative to the tissue, disease and the metabolic state probed. Accordingly, with dynamic nuclear polarization (DNP) and use......In this paper we demonstrate, for the first time, the feasibility of a new imaging concept - combined hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (MRSI) and 18F-FDG-PET imaging. This procedure was performed in a clinical PET/MRI scanner with a canine cancer patient. We have...... of 13C-pyruvate it is now possible to directly study the Warburg Effect through the rate of conversion of 13C-pyruvate to 13C-lactate. In this study, we combined it with 18F-FDG-PET that studies uptake of glucose in the cells. A canine cancer patient with a histology verified local recurrence...

  9. “Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

    Science.gov (United States)

    The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1alpha subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated...

  10. An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Oud, B.; Flores, C.L.; Gancedo, C.; Zhang, X.; Trueheart, J.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.

    2012-01-01

    Background Pyruvate-decarboxylase negative (Pdc-) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards

  11. Leigh syndrome associated with a deficiency of the pyruvate dehydrogenase complex: results of treatment with a ketogenic diet

    NARCIS (Netherlands)

    Wijburg, F. A.; Barth, P. G.; Bindoff, L. A.; Birch-Machin, M. A.; van der Blij, J. F.; Ruitenbeek, W.; TURNBULL, D. M.; Schutgens, R. B.

    1992-01-01

    A one-year-old boy suffering from intermittent lactic acidosis, muscular hypotonia, horizontal gaze paralysis and spasticity in both legs had low activity of the pyruvate dehydrogenase complex associated with low amounts of immunoreactive E 1 alpha and E 1 beta. Leigh syndrome was diagnosed on the

  12. Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites

    Science.gov (United States)

    Cooper, George; Reed, Chris; Nguyen, Dang; Carter, Malika; Wang, Yi

    2011-01-01

    Carbonaceous meteorites deliver a variety of organic compounds to Earth that may have played a role in the origin and/or evolution of biochemical pathways. Some apparently ancient and critical metabolic processes require several compounds, some of which are relatively labile such as keto acids. Therefore, a prebiotic setting for any such individual process would have required either a continuous distant source for the entire suite of intact precursor molecules and/or an energetic and compact local synthesis, particularly of the more fragile members. To date, compounds such as pyruvic acid, oxaloacetic acid, citric acid, isocitric acid, and α-ketoglutaric acid (all members of the citric acid cycle) have not been identified in extraterrestrial sources or, as a group, as part of a “one pot” suite of compounds synthesized under plausibly prebiotic conditions. We have identified these compounds and others in carbonaceous meteorites and/or as low temperature (laboratory) reaction products of pyruvic acid. In meteorites, we observe many as part of three newly reported classes of compounds: keto acids (pyruvic acid and homologs), hydroxy tricarboxylic acids (citric acid and homologs), and tricarboxylic acids. Laboratory syntheses using 13C-labeled reactants demonstrate that one compound alone, pyruvic acid, can produce several (nonenzymatic) members of the citric acid cycle including oxaloacetic acid. The isotopic composition of some of the meteoritic keto acids points to interstellar or presolar origins, indicating that such compounds might also exist in other planetary systems. PMID:21825143

  13. Modeling non-linear kinetics of hyperpolarized [1-(13)C] pyruvate in the crystalloid-perfused rat heart

    NARCIS (Netherlands)

    Mariotti, E.; Orton, M. R.; Eerbeek, O.; Ashruf, J. F.; Zuurbier, C. J.; Southworth, R.; Eykyn, T. R.

    2016-01-01

    Hyperpolarized (13)C MR measurements have the potential to display non-linear kinetics. We have developed an approach to describe possible non-first-order kinetics of hyperpolarized [1-(13)C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass

  14. The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E.

    NARCIS (Netherlands)

    Boxma, B.; Voncken, F.L.M.; Jannink, S.A.; Alen, T.A. van; Akhmanova, A.S.; Weelden, S.W. van; Hellemond, J.J. van; Ricard, G.N.S.; Huynen, M.A.; Tielens, A.G.; Hackstein, J.H.P.

    2004-01-01

    Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of

  15. The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

    Science.gov (United States)

    Stamm, Irmela; Lottspeich, Friedrich; Plaga, Wulf

    2005-06-01

    Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

  16. The effects of creatine pyruvate and creatine citrate on performance during high intensity exercise

    Directory of Open Access Journals (Sweden)

    Purpura Martin

    2008-02-01

    Full Text Available Abstract Background A double-blind, placebo-controlled, randomized study was performed to evaluate the effect of oral creatine pyruvate (Cr-Pyr and creatine citrate (Cr-Cit supplementation on exercise performance in healthy young athletes. Methods Performance during intermittent handgrip exercise of maximal intensity was evaluated before (pretest and after (posttest 28 days of Cr-Pyr (5 g/d, n = 16, Cr-Cit (5 g/d, n = 16 or placebo (pla, 5 g/d, n = 17 intake. Subjects performed ten 15-sec exercise intervals, each followed by 45 sec rest periods. Results Cr-Pyr (p Conclusion It is concluded that four weeks of Cr-Pyr and Cr-Cit intake significantly improves performance during intermittent handgrip exercise of maximal intensity and that Cr-Pyr might benefit endurance, due to enhanced activity of the aerobic metabolism.

  17. Effects of IL-6 on pyruvate dehydrogenase regulation in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Biensø, Rasmus Sjørup; Knudsen, Jakob Grunnet; Brandt, Nina

    2014-01-01

    Skeletal muscle regulates substrate choice according to demand and availability and pyruvate dehydrogenase (PDH) is central in this regulation. Circulating interleukin (IL)-6 increases during exercise and IL-6 has been suggested to increase whole body fat oxidation. Furthermore, IL-6 has been...... reported to increase AMP-activated protein kinase (AMPK) phosphorylation and AMPK suggested to regulate PDHa activity. Together, this suggests that IL-6 may be involved in regulating PDH. The aim of this study was to investigate the effect of a single injection of IL-6 on PDH regulation in skeletal muscle...... in fed and fasted mice. Fed and 16-18 h fasted mice were injected with either 3 ng · g(-1) recombinant mouse IL-6 or PBS as control. Fasting markedly reduced plasma glucose, muscle glycogen, muscle PDHa activity, as well as increased PDK4 mRNA and protein content in skeletal muscle. IL-6 injection did...

  18. Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

    DEFF Research Database (Denmark)

    Ostergaard, Elsebet; Duno, Morten; Møller, Lisbeth Birk

    2013-01-01

    We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first...... possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site...... mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense...

  19. Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

    Science.gov (United States)

    Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki; Duschene, Kaitlin S; Crain, Adam V; Byer, Amanda S; Shepard, Eric M; Rasmussen, Ashley; Yang, Jian; Broderick, William E; Vey, Jessica L; Drennan, Catherine L; Hoffman, Brian M; Broderick, Joan B

    2017-08-30

    Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.

  20. Mechanistic photodecarboxylation of pyruvic acid: Excited-state proton transfer and three-state intersection

    Science.gov (United States)

    Chang, Xue-Ping; Fang, Qiu; Cui, Ganglong

    2014-10-01

    Photodissociation dynamics of pyruvic acid experimentally differs from that of commonly known ketones. We have employed the complete active space self-consistent field and its multi-state second-order perturbation methods to study its photodissociation mechanism in the S0, T1, and S1 states. We have uncovered four nonadiabatic photodecarboxylation paths. (i) The S1 system relaxes via an excited-state intramolecular proton transfer (ESIPT) to a hydrogen-transferred tautomer, near which an S1/S0 conical intersection funnels the S1 to S0 state. Then, some trajectories continue completing the decarboxylation reaction in the S0 state; the remaining trajectories via a reverse hydrogen transfer return to the S0 minimum, from which a thermal decarboxylation reaction occurs. (ii) Due to a small S1 -T1 energy gap and a large S1/T1 spin-orbit coupling, an efficient S1 → T1 intersystem crossing process happens again near this S1/S0 conical intersection. When decaying to T1 state, a direct photodecarboxylation proceeds. (iii) Prior to ESIPT, the S1 system first decays to the T1 state via an S1 → T1 intersystem crossing; then, the T1 system evolves to a hydrogen-transferred tautomer. Therefrom, an adiabatic T1 decarboxylation takes place due to a small barrier of 7.7 kcal/mol. (iv) Besides the aforementioned T1 ESIPT process, there also exists a comparable Norrish type I reaction in the T1 state, which forms the ground-state products of CH3CO and COOH. Finally, we have found that ESIPT plays an important role. It closes the S1-T1 and S1-S0 energy gaps, effecting an S1/T1/S0 three-state intersection region, and mediating nonadiabatic photodecarboxylation reactions of pyruvic acid.

  1. In vitro bioconversion of chitin to pyruvate with thermophilic enzymes.

    Science.gov (United States)

    Honda, Kohsuke; Kimura, Keisuke; Ninh, Pham Huynh; Taniguchi, Hironori; Okano, Kenji; Ohtake, Hisao

    2017-09-01

    Chitin is the second most abundant organic compound on the planet and thus has been regarded as an alternative resource to petroleum feedstocks. One of the key challenges in the biological conversion of biomass-derived polysaccharides, such as cellulose and chitin, is to close the gap between optimum temperatures for enzymatic saccharification and microbial fermentation and to implement them in a single bioreactor. To address this issue, in the present study, we aimed to perform an in vitro, one-pot bioconversion of chitin to pyruvate, which is a precursor of a wide range of useful metabolites. Twelve thermophilic enzymes, including that for NAD + regeneration, were heterologously produced in Escherichia coli and semi-purified by heat treatment of the crude extract of recombinant cells. When the experimentally decided concentrations of enzymes were incubated with 0.5 mg mL -1 colloidal chitin (equivalent to 2.5 mM N-acetylglucosamine unit) and an adequate set of cofactors at 70°C, 0.62 mM pyruvate was produced in 5 h. Despite the use of a cofactor-balanced pathway, determination of the pool sizes of cofactors showed a rapid decrease in ATP concentration, most probably due to the thermally stable ATP-degrading enzyme(s) derived from the host cell. Integration of an additional enzyme set of thermophilic adenylate kinase and polyphosphate kinase led to the deceleration of ATP degradation, and the final product titer was improved to 2.1 mM. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Glutamate decarboxylase and. gamma. -aminobutyric acid transaminase activity in brain structures during action of high concentrated sulfide gas on a background of hypo- and hypercalcemia

    Energy Technology Data Exchange (ETDEWEB)

    Kadyrov, G.K.; Aliyev, A.M.

    Activity of the following enzymes was studied on the background of hypo- and hypercalcemia and exposure to high concentration of sulfide gas: glutamate decarboxylase (GDC) and {gamma}-aminobutyric acid transaminase (GABA-T). These enzymes regulate metabolism of GABA. The results showed that a 3.5 hr exposure to sulfide gas at a concentration of 0.3 mg/1 led to significantly increased activity of GDC in cerebral hemispheres, cerebellum and in brain stem. Activity of GABA-T dropped correspondingly. On the background of hypercalcemia induced by im. injection of 10% calcium gluconate (0.6 m1/200 g body weight of experimental rats) the negative effect caused by the exposure to sulfide gas was diminished. Under conditions of hypocalcemia (im. injection of 10 mg/200 g body weight of sodium oxalate), exposure to sulfide gas led to a significantly decreased activity of GDC and GABA-T in the hemispheres and in the brain stem, but in the cerebellum the activity of GDC increased sharply while that of GABA-T decreased correspondingly. 8 refs.

  3. γ-Aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell wall defects during salt stress in Arabidopsis roots.

    Science.gov (United States)

    Renault, Hugues; El Amrani, Abdelhak; Berger, Adeline; Mouille, Grégory; Soubigou-Taconnat, Ludivine; Bouchereau, Alain; Deleu, Carole

    2013-05-01

    Environmental constraints challenge cell homeostasis and thus require a tight regulation of metabolic activity. We have previously reported that the γ-aminobutyric acid (GABA) metabolism is crucial for Arabidopsis salt tolerance as revealed by the NaCl hypersensitivity of the GABA transaminase (GABA-T, At3g22200) gaba-t/pop2-1 mutant. In this study, we demonstrate that GABA-T deficiency during salt stress causes root and hypocotyl developmental defects and alterations of cell wall composition. A comparative genome-wide transcriptional analysis revealed that expression levels of genes involved in carbon metabolism, particularly sucrose and starch catabolism, were found to increase upon the loss of GABA-T function under salt stress conditions. Consistent with the altered mutant cell wall composition, a number of cell wall-related genes were also found differentially expressed. A targeted quantitative analysis of primary metabolites revealed that glutamate (GABA precursor) accumulated while succinate (the final product of GABA metabolism) significantly decreased in mutant roots after 1 d of NaCl treatment. Furthermore, sugar concentration was twofold reduced in gaba-t/pop2-1 mutant roots compared with wild type. Together, our results provide strong evidence that GABA metabolism is a major route for succinate production in roots and identify GABA as a major player of central carbon adjustment during salt stress. © 2012 Blackwell Publishing Ltd.

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

    Science.gov (United States)

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

    2006-08-01

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

  5. Serum and Plasma Metabolomic Biomarkers for Lung Cancer.

    Science.gov (United States)

    Kumar, Nishith; Shahjaman, Md; Mollah, Md Nurul Haque; Islam, S M Shahinul; Hoque, Md Aminul

    2017-01-01

    In drug invention and early disease prediction of lung cancer, metabolomic biomarker detection is very important. Mortality rate can be decreased, if cancer is predicted at the earlier stage. Recent diagnostic techniques for lung cancer are not prognosis diagnostic techniques. However, if we know the name of the metabolites, whose intensity levels are considerably changing between cancer subject and control subject, then it will be easy to early diagnosis the disease as well as to discover the drug. Therefore, in this paper we have identified the influential plasma and serum blood sample metabolites for lung cancer and also identified the biomarkers that will be helpful for early disease prediction as well as for drug invention. To identify the influential metabolites, we considered a parametric and a nonparametric test namely student׳s t-test as parametric and Kruskal-Wallis test as non-parametric test. We also categorized the up-regulated and down-regulated metabolites by the heatmap plot and identified the biomarkers by support vector machine (SVM) classifier and pathway analysis. From our analysis, we got 27 influential (p-value<0.05) metabolites from plasma sample and 13 influential (p-value<0.05) metabolites from serum sample. According to the importance plot through SVM classifier, pathway analysis and correlation network analysis, we declared 4 metabolites (taurine, aspertic acid, glutamine and pyruvic acid) as plasma biomarker and 3 metabolites (aspartic acid, taurine and inosine) as serum biomarker.

  6. Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions

    OpenAIRE

    Vaartjes, W.J.; Breejen, J.N. den; Geelen, M.J.H.; Bergh, S.G. van den

    1980-01-01

    1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite direction of the activity of pyruvate dehydrogenase (EC. 1.2.4.1). 3. Changes of the transmembrane pH gradient and of the membrane potential, brought about by the pretreatments of the mitochondria, c...

  7. Glutamine synthetase and alanine transaminase expression are decreased in livers of aged vs. young beef cows and GS can be upregulated by 17β-estradiol implants.

    Science.gov (United States)

    Miles, E D; McBride, B W; Jia, Y; Liao, S F; Boling, J A; Bridges, P J; Matthews, J C

    2015-09-01

    Aged beef cows (≥ 8 yr of age) produce calves with lower birth and weaning weights. In mammals, aging is associated with reduced hepatic expression of glutamine synthetase (GS) and alanine transaminase (ALT), thus impaired hepatic Gln-Glu cycle function. To determine if the relative protein content of GS, ALT, aspartate transaminase (AST), glutamate transporters (EAAC1, GLT-1), and their regulating protein (GTRAP3-18) differed in biopsied liver tissue of (a) aged vs. young (3 to 4 yr old) nonlactating, nongestating Angus cows (Exp. 1 and 2) and (b) aged mixed-breed cows with and without COMPUDOSE (17β-estradiol) ear implants (Exp. 3), Western blot analyses were performed. In Exp. 1, 12 young (3.62 ± 0.01 yr) and 13 aged (10.08 ± 0.42 yr) cows grazed the same mixed forage for 42 d (August-October). In Exp. 2, 12 young (3.36 ± 0.01 yr) and 12 aged (10.38 ± 0.47 yr) cows were individually fed (1.03% of BW) a corn-silage-based diet to maintain BW for 20 d. For both Exp. 1 and 2, the effect of cow age was assessed by ANOVA using the MIXED procedure of SAS. Cow BW did not change ( ≥ 0.17). Hepatic ALT (78% and 61%) and GS (52% and 71%) protein content (Exp. 1 and 2, respectively) was decreased ( ≤ 0.01), whereas GTRAP3-18 (an inhibitor of EAAC1 activity) increased ( ≤ 0.01; 170% and 136%) and AST, GLT-1, and EAAC1 contents did not differ ( ≥ 0.17) in aged vs. young cows. In Exp. 2, free concentrations (nmol/g) of Glu, Ala, Gln, Arg, and Orn in liver homogenates were determined. Aged cows tended to have less ( = 0.10) free Gln (15.0%) than young cows, whereas other AA concentrations did not differ ( 0.26). In Exp. 3, 14 aged (> 10 yr) cows were randomly allotted ( = 7) to sham or COMPUDOSE (25.7 mg of 17β-estradiol) implant treatment (TRT), and had ad libitum access to alfalfa hay for 28 d. Blood and liver biopsies were collected 14 and 28 d after implant treatment. Treatment, time after implant (DAY), and TRT × DAY effects were assessed by ANOVA using

  8. [Prevalence of non-alcoholic fatty liver disease in a population with elevated transaminases and level of accuracy of the diagnosis in Primary Care].

    Science.gov (United States)

    Samperio-González, María Amelia; Selvi-Blasco, Marta; Manzano-Montero, Mónica; Méndez-Gómez, Judit; Gil-Prades, Montserrat; Azagra, Rafael

    2016-05-01

    Nonalcoholic fatty liver disease (NAFLD) is the most common cause of elevated transaminases in adults. Determine the prevalence of NASH in patients with sustained hypertransaminasemia, and Know the adequacy of the registered in Primary Care (AP) diagnosis. 1) Cross-sectional study with a random sample of patients with elevated alanine aminotransferase (ALT) held (ALT> 32 for ≥6 months), ruling out other causes of liver disease, according to clinical, laboratory and ultrasound scan criteria in AP and 2) cross-sectional description of all cases diagnosed with NASH recorded (K76 - ICD10) with diagnostic adequacy analysis according to standard criteria. 290 patients were analyzed: 76 were diagnosed as NASH (26.1%), 44 women (57.9%). Multivariate analysis adjusted for age and sex showed no association between NASH and male gender (OR: 0.5; CI95%: 0.3-0.9), diabetes mellitus (DM) (OR: 2.42; CI95%: 1.2-4.9) and hypertension blood pressure (HBP) (OR: 3.07; CI 95% 1.6-5.6). Of the 209 diagnosed with NASH record: 51 (24.4%) met the criteria for NASH. The rest had insufficient records. 53.1% lacked sustained hypertransaminasemia; 48% of viral serology; 11% supported and 53.1% abdominal ultrasound registration of alcohol. Severe NASH is frequent among patients with sustained hypertransaminasemia. The DM and hypertension significantly increase the risk of NASH. The diagnosis of NASH is recorded without considering all criteria and mainly NASH made by ultrasonography. They should unify diagnostic criteria in the register of NASH. Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

  9. The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid.

    Science.gov (United States)

    Ringel, Michael T; Dräger, Gerald; Brüser, Thomas

    2017-11-10

    The periplasmic conversion of ferribactin to pyoverdine is essential for siderophore biogenesis in fluorescent pseudomonads, such as pathogenic Pseudomonas aeruginosa or plant growth-promoting Pseudomonas fluorescens The non-ribosomal peptide ferribactin undergoes cyclizations and oxidations that result in the fluorophore, and a strictly conserved fluorophore-bound glutamic acid residue is converted to a range of variants, including succinamide, succinic acid, and α-ketoglutaric acid residues. We recently discovered that the pyridoxal phosphate-containing enzyme PvdN is responsible for the generation of the succinamide, which can be hydrolyzed to succinic acid. Based on this, a distinct unknown enzyme was postulated to be responsible for the conversion of the glutamic acid to α-ketoglutaric acid. Here we report the identification and characterization of this enzyme in P. fluorescens strain A506. In silico analyses indicated a periplasmic transaminase in fluorescent pseudomonads and other proteobacteria that we termed PtaA for " p eriplasmic t ransaminase A " An in-frame-deleted ptaA mutant selectively lacked the α-ketoglutaric acid form of pyoverdine, and recombinant PtaA complemented this phenotype. The ptaA / pvdN double mutant produced exclusively the glutamic acid form of pyoverdine. PtaA is homodimeric and contains a pyridoxal phosphate cofactor. Mutation of the active-site lysine abolished PtaA activity and affected folding as well as Tat-dependent transport of the enzyme. In pseudomonads, the occurrence of ptaA correlates with the occurrence of α-ketoglutaric acid forms of pyoverdines. As this enzyme is not restricted to pyoverdine-producing bacteria, its catalysis of periplasmic transaminations is most likely a general tool for specific biosynthetic pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Changes of ammonia, urea contents and transaminase activity in the body during aerial exposure and ammonia loading in Chinese loach Paramisgurnus dabryanus.

    Science.gov (United States)

    Zhang, Yun-Long; Zhang, Hai-Long; Wang, Ling-Yu; Gu, Bei-Yi; Fan, Qi-Xue

    2017-04-01

    The Paramisgurnus dabryanus was exposed to 30 mmol L -1 NH 4 Cl solution and air to assessing the change of body ammonia and urea contents and the activities of alanine aminotransferase (ALT) and aspartate transaminase (AST). After 48 h of ammonia exposure, ammonia concentration in the plasma, brain, liver and muscle were 3.3-fold, 5.6-fold, 3.5-fold and 4.2-fold, respectively, those of the control values. Plasma, brain, liver and muscle ammonia concentrations increased to 2.2-fold, 3.3-fold, 2.5-fold and 2.9-fold, respectively, those of control values in response to 48 h of aerial exposure. Within the given treatment (ammonia or aerial exposure), there was no change in plasma, brain and liver urea concentrations between exposure durations. The plasma ALT activity was significantly affected by exposure time during aerial exposure, while the liver ALT activity was not affected by ammonia or aerial exposure. Exposure to NH 4 Cl or air had no effect on either plasma or liver AST activity. Our results suggested that P. dabryanus could accumulate quite high level of internal ammonia because of the high ammonia tolerance in its cells and tissues, and NH 3 volatilization would be a possible ammonia detoxification strategy in P. dabryanus. Urea synthesis was not an effective mechanism to deal with environmental or internal ammonia problem. The significant increase of ALT activity in plasma during aerial exposure, indicating that alanine synthesis through certain amino acid catabolism may be subsistent in P. dabryanus.

  11. Pyruvate dehydrogenase complex and lactate dehydrogenase as targets for therapy of acute liver failure.

    Science.gov (United States)

    Ferriero, Rosa; Nusco, Edoardo; De Cegli, Rossella; Carissimo, Annamaria; Manco, Giuseppe; Brunetti-Pierri, Nicola

    2018-03-23

    Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate in the nucleus to regulate histone acetylation and gene expression. Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-Ab, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by Gene Ontology Enrichment Analysis. Efficacy of histone acetyltransferase inhibitor garcinol and LDH inhibitor galloflavin at reducing liver damage was evaluated in mice with induced hepatotoxicity. Levels and activities of PDHC and LDH were increased in cytoplasmatic and nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-coA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to response to damage. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. PDHC and LDH translocate to the nucleus and are targets for therapy of acute liver failure. Acute liver failure is a rapidly progressive and life-threatening deterioration of liver function resulting in high mortality and

  12. Mechanistic photodecarboxylation of pyruvic acid: Excited-state proton transfer and three-state intersection

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Xue-Ping; Fang, Qiu, E-mail: fangqiu917@bnu.edu.cn; Cui, Ganglong, E-mail: ganglong.cui@bnu.edu.cn [Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875 (China)

    2014-10-21

    Photodissociation dynamics of pyruvic acid experimentally differs from that of commonly known ketones. We have employed the complete active space self-consistent field and its multi-state second-order perturbation methods to study its photodissociation mechanism in the S{sub 0}, T{sub 1}, and S{sub 1} states. We have uncovered four nonadiabatic photodecarboxylation paths. (i) The S{sub 1} system relaxes via an excited-state intramolecular proton transfer (ESIPT) to a hydrogen-transferred tautomer, near which an S{sub 1}/S{sub 0} conical intersection funnels the S{sub 1} to S{sub 0} state. Then, some trajectories continue completing the decarboxylation reaction in the S{sub 0} state; the remaining trajectories via a reverse hydrogen transfer return to the S{sub 0} minimum, from which a thermal decarboxylation reaction occurs. (ii) Due to a small S{sub 1} −T{sub 1} energy gap and a large S{sub 1}/T{sub 1} spin-orbit coupling, an efficient S{sub 1} → T{sub 1} intersystem crossing process happens again near this S{sub 1}/S{sub 0} conical intersection. When decaying to T{sub 1} state, a direct photodecarboxylation proceeds. (iii) Prior to ESIPT, the S{sub 1} system first decays to the T{sub 1} state via an S{sub 1} → T{sub 1} intersystem crossing; then, the T{sub 1} system evolves to a hydrogen-transferred tautomer. Therefrom, an adiabatic T{sub 1} decarboxylation takes place due to a small barrier of 7.7 kcal/mol. (iv) Besides the aforementioned T{sub 1} ESIPT process, there also exists a comparable Norrish type I reaction in the T{sub 1} state, which forms the ground-state products of CH{sub 3}CO and COOH. Finally, we have found that ESIPT plays an important role. It closes the S{sub 1}-T{sub 1} and S{sub 1}-S{sub 0} energy gaps, effecting an S{sub 1}/T{sub 1}/S{sub 0} three-state intersection region, and mediating nonadiabatic photodecarboxylation reactions of pyruvic acid.

  13. Zinc in human serum

    International Nuclear Information System (INIS)

    Kiilerich, S.

    1987-01-01

    The zinc ion is essential for the living organism. Many pathological conditions have been described as a consequence of zinc deficiency. As zinc constitutes less than 0.01 per cent of the body weight, it conventionally belongs to the group of trace elements. The method of atomic absorption spectrophotometry is used to measure the concentration of zinc in serum and urine from healthy persons. The assumptions of the method is discussed. The importance of proteinbinding, diet and the diurnal variation of serum zinc concentration is presented. Serum versus plasma zinc concentration is discussed. Reference serum zinc values from 104 normal subjects are given. Zinc in serum is almost entirely bound to proteins. A preliminary model for the estimation of the distribution of zinc between serum albumin and α 2 -macroglobulin is set up. This estimate has been examined by an ultracentrufugation method. The binding of zinc to a α 2 -macroglobulin in normal persons is appoximately 7 per cent, in patients with cirrhosis of the liver of alcoholic origin approximately 6 per cent, in patients with insulin dependent diabetes mellitus approximately 5 per cent, and in patients with chronic renal failure approximately 2 per cent. It is concluded, therefore, that for clinical purposes it is sufficient to use the concentration of total serum zinc corrected for the concentration of serum albumin. (author)

  14. Data regarding the growth of Lactobacillus acidophilus NCFM on different carbohydrates and recombinant production of elongation factor G and pyruvate kinase

    DEFF Research Database (Denmark)

    Celebioglu, Hasan Ufuk; Olesen, Sita Vaag; Prehn, Kennie

    2017-01-01

    The present study describes the growth of the very well-known probiotic bacterium Lactobacillus acidophilus NCFM on different carbohydrates. Furthermore, recombinant production of putative moonlighting proteins elongation factor G and pyruvate kinase from this bacterium is described. For further...

  15. Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

    DEFF Research Database (Denmark)

    Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian

    2015-01-01

    , and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain...... pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide...... dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes....

  16. Formation and utilization of acetoin, an unusual product of pyruvate metabolism by Ehrlich and AS30-D tumor mitochondria.

    Science.gov (United States)

    Baggetto, L G; Lehninger, A L

    1987-07-15

    [14C]Pyruvate was rapidly non-oxidatively decarboxylated by Ehrlich tumor mitochondria at a rate of 40 nmol/min/mg of protein in the presence or absence of ADP. A search for decarboxylation products led to significant amounts of acetoin formed when Ehrlich tumor mitochondria were incubated with 1 mM [14C] pyruvate in the presence of ATP. Added acetoin to aerobic tumor mitochondria was rapidly utilized in the presence of ATP at a rate of 65 nmol/min/mg of protein. Citrate has been found as a product of acetoin utilization and was exported from the tumor mitochondria. Acetoin has been found in the ascitic liquid of Ehrlich and AS30-D tumor-bearing animals. These unusual reactions were not observed in control rat liver mitochondria.

  17. Blood glucose, lactate, pyruvate, glycerol, 3-hydroxybutyrate and acetoacetate measurements in man using a centrifugal analyser with a fluorimetric attachment.

    Science.gov (United States)

    Harrison, J; Hodson, A W; Skillen, A W; Stappenbeck, R; Agius, L; Alberti, K G

    1988-03-01

    Methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, 3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood, using the Cobas Bio centrifugal analyser fitted with a fluorimetric attachment. Intra-assay and inter-assay coefficients of variation ranged from 1.9 to 7.9% and from 1.0 to 7.2% respectively. Correlation coefficients ranged from 0.96 to 0.99 against established continuous-flow and manual spectrophotometric methods. All seven metabolites can be measured using a single perchloric acid extract of 20 microliter of blood. The versatility of the assays is such that as little as 100 pmol pyruvate, 3-hydroxybutyrate or as much as 15 nmol glucose can be measured in the same 20 microliter extract.

  18. Pyruvic Oxime Nitrification and Copper and Nickel Resistance by a Cupriavidus pauculus, an Active Heterotrophic Nitrifier-Denitrifier

    OpenAIRE

    Ramirez, Miguel; Obrzydowski, Jennifer; Ayers, Mary; Virparia, Sonia; Wang, Meijing; Stefan, Kurtis; Linchangco, Richard; Castignetti, Domenic

    2014-01-01

    Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion. A Cupriavidus pauculus, previously thought an Alcaligenes sp. and noted as an active heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2) and nitrous oxide (N2O) while heterotrophically nitrifying the organic substrate pyruvic oxime [CH3–C(NOH)–COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic analyses indicated that the organism is a member of a g...

  19. Performance during a strenuous swimming session is associated with high blood lactate: pyruvate ratio and hypoglycemia in fasted rats.

    Science.gov (United States)

    Travassos, P B; Godoy, G; De Souza, H M; Curi, R; Bazotte, R B

    2018-03-26

    The aim of this study was to investigate the effect of lactatemia elevation and glycemia reduction on strenuous swimming performance in fasted rats. Three rats were placed in a swimming tank at the same time. The first rat was removed immediately (control group) and the remaining ones were submitted to a strenuous swimming session. After the second rat was exhausted (Exh group), the third one was immediately removed from the water (Exe group). According to the period of time required for exhaustion, the rats were divided into four groups: low performance (3-7 min), low-intermediary performance (8-12 min), high-intermediary performance (13-17 min), and high performance (18-22 min). All rats were removed from the swimming tanks and immediately killed by decapitation for blood collection or anesthetized for liver perfusion experiments. Blood glucose, lactate, and pyruvate concentrations, blood lactate/pyruvate ratio, and liver lactate uptake and its conversion to glucose were evaluated. Exhaustion in low and low-intermediary performance were better associated with higher lactate/pyruvate ratio. On the other hand, exhaustion in high-intermediary and high performance was better associated with hypoglycemia. Lactate uptake and glucose production from lactate in livers from the Exe and Exh groups were maintained. We concluded that there is a time sequence in the participation of lactate/pyruvate ratio and hypoglycemia in performance during an acute strenuous swimming section in fasted rats. The liver had an important participation in preventing hyperlactatemia and hypoglycemia during swimming through lactate uptake and its conversion to glucose.

  20. Effects of insulin on perfused liver from streptozotocin-diabetic and untreated rats: 13C NMR assay of pyruvate kinase flux

    International Nuclear Information System (INIS)

    Cohen, S.M.

    1987-01-01

    The effects of insulin in vitro on perfused liver from streptozotocin-diabetic rats and their untreated littermates during gluconeogenesis from either [3- 13 C]alanine + ethanol or [2- 13 C]pyruvate + NH 4 Cl + ethanol were studied by 13 C NMR. A 13 C NMR determination of the rate of pyruvate kinase flux under steady-state conditions of active gluconeogenesis was developed; this assay includes a check on the reuse of recycled pyruvate. The preparations studied provided gradations of pyruvate kinase flux within the confines of the assay's requirement of active gluconeogenesis. By this determination, the rate of pyruvate kinase flux was 0.74 +/- 0.04 of the gluconeogenic rate in liver from 24-h-fasted controls; in liver from 12-h fasted controls, relative pyruvate kinase flux increased to 1.0 +/- 0.2. In diabetic liver, this flux was undetectable by the authors NMR method. Insulin's hepatic influence in vitro was greatest in the streptozotocin model of type 1 diabetes: upon treatment of diabetic liver with 7 nM insulin in vitro, a partial reversal of many of the differences noted between diabetic and control liver was demonstrated by 13 C NMR. A major effect of insulin in vitro upon diabetic liver was the induction of a large increase in the rate of pyruvate kinase flux, bringing relative and absolute fluxes up to the levels measured in 24-h-fasted controls. By way of comparison, the effects of ischemia on diabetic liver were studied by 13 C NMR to test whether changes in allosteric effectors under these conditions could also increase pyruvate kinase flux. A large increase in this activity was demonstrated in ischemic diabetic liver

  1. Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide.

    OpenAIRE

    Thomas, A P; Halestrap, A P

    1981-01-01

    1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis...

  2. Application of mitochondrial pyruvate carrier blocker UK5099 creates metabolic reprogram and greater stem-like properties in LnCap prostate cancer cells in vitro

    OpenAIRE

    Zhong, Yali; Li, Xiaoran; Yu, Dandan; Li, Xiaoli; Li, Yaqing; Long, Yuan; Yuan, Yuan; Ji, Zhenyu; Zhang, Mingzhi; Wen, Jian-Guo; Nesland, Jahn M.; Suo, Zhenhe

    2015-01-01

    Aerobic glycolysis is one of the important hallmarks of cancer cells and eukaryotic cells. In this study, we have investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with UK5099 and the metabolic alteration as well as stemness phenotype of prostatic cancer cells. It was found that blocking pyruvate transportation into mitochondrial attenuated mitochondrial oxidative phosphorylation (OXPHOS) and increased glycolysis. The UK5099 treated cells showed significantly...

  3. Role of pyruvate dehydrogenase inhibition in the development of hypertrophy in the hyperthyroid rat heart: a combined magnetic resonance imaging and hyperpolarized magnetic resonance spectroscopy study.

    Science.gov (United States)

    Atherton, Helen J; Dodd, Michael S; Heather, Lisa C; Schroeder, Marie A; Griffin, Julian L; Radda, George K; Clarke, Kieran; Tyler, Damian J

    2011-06-07

    Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg x kg(-1) x d(-1)). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022 ± 0.0002 versus 0.0055 ± 0.0005 second(-1); P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-(13)C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100 ± 20 versus 200 ± 30 mg; P=0.04) despite no change in the increase observed in cardiac output. This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic

  4. Optimized methods to measure acetoacetate, 3-hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment

    OpenAIRE

    Stappenbeck, R.; Hodson, A. W.; Skillen, A. W.; Agius, L.; Alberti, K. G. M. M.

    1990-01-01

    Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were ...

  5. Crystallization and preliminary X-ray analysis of dihydrodipicolinate synthase from Clostridium botulinum in the presence of its substrate pyruvate

    International Nuclear Information System (INIS)

    Atkinson, Sarah C.; Dobson, Renwick C. J.; Newman, Janet M.; Gorman, Michael A.; Dogovski, Con; Parker, Michael W.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyzes an important step in lysine biosynthesis. Here, the crystallization and preliminary diffraction analysis to 1.2 Å resolution of DHDPS from C. botulinum in the presence of its substrate pyruvate is reported. In this paper, the crystallization and preliminary X-ray diffraction analysis to near-atomic resolution of DHDPS from Clostridium botulinum crystallized in the presence of its substrate pyruvate are presented. The enzyme crystallized in a number of forms using a variety of PEG precipitants, with the best crystal diffracting to 1.2 Å resolution and belonging to space group C2, in contrast to the unbound form, which had trigonal symmetry. The unit-cell parameters were a = 143.4, b = 54.8, c = 94.3 Å, β = 126.3°. The crystal volume per protein weight (V M ) was 2.3 Å 3 Da −1 (based on the presence of two monomers in the asymmetric unit), with an estimated solvent content of 46%. The high-resolution structure of the pyruvate-bound form of C. botulinum DHDPS will provide insight into the function and stability of this essential bacterial enzyme

  6. Scanning mutagenesis of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

    Directory of Open Access Journals (Sweden)

    Nagib eAhsan

    2012-07-01

    Full Text Available The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform scanning mutagenesis of the residues flanking the site of phosphorylation. Consistent with the results from phylogenetic analysis of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

  7. Pyruvate remediation of cell stress and genotoxicity induced by haloacetic acid drinking water disinfection by-products.

    Science.gov (United States)

    Dad, Azra; Jeong, Clara H; Pals, Justin A; Wagner, Elizabeth D; Plewa, Michael J

    2013-10-01

    Monohaloacetic acids (monoHAAs) are a major class of drinking water disinfection by-products (DBPs) and are cytotoxic, genotoxic, mutagenic, and teratogenic. We propose a model of toxic action based on monoHAA-mediated inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a target cytosolic enzyme. This model predicts that GAPDH inhibition by the monoHAAs will lead to a severe reduction of cellular ATP levels and repress the generation of pyruvate. A loss of pyruvate will lead to mitochondrial stress and genomic DNA damage. We found a concentration-dependent reduction of ATP in Chinese hamster ovary cells after monoHAA treatment. ATP reduction per pmol monoHAA followed the pattern of iodoacetic acid (IAA) > bromoacetic acid (BAA) > chloroacetic acid (CAA), which is the pattern of potency observed with many toxicological endpoints. Exogenous supplementation with pyruvate enhanced ATP levels and attenuated monoHAA-induced genomic DNA damage as measured with single cell gel electrophoresis. These data were highly correlated with the SN 2 alkylating potentials of the monoHAAs and with the induction of toxicity. The results from this study strongly support the hypothesis that GAPDH inhibition and the possible subsequent generation of reactive oxygen species is linked with the cytotoxicity, genotoxicity, teratogenicity, and neurotoxicity of these DBPs. Copyright © 2013 Wiley Periodicals, Inc.

  8. Coordination of manganous ion at the active site of pyruvate, phosphate dikinase: the complex of oxalate with the phosphorylated enzyme

    International Nuclear Information System (INIS)

    Kofron, J.L.; Ash, D.E.; Reed, G.H.

    1988-01-01

    Electron paramagnetic resonance spectroscopy has been used to investigate the structure of the complex of manganous ion with the phosphorylated form of pyruvate, phosphate dikinase (E/sub p/) and the inhibitor oxalate. Oxalate, an analogue of the enolate of pyruvate, is competitive with respect to pyruvate in binding to the phosphorylated form of the enzyme. Superhyperfine coupling between the unpaired electrons of Mn(I) and ligands specifically labeled with 17 O has been used to identify oxygen ligands to Mn(II) in the complex with oxalate and the phosphorylated form of the enzyme. Oxalate binds at the active site as a bidentate chelate with Mn(II). An oxygen from the 3'-N-phosphohistidyl residue of the protein is in the coordination sphere of Mn(II), and at least two water molecules are also bound to Mn(II) in the complex. Oxalate also binds directly to Mn(II) in a complex with nonphosphorylated enzyme. The structure for the E/sub p/-Mn(II)-oxalate complex implies that simultaneous coordination of a phospho group and of the attacking nucleophile to the divalent cation is likely an important factor in catalysis of this phospho-transfer reaction

  9. Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT--relationship to newly identified mitochondrial pyruvate carrier proteins.

    Directory of Open Access Journals (Sweden)

    Jerry R Colca

    Full Text Available Thiazolidinedione (TZD insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44 and BRP44 Like (BRP44L, which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of (13C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

  10. Hepatic Mitochondrial Pyruvate Carrier 1 Is Required for Efficient Regulation of Gluconeogenesis and Whole-Body Glucose Homeostasis.

    Science.gov (United States)

    Gray, Lawrence R; Sultana, Mst Rasheda; Rauckhorst, Adam J; Oonthonpan, Lalita; Tompkins, Sean C; Sharma, Arpit; Fu, Xiaorong; Miao, Ren; Pewa, Alvin D; Brown, Kathryn S; Lane, Erin E; Dohlman, Ashley; Zepeda-Orozco, Diana; Xie, Jianxin; Rutter, Jared; Norris, Andrew W; Cox, James E; Burgess, Shawn C; Potthoff, Matthew J; Taylor, Eric B

    2015-10-06

    Gluconeogenesis is critical for maintenance of euglycemia during fasting. Elevated gluconeogenesis during type 2 diabetes (T2D) contributes to chronic hyperglycemia. Pyruvate is a major gluconeogenic substrate and requires import into the mitochondrial matrix for channeling into gluconeogenesis. Here, we demonstrate that the mitochondrial pyruvate carrier (MPC) comprising the Mpc1 and Mpc2 proteins is required for efficient regulation of hepatic gluconeogenesis. Liver-specific deletion of Mpc1 abolished hepatic MPC activity and markedly decreased pyruvate-driven gluconeogenesis and TCA cycle flux. Loss of MPC activity induced adaptive utilization of glutamine and increased urea cycle activity. Diet-induced obesity increased hepatic MPC expression and activity. Constitutive Mpc1 deletion attenuated the development of hyperglycemia induced by a high-fat diet. Acute, virally mediated Mpc1 deletion after diet-induced obesity decreased hyperglycemia and improved glucose tolerance. We conclude that the MPC is required for efficient regulation of gluconeogenesis and that the MPC contributes to the elevated gluconeogenesis and hyperglycemia in T2D. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. MCT1 modulates cancer cell pyruvate export and growth of tumors that co-express MCT1 and MCT4

    Science.gov (United States)

    Hong, Candice Sun; Graham, Nicholas A.; Gu, Wen; Camacho, Carolina Espindola; Mah, Vei; Maresh, Erin L.; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A. L.; Gardner, Brian K.; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K.; Hurvitz, Sara A.; Dubinett, Steven M.; Critchlow, Susan E.; Kurdistani, Siavash K.; Goodglick, Lee; Braas, Daniel; Graeber, Thomas G.; Christofk, Heather R.

    2016-01-01

    SUMMARY Monocarboxylate Transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export, which when inhibited enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors further supporting their use as anti-cancer therapeutics. PMID:26876179

  12. The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

    Science.gov (United States)

    Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

    2014-01-01

    Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile. Copyright © 2013. Published by Elsevier GmbH.

  13. A case of pyruvate dehydrogenase deficiency with low density areas in white matter noticed by CT scan

    International Nuclear Information System (INIS)

    Kimura, Akiko; Kyoya, Seizo; Matsushima, Akihiro; Irimichi, Hideki; Koike, Yoshiko.

    1985-01-01

    The patient was a 4-month-old boy, the first child of healthy, non-consanguineous patient. He was mildly asphyxiated at birth and developed severe convulsions at two days of age. At 4 months of age, he was referred to us because of infantile spasms and motor retardation. The EEG showed hypsarhythmia, ACTH and anticonvulsants were started, but his seizures were not controlled completely. At 8 months of age, the CT scan demonstrated a cerebral atrophy with enlarged ventricles and a diffuse low density of cerebral white matter, and lactic acidosis was first noticed. The glucose, glucagon, fructose, and alanine tolerance tests revealed almost normal responses in blood glucose levels and elevation of lactate levels above the initial value. Enzyme studies revealed a severe deficiency of pyruvate dehydrogenase complex and pyruvate dehydrogenase (E 1 ), and a normal activity of pyruvate carboxylase in liver obtained by biopsy. In biopsied muscle, mitochondria appeared normal. Treatment with thiamine, lipoic acid and anticonvulsants was not effective. The clinical picture of PDC deficiency has been correlated with the amount of the residual activity, and this case confirmed to the ''severe'' category. Several pathologic entities may be associated with PDHC deficiency, and CT findings in our case demonstrated the demyelinating condition. The precise relationship between the defect and the pathogenesis remains to be elucidated. (author)

  14. Improvement of ethanol yield from glycerol via conversion of pyruvate to ethanol in metabolically engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Yu, Kyung Ok; Jung, Ju; Ramzi, Ahmad Bazli; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2012-02-01

    The conversion of low-priced glycerol to higher value products has been proposed as a way to improve the economic viability of the biofuels industry. In a previous study, the conversion of glycerol to ethanol in a metabolically engineered strain of Saccharomyces cerevisiae was accomplished by minimizing the synthesis of glycerol, the main by-product in ethanol fermentation processing. To further improve ethanol production, overexpression of the native genes involved in conversion of pyruvate to ethanol in S. cerevisiae was successfully accomplished. The overexpression of an alcohol dehydrogenase (adh1) and a pyruvate decarboxylase (pdc1) caused an increase in growth rate and glycerol consumption under fermentative conditions, which led to a slight increase of the final ethanol yield. The overall expression of the adh1 and pdc1 genes in the modified strains, combined with the lack of the fps1 and gpd2 genes, resulted in a 1.4-fold increase (about 5.4 g/L ethanol produced) in fps1Δgpd2Δ (pGcyaDak, pGupCas) (about 4.0 g/L ethanol produced). In summary, it is possible to improve the ethanol yield by overexpression of the genes involved in the conversion of pyruvate to ethanol in engineered S. cerevisiae using glycerol as substrate.

  15. Regulation of Muscle Pyruvate Dehydrogenase Complex in Insulin Resistance: Effects of Exercise and Dichloroacetate

    Directory of Open Access Journals (Sweden)

    Dumitru Constantin-Teodosiu

    2013-10-01

    Full Text Available Since the mitochondrial pyruvate dehydrogenase complex (PDC controls the rate of carbohydrate oxidation, impairment of PDC activity mediated by high-fat intake has been advocated as a causative factor for the skeletal muscle insulin resistance, metabolic syndrome, and the onset of type 2 diabetes (T2D. There are also situations where muscle insulin resistance can occur independently from high-fat dietary intake such as sepsis, inflammation, or drug administration though they all may share the same underlying mechanism, i.e., via activation of forkhead box family of transcription factors, and to a lower extent via peroxisome proliferator-activated receptors. The main feature of T2D is a chronic elevation in blood glucose levels. Chronic systemic hyperglycaemia is toxic and can lead to cellular dysfunction that may become irreversible over time due to deterioration of the pericyte cell's ability to provide vascular stability and control to endothelial proliferation. Therefore, it may not be surprising that T2D's complications are mainly macrovascular and microvascular related, i.e., neuropathy, retinopathy, nephropathy, coronary artery, and peripheral vascular diseases. However, life style intervention such as exercise, which is the most potent physiological activator of muscle PDC, along with pharmacological intervention such as administration of dichloroacetate or L-carnitine can prove to be viable strategies for treating muscle insulin resistance in obesity and T2D as they can potentially restore whole body glucose disposal.

  16. Role of plastidic pyruvate dehydrogenase complex (pl. PDC) in chloroplast metabolism of spinach

    International Nuclear Information System (INIS)

    Schulze-Siebert, D.; Homeyer, U.; Schultz, G.

    1986-01-01

    Labeling experiments of chloroplasts in the light ( 14 CO 2 , 2- 14 C-pyruvate etc.) revealed that pl. PDC is predominantly involved in the synthesis of branched chain amino acids and pl. isoprenoids (carotenes, PQ, α-T). In this context, pl. phosphoglycerate mutase as missing link in the C 3 → C 2 metabolism of chloroplasts was identified by latency experiments. This indicates a direct pathway from Calvin cycle to pl. PDC. Using protoplasts, maximal rates in pl. PDC metabolism were obtained. On the other hand, mitochondrial PDC in protoplasts is mainly involved in fatty acid synthesis by known mechanism. Additionally, cytosolic-ER-isoprenoids were formed (e.g. sterols). When 14 CO 2 was simultaneously applied with unlabeled acetate to protoplasts in the light an isotopic dilution of fatty acids were found but not of pl. isoprenoids. This may indicate an partially channeling of pl. PDC and mevalonate pathway for pl. isoprenoid synthesis. Inhibitory studies with DCMU point in the same direction

  17. An improved strategy for the crystallization of Leishmania mexicana pyruvate kinase

    International Nuclear Information System (INIS)

    Morgan, Hugh P.; McNae, Iain W.; Hsin, Kun-Yi; Michels, Paul A. M.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.

    2010-01-01

    The first crystal structure of Leishmania mexicana pyruvate kinase (LmPYK) obtained at a neutral pH. LmPYK was co-crystallized with the small molecule 1,3,6,8-pyrenetetrasulfonic acid, which provides a helpful intermolecular bridge between macromolecules. The inclusion of novel small molecules in crystallization experiments has provided very encouraging results and this method is now emerging as a promising alternative strategy for crystallizing ‘problematic’ biological macromolecules. These small molecules have the ability to promote lattice formation through stabilizing intermolecular interactions in protein crystals. Here, the use of 1,3,6,8-pyrenetetrasulfonic acid (PTS), which provides a helpful intermolecular bridge between Leishmania mexicana PYK (LmPYK) macromolecules in the crystal, is reported, resulting in the rapid formation of a more stable crystal lattice at neutral pH and greatly improved X-ray diffraction results. The refined structure of the LmPYK–PTS complex revealed the negatively charged PTS molecule to be stacked between positively charged (surface-exposed) arginine side chains from neighbouring LmPYK molecules in the crystal lattice

  18. A catalyzing phantom for reproducible dynamic conversion of hyperpolarized [1-¹³C]-pyruvate.

    Science.gov (United States)

    Walker, Christopher M; Lee, Jaehyuk; Ramirez, Marc S; Schellingerhout, Dawid; Millward, Steven; Bankson, James A

    2013-01-01

    In vivo real time spectroscopic imaging of hyperpolarized ¹³C labeled metabolites shows substantial promise for the assessment of physiological processes that were previously inaccessible. However, reliable and reproducible methods of measurement are necessary to maximize the effectiveness of imaging biomarkers that may one day guide personalized care for diseases such as cancer. Animal models of human disease serve as poor reference standards due to the complexity, heterogeneity, and transient nature of advancing disease. In this study, we describe the reproducible conversion of hyperpolarized [1-¹³C]-pyruvate to [1-¹³C]-lactate using a novel synthetic enzyme phantom system. The rate of reaction can be controlled and tuned to mimic normal or pathologic conditions of varying degree. Variations observed in the use of this phantom compare favorably against within-group variations observed in recent animal studies. This novel phantom system provides crucial capabilities as a reference standard for the optimization, comparison, and certification of quantitative imaging strategies for hyperpolarized tracers.

  19. A catalyzing phantom for reproducible dynamic conversion of hyperpolarized [1-¹³C]-pyruvate.

    Directory of Open Access Journals (Sweden)

    Christopher M Walker

    Full Text Available In vivo real time spectroscopic imaging of hyperpolarized ¹³C labeled metabolites shows substantial promise for the assessment of physiological processes that were previously inaccessible. However, reliable and reproducible methods of measurement are necessary to maximize the effectiveness of imaging biomarkers that may one day guide personalized care for diseases such as cancer. Animal models of human disease serve as poor reference standards due to the complexity, heterogeneity, and transient nature of advancing disease. In this study, we describe the reproducible conversion of hyperpolarized [1-¹³C]-pyruvate to [1-¹³C]-lactate using a novel synthetic enzyme phantom system. The rate of reaction can be controlled and tuned to mimic normal or pathologic conditions of varying degree. Variations observed in the use of this phantom compare favorably against within-group variations observed in recent animal studies. This novel phantom system provides crucial capabilities as a reference standard for the optimization, comparison, and certification of quantitative imaging strategies for hyperpolarized tracers.

  20. Metabolic modeling of energy balances in Mycoplasma hyopneumoniae shows that pyruvate addition increases growth rate.

    Science.gov (United States)

    Kamminga, Tjerko; Slagman, Simen-Jan; Bijlsma, Jetta J E; Martins Dos Santos, Vitor A P; Suarez-Diez, Maria; Schaap, Peter J

    2017-10-01

    Mycoplasma hyopneumoniae is cultured on large-scale to produce antigen for inactivated whole-cell vaccines against respiratory disease in pigs. However, the fastidious nutrient requirements of this minimal bacterium and the low growth rate make it challenging to reach sufficient biomass yield for antigen production. In this study, we sequenced the genome of M. hyopneumoniae strain 11 and constructed a high quality constraint-based genome-scale metabolic model of 284 chemical reactions and 298 metabolites. We validated the model with time-series data of duplicate fermentation cultures to aim for an integrated model describing the dynamic profiles measured in fermentations. The model predicted that 84% of cellular energy in a standard M. hyopneumoniae cultivation was used for non-growth associated maintenance and only 16% of cellular energy was used for growth and growth associated maintenance. Following a cycle of model-driven experimentation in dedicated fermentation experiments, we were able to increase the fraction of cellular energy used for growth through pyruvate addition to the medium. This increase in turn led to an increase in growth rate and a 2.3 times increase in the total biomass concentration reached after 3-4 days of fermentation, enhancing the productivity of the overall process. The model presented provides a solid basis to understand and further improve M. hyopneumoniae fermentation processes. Biotechnol. Bioeng. 2017;114: 2339-2347. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Role of isoenzyme M2 of pyruvate kinase in urothelial tumorigenesis.

    Science.gov (United States)

    Zhou, Haiping; Wang, Xing; Mo, Lan; Liu, Yan; He, Feng; Zhang, Fenglin; Huang, Kuo-How; Wu, Xue-Ru

    2016-04-26

    The conversion of precancerous lesions to full-fledged cancers requires the affected cells to surpass certain rate-limiting steps. We recently showed that activation of HRAS proto-oncogene in urothelial cells of transgenic mice causes simple urothelial hyperplasia (SUH) which is persistent and whose transition to low-grade papillary urothelial carcinoma (UC) must undergo nodular urothelial hyperplasia (NUH). We hypothesized that NUH, which has acquired fibrovascular cores, plays critical roles in mesenchymal-to-epithelial signaling, breaching the barriers of urothelial tumor initiation. Using proteomics involving two-dimensional gel electrophoresis, immunoblotting with pan-phosphotyrosine antibody and MALDI-mass spectrometry, we identified isoform 2 of pyruvate kinase (PKM2) as the major tyrosine-phosphorylated protein switched on during NUH. We extended this finding using specimens from transgenic mice, human UC and UC cell lines, establishing that PKM2, but not its spliced variant PKM1, was over-expressed in low-grade and, more prominently, high-grade UC. In muscle-invasive UC, PKM2 was co-localized with cytokeratins 5 and 14, UC progenitor markers. Specific inhibition of PKM2 by siRNA or shRNA suppressed UC cell proliferation via increased apoptosis, autophagy and unfolded protein response. These results strongly suggest that PKM2 plays an important role in the genesis of low-grade non-invasive and high-grade invasive urothelial carcinomas.

  2. Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

    Science.gov (United States)

    Salzillo, Marzia; Vastano, Valeria; Capri, Ugo; Muscariello, Lidia; Marasco, Rosangela

    2017-04-01

    Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

    Science.gov (United States)

    Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

    2017-07-14

    The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Posttranslational Modifications of Pyruvate Kinase M2: Tweaks that Benefit Cancer

    Directory of Open Access Journals (Sweden)

    Gopinath Prakasam

    2018-02-01

    Full Text Available Cancer cells rewire metabolism to meet biosynthetic and energetic demands. The characteristic increase in glycolysis, i.e., Warburg effect, now considered as a hallmark, supports cancer in various ways. To attain such metabolic reshuffle, cancer cells preferentially re-express the M2 isoform of pyruvate kinase (PKM2, M2-PK and alter its quaternary structure to generate less-active PKM2 dimers. The relatively inactive dimers cause the accumulation of glycolytic intermediates that are redirected into anabolic pathways. In addition, dimeric PKM2 also benefits cancer cells through various non-glycolytic moonlight functions, such as gene transcription, protein kinase activity, and redox balance. A large body of data have shown that several distinct posttranslation modifications (PTMs regulate PKM2 in a way that benefits cancer growth, e.g., formation of PKM2 dimers. This review discusses the recent advancements in our understanding of various PTMs and the benefits they impart to the sustenance of cancer. Understanding the PTMs in PKM2 is crucial to assess their therapeutic potential and to design novel anticancer strategies.

  5. Mitochondrial pyruvate carrier function determines cell stemness and metabolic reprogramming in cancer cells

    Science.gov (United States)

    Li, Xiaoran; Kan, Quancheng; Fan, Zhirui; Li, Yaqing; Ji, Yasai; Zhao, Jing; Zhang, Mingzhi; Grigalavicius, Mantas; Berge, Viktor; Goscinski, Mariusz Adam; M. Nesland, Jahn; Suo, Zhenhe

    2017-01-01

    One of the remarkable features of cancer cells is aerobic glycolysis, a phenomenon known as the “Warburg Effect”, in which cells rely preferentially on glycolysis instead of oxidative phosphorylation (OXPHOS) as the main energy source even in the presence of high oxygen tension. Cells with dysfunctional mitochondria are unable to generate sufficient ATP from mitochondrial OXPHOS, and then are forced to rely on glycolysis for ATP generation. Here we report our results in a prostate cancer cell line in which the mitochondrial pyruvate carrier 1 (MPC1) gene was knockout. It was discovered that the MPC1 gene knockout cells revealed a metabolism reprogramming to aerobic glycolysis with reduced ATP production, and the cells became more migratory and resistant to both chemotherapy and radiotherapy. In addition, the MPC1 knockout cells expressed significantly higher levels of the stemness markers Nanog, Hif1α, Notch1, CD44 and ALDH. To further verify the correlation of MPC gene function and cell stemness/metabolic reprogramming, MPC inhibitor UK5099 was applied in two ovarian cancer cell lines and similar results were obtained. Taken together, our results reveal that functional MPC may determine the fate of metabolic program and the stemness status of cancer cells in vitro. PMID:28624784

  6. Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus

    International Nuclear Information System (INIS)

    Suzuki, Kenichiro; Ito, Sohei; Shimizu-Ibuka, Akiko; Sakai, Hiroshi

    2005-01-01

    This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6 2 22 and diffracted to a resolution of 2.4 Å. Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. A crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6 2 22, with unit-cell parameters a = b = 145.97, c = 118.03 Å

  7. Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.

    Science.gov (United States)

    Modig, Tobias; Lidén, Gunnar; Taherzadeh, Mohammad J

    2002-01-01

    The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition. PMID:11964178

  8. Ameliorative effect of ethyl pyruvate in neuropathic pain induced by chronic constriction injury of sciatic nerve

    Directory of Open Access Journals (Sweden)

    Varsha J. Bansode

    2014-01-01

    Full Text Available Objective: The present study was designed to investigate the ameliorative effects of ethyl pyruvate (EP in chronic constriction injury (CCI-induced painful neuropathy in rats. Materials and Methods: EP 50 and 100 mg/kg was administered for 21 consecutive days starting from the day of surgery. The effects of EP in the paw pressure, acetone drop, and tail heat immersion tests were assessed, reflecting the degree of mechanical hyperalgesia, cold allodynia, and spinal thermal sensation, respectively. Axonal degeneration of the sciatic nerve was assessed histopathologically. The levels of thiobarbituric acid reactive species, reduced glutathione (GSH, catalase (CAT, and superoxide dismutase (SOD were determined to assess oxidative stress. Key Findings: Administration of 50 and 100 mg/kg EP attenuated the reduction of nociceptive threshold in the paw pressure, acetone drop, and tail heat immersion tests. EP 100 mg/kg significantly attenuated reactive changes in histopathology and increase in oxidative stress. Conclusion: EP 100 mg/kg showed beneficial activity against nerve trauma-induced neuropathy. Hence, it can be used as a better treatment option in neuropathic pain (NP. The observed antinociceptive effects of EP may possibly be attributed to its antioxidant and anti-inflammatory activity.

  9. Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

    Science.gov (United States)

    Song, Hyun-Ouk

    2016-02-01

    Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

  10. Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

    Science.gov (United States)

    Lazar, Nathaniel; Fay, Allison; Nandakumar, Madhumitha; Boyle, Kerry E; Xavier, Joao; Rhee, Kyu; Glickman, Michael S

    2017-12-01

    Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism. © 2017 John Wiley & Sons Ltd.

  11. Functional Characterization of Waterlogging and Heat Stresses Tolerance Gene Pyruvate decarboxylase 2 from Actinidia deliciosa

    Directory of Open Access Journals (Sweden)

    Hui-Ting Luo

    2017-11-01

    Full Text Available A previous report showed that both Pyruvate decarboxylase (PDC genes were significantly upregulated in kiwifruit after waterlogging treatment using Illumina sequencing technology, and that the kiwifruit AdPDC1 gene was required during waterlogging, but might not be required during other environmental stresses. Here, the function of another PDC gene, named AdPDC2, was analyzed. The expression of the AdPDC2 gene was determined using qRT-PCR, and the results showed that the expression levels of AdPDC2 in the reproductive organs were much higher than those in the nutritive organs. Waterlogging, NaCl, and heat could induce the expression of AdPDC2. Overexpression of kiwifruit AdPDC2 in transgenic Arabidopsis enhanced resistance to waterlogging and heat stresses in five-week-old seedlings, but could not enhance resistance to NaCl and mannitol stresses at the seed germination stage and in early seedlings. These results suggested that the kiwifruit AdPDC2 gene may play an important role in waterlogging resistance and heat stresses in kiwifruit.

  12. Brain Glycogenolysis, Adrenoceptors, Pyruvate Carboxylase, Na+,K+-ATPase and Marie E. Gibbs’ Pioneering Learning Studies

    Directory of Open Access Journals (Sweden)

    Leif eHertz

    2013-04-01

    Full Text Available The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., JCBFM, in press. According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present ‘perspective’ suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either i activation of astrocytic receptors, perhaps especially beta-adrenergic, or ii even small increases in extracellular K+ concentration above its normal resting level. It discusses i the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; ii the established role of K+-stimulated glycogenolysis for K+ uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K+ homeostasis in the brain in vivo; and iii the plausible role of transmitter-induced glycogenolysis, stimulating Na+,K+-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K+ concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP.

  13. Acute hypertensive stress imaged by cardiac hyperpolarized [1-C]pyruvate magnetic resonance

    DEFF Research Database (Denmark)

    Tougaard, Rasmus Stilling; Hansen, Esben Søvsø Szocska; Laustsen, Christoffer

    2018-01-01

    PURPOSE: Deranged metabolism is now recognized as a key causal factor in a variety of heart diseases, and is being studied extensively. However, invasive methods may alter metabolism, and conventional imaging techniques measure tracer uptake but not downstream metabolism. These challenges may...... be overcome by hyperpolarized MR, a noninvasive technique currently crossing the threshold into human trials. The aim of this study was to image metabolic changes in the heart in response to endogastric glucose bolus and to acute hypertension. METHODS: Five postprandial pigs were scanned with hyperpolarized.......008) and ejection fraction decreased from 54 ± 2% to 47 ± 6% (P = 0.03) The hemodynamic changes were accompanied by increases in the hyperpolarized [1-13C]pyruvate MR derived ratios of lactate/alanine (from 0.58 ± 0.13 to 0.78 ± 0.06, P = 0.03) and bicarbonate/alanine (from 0.55 ± 0.12 to 0.91 ± 0.14, P = 0...

  14. SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma.

    Science.gov (United States)

    Tai, Wei-Tien; Hung, Man-Hsin; Chu, Pei-Yi; Chen, Yao-Li; Chen, Li-Ju; Tsai, Ming-Hsien; Chen, Min-Husan; Shiau, Chung-Wai; Boo, Yin-Pin; Chen, Kuen-Feng

    2016-04-19

    Pyruvate kinase M2 (PKM2) is known to promote tumourigenesis through dimer formation of p-PKM2Y105. Here, we investigated whether SH2-containing protein tyrosine phosphatase 1 (SHP-1) decreases p-PKM2Y105 expression and, thus, determines the sensitivity of sorafenib through inhibiting the nuclear-related function of PKM2. Immunoprecipitation and immunoblot confirmed the effect of SHP-1 on PKM2Y105 dephosphorylation. Lactate production was assayed in cells and tumor samples to determine whether sorafenib reversed the Warburg effect. Clinical hepatocellular carcinoma (HCC) tumor samples were assessed for PKM2 expression. SHP-1 directly dephosphorylated PKM2 at Y105 and further decreased the proliferative activity of PKM2; similar effects were found in sorafenib-treated HCC cells. PKM2 was also found to determine the sensitivity of targeted drugs, such as sorafenib, brivanib, and sunitinib, by SHP-1 activation. Significant sphere-forming activity was found in HCC cells stably expressing PKM2. Clinical findings suggest that PKM2 acts as a predicting factor of early recurrence in patients with HCC, particularly those without known risk factors (63.6%). SHP-1 dephosphorylates PKM2 at Y105 to inhibit nuclear function of PKM2 and determines the efficacy of targeted drugs. Targeting PKM2 by SHP-1 might provide new therapeutic insights for patients with HCC.

  15. Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

    International Nuclear Information System (INIS)

    Voss, Jarrod E.; Scally, Stephen W.; Taylor, Nicole L.; Dogovski, Con; Alderton, Malcolm R.; Hutton, Craig A.; Gerrard, Juliet A.; Parker, Michael W.; Dobson, Renwick C. J.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyses an important step in lysine biosynthesis. Here, the expression, purification, crystallization and preliminary diffraction analysis to 2.15 Å resolution of DHDPS from B. anthracis soaked with the substrate pyruvate are reported. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 Å are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme

  16. Serial changes in selected serum constituents in low birth weight infants on peripheral parenteral nutrition with different zinc and copper supplements.

    Science.gov (United States)

    Lockitch, G; Pendray, M R; Godolphin, W J; Quigley, G

    1985-07-01

    One hundred and five infants of birth weight 2000 g or less who received peripherally administered parenteral nutrition for periods of three or more weeks, were randomly assigned to groups receiving different amounts of zinc and copper supplement. The blood concentrations of zinc, copper, retinol-binding protein, prealbumin, alkaline phosphatase and aspartate transaminase were followed weekly. Mean serum zinc, retinol-binding protein and prealbumin declined significantly over time while alkaline phosphatase rose. Only the group receiving the highest zinc supplement maintained a mean serum zinc concentration within the normal range at seven weeks. No difference in the protein or enzyme concentrations was found between the different zinc supplement groups. No difference was seen in serum copper or ceruloplasmin between copper dose groups although one intravenous supplement was double that of the other.

  17. Attenuation of Methotrexate-Induced Embryotoxicity and Oxidative Stress by Ethyl Pyruvate

    Directory of Open Access Journals (Sweden)

    Najafi Gholamreza

    2016-07-01

    Full Text Available Background Methotrexate (MTX, as an anti-folate agent, is widely used in the treatment of rheumatic disorders and malignant tumors, however it damages reproductive sys- tem in mice. The aim of this research was to study the effects of ethyl pyruvate (EP on embryo development and oxidative stress changes in the testis of mice treated with MTX. Materials and Methods In this experimental study, thirty-two adult male Naval Medical Research Institute mice, with average weight of 26 ± 2 g, were divided into four groups. The first group (control received distilled water (0.1 ml/mice/day, while the second group was intraperitoneally (IP treated with 20 mg/kg MTX once per week. The third group was IP treated with 40 mg/kg/day EP, and the fourth group was IP treated with both 20 mg/kg MTX and 40 mg/kg/day EP for 30 days. At the end of treatment fertilization rate and embryonic development were evaluated. Differences between these groups were assessed by ANOVA using the SPSS software package for Windows with a Tukey-Kramer multiple post-hoc comparison test. Results MTX treatment caused significant (P<0.05 increase in malondialdehyde (MDA and reduced catalase (CAT, as well as leading to in vitro fertilization (IVF and embryonic development. The improved effects of EP on the IVF were determined by the reduced level of MDA (index of oxidative stress and significant increased level of CAT (a key antioxidant. We observed significant increase in fertilization rate and embryonic development in the treated group with both MTX and EP. Conclusion It is suggested that EP can be useful in ameliorating testicular damages and embryotoxicity induced by MTX. These effects could be attributed to its antioxidant properties.

  18. miR-122 targets pyruvate kinase M2 and affects metabolism of hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Angela M Liu

    Full Text Available In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg's effect, with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122 is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC. Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK gene showed the strongest anti-correlation coefficient (Pearson r = -0.6938, p = <0.0001. In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2 is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.

  19. An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor.

    Science.gov (United States)

    Yuan, Meng; McNae, Iain W; Chen, Yiyuan; Blackburn, Elizabeth A; Wear, Martin A; Michels, Paul A M; Fothergill-Gilmore, Linda A; Hupp, Ted; Walkinshaw, Malcolm D

    2018-05-10

    We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer-monomer dissociation K d is estimated to be ~0.9 µM with a slow dissociation rate (t 1/2 ~ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R-states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such 'allostatic' regulation may be important in metabolic reprogramming and influencing cell fate. ©2018 The Author(s).

  20. The effect of ethyl pyruvate on oxidative stress in intestine and bacterial translocation after thermal injury.

    Science.gov (United States)

    Karabeyoğlu, Melih; Unal, Bülent; Bozkurt, Betül; Dolapçi, Iştar; Bilgihan, Ayşe; Karabeyoğlu, Işil; Cengiz, Omer

    2008-01-01

    Thermal injury causes a breakdown in the intestinal mucosal barrier due to ischemia reperfusion injury, which can induce bacterial translocation (BT), sepsis, and multiple organ failure in burn patients. The aim of this study was to investigate the effect of ethyl pyruvate (EP) on intestinal oxidant damage and BT in burn injury. Thirty-two rats were randomly divided into four groups. The sham group was exposed to 21 degrees C water and injected intraperitoneal with saline (1 mL/100 g). The sham + EP group received EP (40 mg/kg) intraperitoneally 6 h after the sham procedure. The burn group was exposed to thermal injury and given intraperitoneal saline injection (1 mL/100 g). The burn + EP group received EP (40 mg/kg) intraperitoneally 6 h after thermal injury. Twenty-four hours later, tissue samples were obtained from mesenteric lymph nodes, spleen, and liver for microbiological analysis and ileum samples were harvested for biochemical analysis. Thermal injury caused severe BT in burn group. EP supplementation decreased BT in mesenteric lymph nodes and spleen in the burn + EP group compared with the burn group (P < 0.05). Also, burn caused BT in liver, but this finding was not statistically significant among all groups. Thermal injury caused a statistically significant increase in malondialdehyde and myeloperoxidase levels, and EP prevented this effects in the burn + EP group compared with the burn group (P < 0.05). Our data suggested that EP can inhibit the BT and myeloperoxidase and malondialdehyde production in intestine following thermal injury, suggesting anti-inflammatory and anti-oxidant properties of EP.

  1. Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells.

    Directory of Open Access Journals (Sweden)

    Ansaya Thonpho

    Full Text Available Pyruvate carboxylase (PC is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5'-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an activator element(s for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y, as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription.

  2. Reduced brain/serum glucose ratios predict cerebral metabolic distress and mortality after severe brain injury.

    Science.gov (United States)

    Kurtz, Pedro; Claassen, Jan; Schmidt, J Michael; Helbok, Raimund; Hanafy, Khalid A; Presciutti, Mary; Lantigua, Hector; Connolly, E Sander; Lee, Kiwon; Badjatia, Neeraj; Mayer, Stephan A

    2013-12-01

    The brain is dependent on glucose to meet its energy demands. We sought to evaluate the potential importance of impaired glucose transport by assessing the relationship between brain/serum glucose ratios, cerebral metabolic distress, and mortality after severe brain injury. We studied 46 consecutive comatose patients with subarachnoid or intracerebral hemorrhage, traumatic brain injury, or cardiac arrest who underwent cerebral microdialysis and intracranial pressure monitoring. Continuous insulin infusion was used to maintain target serum glucose levels of 80-120 mg/dL (4.4-6.7 mmol/L). General linear models of logistic function utilizing generalized estimating equations were used to relate predictors of cerebral metabolic distress (defined as a lactate/pyruvate ratio [LPR] ≥ 40) and mortality. A total of 5,187 neuromonitoring hours over 300 days were analyzed. Mean serum glucose was 133 mg/dL (7.4 mmol/L). The median brain/serum glucose ratio, calculated hourly, was substantially lower (0.12) than the expected normal ratio of 0.40 (brain 2.0 and serum 5.0 mmol/L). In addition to low cerebral perfusion pressure (P = 0.05) and baseline Glasgow Coma Scale score (P brain/serum glucose ratios below the median of 0.12 were independently associated with an increased risk of metabolic distress (adjusted OR = 1.4 [1.2-1.7], P brain/serum glucose ratios were also independently associated with in-hospital mortality (adjusted OR = 6.7 [1.2-38.9], P brain/serum glucose ratios, consistent with impaired glucose transport across the blood brain barrier, are associated with cerebral metabolic distress and increased mortality after severe brain injury.

  3. [The National Serum Bank].

    Science.gov (United States)

    Magos-López, C; Sánchez-Villarreal, F; Gutiérrez, G; Tapia-Conyer, R

    1992-01-01

    A National Serum Bank was established to store sera obtained during the National Seroepidemiological Survey performed in Mexico in 1987. More than 70,000 serum samples were obtained from subjects of either sex 1-99 years of age in each of the 32 states of the country. The current collection of sera includes 28,704 male samples and 40,629 female samples. This paper describes the procedures for handling serum samples, including reception registry, storage and distribution to several laboratories for detection of measles, rubella, poliomyelitis, AIDS, diphtheria, pertussis, tetanus, brucella, salmonella, amoeba, toxoplasma, American trypanosomiasis and cysticercus. Determinations of total cholesterol were also made in order to describe its distribution and to identify the prevalence of hypercholesterolemia.

  4. Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogues

    Science.gov (United States)

    Lietzan, Adam D.; St. Maurice, Martin

    2014-01-01

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates. PMID:24157795

  5. GABAergic transmission and chloride equilibrium potential are not modulated by pyruvate in the developing optic tectum of Xenopus laevis tadpoles.

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    Arseny S Khakhalin

    Full Text Available In the developing mammalian brain, gamma-aminobutyric acid (GABA is thought to play an excitatory rather than an inhibitory role due to high levels of intracellular Cl(- in immature neurons. This idea, however, has been questioned by recent studies which suggest that glucose-based artificial cerebrospinal fluid (ACSF may be inadequate for experiments on immature and developing brains. These studies suggest that immature neurons may require alternative energy sources, such as lactate or pyruvate. Lack of these other energy sources is thought to result in artificially high intracellular Cl(- concentrations, and therefore a more depolarized GABA receptor (GABAR reversal potential. Since glucose metabolism can vary widely among different species, it is important to test the effects of these alternative energy sources on different experimental preparations. We tested whether pyruvate affects GABAergic transmission in isolated brains of developing wild type Xenopus tadpoles in vitro by recording the responsiveness of tectal neurons to optic nerve stimulation, and by measuring currents evoked by local GABA application in a gramicidin perforated patch configuration. We found that, in contrast with previously reported results, the reversal potential for GABAR-mediated currents does not change significantly between developmental stages 45 and 49. Partial substitution of glucose by pyruvate had only minor effects on both the GABA reversal potential, and the responsiveness of tectal neurons at stages 45 and 49. Total depletion of energy sources from the ACSF did not affect neural responsiveness. We also report a strong spatial gradient in GABA reversal potential, with immature cells adjacent to the lateral and caudal proliferative zones having more positive reversal potentials. We conclude that in this experimental preparation standard glucose-based ACSF is an appropriate extracellular media for in vitro experiments.

  6. Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

    Science.gov (United States)

    Choutko, Alexandra; Eichenberger, Andreas P; Gunsteren, Wilfred F; Dolenc, Jožica

    2013-01-01

    The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues. PMID:23595942

  7. Exploration of swapping enzymatic function between two proteins: a simulation study of chorismate mutase and isochorismate pyruvate lyase.

    Science.gov (United States)

    Choutko, Alexandra; Eichenberger, Andreas P; van Gunsteren, Wilfred F; Dolenc, Jožica

    2013-06-01

    The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues. © 2013 The Protein Society.

  8. Pyruvate dehydrogenase complexes from the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum, helminths exhibiting anaerobic mitochondrial metabolism.

    Science.gov (United States)

    Diaz, F; Komuniecki, R W

    1994-10-01

    The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from 2 parasitic helminths exhibiting anaerobic mitochondrial metabolism, the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum. The P. equorum PDC yielded 7 major bands when separated by SDS-PAGE. The bands of 72, 55-53.5, 41 and 36 kDa corresponded to E2, E3, E1 alpha and E1 beta, respectively. The complex also contained additional unidentified proteins of 43 and 45 kDa. Incubation of the complex with [2-14C]pyruvate resulted in the acetylation of only E2. These results suggest that the P. equorum PDC lacks protein X and exhibits an altered subunit composition, as has been described previously for the PDC of the related nematode, Ascaris suum. In contrast, the D. caninum PDC yielded only four major bands after SDS-PAGE of 59, 58, 39 and 34 kDa, which corresponded to E3, E2, E1 alpha and E1 beta, respectively. Incubation of the D. caninum complex with [2-14C]pyruvate resulted in the acetylation of E2 and a second protein which comigrated with E3, suggesting that the D. caninum complex contained protein X and had a subunit composition similar to PDCs from other eukaryotic organisms. Both helminth complexes appeared less sensitive to inhibition by elevated NADH/NAD+ ratios than complexes isolated from aerobic organisms, as would be predicted for PDCs from organisms exploiting microaerobic habitats. These results suggest that although these helminths have similar anaerobic mitochondrial pathways, they contain significantly different PDCs.

  9. Effects of hypoxia and pyruvate infusion on myocardial fatty acid oxidation measured with 123I heptadecanoic acid

    International Nuclear Information System (INIS)

    Comans, E.F.I.; Visser, F.C.; Elzinga, Gijs

    1993-01-01

    Radio-iodinated fatty acids like 123 I heptadecanoic acid (HDA) can be used for the non-invasive delineation of myocardial non-esterified fatty acid (FA) metabolism. In this study the quantitative value of HDA was assessed for the measurement of myocardial FA oxidation. In an isolated saline perfused rat heart preparation myocardial time-activity curves were made during control perfusion and after inhibition of FA oxidation by hypoxia and infusion of 10.0 mM pyruvate, respectively. Control experiments were performed using 1- 14 C palmitate as the 'golden standard' for myocardial FA oxidation. Myocardial HDA oxidation was calculated from the amplitude of the third exponential term of the time-activity curve. During control perfusion no differences were observed between the calculated oxygen equivalents (from HDA oxidation) and the measured (A-V oxygen content difference) and the estimated ( 14 CO 2 production) values. Inhibition of palmitate oxidation with pyruvate was accurately detected with HDA. During hypoxic perfusion, an overestimation of palmitate oxidation was calculated on the basic of HDA oxidation. Infusion of pyruvate did not influence the time constants of the time-activity curves, whereas during hypoxic perfusion an increase of the time constant of the third exponential term was observed, probably caused by the presence of back-diffusion of non-metabolized HDA. We conclude that HDA can be used as a quantitative tool for the measurement of myocardial FA oxidation under various metabolic conditions. During periods of a decreased oxygen availability back-diffusion of FA needs to be taken into account for the interpretation of the myocardial time-activity curves. (author)

  10. Gluconeogenesis is associated with high rates of tricarboxylic acid and pyruvate cycling in fasting northern elephant seals.

    Science.gov (United States)

    Champagne, Cory D; Houser, Dorian S; Fowler, Melinda A; Costa, Daniel P; Crocker, Daniel E

    2012-08-01

    Animals that endure prolonged periods of food deprivation preserve vital organ function by sparing protein from catabolism. Much of this protein sparing is achieved by reducing metabolic rate and suppressing gluconeogenesis while fasting. Northern elephant seals (Mirounga angustirostris) endure prolonged fasts of up to 3 mo at multiple life stages. During these fasts, elephant seals maintain high levels of activity and energy expenditure associated with breeding, reproduction, lactation, and development while maintaining rates of glucose production typical of a postabsorptive mammal. Therefore, we investigated how fasting elephant seals meet the requirements of glucose-dependent tissues while suppressing protein catabolism by measuring the contribution of glycogenolysis, glycerol, and phosphoenolpyruvate (PEP) to endogenous glucose production (EGP) during their natural 2-mo postweaning fast. Additionally, pathway flux rates associated with the tricarboxylic acid (TCA) cycle were measured specifically, flux through phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate cycling. The rate of glucose production decreased during the fast (F(1,13) = 5.7, P = 0.04) but remained similar to that of postabsorptive mammals. The fractional contributions of glycogen, glycerol, and PEP did not change with fasting; PEP was the primary gluconeogenic precursor and accounted for ∼95% of EGP. This large contribution of PEP to glucose production occurred without substantial protein loss. Fluxes through the TCA cycle, PEPCK, and pyruvate cycling were higher than reported in other species and were the most energetically costly component of hepatic carbohydrate metabolism. The active pyruvate recycling fluxes detected in elephant seals may serve to rectify gluconeogeneic PEP production during restricted anaplerotic inflow in these fasting-adapted animals.

  11. Quantification of in vivo metabolic kinetics of hyperpolarized pyruvate in rat kidneys using dynamic 13C MRSI.

    Science.gov (United States)

    Xu, Tao; Mayer, Dirk; Gu, Meng; Yen, Yi-Fen; Josan, Sonal; Tropp, James; Pfefferbaum, Adolf; Hurd, Ralph; Spielman, Daniel

    2011-10-01

    With signal-to-noise ratio enhancements on the order of 10,000-fold, hyperpolarized MRSI of metabolically active substrates allows the study of both the injected substrate and downstream metabolic products in vivo. Although hyperpolarized [1-(13)C]pyruvate, in particular, has been used to demonstrate metabolic activities in various animal models, robust quantification and metabolic modeling remain important areas of investigation. Enzyme saturation effects are routinely seen with commonly used doses of hyperpolarized [1-(13)C]pyruvate; however, most metrics proposed to date, including metabolite ratios, time-to-peak of metabolic products and single exchange rate constants, fail to capture these saturation effects. In addition, the widely used small-flip-angle excitation approach does not correctly model the inflow of fresh downstream metabolites generated proximal to the target slice, which is often a significant factor in vivo. In this work, we developed an efficient quantification framework employing a spiral-based dynamic spectroscopic imaging approach. The approach overcomes the aforementioned limitations and demonstrates that the in vivo (13)C labeling of lactate and alanine after a bolus injection of [1-(13)C]pyruvate is well approximated by saturatable kinetics, which can be mathematically modeled using a Michaelis-Menten-like formulation, with the resulting estimated apparent maximal reaction velocity V(max) and apparent Michaelis constant K(M) being unbiased with respect to critical experimental parameters, including the substrate dose, bolus shape and duration. Although the proposed saturatable model has a similar mathematical formulation to the original Michaelis-Menten kinetics, it is conceptually different. In this study, we focus on the (13)C labeling of lactate and alanine and do not differentiate the labeling mechanism (net flux or isotopic exchange) or the respective contribution of various factors (organ perfusion rate, substrate transport

  12. Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria

    Directory of Open Access Journals (Sweden)

    Masome Zeinali

    2016-09-01

    Full Text Available Introduction: Ethanol made by a biomass is one of the useful strategies in terms of economic and environmental and as a clean and safe energy to replace fossil fuels considered and examined. Materials and methods: In this study, key enzyme in the production of ethanol (Pyruvate decarboxylase from Zymomonas mobilis bacteria was isolated and cloned at E. coli bacteria by freeze and thaw method. For gene cloning, we used specific primers of pdc and PCR reaction and then pdc gene isolated and pET 28a plasmid double digested with (Sal I and Xho I enzymes. Digestion Products were ligated by T4 DNA ligase in 16 °C for 16 hours. Results: Results of bacteria culture showed that a few colonies containing pET 28a plasmid could grow. Result of colony pcr of pdc gene with specific primers revealed 1700 bp bands in 1% agarose gel electrophoresis. The results of PCR with T7 promotor forward primer and pdc revers primer have proved the accurate direction of integration of pdc gene into plasmid and revealed 1885 bp band. Double digestion of recombinant plasmid with SalI and XhoI enzymes revealed same bands. Finally, RT showed the expected band of 1700 bp that implies the desired gene expression in the samples. Discussion and conclusion: Due to the increased production of ethanol via pyruvate decarboxylase gene cloning in expression plasmids with a strong promoter upstream of the cloning site can conclude that, pyruvate decarboxylase cloning as a key gene would be useful and according to beneficial properties of E. coli bacteria, transfering the gene to bacteria appears to be reasonable.

  13. The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognathi.

    Science.gov (United States)

    Lin, J W; Lu, H C; Chen, H Y; Weng, S F

    1997-10-09

    Partial 3'-end nucleotide sequence of the pkI gene (GenBank accession No. AF019143) from Photobacterium leiognathi ATCC 25521 has been determined, and the encoded pyruvate kinase I is deduced. Pyruvate kinase I is the key enzyme of glycolysis, which converts phosphoenol pyruvate to pyruvate. Alignment and comparison of pyruvate kinase Is from P. leiognathi, E. coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiognathi. The gene order of the pkI and luxZ genes is-pk1-ter-->-R&R"-luxZ-ter"-->, whereas ter is transcriptional terminator for the pkI and related genes, and R&R" is the regulatory region and ter" is transcriptional terminator for the luxZ gene. It clearly elicits that the pkI gene and luxZ gene are divided to two operons. Functional analysis confirms that the potential hairpin loop omega T is the transcriptional terminator for the pkI and related genes. It infers that the pkI and related genes are simply linked to the luxZ gene in P. leiognathi genome.

  14. A Comparison between Radiolabeled Fluorodeoxyglucose Uptake and Hyperpolarized 13C-Labeled Pyruvate Utilization as Methods for Detecting Tumor Response to Treatment

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    Timothy H. Witney

    2009-06-01

    Full Text Available Detection of early tumor responses to treatment can give an indication of clinical outcome. Positron emission tomography measurements of the uptake of the glucose analog, [18F] 2-fluoro-2-deoxy-d-glucose (FDG, have demonstrated their potential for detecting early treatment response in the clinic. We have shown recently that 13C magnetic resonance spectroscopy and spectroscopic imaging measurements of the uptake and conversion of hyperpolarized [1-13C]pyruvate into [1-13C]lactate can be used to detect treatment response in a murine lymphoma model. The present study compares these magnetic resonance measurements with changes in FDG uptake after chemotherapy. A decrease in FDG uptake was found to precede the decrease in flux of hyperpolarized 13C label between pyruvate and lactate, both in tumor cells in vitro and in tumors in vivo. However, the magnitude of the decrease in FDG uptake and the decrease in pyruvate to lactate flux was comparable at 24 hours after drug treatment. In cells, the decrease in FDG uptake was shown to correlate with changes in plasma membrane expression of the facilitative glucose transporters, whereas the decrease in pyruvate to lactate flux could be explained by an increase in poly(ADP-ribose polymerase activity and subsequent depletion of the NAD(H pool. These results show that measurement of flux between pyruvate and lactate may be an alternative to FDG-positron emission tomography for imaging tumor treatment response in the clinic.

  15. Growth of Campylobacter incubated aerobically in fumarate-pyruvate media or media supplemented with dairy, meat, or soy extracts and peptones.

    Science.gov (United States)

    Hinton, Arthur

    2016-09-01

    The ability of Campylobacter to grow aerobically in media supplemented with fumarate-pyruvate or with dairy, meat, or soy extracts or peptones was examined. Optical densities (OD) of Campylobacter cultured in basal media, media supplemented with fumarate-pyruvate or with 1.0, 2.5, 5.0, or 7.5% beef extract was measured. Growth was also compared in media supplemented with other extracts or peptones. Finally, cfu/mL of Campylobacter recovered from basal media or media supplemented with fumarate-pyruvate, casamino acids, beef extract, soytone, or beef extract and soytone was determined. Results indicated that OD of cultures grown in media supplemented with fumarate-pyruvate or with 5.0 or 7.5% beef extract were higher than OD of isolates grown in basal media or media supplemented with lower concentrations of beef extract. Highest OD were produced by isolates grown in media supplemented with beef extract, peptone from meat, polypeptone, proteose peptone, or soytone. Also, more cfu/mL were recovered from media with fumarate-pyruvate, beef extract, soytone, or beef extract-soytone than from basal media or media with casamino acids. Findings indicate that media supplemented with organic acids, vitamins, and minerals and media supplemented with extracts or peptones containing these metabolites can support aerobic growth of Campylobacter. Published by Elsevier Ltd.

  16. Metabolic fate of glucose in rats with traumatic brain injury and pyruvate or glucose treatments: A NMR spectroscopy study.

    Science.gov (United States)

    Shijo, Katsunori; Sutton, Richard L; Ghavim, Sima S; Harris, Neil G; Bartnik-Olson, Brenda L

    2017-01-01

    Administration of sodium pyruvate (SP; 9.08 μmol/kg, i.p.), ethyl pyruvate (EP; 0.34 μmol/kg, i.p.) or glucose (GLC; 11.1 μmol/kg, i.p.) to rats after unilateral controlled cortical impact (CCI) injury has been reported to reduce neuronal loss and improve cerebral metabolism. In the present study these doses of each fuel or 8% saline (SAL; 5.47 nmoles/kg) were administered immediately and at 1, 3, 6 and 23 h post-CCI. At 24 h all CCI groups and non-treated Sham injury controls were infused with [1,2 13 C] glucose for 68 min 13 C nuclear magnetic resonance (NMR) spectra were obtained from cortex + hippocampus tissues from left (injured) and right (contralateral) hemispheres. All three fuels increased lactate labeling to a similar degree in the injured hemisphere. The amount of lactate labeled via the pentose phosphate and pyruvate recycling (PPP + PR) pathway increased in CCI-SAL and was not improved by SP, EP, and GLC treatments. Oxidative metabolism, as assessed by glutamate labeling, was reduced in CCI-SAL animals. The greatest improvement in oxidative metabolism was observed in animals treated with SP and fewer improvements after EP or GLC treatments. Compared to SAL, all three fuels restored glutamate and glutamine labeling via pyruvate carboxylase (PC), suggesting improved astrocyte metabolism following fuel treatment. Only SP treatments restored the amount of [4 13 C] glutamate labeled by the PPP + PR pathway to sham levels. Milder injury effects in the contralateral hemisphere appear normalized by either SP or EP treatments, as increases in the total pool of 13 C lactate and labeling of lactate in glycolysis, or decreases in the ratio of PC/PDH labeling of glutamine, were found only for CCI-SAL and CCI-GLC groups compared to Sham. The doses of SP, EP and GLC examined in this study all enhanced lactate labeling and restored astrocyte-specific PC activity but differentially affected neuronal metabolism after CCI injury. The restoration of

  17. Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

    International Nuclear Information System (INIS)

    Lodato, D.T.; Reed, G.H.

    1987-01-01

    The 2 equiv of divalent cation that are required cofactors for pyruvate kinase reside in sites of different affinities for different species of cation. The intrinsic selectivity of the protein-based site for Mn(II) and of the nucleotide-based site for Mg(II) has been exploited in electron paramagnetic resonance (EOR) investigations of ligands for Mn(II) at the protein-based site. Oxalate, a structural analogue of the enolate of pyruvate, has been used as a surrogate for the reactive form of pyruvate in complexes with enzyme, Mn(II), Mg(II), and ATP. Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of 17 O, specifically incorporated into oxalate, shows that oxalate is bound at the active site as a bidentate chelate with Mn(II). Coordination of the γ-phosphate of ATP to this same Mn(II) center is revealed by observation of superhyperfine coupling from 17 O regiospecifically incorporated into the γ-phosphate group of ATP. By contrast, 17 O in the α-phosphate or in the β-phosphate groups of ATP does not influence the spectrum. Experiments in 17 O-enriched water show that there is also a single water ligand bound to the Mn(II). These data indicate that ATP bridges Mn(II) and Mg(II) at the active site. A close spacing of the two divalent cations is also evident from the occurrence of magnetic interactions for complexes in which 2 equiv of Mn(II) are present at the active site. The structure for the enzyme-Mn(II)-oxalate-Mg(II)-ATP complex suggests a scheme for the normal reverse reaction of pyruvate kinase in which the divalent cation at the protein-based site activates the keto acid substrate through chelation and promotes phospho transfer by simultaneous coordination to the enolate oxygen and to a pendant oxygen from the γ-phosphate of ATP

  18. Neonatal pyruvate dehydrogenase deficiency due to a R302H mutation in the PDHA1 gene: MRI findings

    International Nuclear Information System (INIS)

    Soares-Fernandes, Joao P.; Ribeiro, Manuel; Magalhaes, Zita; Rocha, Jaime F.; Teixeira-Gomes, Roseli; Cruz, Romeu; Leijser, Lara M.

    2008-01-01

    Pyruvate dehydrogenase (PDH) deficiency is one of the most common causes of congenital lactic acidosis. Correlations between the genetic defect and neuroimaging findings are lacking. We present conventional and diffusion-weighted MRI findings in a 7-day-old male neonate with PDH deficiency due to a mosaicism for the R302H mutation in the PDHA1 gene. Corpus callosum dysgenesis, widespread increased diffusion in the white matter, and bilateral subependymal cysts were the main features. Although confirmation of PDH deficiency depends on specialized biochemical analyses, neonatal MRI plays a role in evaluating the pattern and extent of brain damage, and potentially in early diagnosis and clinical decision making. (orig.)

  19. Coupling between the blood lactate-to-pyruvate ratio and MCA Vmean at the onset of exercise in humans

    DEFF Research Database (Denmark)

    Rasmussen, Peter; Madsen, Camilla A; Nielsen, Henning B

    2009-01-01

    Activation-induced increase in cerebral blood flow is coupled to enhanced metabolic activity, maybe with brain tissue redox state and oxygen tension as key modulators. To evaluate this hypothesis at the onset of exercise in humans, blood was sampled at 0.1 to 0.2 Hz from the radial artery and right...... internal jugular vein, while middle cerebral artery mean flow velocity (MCA V(mean)) was recorded. Both the arterial and venous lactate-to-pyruvate ratio increased after 10 s (P capillary...... state and oxygenation as potential modulators of an increase in cerebral blood flow at the onset of exercise....

  20. Decreased expression of pyruvate dehydrogenase A1 predicts an unfavorable prognosis in ovarian carcinoma.

    Science.gov (United States)

    Li, Yaqing; Huang, Ruixia; Li, Xiaoli; Li, Xiaoran; Yu, Dandan; Zhang, Mingzhi; Wen, Jianguo; Goscinski, Mariusz Adam; Trope, Claes G; Nesland, Jahn M; Suo, Zhenhe

    2016-01-01

    Pyruvate dehydrogenase A1 (PDHA1) serves as a gate-keeper enzyme link between glycolysis and the mitochondrial citric acid cycle. The inhibition of PDHA1 in cancer cells can result in an increased Warburg effect and a more aggressive phenotype in cancer cells. This study was conducted to investigate the expression of PDHA1 in ovarian cancer and the correlation between PDHA1 expression and the prognosis of patients. The PDHA1 protein expression in 3 ovarian cancer cell lines (OVCAR-3, SKOV-3 and ES-2) and 248 surgically removed ovarian carcinoma samples was immunocytochemically examined. Statistical analyses were performed to evaluate the correlations between PDHA1 expression and the clinicopathological characteristics of the patients as well as the predictive value of PDHA1. The results showed the presence of variable expression of PDHA1 in the three ovarian cancer cell lines. Of the 248 ovarian cancer tissue specimens, 45 cases (18.1%) were negative in tumor cells for PDHA1, 162 cases (65.3%) displayed a low expression level, and 41 cases (16.5%) had a relatively high PDHA1 staining. The expression of PDHA1 was associated with the histological subtype ( P =0.004) and FIGO stage ( P =0.002). The median OS time in the PDHA1 negative group, low expression group and high expression group were 0.939 years, 1.443 years and 9.900 years, respectively. The median PFS time in the above three groups were 0.287 years, 0.586 years and 9.900 years, respectively. Furthermore, the high expression of PDHA1 in ovarian carcinoma cells was significantly associated with better OS and PFS by statistical analyses. Multivariate analyses showed that PDHA1 expression was also an independent prognostic factor for higher OS in ovarian cancer patients (HR=0.705, 95% CI 0.541-0.918, P =0.01). Our study indicated that the decreased expression of PDHA1 might be an independent prognostic factor in unfavorable outcomes.

  1. Plasticity of the Pyruvate Node Modulates Hydrogen Peroxide Production and Acid Tolerance in Multiple Oral Streptococci.

    Science.gov (United States)

    Cheng, Xingqun; Redanz, Sylvio; Cullin, Nyssa; Zhou, Xuedong; Xu, Xin; Joshi, Vrushali; Koley, Dipankar; Merritt, Justin; Kreth, Jens

    2018-01-15

    Commensal Streptococcus sanguinis and Streptococcus gordonii are pioneer oral biofilm colonizers. Characteristic for both is the SpxB-dependent production of H 2 O 2 , which is crucial for inhibiting competing biofilm members, especially the cariogenic species Streptococcus mutans H 2 O 2 production is strongly affected by environmental conditions, but few mechanisms are known. Dental plaque pH is one of the key parameters dictating dental plaque ecology and ultimately oral health status. Therefore, the objective of the current study was to characterize the effects of environmental pH on H 2 O 2 production by S. sanguinis and S. gordonii S. sanguinis H 2 O 2 production was not found to be affected by moderate changes in environmental pH, whereas S. gordonii H 2 O 2 production declined markedly in response to lower pH. Further investigation into the pyruvate node, the central metabolic switch modulating H 2 O 2 or lactic acid production, revealed increased lactic acid levels for S. gordonii at pH 6. The bias for lactic acid production at pH 6 resulted in concomitant improvement in the survival of S. gordonii at low pH and seems to constitute part of the acid tolerance response of S. gordonii Differential responses to pH similarly affect other oral streptococcal species, suggesting that the observed results are part of a larger phenomenon linking environmental pH, central metabolism, and the capacity to produce antagonistic amounts of H 2 O 2 IMPORTANCE Oral biofilms are subject to frequent and dramatic changes in pH. S. sanguinis and S. gordonii can compete with caries- and periodontitis-associated pathogens by generating H 2 O 2 Therefore, it is crucial to understand how S. sanguinis and S. gordonii adapt to low pH and maintain their competitiveness under acid stress. The present study provides evidence that certain oral bacteria respond to environmental pH changes by tuning their metabolic output in favor of lactic acid production, to increase their acid survival

  2. Enhanced pyruvate dehydrogenase activity improves cardiac outcomes in a murine model of cardiac arrest.

    Directory of Open Access Journals (Sweden)

    Lin Piao

    Full Text Available Post-ischemic changes in cellular metabolism alter myocardial and neurological function. Pyruvate dehydrogenase (PDH, the limiting step in mitochondrial glucose oxidation, is inhibited by increased expression of PDH kinase (PDK during ischemia/reperfusion injury. This results in decreased utilization of glucose to generate cellular ATP. Post-cardiac arrest (CA hypothermia improves outcomes and alters metabolism, but its influence on PDH and PDK activity following CA are unknown. We hypothesized that therapeutic hypothermia (TH following CA is associated with the inhibition of PDK activity and increased PDH activity. We further hypothesized that an inhibitor of PDK activity, dichloroacetate (DCA, would improve PDH activity and post-CA outcomes.Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 12-minute KCl-induced CA followed by cardiopulmonary resuscitation. Compared to normothermic (37°C CA controls, administering TH (30°C improved overall survival (72-hour survival rate: 62.5% vs. 28.6%, P<0.001, post-resuscitation myocardial function (ejection fraction: 50.9±3.1% vs. 27.2±2.0%, P<0.001; aorta systolic pressure: 132.7±7.3 vs. 72.3±3.0 mmHg, P<0.001, and neurological scores at 72-hour post CA (9.5±1.3 vs. 5.4±1.3, P<0.05. In both heart and brain, CA increased lactate concentrations (1.9-fold and 3.1-fold increase, respectively, P<0.01, decreased PDH enzyme activity (24% and 50% reduction, respectively, P<0.01, and increased PDK protein expressions (1.2-fold and 1.9-fold, respectively, P<0.01. In contrast, post-CA treatment with TH normalized lactate concentrations (P<0.01 and P<0.05 and PDK expressions (P<0.001 and P<0.05, while increasing PDH activity (P<0.01 and P<0.01 in both the heart and brain. Additionally, treatment with DCA (0.2 mg/g body weight 30 min prior to CA improved both myocardial hemodynamics 2 hours post-CA (aortic systolic pressure: 123±3 vs. 96±4 mmHg, P<0.001 and 72-hour survival rates

  3. Expression of pyruvate dehydrogenase is an independent prognostic marker in gastric cancer

    Science.gov (United States)

    Sun, Xu-Ren; Sun, Zhe; Zhu, Zhi; Guan, Hai-Xia; Li, Chen-Yan; Zhang, Jun-Yan; Zhang, Yi-Ning; Zhou, Huan; Zhang, Hui-Jing; Xu, Hui-Mian; Sun, Ming-Jun

    2015-01-01

    AIM: To investigate the expression and prognostic role of pyruvate dehydrogenase (PDH) in gastric cancer (GC). METHODS: This study included 265 patients (194 male, 71 female, mean age 59 years (range, 29-81 years) with GC who underwent curative surgery at the First Affiliated Hospital of China Medical University from January 2006 to May 2007. All patients were followed up for more than 5 years. Patient-derived paraffin embedded GC specimens were collected for tissue microarrays (TMAs). We examined PDH expression by immunohistochemistry in TMAs containing tumor tissue and matched non-neoplastic mucosa. Immunoreactivity was evaluated independently by two researchers. Overall survival (OS) rates were determined using the Kaplan-Meier estimator. Correlations with other clinicopathologic factors were evaluated by two-tailed χ2 tests or a two-tailed t-test. The Cox proportional-hazard model was used in univariate analysis and multivariate analysis to identify factors significantly correlated with prognosis. RESULTS: Immunohistochemistry showed that 35.47% of total cancer tissue specimens had cytoplasmic PDH staining. PDH expression was much higher in normal mucosa specimens (75.09%; P = 0.001). PDH expression was correlated with Lauren grade (70.77% in intestinal type vs 40.0% in diffuse type; P = 0.001), lymph node metastasis (65.43% with no metastasis vs 51.09% with metastasis; P = 0.033), lymphatic invasion (61.62% with no invasion vs 38.81% with invasion; P = 0.002), histologic subtypes (70.77% in intestinal type vs 40.0% in diffuse type; P = 0.001) and tumor-node-metastasis (TNM) stage (39% in poorly differentiated vs 65.91% in well differentiated and 67.11% in moderately differentiated; P = 0.001) in GC. PDH expression in cancer tissue was significantly associated with higher OS (P < 0.001). The multivariate analysis adjusted for age, Lauren classification, TNM stage, lymph node metastasis, histological type, tumor size, depth of invasion and lymphatic invasion

  4. Radioimmunoassay of haloperidol in human serum: correlation of serum haloperidol with serum prolactin

    International Nuclear Information System (INIS)

    Poland, R.E.; Rubin, R.T.

    1981-01-01

    A radioimmunoassay (RIA) for measurement of serum haloperidol is described. Compared to gaschromatography (GC), RIA vaues average 40% higher. However, a simple organic extraction of serum yields statistically equivalent RIA and GC haloperidol determinations. For both men and women combined, there was a positive correlation between dose (mg/kg/day) and steady-state serum haloperidol level (r = +0.86) and between steady-state serum haloperidol and serum prolactin (PRL) concentration

  5. Serum YKL-40

    DEFF Research Database (Denmark)

    Mylin, Anne K; Abildgaard, Niels; Johansen, Julia S

    2015-01-01

    to clinical progression, i.e. skeletal-related events (SREs), in 230 newly diagnosed patients with multiple myeloma receiving intravenous bisphosphonates. Serum concentrations of YKL-40 and biochemical bone markers (CTX-MMP, CTX-I, PINP) were measured at diagnosis. Patients were evaluated every third month...

  6. 79 - 81_Wali - Serum

    African Journals Online (AJOL)

    DR. AMINU

    2013-06-01

    Jun 1, 2013 ... SERUM ANTIOXIDANT VITAMINS LEVELS IN CHILDREN WITH SICKLE. CELL ANAEMIA IN ... play a role in the development of sickle cell anaemic complications. In the current study, ... vaso-occlusive episodes, acute chest syndrome and impaired ... frequency of sickling crises accompanied by tissue or.

  7. Serum Lipoprotein (a Levels in Chronic Renal Failure and Liver Cirrhosis Patients. Relationship with Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Essam Mady

    1999-01-01

    Full Text Available This study was carried out to investigate the relationship between lipoprotein (a levels and the development of atherosclerosis in chronic renal failure (CRF patients with the possible role of the liver. Serum Lp (a levels were measured in samples from 20 CRF patients on hemodialysis (HD, 20 liver cirrhosis (LC patients, 20 patients having both CRF and LC and undergoing HD, and 20 normal control subjects. Renal function (blood urea nitrogen (BUN and creatinine, hepatic function (transaminases (ALT and AST, alkaline phosphatase (ALP and total bilirubin investigations and serum cholesterol were carried out for all the subjects enrolled in this study. Serum Lp (a concentration in CRF patients without LC was 87.25 ± 6.17 mg/dl, which was significantly higher than all the investigated groups (P < 0.001. Lp(a concentration in patients with both CRF and LC was 24.65 ± 1.98 mg/dl, which was not significantly different from the controls, but was significantly higher than that in the subjects with LC only (P < 0.001 where the latter group had significantly low Lp (a values (11.1 ± 0.99 relative to all the other groups (P < 0.001. Lp (a correlated positively with cholesterol in all groups except the LC subjects, but did not correlate with age, or renal function in both CRF groups.

  8. Methylmercury exposure for 14 days (short-term) produces behavioral and biochemical changes in mouse cerebellum, liver, and serum.

    Science.gov (United States)

    Macedo-Júnior, Sérgio José; Luiz-Cerutti, Murilo; Nascimento, Denise B; Farina, Marcelo; Soares Santos, Adair Roberto; de Azevedo Maia, Alcíbia Helena

    2017-01-01

    Various studies on methylmercury (MeHg)-induced toxicity focused on the central nervous system (CNS) as a primary target. However, MeHg-mediated toxicity is related to metallic interaction with electrophilic groups, which are not solely restricted to the CNS, but these reactive groups are present ubiquitously in several systems/organs. The aim of this study was thus to examine MeHg-induced systemic toxicity in mice using a standardized neurotoxicology testing exposure model to measure cerebellar neurotoxicity by determining biochemical and behavioral parameters in the cerebellum. After 2 weeks exposure to MeHg (40 µg/ml; diluted in drinking water; ad libitum), adult male Swiss mice showed a marked motor impairment characteristic of cerebellar toxicity as noted in the following tests: rotarod, beam walking, pole, and hind limb clasping. MeHg treatment resulted in Hg deposition in the cerebellum as well as reduction in cerebellar weight, glutathione peroxidase (GPx) activity, and interleukin (IL)-6 levels. MeHg ingestion increased cerebellar glutathione reductase (GR) activity and brain-derived neurotrophic factor (BDNF) levels. In addition to cerebellar toxicity, MeHg treatment also elevated total and non-high density lipoprotein (non-HDL) cholesterol levels, as well as serum aspartate transaminase (AST) and alanine transaminase (ALT) enzymatic activities, systemic parameters. Increased liver weight and reduced serum urea levels were also noted in MeHg-exposed mice. Taken together, our findings demonstrated that a well-standardized exposure protocol to examine MeHg-induced neurotoxicity also produced systemic toxicity in mice, which was characterized by changes in markers of hepatic function as well as serum lipid homeostasis.

  9. Effects of adrenergic agents on intracellular ca(2+) homeostasis and metabolism of glucose in astrocytes with an emphasis on pyruvate carboxylation, oxidative decarboxylation and recycling

    DEFF Research Database (Denmark)

    Obel, Linea Lykke Frimodt; Andersen, Karen M H; Bak, Lasse Kristoffer

    2012-01-01

    and oxidative decarboxylation in astrocytic glucose metabolism. Importantly, pyruvate carboxylation was best visualized at 10 min of incubation. The abundance and pattern of labeling in lactate and alanine indicated not only an extensive activity of malic enzyme (initial step for pyruvate recycling) but also...... a high degree of compartmentalization of the pyruvate pool. Stimulating with 1 µM NE had no effect on labeling patterns and glycogen metabolism, whereas 100 µM NE increased glutamate labeling and decreased labeling in alanine, the latter supposedly due to dilution from degradation of non-labeled glycogen....... It is suggested that further experiments uncovering the correlation between adrenergic and glutamatergic pathways should be performed in order to gain further insight into the role of astrocytes in brain function and dysfunction, the latter including excitotoxicity....

  10. Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose.

    Science.gov (United States)

    Ishchuk, Olena P; Voronovsky, Andriy Y; Stasyk, Oleh V; Gayda, Galina Z; Gonchar, Mykhailo V; Abbas, Charles A; Sibirny, Andriy A

    2008-11-01

    Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

  11. Intraperitoneal lactate/pyruvate ratio and the level of glucose and glycerol concentration differ between patients surgically treated for upper and lower perforations of the gastrointestinal tract

    DEFF Research Database (Denmark)

    Sabroe, Jonas E; Axelsen, Anne R; Ellebæk, Mark B

    2017-01-01

    collected every 4th hour for up to 7 postoperative days. Samples were analysed for concentrations of glucose, lactate, pyruvate and glycerol. RESULTS: Microdialysis results showed that patients with upper gastrointestinal tract lesions had significantly higher levels of postoperative intraperitoneal glucose...... and glycerol concentrations, as well as lower lactate/pyruvate ratios and lactate/glucose ratios. In the group with perforation of the lower gastrointestinal tract, those patients with a complicated course showed lower levels of postoperative intraperitoneal glucose concentration and glycerol concentration...... and higher lactate/pyruvate ratios and lactate/glucose ratios than those patients with an uncomplicated course. CONCLUSION: Patients with upper and lower gastrointestinal tract lesions showed differences in postoperative biomarker levels. A difference was also seen between patients with complicated...

  12. Molecular identification and characterization of the pyruvate decarboxylase gene family associated with latex regeneration and stress response in rubber tree.

    Science.gov (United States)

    Long, Xiangyu; He, Bin; Wang, Chuang; Fang, Yongjun; Qi, Jiyan; Tang, Chaorong

    2015-02-01

    In plants, ethanolic fermentation occurs not only under anaerobic conditions but also under aerobic conditions, and involves carbohydrate and energy metabolism. Pyruvate decarboxylase (PDC) is the first and the key enzyme of ethanolic fermentation, which branches off the main glycolytic pathway at pyruvate. Here, four PDC genes were isolated and identified in a rubber tree, and the protein sequences they encode are very similar. The expression patterns of HbPDC4 correlated well with tapping-simulated rubber productivity in virgin rubber trees, indicating it plays an important role in regulating glycometabolism during latex regeneration. HbPDC1, HbPDC2 and HbPDC3 had striking expressional responses in leaves and bark to drought, low temperature and high temperature stresses, indicating that the HbPDC genes are involve in self-protection and defense in response to various abiotic and biotic stresses during rubber tree growth and development. To understand ethanolic fermentation in rubber trees, it will be necessary to perform an in-depth study of the regulatory pathways controlling the HbPDCs in the future. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  13. Mitochondrial pyruvate carrier function is negatively linked to Warburg phenotype in vitro and malignant features in esophageal squamous cell carcinomas

    Science.gov (United States)

    Li, Yaqing; Li, Xiaoran; Kan, Quancheng; Zhang, Mingzhi; Li, Xiaoli; Xu, Ruiping; Wang, Junsheng; Yu, Dandan; Goscinski, Mariusz Adam; Wen, Jian-Guo; Nesland, Jahn M.; Suo, Zhenhe

    2017-01-01

    Aerobic glycolysis is one of the emerging hallmarks of cancer cells. In this study, we investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with MPC blocker UK5099 and the metabolic alteration as well as aggressive features of esophageal squamous carcinoma. It was found that blocking pyruvate transportation into mitochondria attenuated mitochondrial oxidative phosphorylation (OXPHOS) and triggered aerobic glycolysis, a feature of Warburg effect. In addition, the HIF-1α expression and ROS production were also activated upon UK5099 application. It was further revealed that the UK5099-treated cells became significantly more resistant to chemotherapy and radiotherapy, and the UK5099-treated tumor cells also exhibited stronger invasive capacity compared to the parental cells. In contrast to esophageal squamous epithelium cells, decreased MPC protein expression was observed in a series of 157 human squamous cell carcinomas, and low/negative MPC1 expression predicted an unfavorable clinical outcome. All these results together revealed the potential connection of altered MPC expression/activity with the Warburg metabolic reprogramming and tumor aggressiveness in cell lines and clinical samples. Collectively, our findings highlighted a therapeutic strategy targeting Warburg reprogramming of human esophageal squamous cell carcinomas. PMID:27911865

  14. Escherichia coli pyruvate dehydrogenase complex: particle masses of the complex and component enzymes measured by scanning transmission electron microscopy

    International Nuclear Information System (INIS)

    CaJacob, C.A.; Frey, P.A.; Hainfeld, J.F.; Wall, J.S.; Yang, H.

    1985-01-01

    Particle masses of the Escherichia coli pyruvate dehydrogenase (PDH) complex and its component enzymes have been measured by scanning transmission electron microscopy (STEM). The particle mass of PDH complex measured by STEM is 5.28 X 10(6) with a standard deviation of 0.40 X 10(6). The masses of the component enzymes are 2.06 X 10(5) for the dimeric pyruvate dehydrogenase (E1), 1.15 X 10(5) for dimeric dihydrolipoyl dehydrogenase (E3), and 2.20 X 10(6) for dihydrolipoyl transacetylase (E2), the 24-subunit core enzyme. STEM measurements on PDH complex incubated with excess E3 or E1 failed to detect any additional binding of E3 but showed that the complex would bind additional E1 under forcing conditions. The additional E1 subunits were bound too weakly to represent binding sites in an isolated or isolable complex. The mass measurements by STEM are consistent with the subunit composition 24:24:12 when interpreted in the light of the flavin content of the complex and assuming 24 subunits in the core enzyme (E2)

  15. Effect of Pyruvate Decarboxylase Knockout on Product Distribution Using Pichia pastoris (Komagataella phaffii) Engineered for Lactic Acid Production.

    Science.gov (United States)

    Melo, Nadiele T M; Mulder, Kelly C L; Nicola, André Moraes; Carvalho, Lucas S; Menino, Gisele S; Mulinari, Eduardo; Parachin, Nádia S

    2018-02-16

    Lactic acid is the monomer unit of the bioplastic poly-lactic acid (PLA). One candidate organism for lactic acid production is Pichia pastoris , a yeast widely used for heterologous protein production. Nevertheless, this yeast has a poor fermentative capability that can be modulated by controlling oxygen levels. In a previous study, lactate dehydrogenase (LDH) activity was introduced into P. pastoris, enabling this yeast to produce lactic acid. The present study aimed to increase the flow of pyruvate towards the production of lactic acid in P. pastoris . To this end, a strain designated GLp was constructed by inserting the bovine lactic acid dehydrogenase gene (LDHb) concomitantly with the interruption of the gene encoding pyruvate decarboxylase (PDC). Aerobic fermentation, followed by micro-aerophilic culture two-phase fermentations, showed that the GLp strain achieved a lactic acid yield of 0.65 g/g. The distribution of fermentation products demonstrated that the acetate titer was reduced by 20% in the GLp strain with a concomitant increase in arabitol production: arabitol increased from 0.025 g/g to 0.174 g/g when compared to the GS115 strain. Taken together, the results show a significant potential for P. pastoris in producing lactic acid. Moreover, for the first time, physiological data regarding co-product formation have indicated the redox balance limitations of this yeast.

  16. miR-378 Activates the Pyruvate-PEP Futile Cycle and Enhances Lipolysis to Ameliorate Obesity in Mice

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    2016-03-01

    Full Text Available Obesity has been linked to many health problems, such as diabetes. However, there is no drug that effectively treats obesity. Here, we reveal that miR-378 transgenic mice display reduced fat mass, enhanced lipolysis, and increased energy expenditure. Notably, administering AgomiR-378 prevents and ameliorates obesity in mice. We also found that the energy deficiency seen in miR-378 transgenic mice was due to impaired glucose metabolism. This impairment was caused by an activated pyruvate-PEP futile cycle via the miR-378-Akt1-FoxO1-PEPCK pathway in skeletal muscle and enhanced lipolysis in adipose tissues mediated by miR-378-SCD1. Our findings demonstrate that activating the pyruvate-PEP futile cycle in skeletal muscle is the primary cause of elevated lipolysis in adipose tissues of miR-378 transgenic mice, and it helps orchestrate the crosstalk between muscle and fat to control energy homeostasis in mice. Thus, miR-378 may serve as a promising agent for preventing and treating obesity in humans.

  17. Diagnostic relevance of radioiron-absorption-measurements and immunoradiometric serum-ferritin-assay in the evaluation of iron stores

    International Nuclear Information System (INIS)

    Heinrich, H.C.

    1978-01-01

    Negative iron balance and enhanced iron demand respectively causes deficient iron stores (prelatent iron deficiency) with increased iron absorption, later on decrease of serum iron and increase of transferrin (latent Fe deficiency) and at least iron deficient anemia (manifest iron deficiency). In prelatend iron deficiency diagnostic 59 Fe 2+ absorption is increased and the RES cells do not show storage iron cytochemically. In latent iron deficiency in addition serum iron, transferrin iron saturation and serum ferritin is decreased and hypochromic mikrocytic anemia completes the signs of manifest iron deficiency. Besides rare cases of primary hemochromatosis and marked hyperdasia of ineffective erythropoiesis in homocygotic beta-thalassemia, hereditary non-spherocytic hemolytic anemia caused by pyruvate kinase deficiency and some sideroblastic anemias increased 59 Fe 2+ absorption is a reliable measure of exhausted iron stores. In these exceptional cases differential diagnosis between sideroachrestic and siderosensitive iron deficiency anemia can be made by measurement of serum iron and serum ferritin respectively. The etiology of iron deficiency is to be cleared by measurement of 59 Fe absorption from 59 Fe 2+ and 59 Fe-marked meat with consecutive estimation of whole body 59 Fe elimination. Shortly after completion or during oral iron therapy serum ferritin concentration is not suitable to evaluate the content of iron stores. (orig.) [de

  18. SERUM ELECTROLYTES AND ENZYMES IN ENDOMETRITIC NILI-RAVI BUFF ALOES OF TWO AGE GROUPS AND AT TWO STAGES OF LACTATION

    Directory of Open Access Journals (Sweden)

    A. Sarwar, R. U. Shahid, S. Masood, R. Kausar and S. G. Sha

    2002-04-01

    Full Text Available Serum concentrations of tour electrol~1es including sodium (Na, potassium (K, calcium (Ca and Chloride ( Cl and activities of two enzymes i.e. aspartate transaminase ( AST and alanine transaminase (ALT were compared amongst 80 pregnant Nili-Ravi buffaloes in a 23 factorial experiment: between endometritic and health~ lots of two age groups and at two stages of lactation. The analysis of variance revealed that: endometritic buffaloes showing muco-purulent vaginal discharge exhibited raise in ALT, AST and Cl, while decline in Ca. Milking stage affected two parameters namely, serum Cl and AL T. Both Cl and ALT were found to be decreased up to 11 months of lactation. Age groups remained inert on all of the parameters studied. This data indicates that electrolytes and enzymes clearly deviate from their normal levels in endometritic buffaloes which may in turn affect reproductive performance negatively. Thus maintenance of optimal uterine health and balanced nutrition, particularly with reference to blood constituents are critical for better reproductive performance in animals of this species.

  19. Comparative kinetic studies of Mn2+-activated and fructose-1,6-P-modified Mg2+-activated pyruvate kinase from Concholepas concholepas.

    Science.gov (United States)

    Carvajal, N; González, R; Morán, A; Oyarce, A M

    1985-01-01

    Initial velocity and product inhibition studies of Mn2+-activated and FDP-modified Mg2+-activated pyruvate kinase from Concholepas concholepas, were performed. Evidence is presented to show that the Mn2+-enzyme catalyzes an ordered sequential mechanism, with ADP being the first substrate and pyruvate the last product. The results presented are consistent with a random combination of reactants with the FDP-modified Mg2+-activated enzyme and the formation of the dead-end complexes enzyme ADP-ATP and enzyme-PEP-ATP.

  20. Serum Creatinine: Not So Simple!

    OpenAIRE

    DELANAYE, Pierre; Cavalier, Etienne; Pottel, Hans

    2017-01-01

    Measuring serum creatinine is cheap and commonly done in daily practice. However, interpretation of serum creatinine results is not always easy. In this review, we will briefly remind the physiological limitations of serum creatinine due notably to its tubular secretion and the influence of muscular mass or protein intake on its concentration. We mainly focus on the analytical limitations of serum creatinine, insisting on important concept such as reference intervals, standardization (and IDM...

  1. A New Theory for Calculation of Some Biochemical Parameters Concentration in Human Serum

    International Nuclear Information System (INIS)

    Moustafa, K.A.; Amien, A.I.

    2009-01-01

    The serum volumes of the blood samples are varied from one patient to another according to the packed cell volume (PCV %), so if the patient sample has low PCV %, it will have high serum volume and vice versa. To calculate a certain clinical parameter such as glucose in the serum of patients using the conventional calculation, it will give the concentration in units per deci liter serum, while by calculating the concentration according to the ratio of serum volume to the total volume of the blood, it will give different results. Thus, the present study aimed to find a new theory used for calculation of some biochemical parameters concentration taking into consideration the ratio of plasma volume to the total blood volume. The present study was conducted on 122 subjects. These subjects were categorized into 4 groups. Group 1 (G1) comprised 40 healthy subjects as control group, group 2 (G2) comprised 30 low PCV % patients, group 3 (G3) comprised 30 subjects with relatively high PCV % and group 4 comprised 22 diabetic patients. Each group of the previous groups was further subdivided into group a (G a ) and group b (G b ). In the later group, the results were multiplied by the correction factor (V p /V b ), which is the ratio of plasma volume (V p ) to the blood volume (V b ) TSH hormone, glucose, cholesterol, triglycerides, urea, creatinine, uric acid, alanine aminotransferase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) were estimated and the results were multiplied by the correction factor (V p /V b ) to get the results after correction. Compared the results before and after correction, there was a very highly significant (ρ p /V b ), thus we recommended that biochemical parameters results must be calculated as indicated in the present study to obtain actual results which might be useful in the correct diagnosis, monitoring and follow up of the different diseases

  2. The Human Serum Metabolome

    Science.gov (United States)

    Psychogios, Nikolaos; Hau, David D.; Peng, Jun; Guo, An Chi; Mandal, Rupasri; Bouatra, Souhaila; Sinelnikov, Igor; Krishnamurthy, Ramanarayan; Eisner, Roman; Gautam, Bijaya; Young, Nelson; Xia, Jianguo; Knox, Craig; Dong, Edison; Huang, Paul; Hollander, Zsuzsanna; Pedersen, Theresa L.; Smith, Steven R.; Bamforth, Fiona; Greiner, Russ; McManus, Bruce; Newman, John W.; Goodfriend, Theodore; Wishart, David S.

    2011-01-01

    Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca. PMID:21359215

  3. Physiological studies on the effect of a phosphodiesterase inhibitor on the blood obtained from hypothyroid rats

    International Nuclear Information System (INIS)

    Abdel-Ghany, I.Y.

    1997-01-01

    In the present study 300 male albino rats (100-120 g) were used. The search was planned to evaluate the biochemical effects of the therapeutic drug (pentoxifylline) in combination with thyroxine on the hypothyroid mammals. The biochemical determinations were serum: Tri-iodothyronine (T-3), thyroxine (T-4), Glucose, total protein, urea, creatinine, along with the enzymatic activities of : alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT). The study included also glycogen, lactic acid, pyruvic acid, along with the enzymatic activities of : LDH, GOT and GPT in liver tissue homogenate. The data obtained, revealed significant alterations in assayed parameters reflecting disturbance in the metabolism due to hypothyroid state. Treatment of rats with thyroxine and / or pentoxifylline revealed significant amelioration in most of the tested parameters indicating the beneficial effect of these drugs. 17 tabs.,17 figs.,123 refs

  4. An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae.

    Science.gov (United States)

    Oud, Bart; Flores, Carmen-Lisset; Gancedo, Carlos; Zhang, Xiuying; Trueheart, Joshua; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

    2012-09-15

    Pyruvate-decarboxylase negative (Pdc⁻) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc⁻S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc⁻ strains cannot grow on high glucose concentrations and require C₂-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Genetic analysis of a Pdc⁻ strain previously evolved to overcome these deficiencies revealed a 225 p in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc⁻ strain enabled growth on 20 g l⁻¹ glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h⁻¹) similar to that of the evolved Pdc⁻ strain (0.23 h⁻¹). Furthermore, the reverse engineered Pdc⁻ strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h⁻¹) than the evolved strain (0.20 h⁻¹). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc⁻S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc⁻ strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C₂-compound auxotrophy. In this study we have discovered and characterised a

  5. An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Oud Bart

    2012-09-01

    Full Text Available Abstract Background Pyruvate-decarboxylase negative (Pdc- strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc- strains cannot grow on high glucose concentrations and require C2-compounds (ethanol or acetate for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Results Genetic analysis of a Pdc- strain previously evolved to overcome these deficiencies revealed a 225bp in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc- strain enabled growth on 20 g l-1 glucose and 0.3% (v/v ethanol at a maximum specific growth rate (0.24 h-1 similar to that of the evolved Pdc- strain (0.23 h-1. Furthermore, the reverse engineered Pdc- strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h-1 than the evolved strain (0.20 h-1. The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc-S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc- strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C2-compound auxotrophy. Conclusions In this study we have discovered and

  6. Simultaneous hyperpolarized 13C-pyruvate MRI and 18F-FDG-PET in cancer (hyperPET)

    DEFF Research Database (Denmark)

    Borgwardt, Henrik Gutte; Hansen, Adam Espe; Henriksen, Sarah T.

    2015-01-01

    have named this concept hyper PET. Intravenous injection of the hyperpolarized (13)C-pyruvate results in an increase of (13)C-lactate, (13)C-alanine and (13)C-CO2 ((13)C-HCO3) resonance peaks relative to the tissue, disease and the metabolic state probed. Accordingly, with dynamic nuclear polarization......In this paper we demonstrate, for the first time, the feasibility of a new imaging concept - combined hyperpolarized (13)C-pyruvate magnetic resonance spectroscopic imaging (MRSI) and (18)F-FDG-PET imaging. This procedure was performed in a clinical PET/MRI scanner with a canine cancer patient. We...... (DNP) and use of (13)C-pyruvate it is now possible to directly study the Warburg Effect through the rate of conversion of (13)C-pyruvate to (13)C-lactate. In this study, we combined it with (18)F-FDG-PET that studies uptake of glucose in the cells. A canine cancer patient with a histology verified...

  7. Interaction between the thyroid hormone receptor and co-factors on the promoter of the gene encoding phospho enol pyruvate carboxykinase

    NARCIS (Netherlands)

    Schmidt, E. D.; van Beeren, M.; Glass, C. K.; Wiersinga, W. M.; Lamers, W. H.

    1993-01-01

    Using transient transfection studies we localized a thyroid hormone-responsive element on the promoter of the rat phospho-enol pyruvate carboxykinase gene between 355 and 174 bp upstream of the transcription start site. DNAse 1 footprinting analysis within this region showed that a 28 bp fragment at

  8. The MDM2-p53-pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    DEFF Research Database (Denmark)

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao

    2016-01-01

    deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates...

  9. Studies on the formation of lactate and pyruvate from glucose in cultured skin fibroblasts: implications for detection of respiratory chain defects

    NARCIS (Netherlands)

    Wijburg, F. A.; Feller, N.; Scholte, H. R.; Przyrembel, H.; Wanders, R. J.

    1989-01-01

    We investigated the time course of the formation of lactate and pyruvate from glucose in cultured skin fibroblasts from controls, from a patient with a cytochrome c oxidase deficiency and from controls treated with inhibitors of the individual respiratory chain complexes. Fibroblasts from the

  10. Discovery of a 1,2-bis(3-indolyl)ethane that selectively inhibits the pyruvate kinase of methicillin-resistant Staphylococcus aureus over human isoforms.

    Science.gov (United States)

    Zoraghi, Roya; Campbell, Sara; Kim, Catrina; Dullaghan, Edie M; Blair, Lachlan M; Gillard, Rachel M; Reiner, Neil E; Sperry, Jonathan

    2014-11-01

    Methicillin-resistant Staphylococcus aureus pyruvate kinase (MRSA PK) has recently been identified as a target for development of novel antibacterial agents. Testing a series of 1,2-bis(3-indolyl)ethanes against MRSA PK has led to the discovery of a potent inhibitor that is selective over human isoforms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Functional pyruvate formate lyase pathway expressed with two different electron donors in Saccharomyces cerevisiae at aerobic growth

    DEFF Research Database (Denmark)

    Zhang, Yiming; Dai, Zongjie; Krivoruchko, Anastasia

    2015-01-01

    pyruvate decarboxylase and having a reduced glucose uptake rate due to a mutation in the transcriptional regulator Mth1, IMI076 (Pdc-MTH1-ΔT ura3-52). PFL was expressed with two different electron donors, reduced ferredoxin or reduced flavodoxin, respectively, and it was found that the coexpression...

  12. Elevated levels of 14-3-3 proteins, serotonin, gamma enolase and pyruvate kinase identified in clinical samples from patients diagnosed with colorectal cancer

    Czech Academy of Sciences Publication Activity Database

    Dowling, P.; Hughes, D. J.; Larkin, A.M.; Meiller, J.; Henry, M.; Meleady, P.; Lynch, V.; Pardini, B.; Naccarati, A.; Levý, M.; Vodička, Pavel; Neary, P.; Clynes, M.

    2015-01-01

    Roč. 441, feb. (2015), s. 133-141 ISSN 0009-8981 Institutional support: RVO:68378041 Keywords : biomarkers * colorectal cancer * proteomics * mass spectrometry * 14-3-3 proteins * pyruvate kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.799, year: 2015

  13. Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress.

    Science.gov (United States)

    Delgado, María Graciela; Oliva, Carlos; López, Estefanía; Ibacache, Andrés; Galaz, Alex; Delgado, Ricardo; Barros, L Felipe; Sierralta, Jimena

    2018-01-19

    The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.

  14. Computer-assisted study on the reaction between pyruvate and ylide in the pathway leading to lactyl-ThDP.

    Science.gov (United States)

    Alvarado, Omar; Jaña, Gonzalo; Delgado, Eduardo J

    2012-08-01

    In this study the formation of the lactyl-thiamin diphosphate intermediate (L-ThDP) is addressed using density functional theory calculations at X3LYP/6-31++G(d,p) level of theory. The study includes potential energy surface scans, transition state search, and intrinsic reaction coordinate calculations. Reactivity is analyzed in terms of Fukui functions. The results allow to conclude that the reaction leading to the formation of L-ThDP occurs via a concerted mechanism, and during the nucleophilic attack on the pyruvate molecule, the ylide is in its AP form. The calculated activation barrier for the reaction is 19.2 kcal/mol, in agreement with the experimental reported value.

  15. Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

    2015-01-01

    Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). RESULTS/METHODOLOGY: We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease.

  16. Alu element insertion in PKLR gene as a novel cause of pyruvate kinase deficiency in Middle Eastern patients.

    Science.gov (United States)

    Lesmana, Harry; Dyer, Lisa; Li, Xia; Denton, James; Griffiths, Jenna; Chonat, Satheesh; Seu, Katie G; Heeney, Matthew M; Zhang, Kejian; Hopkin, Robert J; Kalfa, Theodosia A

    2018-03-01

    Pyruvate kinase deficiency (PKD) is the most frequent red blood cell enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Over 250 PKLR-gene mutations have been described, including missense/nonsense, splicing and regulatory mutations, small insertions, small and gross deletions, causing PKD and hemolytic anemia of variable severity. Alu retrotransposons are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. Alu insertions have been associated with a number of human diseases either by disrupting a coding region or a splice signal. Here, we report on two unrelated Middle Eastern patients, both born from consanguineous parents, with transfusion-dependent hemolytic anemia, where sequence analysis revealed a homozygous insertion of AluYb9 within exon 6 of the PKLR gene, causing precipitous decrease of PKLR RNA levels. This Alu element insertion consists a previously unrecognized mechanism underlying pathogenesis of PKD. © 2017 Wiley Periodicals, Inc.

  17. Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells

    Science.gov (United States)

    Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

    2015-01-01

    Background Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). Results/Methodology We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Conclusion Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease. PMID:25826140

  18. Free serum thyroxine

    International Nuclear Information System (INIS)

    Degrossi, O.J.; Altschuler, Noe; Cabrejas, M.L. de; Pinkas, Mirta; Garcia del Rio, Hernan

    1982-01-01

    The use of radiommunoassay (RIA) tehcniques has increased the diagnosis of thyroid functional alterations. A solid phase RIA method for free thyroxine (FT4) measurement was tested. Serum FT4, Total T4, T3 and TSH were determined by radioimmunoassay in 179 subjects. One hundred twenty two patients were normal (8 to 75 years old); FT4 was 1.42 +- 0.03 ng/100 ml (avg. value and std. error). In 27 cases of thyrotoxicosis the values were 4.66 +- 0.48 ng/100 ml and in 15 cases of hypothyroidism 0.50 +- 0.06 ng/100 ml (statistics probability [es

  19. Evaluation of Serum Lactate Dehydrogenase Activity in a Virtual Environment

    Directory of Open Access Journals (Sweden)

    V.M.T. Trindade

    2013-05-01

    Full Text Available Introduction: Lactate dehydrogenase is a citosolic enzyme involved in reversible transformation of pyruvate to lactate. It participates in anaerobic glycolysis of skeletal muscle and red blood cells, in liver gluconeogenesis and in aerobic metabolism of heart muscle. The determination of its activity helps in the diagnosis of various diseases, because it is increased in serum of patients suffering from myocardial infarction, acute hepatitis, muscular dystrophy and cancer. This paper presents a learning object, mediated by computer, which contains the simulation of the laboratory determination serum lactate dehydrogenase activity measured by the spectrophotometric method, based in the decrease of absorbance at 340 nm. Materials and Methods: Initially, pictures and videos were obtained recording the procedure of the methodology. The most representative images were selected, edited and inserted into an animation developed with the aid of the tool Adobe ® Flash ® CS3. The validation of the object was performed by the students of Biochemistry I (Pharmacy-UFRGS from the second semester of 2009 and both of 2010. Results and Discussion: The analysis of students' answers revealed that 80% attributed the excellence of the navigation program, the display format and to aid in learning. Conclusion: Therefore, this software can be considered an adequate teaching resource as well as an innovative support in the construction of theoretical and practical knowledge of Biochemistry. Available at: http://www6.ufrgs.br/gcoeb/LDH

  20. Lactate/pyruvate transporter MCT-1 is a direct Wnt target that confers sensitivity to 3-bromopyruvate in colon cancer.

    Science.gov (United States)

    Sprowl-Tanio, Stephanie; Habowski, Amber N; Pate, Kira T; McQuade, Miriam M; Wang, Kehui; Edwards, Robert A; Grun, Felix; Lyou, Yung; Waterman, Marian L

    2016-01-01

    There is increasing evidence that oncogenic Wnt signaling directs metabolic reprogramming of cancer cells to favor aerobic glycolysis or Warburg metabolism. In colon cancer, this reprogramming is due to direct regulation of pyruvate dehydrogenase kinase 1 ( PDK1 ) gene transcription. Additional metabolism genes are sensitive to Wnt signaling and exhibit correlative expression with PDK1. Whether these genes are also regulated at the transcriptional level, and therefore a part of a core metabolic gene program targeted by oncogenic WNT signaling, is not known. Here, we identify monocarboxylate transporter 1 (MCT-1; encoded by SLC16A1 ) as a direct target gene supporting Wnt-driven Warburg metabolism. We identify and validate Wnt response elements (WREs) in the proximal SLC16A1 promoter and show that they mediate sensitivity to Wnt inhibition via dominant-negative LEF-1 (dnLEF-1) expression and the small molecule Wnt inhibitor XAV939. We also show that WREs function in an independent and additive manner with c-Myc, the only other known oncogenic regulator of SLC16A1 transcription. MCT-1 can export lactate, the byproduct of Warburg metabolism, and it is the essential transporter of pyruvate as well as a glycolysis-targeting cancer drug, 3-bromopyruvate (3-BP). Using sulforhodamine B (SRB) assays to follow cell proliferation, we tested a panel of colon cancer cell lines for sensitivity to 3-BP. We observe that all cell lines are highly sensitive and that reduction of Wnt signaling by XAV939 treatment does not synergize with 3-BP, but instead is protective and promotes rapid recovery. We conclude that MCT-1 is part of a core Wnt signaling gene program for glycolysis in colon cancer and that modulation of this program could play an important role in shaping sensitivity to drugs that target cancer metabolism.

  1. Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

    Science.gov (United States)

    2012-01-01

    Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene

  2. Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

    Science.gov (United States)

    Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

    2011-01-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

  3. Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

    Directory of Open Access Journals (Sweden)

    Milanovic Vesna

    2012-02-01

    Full Text Available Abstract Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1 and alcohol dehydrogenase (Adh1 were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation

  4. Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).

    Science.gov (United States)

    Plaga, W; Frank, R; Knappe, J

    1988-12-15

    Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

  5. Triiodothyronine increases myocardial function and pyruvate entry into the citric acid cycle after reperfusion in a model of infant cardiopulmonary bypass

    Science.gov (United States)

    Olson, Aaron K.; Bouchard, Bertrand; Ning, Xue-Han; Isern, Nancy; Rosiers, Christine Des

    2012-01-01

    Triiodothyronine (T3) supplementation improves clinical outcomes in infants after cardiac surgery using cardiopulmonary bypass by unknown mechanisms. We utilized a translational model of infant cardiopulmonary bypass to test the hypothesis that T3 modulates pyruvate entry into the citric acid cycle (CAC), thereby providing the energy support for improved cardiac function after ischemia-reperfusion (I/R). Neonatal piglets received intracoronary [2-13Carbon(13C)]pyruvate for 40 min (8 mM) during control aerobic conditions (control) or immediately after reperfusion (I/R) from global hypothermic ischemia. A third group (I/R-Tr) received T3 (1.2 μg/kg) during reperfusion. We assessed absolute CAC intermediate levels and flux parameters into the CAC through oxidative pyruvate decarboxylation (PDC) and anaplerotic carboxylation (PC) using [2-13C]pyruvate and isotopomer analysis by gas and liquid chromatography-mass spectrometry and 13C-nuclear magnetic resonance spectroscopy. When compared with I/R, T3 (group I/R-Tr) increased cardiac power and oxygen consumption after I/R while elevating flux of both PDC and PC (∼4-fold). Although neither I/R nor I/R-Tr modified absolute CAC levels, T3 inhibited I/R-induced reductions in their molar percent enrichment. Furthermore, 13C-labeling of CAC intermediates suggests that T3 may decrease entry of unlabeled carbons at the level of oxaloacetate through anaplerosis or exchange reaction with asparate. T3 markedly enhances PC and PDC fluxes, thereby providing potential substrate for elevated cardiac function after reperfusion. This T3-induced increase in pyruvate fluxes occurs with preservation of the CAC intermediate pool. Our labeling data raise the possibility that T3 reduces reliance on amino acids for anaplerosis after reperfusion. PMID:22180654

  6. The investigation of anti-inflammatory activity of Yi Guanjian decoction by serum metabonomics approach.

    Science.gov (United States)

    Shui, Sufang; Cai, Xiaorong; Huang, Rongqing; Xiao, Bingkun; Yang, Jianyun

    2017-01-30

    Yi Guanjian (YGJ), one of the Chinese herbal medicines most commonly used in western countries, reported to possess significant anti-inflammatary effects that inhibit the process of inflammation. However, the mechanisms underlying its anti-inflammation effects remain largely unresolved. This study was aimed to investigate the anti-inflammatory activity of YGJ and to explore its potential anti-inflammatory mechanisms by serum metabonomics approach. An xylene-induced mouse right-ear-edema model was used as an inflammatory response in vivo model. Ear edema, prostaglandin E2 (PGE 2 ) and Tumor-Necrosis-Factor-alpha (TNF-α) were detected. Then, serum metabolic profiling was analyzed and pathway analysis performed on the biomarkers reversed after YGJ administration and further integration of metabolic networks. The results showed that YGJ alleviated ear edema and decreased serum PGE 2 and TNF-α levels. Fourteen biomarkers were screened, and the levels were all reversed to different degrees after YGJ administration. These biomarkers were mainly related to linoleic acid metabolism, taurine and hypotaurine metabolism, glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism and citrate cycle (TCA cycle). In metabolic networks, glycine and pyruvate were node molecules. This indicated that YGJ could significantly inhibit inflammatory response triggered by acute local stimulation and exerted anti-inflammatory activity mainly by regulating node molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Serum metabonomic analysis of protective effects of Curcuma aromatica oil on renal fibrosis rats.

    Science.gov (United States)

    Zhao, Liangcai; Zhang, Haiyan; Yang, Yunjun; Zheng, Yongquan; Dong, Minjian; Wang, Yaqiang; Bai, Guanghui; Ye, Xinjian; Yan, Zhihan; Gao, Hongchang

    2014-01-01

    Curcuma aromatica oil is a traditional herbal medicine demonstrating protective and anti-fibrosis activities in renal fibrosis patients. However, study of its mechanism of action is challenged by its multiple components and multiple targets that its active agent acts on. Nuclear magnetic resonance (NMR)-based metabonomics combined with clinical chemistry and histopathology examination were performed to evaluate intervening effects of Curcuma aromatica oil on renal interstitial fibrosis rats induced by unilateral ureteral obstruction. The metabolite levels were compared based on integral values of serum 1H NMR spectra from rats on 3, 7, 14, and 28 days after the medicine administration. Time trajectory analysis demonstrated that metabolic profiles of the agent-treated rats were restored to control levels after 7 days of dosage. The results confirmed that the agent would be an effective anti-fibrosis medicine in a time-dependent manner, especially in early renal fibrosis stage. Targeted metabolite analysis showed that the medicine could lower levels of lipid, acetoacetate, glucose, phosphorylcholine/choline, trimethylamine oxide and raise levels of pyruvate, glycine in the serum of the rats. Serum clinical chemistry and kidney histopathology examination dovetailed well with the metabonomics data. The results substantiated that Curcuma aromatica oil administration can ameliorate renal fibrosis symptoms by inhibiting some metabolic pathways, including lipids metabolism, glycolysis and methylamine metabolism, which are dominating targets of the agent working in vivo. This study further strengthens the novel analytical approach for evaluating the effect of traditional herbal medicine and elucidating its molecular mechanism.

  8. Serum Creatinine: Not So Simple!

    Science.gov (United States)

    Delanaye, Pierre; Cavalier, Etienne; Pottel, Hans

    2017-01-01

    Measuring serum creatinine is cheap and commonly done in daily practice. However, interpretation of serum creatinine results is not always easy. In this review, we will briefly remind the physiological limitations of serum creatinine due notably to its tubular secretion and the influence of muscular mass or protein intake on its concentration. We mainly focus on the analytical limitations of serum creatinine, insisting on important concept such as reference intervals, standardization (and IDMS traceability), analytical interferences, analytical coefficient of variation (CV), biological CV and critical difference. Because the relationship between serum creatinine and glomerular filtration rate is hyperbolic, all these CVs will impact not only the precision of serum creatinine but still more the precision of different creatinine-based equations, especially in low or normal-low creatinine levels (or high or normal-high glomerular filtration rate range). © 2017 S. Karger AG, Basel.

  9. Radioimmunoassay of cholylglycine in serum

    International Nuclear Information System (INIS)

    Wakushima, Tadashi; Yamanishi, Yasuhito; Hirayama, Chisato

    1979-01-01

    Serum levels of cholylglycines (CG) were determined by radioimmunoassay and that of total bile acids (TBA) by enzymatic method. In normal subjects, serum levels of CG, TBA and CG/TBA ratio were 0.6 +- 0.4 μM, 7 +- 2 μM, and 0.08 +- 0.06, respectively. They were increased markedly in acute hepatitis and moderately in chronic hepatitis and cirrhosis. Thus, measurement of serum CG as compared with serum TBA appears to be a sensitive liver test. (author)

  10. Variation of transaminases, HCV-RNA levels and Th1/Th2 cytokine production during the post-partum period in pregnant women with chronic hepatitis C.

    Directory of Open Access Journals (Sweden)

    Angeles Ruiz-Extremera

    Full Text Available This study analyses the evolution of liver disease in women with chronic hepatitis C during the third trimester of pregnancy and the post-partum period, as a natural model of immune modulation and reconstitution. Of the 122 mothers recruited to this study, 89 were HCV-RNA+ve/HIV-ve and 33 were HCV-RNA-ve/HIV-ve/HCVantibody+ve and all were tested during the third trimester of pregnancy, at delivery and post-delivery. The HCV-RNA+ve mothers were categorized as either Type-A (66%, with an increase in ALT levels in the post-partum period (>40 U/L; P<0.001 or as Type-B (34%, with no variation in ALT values. The Type-A mothers also presented a significant decrease in serum HCV-RNA levels in the post-delivery period (P<0.001 and this event was concomitant with an increase in Th1 cytokine levels (INFγ, P = 0.04; IL12, P = 0.01 and IL2, P = 0.01. On the other hand, the Type-B mothers and the HCV-RNA-ve women presented no variations in either of these parameters. However, they did present higher Th1 cytokine levels in the partum period (INFγ and IL2, P<0.05 than both the Type-A and the HCV-RNA-ve women. Cytokine levels at the moment of delivery do not constitute a risk factor associated with HCV vertical transmission. It is concluded that differences in the ALT and HCV-RNA values observed in HCV-RNA+ve women in the postpartum period might be due to different ratios of Th1 cytokine production. In the Type-B women, the high partum levels of Th1 cytokines and the absence of post-partum variation in ALT and HCV-RNA levels may be related to permanent Th1 cytokine stimulation.

  11. Investigation of the biosynthesis of acetyl-CoA and oxaloacetic acid from pyruvic acid and the quantitative evaluation of incorporated 13C-labeled l-alanine in Arthrobacter hyalinus

    International Nuclear Information System (INIS)

    Katsumi Iida

    2014-01-01

    Studies on the contribution to acetyl-CoA and oxaloacetic acid from the pyruvic acid transformation from l-alanine in Arthrobacter hyalinus were conducted by means of feeding experiments with l-[1- 13 C]alanine and l-[3- 13 C]alanine, followed by an analysis of the labeling patterns of coproporphyrinogen III using 13 C NMR spectroscopy. The results demonstrated that l-alanine was transformed via pyruvic acid to both acetyl-CoA and oxaloacetic acid. Additionally, the quantitative analysis indicated that pyruvic acid was transformed to acetyl-CoA and oxaloacetic acid in the ratio of 1:0.8. (author)

  12. Relationship between serum total magnesium and serum potassium ...

    African Journals Online (AJOL)

    Relationship between serum total magnesium and serum potassium in emergency surgical patients in a tertiary hospital in Ghana. Robert Djagbletey, Brenda Phillips, Frank Boni, Christian Owoo, Ebenezer Owusu-Darkwa, Papa Kobina Gyakye deGraft-Johnson, Alfred E. Yawson ...

  13. Antioxidant and hepatoprotective activity of ethanolic and aqueous extracts of Limonia elephantum leaves

    Directory of Open Access Journals (Sweden)

    K. Kamalakannan

    2014-08-01

    Full Text Available Limonia elephantum (wood apple has been widely used in an Indian folk medicine system. In the present study, the acute toxicity studies were carried out to determine the safety of the compounds in liver diseases. The antioxidant and the hepatoprotective properties of the L. elephantum are evaluated against paracetamol induced hepatic damage in rats. Liver superoxide dismutase, lipid peroxidation, glutathione peroxidase, catalase levels and serum biochemical profile such as serum glutamate oxalate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, bilirubin and total protein were examined for the antioxidant and hepatoprotective effects of these treatments. The results of the present studies strongly indicate that the higher dose (400 mg/kg of ethanolic extract of L. elephantum proved against hepatoprotective effects and also the antioxidant properties.

  14. Avaliação dos níveis das transaminases dos pacientes com infecção pelo Plasmodium vivax atendidos em um centro de referência para tratamento da malária no Estado de Mato Grosso

    Directory of Open Access Journals (Sweden)

    Elisane de Freitas Pereira

    2017-11-01

    Full Text Available Objetivo: Analisar os marcadores hepáticos (transaminases em pacientes com malária causada pelo Plasmodium vivax. Métodos: Trata-se de um estudo transversal retrospectivo, incluindo pacientes que apresentaram monoinfecção pelo P. vivax. O teste de Mann-Whitney U ou o teste t de Student foram utilizados para comparar a atividade média das transaminases de acordo com a classificação clínica e epidemiológica do paciente infectado. O teste de Kruskal-Wallis foi utilizado para comparar a média da atividade das transaminases de acordo com a classificação da infecção. Valores de p<0,05 foram considerados significativos. Resultados: Indivíduos menores de 7 anos apresentaram maior atividade da transaminase glutâmico oxalacética (TGO e da transaminase glutâmico pirúvica (TGP — respectivamente, 68,7 U/L e 79,8 U/L — em relação a pacientes com idade de 8 a 59 anos (TGO 29,8 U/L e TGP 39,2 U/L e acima de 60 anos (TGO 32,5 U/L e TGP 34 U/L. A média da dosagem sérica de TGO de todos os pacientes incluídos no estudo foi de 31,99 U/L e de TGP foi de 41,1 U/L. A atividade de TGP nos pacientes primoinfectados foi maior que nos reinfectados ou com recaída, sendo 65,6 U/L contra 36,2 U/L e 32,7 U/L, respectivamente. Conclusão: Crianças apresentam maior atividade de transaminases que os indivíduos maiores de sete anos de idade. Pacientes com infecção prévia pelo P. vivax apresentam menores valores séricos da atividade das transaminases do que os primoinfectados. Indivíduos com episódios de recaída apresentam menores valores das enzimas avaliadas do que aqueles com reinfecção ou primoinfecção.

  15. STUDY OF SERUM HAPTOGLOBIN LEVEL AND ITS RELATION TO ERYTHROPOIETIC ACTIVITY IN BETA THALASSEMIA CHILDREN .

    Directory of Open Access Journals (Sweden)

    Seham Ragab

    2015-02-01

    Full Text Available Background  :Serum haptoglobin (Hp is a reliable marker for hemolysis regardless the inflammatory state.  Objective: We investigated the possible relation between Hp depletion and hemolysis severity, hepatitis C virus (HCV infection and iron load in β-thalassemia children. Methods: Twenty  two β-thalassemia major (TM ,20 β-thalassemia  intermedia (TI children with 20 age and sex matched healthy controls were involved. Pre-transfusion hemoglobin level was considered . Serum ferritin , Hp  and transferrin receptor  levels (sTfR  (by ELISA , alanine aminotransferase (ALT and  aspartate aminotransferase (AST  (by colorimetric method were assayed. Markers of hepatitis C virus  (HCV  were done by PCR. Results:  The mean Hp levels among the studied groups were as follows; 8.02 ± 0.93 (mg/dl , 8.6 ±0.72 (mg/dl  and 122  ± 18.5(mg/dl   for TM ,TI and the controls respectively . Both patient groups had significantly lower Hp level compared to the controls (P<0.0001  with significant lower level in TM compared to TI  children ( P= 0.034  .Significant inverse correlations were  found between serum Hp and sTfR levels in thalassemia children combined and in each group (TM and TI as well as among HCV infected children. STfR   was the only significant independent predictor for  serum Hp level (t= -5.585 , P<0.0001 . Among  HCV infected patients , no significant correlation was found between serum Hp and serum transaminases  .Conclusion:  Serum Hp depletion in thalassemia had significant relation to disease severity and correlated   well with their erythropoietic activity, as assessed by the measurement of  sTfR without significant relation  HCV infection . Large sample  multicenter studies are  recommended.

  16. DETERMINATION OF SERUM ALBUMIN WITH ...

    African Journals Online (AJOL)

    The reaction of tribromoarsenazo(TB-ASA) with serum albumin in the presence of emulgent OP was studied by spectrophotometry. In a Britton-Robinson buffer solution at pH 2.9, tribromoarsenazo and bovine serum albumin can immediately form a red compound in the presence of emulgent OP with a maximum absorption ...

  17. Inhibiting sperm pyruvate dehydrogenase complex and its E3 subunit, dihydrolipoamide dehydrogenase affects fertilization in Syrian hamsters.

    Directory of Open Access Journals (Sweden)

    Archana B Siva

    Full Text Available BACKGROUND/AIMS: The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc and its E3 subunit, dihydrolipoamide dehydrogenase (DLD in hamster in vitro fertilization (IVF via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. METHODOLOGY AND PRINCIPAL FINDINGS: Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid. Oocytes fertilized with MICA-treated (MT [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. CONCLUSIONS: This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In

  18. Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex*

    Science.gov (United States)

    Wang, Junjie; Nemeria, Natalia S.; Chandrasekhar, Krishnamoorthy; Kumaran, Sowmini; Arjunan, Palaniappa; Reynolds, Shelley; Calero, Guillermo; Brukh, Roman; Kakalis, Lazaros; Furey, William; Jordan, Frank

    2014-01-01

    The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s−1, comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP). PMID:24742683

  19. Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.

    Science.gov (United States)

    Wang, Junjie; Nemeria, Natalia S; Chandrasekhar, Krishnamoorthy; Kumaran, Sowmini; Arjunan, Palaniappa; Reynolds, Shelley; Calero, Guillermo; Brukh, Roman; Kakalis, Lazaros; Furey, William; Jordan, Frank

    2014-05-30

    The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP). © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Effect of L-carnitine and pyruvate on equine sperm maintained at 5 ºC and 15 ºC during 24 h: preliminary results

    Directory of Open Access Journals (Sweden)

    Avila G

    2016-12-01

    Full Text Available The aim of this study was to evaluate if the addition of L-carnitine and pyruvate to two semen transport extenders (Kenney and Kenney modified by Tyrodes is able to maintain sperm parameters for 24 h at 5 ºC and 15 ºC. Semen was obtained from 3 stallions (n=3; r=2 and at time 0 and after 24 h of cooling, the following parameters evaluated: total and progressive motility (CASA, viability and acrosome status (FITC-PNA-PI, membrane function (HOS, and DNA with Toluidine Blue stain (TB and the Sperm Chromatin Dispersion assay (SCD. Each temperature was individually analyzed using a factorial design with a 5% significance level. No interactions were observed. For the moment, the Kenney extender with the addition of L-carnitine and pyruvate showed the best results for maintaining most sperm parameters for 24 h at both 5 ºC and 15 ºC.

  1. Optimized methods to measure acetoacetate, 3-hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment.

    Science.gov (United States)

    Stappenbeck, R; Hodson, A W; Skillen, A W; Agius, L; Alberti, K G

    1990-01-01

    Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were 7.1 and 12% respectively. All seven metabolites can be measured in a perchloric acid extract of 20 mul of blood. The methods have been optimized with regard to variation in the perchloric acid content of the samples. These variations arise from the method of sample preparation used to minimize changes occurring in metabolite concentration after venepuncture.

  2. Protective effect of indole-3-pyruvate against ultraviolet b-induced damage to cultured HaCaT keratinocytes and the skin of hairless mice.

    Directory of Open Access Journals (Sweden)

    Reiji Aoki

    Full Text Available Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr, 4-hydroxyphenylpyruvate (HPPyr, and indole-3-pyruvate (IPyr against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm2 and maintained with or without test compounds (1-25 mM.In addition, the dorsal skin of hairless mice (HR-1 was treated with test compounds (10 μmol and exposed to UVB light (1 J/cm2 twice [corrected]. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1β (IL-1β and interleukin 6 (IL-6. IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2 expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1β and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.

  3. Preparation, characterization and thermal behaviour study of 4-dimethyl amino benzal pyruvate of lanthanides (III) and yttrium (III) in solid state

    International Nuclear Information System (INIS)

    Miyano, M.H.

    1990-01-01

    Solid state compounds involving Ln and DMBP, where Ln trivalent lanthanides (except promethium) and yttrium; DMBP 4-dimethyl amino benzylidene pyruvate, were prepared by addition of ligand to the corresponding metal ions chlorides, both in aqueous solution. The precipitates were washed with distilled water and dried at 40 0 C in a forced circulation oven. Complexometry with EDTA, thermogravimetry (TG), differential thermal analysis (DTA), infra-red absorption and X-ray diffraction have been used in the study of these compounds. (author)

  4. Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high-saturated fat diet

    OpenAIRE

    Hwang, Byounghoon; Wu, Pengfei; Harris, Robert A.

    2012-01-01

    Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) might prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it might induce detrimental effects by inhibiting fatty acid oxidation. PPARα agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment with a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of...

  5. Data regarding the growth of Lactobacillus acidophilus NCFM on different carbohydrates and recombinant production of elongation factor G and pyruvate kinase

    Directory of Open Access Journals (Sweden)

    Hasan Ufuk Celebioglu

    2017-10-01

    Full Text Available The present study describes the growth of the very well-known probiotic bacterium Lactobacillus acidophilus NCFM on different carbohydrates. Furthermore, recombinant production of putative moonlighting proteins elongation factor G and pyruvate kinase from this bacterium is described. For further and detailed interpretation of the data presented here, please see the research article “Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM” (Celebioglu et al., 2017 [1].

  6. Increased Interstitial Concentrations of Glutamate and Pyruvate in Vastus Lateralis of Women with Fibromyalgia Syndrome Are Normalized after an Exercise Intervention - A Case-Control Study.

    Directory of Open Access Journals (Sweden)

    Björn Gerdle

    Full Text Available Fibromyalgia syndrome (FMS is associated with central alterations, but controversies exist regarding the presence and role of peripheral factors. Microdialysis (MD can be used in vivo to study muscle alterations in FMS. Furthermore for chronic pain conditions such as FMS, the mechanisms for the positive effects of exercise are unclear. This study investigates the interstitial concentrations of algesics and metabolites in the vastus lateralis muscle of 29 women with FMS and 28 healthy women before and after an exercise intervention.All the participants went through a clinical examination and completed a questionnaire. In addition, their pressure pain thresholds (PPTs in their upper and lower extremities were determined. For both groups, MD was conducted in the vastus lateralis muscle before and after a 15-week exercise intervention of mainly resistance training of the lower limbs. Muscle blood flow and interstitial muscle concentrations of lactate, pyruvate, glutamate, glucose, and glycerol were determined.FMS was associated with significantly increased interstitial concentrations of glutamate, pyruvate, and lactate. After the exercise intervention, the FMS group exhibited significant decreases in pain intensity and in mean interstitial concentrations of glutamate, pyruvate, and glucose. The decrease in pain intensity in FMS correlated significantly with the decreases in pyruvate and glucose. In addition, the FMS group increased their strength and endurance.This study supports the suggestion that peripheral metabolic and algesic muscle alterations are present in FMS patients and that these alterations contribute to pain. After an exercise intervention, alterations normalized, pain intensity decreased (but not abolished, and strength and endurance improved, all findings that suggest the effects of exercise are partially peripheral.

  7. Technique development of 3D dynamic CS-EPSI for hyperpolarized 13 C pyruvate MR molecular imaging of human prostate cancer.

    Science.gov (United States)

    Chen, Hsin-Yu; Larson, Peder E Z; Gordon, Jeremy W; Bok, Robert A; Ferrone, Marcus; van Criekinge, Mark; Carvajal, Lucas; Cao, Peng; Pauly, John M; Kerr, Adam B; Park, Ilwoo; Slater, James B; Nelson, Sarah J; Munster, Pamela N; Aggarwal, Rahul; Kurhanewicz, John; Vigneron, Daniel B

    2018-03-25

    The purpose of this study was to develop a new 3D dynamic carbon-13 compressed sensing echoplanar spectroscopic imaging (EPSI) MR sequence and test it in phantoms, animal models, and then in prostate cancer patients to image the metabolic conversion of hyperpolarized [1- 13 C]pyruvate to [1- 13 C]lactate with whole gland coverage at high spatial and temporal resolution. A 3D dynamic compressed sensing (CS)-EPSI sequence with spectral-spatial excitation was designed to meet the required spatial coverage, time and spatial resolution, and RF limitations of the 3T MR scanner for its clinical translation for prostate cancer patient imaging. After phantom testing, animal studies were performed in rats and transgenic mice with prostate cancers. For patient studies, a GE SPINlab polarizer (GE Healthcare, Waukesha, WI) was used to produce hyperpolarized sterile GMP [1- 13 C]pyruvate. 3D dynamic 13 C CS-EPSI data were acquired starting 5 s after injection throughout the gland with a spatial resolution of 0.5 cm 3 , 18 time frames, 2-s temporal resolution, and 36 s total acquisition time. Through preclinical testing, the 3D CS-EPSI sequence developed in this project was shown to provide the desired spectral, temporal, and spatial 5D HP 13 C MR data. In human studies, the 3D dynamic HP CS-EPSI approach provided first-ever simultaneously volumetric and dynamic images of the LDH-catalyzed conversion of [1- 13 C]pyruvate to [1- 13 C]lactate in a biopsy-proven prostate cancer patient with full gland coverage. The results demonstrate the feasibility to characterize prostate cancer metabolism in animals, and now patients using this new 3D dynamic HP MR technique to measure k PL , the kinetic rate constant of [1- 13 C]pyruvate to [1- 13 C]lactate conversion. © 2018 International Society for Magnetic Resonance in Medicine.

  8. Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production.

    Science.gov (United States)

    Darr, Christa R; Varner, Dickson D; Teague, Sheila; Cortopassi, Gino A; Datta, Sandipan; Meyers, Stuart A

    2016-08-01

    Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity. © 2016 by the Society for the Study of Reproduction, Inc.

  9. Fluorimetric methods for the measurement of intermediate metabolites (lactate, pyruvate, alanine, β-hydroxybutyrate, glycerol) using a COBAS FARA centrifugal analyser

    OpenAIRE

    Monti, L. D.; Sandoli, P. E.; Costa, S.; Phan, V. C.; Piatti, P. M.

    1993-01-01

    Intermediate products of the metabolism of glucose, fat and amino-acid are important in the evaluation of such metabolic disorders as diabetes mellitus, liver disease and metabolic acidosis. In the present study, methods for the measurement of intermediate metabolites (lactate, pyruvate, alanine, β-hydroxybutyrate and glycerol) have been adapted to a fast centrifugal analyzer: the COBAS FARA. Correlation coeffcients rangedfrom 0.90 to 0.99, compared to established manual spectrophotometric me...

  10. Zingiber officinale Roscoe prevents acetaminophen-induced acute hepatotoxicity by enhancing hepatic antioxidant status.

    Science.gov (United States)

    Ajith, T A; Hema, U; Aswathy, M S

    2007-11-01

    A large number of xenobiotics are reported to be potentially hepatotoxic. Free radicals generated from the xenobiotic metabolism can induce lesions of the liver and react with the basic cellular constituents - proteins, lipids, RNA and DNA. Hepatoprotective activity of aqueous ethanol extract of Zingiber officinale was evaluated against single dose of acetaminophen-induced (3g/kg, p.o.) acute hepatotoxicity in rat. Aqueous extract of Z. officinale significantly protected the hepatotoxicity as evident from the activities of serum transaminase and alkaline phosphatase (ALP). Serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and ALP activities were significantly (pHepatic lipid peroxidation was enhanced significantly (pofficinale (200 and 400mg/kg, p.o.) prior to acetaminophen significantly declines the activities of serum transaminases and ALP. Further the hepatic antioxidant status was enhanced in the Z. officinale plus acetaminophen treated group than the control group. The results of the present study concluded that the hepatoprotective effect of aqueous ethanol extract of Z. officinale against acetaminophen-induced acute toxicity is mediated either by preventing the decline of hepatic antioxidant status or due to its direct radical scavenging capacity.

  11. Flux control analysis of mitochondrial oxidative phosphorylation in rat skeletal muscle: pyruvate and palmitoyl-carnitine as substrates give different control patterns

    DEFF Research Database (Denmark)

    Fritzen, Anette J; Grunnet, Niels; Quistorff, Bjørn

    2007-01-01

    was associated with the ADP-generating system, i.e., 0.58 +/- 0.05 with pyruvate, but significantly lower, 0.40 +/- 0.05, with palmitoyl-carnitine as substrate. The flux control coefficients of complex I, III and IV, the ATP synthase, the ATP/ADP carrier and the P(i) carrier were 0.070 +/- 0.03, 0.083 +/- 0.......04, 0.054 +/- 0.01, 0.11 +/- 0.03, 0.090 +/- 0.03 and 0.026 +/- 0.01, respectively, with pyruvate as substrate. With palmitoyl-carnitine all control coefficients were significantly different, except for the P(i) carrier (i.e., 0.024 +/- 0.001, 0.036 +/- 0.01, 0.052 +/- 0.02, 0.020 +/- 0.002, 0.034 +/- 0.......02 and 0.012 +/- 0.002, respectively), probably caused by the shift from NADH to FADH(2) oxidation. The sum of flux control coefficients was not significantly different from unity with pyruvate, while only 0.58 with palmitoyl-carnitine, indicating significant control contributions from the enzymes involved...

  12. Probing early tumor response to radiation therapy using hyperpolarized [1-¹³C]pyruvate in MDA-MB-231 xenografts.

    Directory of Open Access Journals (Sweden)

    Albert P Chen

    Full Text Available Following radiation therapy (RT, tumor morphology may remain unchanged for days and sometimes weeks, rendering anatomical imaging methods inadequate for early detection of therapeutic response. Changes in the hyperpolarized [1-¹³C]lactate signals observed in vivo following injection of pre-polarized [1-¹³C]pyruvate has recently been shown to be a marker for tumor progression or early treatment response. In this study, the feasibility of using ¹³C metabolic imaging with [1-¹³C]pyruvate to detect early radiation treatment response in a breast cancer xenograft model was demonstrated in vivo and in vitro. Significant decreases in hyperpolarized [1-¹³C]lactate relative to [1-¹³C]pyruvate were observed in MDA-MB-231 tumors 96 hrs following a single dose of ionizing radiation. Histopathologic data from the treated tumors showed higher cellular apoptosis and senescence; and changes in the expression of membrane monocarboxylate transporters and lactate dehydrogenase B were also observed. Hyperpolarized ¹³C metabolic imaging may be a promising new tool to develop novel and adaptive therapeutic regimens for patients undergoing RT.

  13. Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Zita Garate

    2015-12-01

    Full Text Available Pyruvate kinase deficiency (PKD is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs from peripheral blood mononuclear cells (PB-MNCs of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR. Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.

  14. Simultaneous Hyperpolarized 13C-Pyruvate MRI and 18F-FDG PET (HyperPET) in 10 Dogs with Cancer

    DEFF Research Database (Denmark)

    Gutte, Henrik; Hansen, Adam E; Larsen, Majbrit M E

    2015-01-01

    with biopsy-verified spontaneous malignant tumors were included for imaging. All dogs underwent a protocol of simultaneous (18)F-FDG PET, anatomic MR, and hyperpolarized dynamic nuclear polarization with (13)C-pyruvate imaging. The data were acquired using a combined clinical PET/MR imaging scanner. We found...... that combined (18)F-FDG PET and (13)C-pyruvate MRS imaging was possible in a single session of approximately 2 h. A continuous workflow was obtained with the injection of (18)F-FDG when the dogs was placed in the PET/MR scanner. (13)C-MRS dynamic acquisition demonstrated in an axial slab increased (13)C......With the introduction of combined PET/MR spectroscopic (MRS) imaging, it is now possible to directly and indirectly image the Warburg effect with hyperpolarized (13)C-pyruvate and (18)F-FDG PET imaging, respectively, via a technique we have named hyperPET. The main purpose of this present study...

  15. Pyruvic oxime nitrification and copper and nickel resistance by a Cupriavidus pauculus, an active heterotrophic nitrifier-denitrifier.

    Science.gov (United States)

    Ramirez, Miguel; Obrzydowski, Jennifer; Ayers, Mary; Virparia, Sonia; Wang, Meijing; Stefan, Kurtis; Linchangco, Richard; Castignetti, Domenic

    2014-01-01

    Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion. A Cupriavidus pauculus, previously thought an Alcaligenes sp. and noted as an active heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2) and nitrous oxide (N2O) while heterotrophically nitrifying the organic substrate pyruvic oxime [CH3-C(NOH)-COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic analyses indicated that the organism is a member of a genus (Cupriavidus) known for its resistance to metals and its metabolism of xenobiotics. The microbe (a Cupriavidus pauculus designated as C. pauculus strain UM1) was examined for its ability to perform heterotrophic nitrification in the presence of Cu(2+) and Ni(2+) and to metabolize the xenobiotic phenol. The bacterium heterotrophically nitrified well when either 1 mM Cu(2+) or 0.5 mM Ni(2+) was present in either enriched or minimal medium. The organism also used phenol as a sole carbon source in either the presence or absence of 1 mM Cu(2+) or 0.5 mM Ni(2+). The ability of this isolate to perform a number of different metabolisms, its noteworthy resistance to copper and nickel, and its potential use as a bioremediation agent are discussed.

  16. Evaluation of hyperpolarized [1-¹³C]-pyruvate by magnetic resonance to detect ionizing radiation effects in real time.

    Science.gov (United States)

    Sandulache, Vlad C; Chen, Yunyun; Lee, Jaehyuk; Rubinstein, Ashley; Ramirez, Marc S; Skinner, Heath D; Walker, Christopher M; Williams, Michelle D; Tailor, Ramesh; Court, Laurence E; Bankson, James A; Lai, Stephen Y

    2014-01-01

    Ionizing radiation (IR) cytotoxicity is primarily mediated through reactive oxygen species (ROS). Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC), an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.

  17. Evaluation of hyperpolarized [1-¹³C]-pyruvate by magnetic resonance to detect ionizing radiation effects in real time.

    Directory of Open Access Journals (Sweden)

    Vlad C Sandulache

    Full Text Available Ionizing radiation (IR cytotoxicity is primarily mediated through reactive oxygen species (ROS. Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP magnetic resonance spectroscopy (MRS and spectroscopic imaging (MRSI can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC, an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.

  18. Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer.

    Science.gov (United States)

    Chao, Tai-Kuang; Huang, Tien-Shuo; Liao, Yu-Ping; Huang, Rui-Lan; Su, Po-Hsuan; Shen, Hueng-Yuan; Lai, Hung-Cheng; Wang, Yu-Chi

    2017-01-01

    Pyruvate kinase M2 (PKM2) regulates glycolysis and oxidative phosphorylation; however, the role of PKM2 in ovarian cancer remains largely unknown. We investigated whether ovarian cancer metabolism could provide insight into the development of therapeutic strategies. We performed immunohistochemical staining for PKM2 on a tissue microarray for multivariate analysis. It revealed that patients exhibiting higher PKM2 expression were significantly associated with malignancy groups (p < 0.001) and pathogenesis models (p < 0.001), had poor progression-free survival rates (p = 0.01) as compared with patients exhibiting lower PKM2 levels, and yielded a hazard ratio of death of 2.02 (95% confidence interval: 0.70-5.85). In cell lines, PKM2 inhibitor significantly inhibited the glycolytic rate according to cellular glucose consumption (p < 0.001). We also utilized Seahorse assays to assess metabolism-related cell-specific factors and the impact of PKM2 inhibitors. Energy shifts as per Seahorse analysis showed attenuation of the extracellular acidification rate (p < 0.05) and no significant difference in oxygen-consumption rate in SKOV3 cells. Treatment with PKM2 inhibitor suppressed ovarian cancer growth and cell migration in vitro and inhibited tumor growth without significant toxicity in a xenograft study. PKM2 inhibition disturbed Warburg effects and inhibited ovarian cancer cell growth. Targeting PKM2 may constitute a promising therapy for patients with ovarian cancer, and clinical trials involving shikonin are warranted.

  19. Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism.

    Science.gov (United States)

    Plaga, W; Vielhaber, G; Wallach, J; Knappe, J

    2000-01-21

    The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969-975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)). Using [1-(14)C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.

  20. Preventing intraperitoneal adhesions with ethyl pyruvate and hyaluronic acid/carboxymethylcellulose: a comparative study in an experimental model.

    Science.gov (United States)

    Caglayan, E Kıyak; Caglayan, K; Erdogan, N; Cinar, H; Güngör, B

    2014-10-01

    To compare the effectiveness of ethyl pyruvate (EP) with that of hyaluronic acid+carboxymethyl cellulose (Seprafilm) for the prevention of intraperitoneal adhesions. Seprafilm has been shown to be effective in many experimental and clinical studies. Thirty rats were divided into three groups at random, and uterine horn abrasion was performed by laparotomy. One group received no treatment (control group), one group received a single intraperitoneal dose of EP 50mg/kg (EP group), and a 2×1-cm patch of Seprafilm was applied in the third group (Seprafilm group). All rats were killed 14 days after surgery. Macroscopic and histopathological evaluation were performed by a surgeon and a pathologist who were blinded to group allocation. Histopathologically, inflammation, fibroblastic activity, foreign body reaction, collagen proliferation, vascular proliferation, Masson-Trichrome score, matrix metalloproteinase-2 score and vascular endothelial growth factor score were studied. Median macroscopic intraperitoneal adhesion scores for the control, EP and Seprafilm groups were 2.8, 1.2 and 1.1, respectively. Multiple comparisons between groups showed a significant difference (p0.05). After histopathological evaluation, significant differences in all parameters were found between the groups (p0.0167). In comparison with the untreated control group, EP and Seprafilm were found to reduce the formation of intraperitoneal adhesions. No significant difference was found between EP and Seprafilm. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Tai-Kuang Chao

    Full Text Available Pyruvate kinase M2 (PKM2 regulates glycolysis and oxidative phosphorylation; however, the role of PKM2 in ovarian cancer remains largely unknown. We investigated whether ovarian cancer metabolism could provide insight into the development of therapeutic strategies. We performed immunohistochemical staining for PKM2 on a tissue microarray for multivariate analysis. It revealed that patients exhibiting higher PKM2 expression were significantly associated with malignancy groups (p < 0.001 and pathogenesis models (p < 0.001, had poor progression-free survival rates (p = 0.01 as compared with patients exhibiting lower PKM2 levels, and yielded a hazard ratio of death of 2.02 (95% confidence interval: 0.70-5.85. In cell lines, PKM2 inhibitor significantly inhibited the glycolytic rate according to cellular glucose consumption (p < 0.001. We also utilized Seahorse assays to assess metabolism-related cell-specific factors and the impact of PKM2 inhibitors. Energy shifts as per Seahorse analysis showed attenuation of the extracellular acidification rate (p < 0.05 and no significant difference in oxygen-consumption rate in SKOV3 cells. Treatment with PKM2 inhibitor suppressed ovarian cancer growth and cell migration in vitro and inhibited tumor growth without significant toxicity in a xenograft study. PKM2 inhibition disturbed Warburg effects and inhibited ovarian cancer cell growth. Targeting PKM2 may constitute a promising therapy for patients with ovarian cancer, and clinical trials involving shikonin are warranted.

  2. Pyruvate kinase M2 overexpression and poor prognosis in solid tumors of digestive system: evidence from 16 cohort studies.

    Science.gov (United States)

    Wu, Jiayuan; Hu, Liren; Chen, Manyu; Cao, Wenjun; Chen, Haicong; He, Taiping

    2016-01-01

    The expression of pyruvate kinase M2 (PKM2) has been linked to tumor formation and invasion. Specifically, the relationship between high PKM2 expression and prognosis has been evaluated in solid tumors of digestive system. However, the prognostic value of PKM2 remains controversial. A literature search of PubMed, Embase, and Cochrane databases was conducted until October 2015. The end point focused on overall survival (OS). The pooled hazard ratio (HR) or odds ratio and the 95% confidence intervals were calculated to correlate PKM2 overexpression with OS and clinicopathological characteristics by employing fixed- or random-effects models, depending on the heterogeneity of the included studies. We identified 18 cohorts in 16 studies involving 2,812 patients for this meta-analysis. Overall, the combined HR for OS in all tumor types was 1.74 (1.44-2.11; Pdigestive system, thereby suggesting that PKM2 might be an indicator of poor prognosis in digestive system cancers.

  3. In silico screening, genotyping, molecular dynamics simulation and activity studies of SNPs in pyruvate kinase M2.

    Directory of Open Access Journals (Sweden)

    Ponnusamy Kalaiarasan

    Full Text Available Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs and 2 mutations of Human Pyruvate Kinase (PK M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431 could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

  4. Pyruvic Oxime Nitrification and Copper and Nickel Resistance by a Cupriavidus pauculus, an Active Heterotrophic Nitrifier-Denitrifier

    Directory of Open Access Journals (Sweden)

    Miguel Ramirez

    2014-01-01

    Full Text Available Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion. A Cupriavidus pauculus, previously thought an Alcaligenes sp. and noted as an active heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2 and nitrous oxide (N2O while heterotrophically nitrifying the organic substrate pyruvic oxime [CH3–C(NOH–COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic analyses indicated that the organism is a member of a genus (Cupriavidus known for its resistance to metals and its metabolism of xenobiotics. The microbe (a Cupriavidus pauculus designated as C. pauculus strain UM1 was examined for its ability to perform heterotrophic nitrification in the presence of Cu2+ and Ni2+ and to metabolize the xenobiotic phenol. The bacterium heterotrophically nitrified well when either 1 mM Cu2+ or 0.5 mM Ni2+ was present in either enriched or minimal medium. The organism also used phenol as a sole carbon source in either the presence or absence of 1 mM Cu2+ or 0.5 mM Ni2+. The ability of this isolate to perform a number of different metabolisms, its noteworthy resistance to copper and nickel, and its potential use as a bioremediation agent are discussed.

  5. SNPs within the beta myosin heavy chain (MYH7 and the pyruvate kinase muscle (PKM2 genes in horse

    Directory of Open Access Journals (Sweden)

    Vincenzo Russo

    2010-01-01

    Full Text Available Two highly expressed skeletal muscle genes (the MYH7 gene encoding the myosin heavy chain slow/β-cardiac isoform and the PKM2 gene encoding the pyruvate kinase muscle isoforms were investigated with the objective to identify DNA markers in horses. A panel of DNA samples from different horse breeds was analysed using a PCR-single strand conformation polymorphism (SSCP approach. Four and two alleles were identified for the MYH7 and PKM2 loci, respectively. Mendelian inheritance of alleles of the two investigated genes was confirmed analysing horse families. Sequencing of PCR products obtained from the MYH7 and PKM2 genes made it possible to characterise two SSCP alleles for each gene. The polymorphisms found in the MYH7 and PKM2 genes were further studied in 61 and 68 horses of three (Italian Heavy Draught Horse, Italian Saddler and Murgese and five (Franches-Montagnes, Haflinger, Italian Heavy Draught Horse, Murgese and Standardbred breeds, respectively. Allele frequencies of the two loci varied among the considered breeds. The SNPs discovery in MYH7 and PKM2 genes makes it possible to locate new molecular markers to ECA1. The identified markers could be used in association analysis with performance traits in horses.

  6. Evaluation of miR-122 as a Serum Biomarker for Hepatotoxicity in Investigative Rat Toxicology Studies.

    Science.gov (United States)

    Sharapova, T; Devanarayan, V; LeRoy, B; Liguori, M J; Blomme, E; Buck, W; Maher, J

    2016-01-01

    MicroRNAs are short noncoding RNAs involved in regulation of gene expression. Certain microRNAs, including miR-122, seem to have ideal properties as biomarkers due to good stability, high tissue specificity, and ease of detection across multiple species. Recent reports have indicated that miR-122 is a highly liver-specific marker detectable in serum after liver injury. The purpose of the current study was to assess the performance of miR-122 as a serum biomarker for hepatotoxicity in short-term (5-28 days) repeat-dose rat toxicology studies when benchmarked against routine clinical chemistry and histopathology. A total of 23 studies with multiple dose levels of experimental compounds were examined, and they included animals with or without liver injury and with various hepatic histopathologic changes. Serum miR-122 levels were quantified by reverse transcription quantitative polymerase chain reaction. Increases in circulating miR-122 levels highly correlated with serum elevations of liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH). Statistical analysis showed that miR-122 outperformed ALT as a biomarker for histopathologically confirmed liver toxicity and was equivalent in performance to AST and GLDH. Additionally, an increase of 4% in predictive accuracy was obtained using a multiparameter approach incorporating miR-122 with ALT, AST, and GLDH. In conclusion, serum miR-122 levels can be utilized as a biomarker of hepatotoxicity in acute and subacute rat toxicology studies, and its performance can rival or exceed those of standard enzyme biomarkers such as the liver transaminases. © The Author(s) 2015.

  7. Elevated serum ferritin concentration is associated with incident type 2 diabetes mellitus in a Chinese population: A prospective cohort study.

    Science.gov (United States)

    Chen, Ling; Li, Yufeng; Zhang, Fang; Zhang, Simin; Zhou, Xianghai; Ji, Linong

    2018-03-08

    We aimed to evaluate the association between serum ferritin levels and incident type 2 diabetes mellitus risk in a Chinese population. This cohort study assessed 2225 Chinese individuals aged 25-75 years. Diabetes mellitus was diagnosed using the 1999 World Health Organization definition with a median follow-up period of 20 months. Cox proportional hazard models were used to estimate adjusted hazard ratios and 95% confidence intervals (CI) for incident diabetes when serum ferritin concentrations increased by one standard deviation. During the follow-up period, 112 cases (62 men and 50 women) of type 2 diabetes mellitus were identified. Baseline serum ferritin levels were higher in the diabetes than the non-diabetes group. After adjusting for age, body mass index, waist circumference, mean arterial pressure, fasting plasma glucose, fasting insulin, hemoglobin A1c, total cholesterol, high-density lipoprotein cholesterol, alanine transaminase and triglyceride levels, family history of diabetes mellitus, pork meat consumption, neutrophil/lymphocyte ratio, education, and annual household income, the hazard ratios for incident diabetes corresponding to one standard deviation increase in serum ferritin levels were 1.17 (95% CI 1.03, 1.34), 1.20 (95% CI 1.003, 1.43), and 1.03 (95% CI 0.82, 1.31) for the total population, men, and women, respectively. High serum ferritin levels were associated with a higher risk of incident type 2 diabetes mellitus independent of traditional risk factors in the total population and men. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Pattern of some risk factors of cardiovascular diseases and liver enzymes among Iranian seafarers

    DEFF Research Database (Denmark)

    Baygi, Fereshteh; Jensen, Olaf Chresten; Qorbani, Mostafa

    2017-01-01

    Background: Little information is available on the trend in cardiovascular risk factors and hepatic enzymes in Iranian seafarers. Thepresent study aimed at assessing the pattern of obesity, hypertension, diabetes, elevated serum glutamic oxaloacetate transaminase(SGOT), and serum glutamate pyruvate...... of antihypertensive drug use. Diabetes (DM) was defined as fasting blood sugar(FBS) > 110 mg/dl, or having a history of oral hypoglycemic agents; and elevated SGOT and SGPT were defined as SGOT > 40 U/Land SGPT > 40 U/L, respectively.Results: The BMI mean±SD values of Iranian seafarers were 24.81±3.07 kg/m2, 25...

  9. Study of Serum Amylase and Serum Cholinesterase in Organophosphorus Poisoning

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    Sharan Badiger

    2016-04-01

    Full Text Available Background: Poisoning due to organophosphorus compounds is most commonly seen. Earlier plasma cholinesterase level was used to assess the severity of poisoning. Presently serum amylase is being recommended as a better indicator of severity. Aims and Objectives: To study plasma cholinesterase and serum amylase levels in acute organophosphorus and to correlate serum amylase levels with clinical severity and outcome. Material and Methods: A total of 80 patients in the study admitted to a tertiary care centre within 24 hours with a history of organophosphorus poisoning were included in study. Estimation of plasma cholinesterase and serum rd amylase was done at the time of admission, and on 3 th day and on 5 day. Results: Occurrence of organophosphorus poisoning was more common among age group 21-30 years and among males (57.5%. They were 25 (31.2% farmers, 23 (28.8% st u d e n ts, a n d 2 2 ( 2 7 . 5% h o u s ewi v e s. Monocrotophos (45.0% was commonly used compound. Mean value of plasma cholinesterase and serum amylase at admission are 3693 U/L, and 185.4 U/L. There was significant inhibition of plasma cholinesterase and elevation of serum amylase at th admission with return to normal values on 5 day. Conclusion: Plasma cholinesterase inhibition 200 U/L has been associated with poor prognosis and proneness to respiratory failure.

  10. Relationships between certain metabolic diseases and selected serum biochemical parameters in seropositive dairy cows against Neospora caninum infection in different stages of lactation

    Science.gov (United States)

    Alekish, Myassar O.; Talafha, Abdelsalam Q; Alshehabat, Musa A; Ismail, Zuhair A Bani

    Neospora caninum is an important cause of abortion in dairy cattle. The general health of affected cows has not been investigated before. Therefore, the main objective of this study was to identify possible relationships between certain metabolic diseases and selected serum biochemical parameters in seropositive dairy cows against N. caninum antibodies in different stages of lactation. The study was carried out using 72 N. caninum seropositive cows and 61 seronegative dairy cows (control). Serum from all cows was tested to determine their N. caninum status (seropositive vs seronegative) using commercially available indirect enzyme-linked immunosorbent assay test kit (iELISA). In addition, serum biochemical parameters including beta-hydroxybutyrate (BHB), glucose, creatinine, blood urea nitrogen, total protein, albumin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH) and gamma-glutamyltranspeptidase (GGT) were determined using routine laboratory methods. The stage of lactation was obtained at the time of sampling from farm records. Student independent t-test showed that there was a significant difference in the serum concentrations of BHB, AST, ALT, and LDH between seropositive and seronegative cows. There was no significant association between seropositivity and the stage of lactation. However, multivariable logistic regression analysis showed that there was a strong association between seropositivity and BHB concentrations. Results of this study indicate a possible relationship between N. caninum seropositivity and certain metabolic diseases such as ketosis and fatty liver syndrome in dairy cows.

  11. Possible curative role of the anti psychotic drug fluphenazine against post-irradiation injury in rats

    International Nuclear Information System (INIS)

    Hassan, S.H.M.; Abu-Ghadeer, A.R.M.; Osman, S.A.A.; Roushdy, H.M.

    1986-01-01

    In the present study, investigation of the possible curative role of the anti psychotic agent ''fluphenazine'' against post irradiation injury of certain sensitive biological targets has been studied in rats. Such investigation includes evaluation of the haematological levels, liver function as manifested by levels of relevant serum enzymes and kidney function as reflected by level of serum creatinine and rate of urine creatinine clearance. Data of the present study indicated that fractionated whole body gamma-irradiation resulted in haematological disorders, significant elevation in serum enzyme activities of both serum glutamic pyruvic transaminase (SGPT) and serum alkaline phosphatase (SALKPH.), significant decrease in serum cholinesterase (SCHE) activity and a significant increase in serum creatinine accompanied by a significant decrease in creatinine clearance. 4 figs., 4 tabs

  12. Phosphate-Catalyzed Hydrogen Peroxide Formation from Agar, Gellan, and κ-Carrageenan and Recovery of Microbial Cultivability via Catalase and Pyruvate.

    Science.gov (United States)

    Kawasaki, Kosei; Kamagata, Yoichi

    2017-11-01

    Previously, we reported that when agar is autoclaved with phosphate buffer, hydrogen peroxide (H 2 O 2 ) is formed in the resulting medium (PT medium), and the colony count on the medium inoculated with environmental samples becomes much lower than that on a medium in which agar and phosphate are autoclaved separately (PS medium) (T. Tanaka et al., Appl Environ Microbiol 80:7659-7666, 2014, https://doi.org/10.1128/AEM.02741-14). However, the physicochemical mechanisms underlying this observation remain largely unknown. Here, we determined the factors affecting H 2 O 2 formation in agar. The H 2 O 2 formation was pH dependent: H 2 O 2 was formed at high concentrations in an alkaline or neutral phosphate buffer but not in an acidic buffer. Ammonium ions enhanced H 2 O 2 formation, implying the involvement of the Maillard reaction catalyzed by phosphate. We found that other gelling agents (e.g., gellan and κ-carrageenan) also produced H 2 O 2 after being autoclaved with phosphate. We then examined the cultivability of microorganisms from a fresh-water sample to test whether catalase and pyruvate, known as H 2 O 2 scavengers, are effective in yielding high colony counts. The colony count on PT medium was only 5.7% of that on PS medium. Catalase treatment effectively restored the colony count of PT medium (to 106% of that on PS medium). In contrast, pyruvate was not as effective as catalase: the colony count on sodium pyruvate-supplemented PT medium was 58% of that on PS medium. Given that both catalase and pyruvate can remove H 2 O 2 from PT medium, these observations indicate that although H 2 O 2 is the main cause of reduced colony count on PT medium, other unknown growth-inhibiting substances that cannot be removed by pyruvate (but can be by catalase) may also be involved. IMPORTANCE The majority of bacteria in natural environments are recalcitrant to laboratory culture techniques. Previously, we demonstrated that one reason for this is the formation of high H 2 O

  13. Serum metabolites, milk yield, and physiological responses during the first week after kidding in Anglo-Nubian, Angora, Baladi, and Damascus goats under subtropical conditions.

    Science.gov (United States)

    Anwar, M M; Ramadan, T A; Taha, T A

    2012-12-01

    This study was carried out to determine the level of certain biochemical variables reflecting the energy metabolic statuses during the first week of lactation in goats. A total of 120 Anglo-Nubian, Angora, Baladi, and Damascus does (30 does per breed) were used throughout 5 consecutive parities (30 does per parity) to investigate the effect of breed, parity, day of lactation, and their interaction on serum metabolites including total protein, albumin, globulin, glucose, total lipids, cholesterol, and transaminases. Blood samples were collected every other day during the first week of lactation. Baladi does had the greatest (P 0.05) after kidding. Baladi goats had the least (P 0.05) by breed whereas both rectal temperature and coefficient of heat tolerance were affected (P Baladi goats expressed an aspect of adaptability where their rectal temperature decreased and coefficient of heat tolerance increased with increasing parity number.

  14. The pyruvic acid analog 3-bromopyruvate interferes with the tetrazolium reagent MTS in the evaluation of cytotoxicity.

    Science.gov (United States)

    Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Vali, Mustafa

    2010-04-01

    3-Bromopyruvate (3BrPA) is a pyruvate analog known for its alkylating property. Recently, several reports have documented the antiglycolytic and anticancer effects of 3BrPA and its potential for therapeutic applications. 3BrPA-mediated cytotoxicity has been evaluated in vitro by various methods including tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)-based assays such as MTT, MTS, and so on. However, growing body of evidences has shown that tetrazolium reagent may interfere with the test compounds. In this study, we investigated whether the tetrazolium reagent interferes with the assessment of 3BrPA cytotoxicity. The results of the tetrazolium-based MTS assay were compared with 3 distinct cell viability detection methods, that is, Trypan Blue staining, ATP depletion, and Annexin V staining in 2 different cell lines, Vx-2 and HepG2. The MTS assay data showed false positive results by indicating increased cell viability at 1 mM and 2 mM 3BrPA whereas the other cell viability assays demonstrated that both Vx-2 and HepG2 cells are not viable at the same treatment conditions. In order to validate the direct interaction of 3BrPA with MTS reagent, we tested cell-free media incubated with different concentrations of 3BrPA. The results of cell-free media showed an increase in absorbance in a dose-dependent manner confirming the interaction of MTS with 3BrPA. Thus, our data clearly demonstrate that 3BrPA interferes with the accuracy of MTS-based cytotoxicity evaluation. Hence, we suggest that employing multiple methods of biochemical as well as morphological cytotoxicity assays is critical to evaluate 3BrPA-mediated cell death.

  15. Diagnostic value of fecal tumor M2-pyruvate kinase for CRC screening: a systematic review and meta-analysis.

    Science.gov (United States)

    Li, Rui; Liu, Jianjun; Xue, Huiping; Huang, Gang

    2012-10-15

    The measurement of fecal tumor M2-pyruvate kinase (PKM2), overexpressed in tumor cells, has been proposed as a novel tool for detecting colorectal cancer (CRC). However, the sensitivity and specificity of this test varied among studies. The aim of this meta-analysis was to determine the diagnostic accuracy of fecal PKM2 for CRC and to evaluate its utility in the CRC screening. It was compared to guaiac fecal occult blood test (gFOBT) or immunological fecal occult blood test (iFOBT). Through comprehensive literature search, 10 studies met the inclusion criteria and were included. Summary estimates for sensitivity and specificity were calculated by using the bivariate random effect model. The hierarchical summary receiver operating characteristic curve was also undertaken. The overall sensitivity and specificity of fecal PKM2 for detecting CRC were 79% (95% CI = 75-83%) and 81% (95% CI = 73-87%), respectively. The summary positive predictive value and negative predictive value were 74% (95% CI = 56-87%) and 86% (95% CI = 79-91%), respectively. The pooled diagnostic odds ratio was 16 (95% CI = 10-26). In head-to-head comparison, the diagnostic odds ratio of PKM2 and gFOBT for CRC were 10.167 (95% CI = 5.992-17.250) and 6.557 (95% CI = 3.467-12.403), respectively. The diagnostic odds ratio of PKM2 and iFOBT for CRC were 9.542 (95% CI = 5.893-15.452) and 67.248 (95% CI = 16.194-279.26), respectively. The fecal PKM2 test was a diagnostic tool with moderate sensitivity and specificity for detecting CRC. Its diagnostic efficiency was similar to that of gFOBT. Because of its relatively low specificity and positive predict value, fecal PKM2 was not recommended used alone as a screening tool for CRC. Copyright © 2012 UICC.

  16. H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells.

    Science.gov (United States)

    Ju, YoungJun; Untereiner, Ashley; Wu, Lingyun; Yang, Guangdong

    2015-11-01

    Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H(2)S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H(2)S S-sulfhydration is now proposed as a major mechanism for H(2)S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H(2)S and its implication in gluconeogenesis in the liver have been unknown. Gene expressions were analyzed by real-time PCR and western blotting, and protein S-sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively. Exogenously applied H(2)S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H(2)S-induced gluconeogenesis. H(2)S had no effect on the expressions of PC mRNA and protein, while H(2)S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H(2)S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H(2)S-induced PC S-sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S-sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression. CSE/H(2)S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver. Tissue-specific regulation of CSE/H(2)S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Effects of high-fat diet and physical activity on pyruvate dehydrogenase kinase-4 in mouse skeletal muscle

    Directory of Open Access Journals (Sweden)

    Rinnankoski-Tuikka Rita

    2012-06-01

    Full Text Available Abstract Background The expression of PDK4 is elevated by diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. It is previously shown that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α, a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4 gene expression via the estrogen-related receptor α (ERRα. We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1α and ERRα and the amount and function of mitochondria in skeletal muscle. Methods Insulin resistance was induced by a high-fat (HF diet for 19 weeks in C57BL/6 J mice, which were either sedentary or with access to running wheels. The skeletal muscle expression levels of PDK4, PGC-1α and ERRα were measured and the quality and quantity of mitochondrial function was assessed. Results The HF mice were more insulin-resistant than the low-fat (LF -fed mice. Upregulation of PDK4 and ERRα mRNA and protein levels were seen after the HF diet, and when combined with running even more profound effects on the mRNA expression levels were observed. Chronic HF feeding and voluntary running did not have significant effects on PGC-1α mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria. Conclusions Our results support the view that insulin resistance is not mediated by the decreased qualitative or quantitative properties of mitochondria. Instead, the role of PDK4 should be contemplated as a possible contributor to high-fat diet-induced insulin resistance.

  18. Distribution of phosphorylated metabolites and magnesium in the red cells of a patient with hyperactive pyruvate kinase

    International Nuclear Information System (INIS)

    Ouwerkerk, R.; van Echteld, C.J.; Staal, G.E.; Rijksen, G.

    1988-01-01

    The intracellular distribution of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) was studied in the red cells of a patient with a high-ATP syndrome by using 31P nuclear magnetic resonance. In this patient, red cell ATP was increased 2.5-fold, whereas 2,3-DPG was decreased fourfold due to the presence of a hyperactive pyruvate kinase. In oxygenated red cells, these abnormal concentrations were reflected to the same extent in all complexes in which ATP and 2,3-DPG take part. The diminished amount of 2,3-DPG bound to hemoglobin was almost completely replaced by ATP-hemoglobin complexes. Therefore, free hemoglobin was only slightly increased. In deoxygenated cells, the relative distribution of ATP and 2,3-DPG complexes was significantly disturbed. The main difference was a shift in the ratio of magnesium ATP (MgATP) over the ATP-hemoglobin complex; 74% of total ATP was complexed to hemoglobin (45% in normal cells), whereas the concentration of MgATP was only slightly increased with respect to normal. The shortage in 2,3-DPG bound to hemoglobin could partially be replenished by an increase in hemoglobin (Mg) ATP complexes. Therefore, the amount of uncomplexed hemoglobin raised from 15% in normal cells to 38% in the patient's cells. As a result, the oxygen-dissociation curve was only moderately shifted to the left. It is concluded that the regulatory role of 2,3-DPG in oxygen transport is taken over in part by (Mg) ATP in this patient. In both aerobic and anaerobic cells, the increase in magnesium bound to ATP, either free or bound to hemoglobin, exceeds the decrease in 2,3-DPG Mg complex. In spite of this, the amount of intracellular free Mg++ was normal or slightly lowered. This suggests the presence of a compensatory mechanism by which the amount of total cellular magnesium could be increased

  19. Skeletal Muscle Pyruvate Dehydrogenase Phosphorylation and Lactate Accumulation During Sprint Exercise in Normoxia and Severe Acute Hypoxia: Effects of Antioxidants

    Directory of Open Access Journals (Sweden)

    David Morales-Alamo

    2018-03-01

    Full Text Available Compared to normoxia, during sprint exercise in severe acute hypoxia the glycolytic rate is increased leading to greater lactate accumulation, acidification, and oxidative stress. To determine the role played by pyruvate dehydrogenase (PDH activation and reactive nitrogen and oxygen species (RNOS in muscle lactate accumulation, nine volunteers performed a single 30-s sprint (Wingate test on four occasions: two after the ingestion of placebo and another two following the intake of antioxidants, while breathing either hypoxic gas (PIO2 = 75 mmHg or room air (PIO2 = 143 mmHg. Vastus lateralis muscle biopsies were obtained before, immediately after, 30 and 120 min post-sprint. Antioxidants reduced the glycolytic rate without altering performance or VO2. Immediately after the sprints, Ser293- and Ser300-PDH-E1α phosphorylations were reduced to similar levels in all conditions (~66 and 91%, respectively. However, 30 min into recovery Ser293-PDH-E1α phosphorylation reached pre-exercise values while Ser300-PDH-E1α was still reduced by 44%. Thirty minutes after the sprint Ser293-PDH-E1α phosphorylation was greater with antioxidants, resulting in 74% higher muscle lactate concentration. Changes in Ser293 and Ser300-PDH-E1α phosphorylation from pre to immediately after the sprints were linearly related after placebo (r = 0.74, P < 0.001; n = 18, but not after antioxidants ingestion (r = 0.35, P = 0.15. In summary, lactate accumulation during sprint exercise in severe acute hypoxia is not caused by a reduced activation of the PDH. The ingestion of antioxidants is associated with increased PDH re-phosphorylation and slower elimination of muscle lactate during the recovery period. Ser293 re-phosphorylates at a faster rate than Ser300-PDH-E1α during the recovery period, suggesting slightly different regulatory mechanisms.

  20. Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo.

    Science.gov (United States)

    Novoa-Herran, Susana; Umaña-Perez, Adriana; Canals, Francesc; Sanchez-Gomez, Myriam

    2016-01-01

    How nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line. The viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test ( n  = 3, significance level 0.10, D > 0.642) and/or ANOVA ( n  = 3, p  depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells. This comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that

  1. Effects of lixisenatide on elevated liver transaminases

    DEFF Research Database (Denmark)

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

    2014-01-01

    ) on lixisenatide versus placebo or active comparators for type 2 diabetes were included. PARTICIPANTS: Individual patient data were retrieved to calculate outcomes for patients with elevated liver blood tests. MAIN OUTCOME MEASURES: Normalisation of alanine aminotransferase (ALT) and aspartate aminotransferase......OBJECTIVE: To evaluate the effects of the glucagon-like peptide-1 receptor agonist lixisenatide on elevated liver blood tests in patients with type 2 diabetes. DESIGN: Systematic review. DATA SOURCES: Electronic and manual searches were combined. STUDY SELECTION: Randomised controlled trials (RCTs...... the proportion of obese or overweight patients with type 2 diabetes who achieve normalisation of ALT. Additional research is needed to determine if the findings translate to clinical outcome measures. TRIAL REGISTRATION NUMBER: PROSPERO; CRD42013005779....

  2. Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design

    Directory of Open Access Journals (Sweden)

    Lee Gyun

    2010-09-01

    Full Text Available Abstract Background Serum-containing medium (SCM, which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs, a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis. Results From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS. Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM. Conclusions The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

  3. Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design.

    Science.gov (United States)

    Jeon, Min Kyoung; Lim, Jong-Baeck; Lee, Gyun Min

    2010-09-21

    Serum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis. From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM. The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

  4. Molecular association of glucose-6-phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells

    International Nuclear Information System (INIS)

    Das, Mahua R.; Bag, Arup K.; Saha, Shekhar; Ghosh, Alok; Dey, Sumit K.; Das, Provas; Mandal, Chitra; Ray, Subhankar; Chakrabarti, Saikat; Ray, Manju; Jana, Siddhartha S.

    2016-01-01

    For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. PKM2

  5. Pyruvate kinase deficiency

    Science.gov (United States)

    ... anemia may need blood transfusions. Removing the spleen ( splenectomy ) may help reduce the destruction of red blood ... donor blood or plasma. Someone who had a splenectomy should receive the pneumococcal vaccine at recommended intervals. ...

  6. Lecithin intake and serum cholesterol.

    NARCIS (Netherlands)

    Knuiman, J.T.; Beynen, A.C.; Katan, M.B.

    1989-01-01

    To find out whether the consumption of lecithin has a more beneficial effect on serum cholesterol than does the consumption of equivalent amounts of polyunsaturated oils, we scrutinized 24 studies on the effect of supplementary lecithin intakes ranging from 1 to 54 mg/d. Most of the studies lacked

  7. Serum transferrin receptor in polycythemia.

    Science.gov (United States)

    Manteiga, R; Remacha, A F; Sardà, M P; Ubeda, J

    1998-10-01

    We measured serum transferrin receptor (sTfR) levels in 22 patients with polycythemia vera and in 26 cases of secondary polycythemia. In our study, raised sTfR levels in both polycythemia groups were related to iron deficiency.

  8. Serum zinc level in thalassemia

    International Nuclear Information System (INIS)

    Keikhaei, B.; Badavi, M.; Pedram, M.; Zandian, K.

    2010-01-01

    To compare serum zinc level between Thalassemia Major (TM) patients and normal population at Shafa Hospital in South West of Iran. A total of 25 male and 36 female of TM patients were enrolled in this study. Out of 61 patients thirty were treated by deferroxamine (DFO) and 31 were on the combination of DFO and deferiprone (DEF) protocol therapy. Sixty normal subjects of the matching age and gender were recruited as controls. From each patient and control group 2 ml of blood was taken in fasting condition. Cell blood count and serum zinc were carried out for both thalassemia patients and normal subjects. The mean age of patients and control group was 15+- 5 years. Mean serum zinc level was 68.97+- 21.12 mu g/dl, 78.10-28.50 mu g/dl, and 80.16+- 26.54 mu g/dl in the TM with DFO, TM with DFO + DEF combination protocol and control group respectively. There was no significant correlation between patients and control group. However 50 percent of TM with DFO, 38.7 percent of TM with DFO + DEF and 32.8 percent of control group had hypozincemia. Nearly 40 to 50 percent of TM patients and one third of normal subjects are suffering from hypozincemia. This study shows that low level of serum zinc is a health problem in both thalassemia patients and normal population in South West of Iran. (author)

  9. HPLC Determination of α-Keto Acids from Human Serum Using 2, 3-Diamino-2, 3- Dimethyl Butane as Derivatizing Reagent

    International Nuclear Information System (INIS)

    Mahar, K.P.; Shar, G.Q.; Khuhawar, M.Y.; Abbasi, K.U.; Azmat, R.; Jameel Ahmed Biag, J.A.

    2013-01-01

    Seven α-keto acids, pyruvic acid (PYR), 2-oxobutyric acid (KB), 3-methyl-2-oxobutyric acid (MKBA), 3-methyl-2-oxovaleric acid (K3MVA), 4-methyl-2-oxovaleric acid (K4MVA), 2-oxoglutaric acid (KG) and Phenyl pyruvic acid (PPY) as derivatives of 2,3-diamino-2,3-dimethybutane (DDB) were separated by HPLC column Zorbax C-18 (4.6x150 mm-id). The compounds were eluted with methanol-water-acetonitrile (40:58:2 V/V/V) with flow rate 1 ml/min. UV detection was carried out by photodiode array at 255 nm. Linear calibration plots were obtained with 0.1 to 60 μg/ml with limits of detection (LoD) within 0.04-0.4 μg/ml. The method was applied for the analysis of α-keto acids from serum of diabetic patients with blood glucose level 430-458 mg/dl and healthy volunteers. The amounts of α-keto acids observed 3.24-9.71 μg/ml with RSD 1.1-1.9 percentage in diabetic patients were higher than healthy volunteer's 0.11-1.3 μg/ml with RSD 0.9-2.6 percentage. (author)

  10. Determination of pyruvate and lactate as potential biomarkers of embryo viability in assisted reproduction by capillary electrophoresis with contactless conductivity detection.

    Science.gov (United States)

    Mádr, Aleš; Celá, Andrea; Klejdus, Bořivoj; Pelcová, Marta; Crha, Igor; Žáková, Jana; Glatz, Zdeněk

    2015-06-01

    Human-assisted reproduction is increasing in importance due to the constantly rising number of couples suffering from infertility issue. A key step in in vitro fertilization is the proper assessment of embryo viability in order to select the embryo with the highest likelihood of resulting in a pregnancy. This study proposes a method based on CE with contactless conductivity detection for the determination of pyruvate and lactate in spent culture media used in human-assisted reproduction. A fused-silica capillary of 64.0 cm total length and 50 μm inner diameter was used. The inner capillary wall was modified by the coating of successive layers of the ionic polymers polybrene and dextran sulfate to reverse EOF. The BGE was composed of 10 mM MES/lithium hydroxide, pH 6.50. The sample was injected by pressure 50 mbar for 18 s, separation voltage was set to -24 kV, and capillary temperature to 15°C. The presented method requires only 2 μL of the culture medium, with LODs for pyruvate and lactate of 0.03 and 0.02 μM, respectively. The results demonstrated the method's suitability for the analysis of spent culture media to support embryo viability assessment by light microscopy, providing information about key metabolites of the energy metabolism of a developing embryo. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as non-invasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate

    Science.gov (United States)

    Matsumoto, Shingo; Saito, Keita; Yasui, Hironobu; Morris, H. Douglas; Munasinghe, Jeeva P.; Lizak, Martin; Merkle, Hellmut; Ardenkjaer-Larsen, Jan Henrik; Choudhuri, Rajani; Devasahayam, Nallathamby; Subramanian, Sankaran; Koretsky, Alan P.; Mitchell, James B.; Krishna, Murali C.

    2012-01-01

    The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation, and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate (3-BP) blocks glycolysis pathway by inhibiting hypoxia inducible enzymes, and enhanced cytotoxicity of 3-BP under hypoxic conditions has been reported in vitro. However, the efficacy of 3-BP was substantially attenuated in hypoxic tumor regions (pO2 < 10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 (MCT1) is the major transporter for pyruvate and the analog 3-BP in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of MCT1 in vivo. Expression of MCT1 was enhanced in moderately hypoxic (8–15 mmHg) tumor regions, but down regulated in severely hypoxic (< 5 mmHg) tumor regions. These results emphasize the importance of non-invasive imaging biomarkers to confirm the action of hypoxia-activated drugs. PMID:22692861

  12. Assessing the survival of MRC5 and a549 cell lines upon exposure to pyruvic Acid, sodium citrate and sodium bicarbonate - biomed 2013.

    Science.gov (United States)

    Farah, Ibrahim O; Lewis, Veshell L; Ayensu, Wellington K; Cameron, Joseph A

    2013-01-01

    Lung cancer is among the most prevalent and deadly cancers in United States. In general, cancer cells are known to exhibit higher rates of glycolysis in comparison to normal cells. In attempting to exploit this unique cancer-dependent ATP generation phenomenon, it was our hypothesis that upon exposure to organic inhibitors of glycolysis, cancer cells would not survive normally and that their growth and viability would be vastly decreased; essential glycolytic ATP production will be exhausted to the point of collapsing energy utilization. Furthermore, we hypothesize that no negative effect would be seen with exposures to organic inhibitors for normal lung cells. The human lung fibroblast MRC-5 and the human A549 alveolar epithelial cell lines were used as in vitro models of normal lung and lung cancers respectively. Using standard methods, both cell lines were maintained and exposed to pyruvic acid, sodium citrate and sodium bicarbonate reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging for parallel morphological displays of any changes in the course of their vitality and metabolic activities. Our results indicate that exposure of both cell lines to these organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Pyruvic acid, sodium citrate and sodium bicarbonate showed statistically significant (pcancer biotherapeutics.

  13. Carbon-14 tracer studies in the metabolism of isolated rat-liver parenchymal cells under conditions of gluconeogenesis from lactate and pyruvate

    International Nuclear Information System (INIS)

    Muellhofer, G.; Mueller, C.; Stetten, C. von; Gruber, E.

    1977-01-01

    In rat liver perfusion experiments under conditions of gluconeogenesis from lactate and pyruvate, 14 C-labeling patterns of metabolites with (1- 14 C)-labeled and (2- 14 C)-labeled lactate or pyruvate. [ 14 C]bicarbonate and [1- 14 C]octanoate as tracers have been obtained which do not agree with generally assumed reaction schemes. The experiments have been repeated with incubations of isolated rat-liver parenchymal cells. The results demonstrate that the discrepancies between expected and analysed 14 C-labeling patterns of metabolites were still existent. From this observation, it may be concluded that 14 C-labelling patterns of metabolites are indicative for the existence of still unknown metabolic relationships in liver parenchymal cells. Furthermore, the results of our experiments prove that conclusions based on the exclusive analysis of metabolite levels are of limited value for studying intracellular events, because of uncharacterized compartmentations, which become evident in 14 C-tracer studies. It cannot be answered by our studies whether the apparent existence of differently labelled species of citrate, oxoglutarate, or acetyl-CoA represent intracellular compartmentation, or whether it is the result of metabolic heterogeneity of liver parenchym cells. (orig.) [de

  14. Application of mitochondrial pyruvate carrier blocker UK5099 creates metabolic reprogram and greater stem-like properties in LnCap prostate cancer cells in vitro

    Science.gov (United States)

    Zhong, Yali; Li, Xiaoran; Yu, Dandan; Li, Xiaoli; Li, Yaqing; Long, Yuan; Yuan, Yuan; Ji, Zhenyu; Zhang, Mingzhi; Wen, Jian-Guo; Nesland, Jahn M.; Suo, Zhenhe

    2015-01-01

    Aerobic glycolysis is one of the important hallmarks of cancer cells and eukaryotic cells. In this study, we have investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with UK5099 and the metabolic alteration as well as stemness phenotype of prostatic cancer cells. It was found that blocking pyruvate transportation into mitochondrial attenuated mitochondrial oxidative phosphorylation (OXPHOS) and increased glycolysis. The UK5099 treated cells showed significantly higher proportion of side population (SP) fraction and expressed higher levels of stemness markers Oct3/4 and Nanog. Chemosensitivity examinations revealed that the UK5099 treated cells became more resistant to chemotherapy compared to the non-treated cells. These results demonstrate probably an intimate connection between metabolic reprogram and stem-like phenotype of LnCap cells in vitro. We propose that MPC blocker (UK5099) application may be an ideal model for Warburg effect studies, since it attenuates mitochondrial OXPHOS and increases aerobic glycolysis, a phenomenon typically reflected in the Warburg effect. We conclude that impaired mitochondrial OXPHOS and upregulated glycolysis are related with stem-like phenotype shift in prostatic cancer cells. PMID:26413751

  15. Ethyl pyruvate ameliorates hepatic injury following blunt chest trauma and hemorrhagic shock by reducing local inflammation, NF-kappaB activation and HMGB1 release.

    Science.gov (United States)

    Wagner, Nils; Dieteren, Scott; Franz, Niklas; Köhler, Kernt; Mörs, Katharina; Nicin, Luka; Schmidt, Julia; Perl, Mario; Marzi, Ingo; Relja, Borna

    2018-01-01

    The treatment of patients with multiple trauma including blunt chest/thoracic trauma (TxT) and hemorrhagic shock (H) is still challenging. Numerous studies show detrimental consequences of TxT and HS resulting in strong inflammatory changes, organ injury and mortality. Additionally, the reperfusion (R) phase plays a key role in triggering inflammation and worsening outcome. Ethyl pyruvate (EP), a stable lipophilic ester, has anti-inflammatory properties. Here, the influence of EP on the inflammatory reaction and liver injury in a double hit model of TxT and H/R in rats was explored. Female Lewis rats were subjected to TxT followed by hemorrhage/H (60 min, 35±3 mm Hg) and resuscitation/R (TxT+H/R). Reperfusion was performed by either Ringer`s lactated solution (RL) alone or RL supplemented with EP (50 mg/kg). Sham animals underwent all surgical procedures without TxT+H/R. After 2h, blood and liver tissue were collected for analyses, and survival was assessed after 24h. Resuscitation with EP significantly improved haemoglobin levels and base excess recovery compared with controls after TxT+H/R, respectively (ptrauma and hemorrhagic shock is associated with NF-κB. In particular, the beneficial anti-inflammatory effects of ethyl pyruvate seem to be regulated by the HMGB1/NF-κB axis in the liver, thereby, restraining inflammatory responses and liver injury after double hit trauma in the rat.

  16. Relationship between exposure duration, carboxyhemoglobin, blood glucose, pyruvate and lactate and the severity of intoxication in 39 cases of acute carbon monoxide poisoning in man

    Energy Technology Data Exchange (ETDEWEB)

    Sokal, J.A.; Kralkowska, E.

    1985-08-01

    The relationship between exposure duration, COHb, blood glucose, pyruvate and lactate and the severity of intoxication was investigated in a group of 39 cases of acute CO poisoning treated in the Clinical Toxicology Center in Lodz, Poland. On the basis of clinical criteria the patients were classified into cases of mild, moderate, severe and very severe CO poisoning. COHb and carbohydrate metabolites were estimated in venous blood taken immediately after admission of the patient to hospital prior to treatment. The severity of intoxication did not correlate with blood COHb; variation in exposure duration seems to be responsible for this phenomenon. Severe and very severe poisonings were associated with longer exposures and were accompanied by a markedly higher blood lactate level, compared to mild and moderate cases. Blood pyruvate depended less than lactate on the severity of intoxication. Blood glucose depended neither on exposure duration nor on the severity of intoxication. Among the carbohydrate metabolic parameters studied, blood lactate determination can be helpful in the evaluation of the severity of CO poisoning in man.

  17. Metabonomic profiling of renal cell carcinoma: High-resolution proton nuclear magnetic resonance spectroscopy of human serum with multivariate data analysis

    International Nuclear Information System (INIS)

    Gao Hongchang; Dong Baijun; Liu Xia; Xuan Hanqing; Huang Yiran; Lin Donghai

    2008-01-01

    Metabonomic profiling using proton nuclear magnetic resonance ( 1 H NMR) spectroscopy and multivariate data analysis of human serum samples was used to characterize metabolic profiles in renal cell carcinoma (RCC). We found distinct, easily detectable differences between (a) RCC patients and healthy humans, (b) RCC patients with metastases and without metastases, and (c) RCC patients before and after nephrectomy. Compared to healthy human serum, RCC serum had higher levels of lipid (mainly very low-density lipoproteins), isoleucine, leucine, lactate, alanine, N-acetylglycoproteins, pyruvate, glycerol, and unsaturated lipid, together with lower levels of acetoacetate, glutamine, phosphatidylcholine/choline, trimethylamine-N-oxide, and glucose. This pattern was somewhat reversed after nephrectomy. Altered metabolite concentrations are most likely the result of the cells switching to glycolysis to maintain energy homeostasis following the loss of ATP caused by impaired TCA cycle in RCC. Serum NMR spectra combined with principal component analysis techniques offer an efficient, convenient way of depicting tumour biochemistry and stratifying tumours under different pathophysiological conditions. It may be able to assist early diagnosis and postoperative surveillance of human malignant diseases using single blood samples

  18. Coordinate cis-[Cr(C2O4(pm(OH22]+ Cation as Molecular Biosensor of Pyruvate’s Protective Activity Against Hydrogen Peroxide Mediated Cytotoxity

    Directory of Open Access Journals (Sweden)

    Lech Chmurzyński

    2008-08-01

    Full Text Available In this paper instrumental methods of carbon dioxide (CO2 detection in biological material were compared. Using cis-[Cr(C2O4(pm(OH22]+ cation as a specific molecular biosensor and the stopped-flow technique the concentrations of CO2 released from the cell culture medium as one of final products of pyruvate decomposition caused by hydrogen peroxide were determined. To prove the usefulness of our method of CO2 assessment in the case of biological samples we investigated protective properties of exogenous pyruvate in cultured osteosarcoma 143B cells exposed to 1 mM hydrogen peroxide (H2O2 added directly to culture medium. Pyruvic acid is well known scavenger of H2O2 and, moreover, a molecule which is recognized as one of the major mediator of oxidative stress detected in many diseases and pathological situations like ischemiareperfusion states. The pyruvate's antioxidant activity is described as its rapid reaction with H2O2,which causes nonenzymatic decarboxylation of pyruvate and releases of CO2, water and acetate as final products. In this work for the first time we have correlated the concentration of CO2 dissolved in culture medium with pyruvate's oxidant-scavenging abilities. Moreover, the kinetics of the reaction between aqueous solution of CO2 and coordinate ion, cis-[Cr(C2O4(pm(OH22]+ was analysed. The results obtained enabled determination of the number of steps of the reaction studied. Based on the kinetic equations, rate constants were determined for each step.

  19. Increased serum potassium affects renal outcomes

    DEFF Research Database (Denmark)

    Miao, Y; Dobre, D; Heerspink, H J Lambers

    2011-01-01

    To assess the effect of an angiotensin receptor blocker (ARB) on serum potassium and the effect of a serum potassium change on renal outcomes in patients with type 2 diabetes and nephropathy.......To assess the effect of an angiotensin receptor blocker (ARB) on serum potassium and the effect of a serum potassium change on renal outcomes in patients with type 2 diabetes and nephropathy....

  20. Serum VEGF-A and CCL5 levels as candidate biomarkers for efficacy and toxicity of regorafenib in patients with metastatic colorectal cancer

    Science.gov (United States)

    Suenaga, Mitsukuni; Mashima, Tetsuo; Kawata, Naomi; Wakatsuki, Takeru; Horiike, Yuki; Matsusaka, Satoshi; Dan, Shingo; Shinozaki, Eiji; Seimiya, Hiroyuki; Mizunuma, Nobuyuki; Yamaguchi, Kensei; Yamaguchi, Toshiharu

    2016-01-01

    Regorafenib is an oral multi-kinase inhibitor used as salvage therapy for metastatic colorectal cancer (mCRC). We tested whether serum cytokine levels are associated with clinical outcome in the mCRC patients receiving regorafenib. Serum samples were collected before treatment start, day 21, and progressive disease, and eleven angiogenic and inflammatory cytokine serum levels were examined. Fifty-four patients of a total of 62 enrolled patients were eligible for the analyses. The chemokine ligand 5 (CCL5) levels ≤ cut-off value (59959 pg/ml) at baseline was associated with relative tumor shrinkage (P = 0.021), better progression-free survival (PFS) (P = 0.036) and overall survival (OS) (P = 0.019). Vascular endothelial growth factor A (VEGF-A) levels showing a decrease on day 21 were significantly associated with a better PFS (P = 0.021). CCL5 levels ≤ cut-off was associated with any grade hand-foot skin reaction (HFSR) (P = 0.025) and thrombocytopenia (P = 0.013). Low chemokine ligand 2 levels at baseline were associated with grade 2 ≤ HFSR. High angiopoietin-2 and basic fibroblast growth factor (bFGF) levels at baseline were associated with grade 3 ≤ total bilirubin increase and transaminases increase, respectively. Low bFGF levels at baseline were associated with grade 3 ≤ hypertension. No correlation with severe events was observed. Baseline serum CCL5 levels and decrease of the serum VEGF-A levels may serve as potential predictive markers for survival or treatment-specific toxicities in mCRC patients receiving regorafenib. PMID:27166185

  1. Serum Levels of Interleukin 33 and Soluble ST2 Are Associated with the Extent of Disease Activity and Cutaneous Manifestations in Patients with Active Adult-onset Still's Disease.

    Science.gov (United States)

    Han, Jae Ho; Suh, Chang-Hee; Jung, Ju-Yang; Ahn, Mi-Hyun; Kwon, Ji Eun; Yim, Hyunee; Kim, Hyoun-Ah

    2017-06-01

    Interleukin 33 (IL-33), a member of the IL-1 family and a ligand of the orphan receptor ST2, plays key roles in innate and adaptive immunity. We examined the associations between IL-33/ST2 levels and clinical manifestations of patients with active adult-onset Still's disease (AOSD). Blood samples were collected from 40 patients with active AOSD, 28 patients with rheumatoid arthritis (RA), and 27 healthy controls (HC). The serum levels of IL-33 and soluble ST2 were determined using ELISA. Expression levels of IL-33 and ST2 in biopsy specimens obtained from 34 AOSD patients with rash were immunohistochemically investigated. IL-33 levels of patients with AOSD were higher than those of patients with RA and HC. Soluble ST2 levels of patients with AOSD were higher than those of HC, but not of patients with RA. Serum IL-33 levels correlated with systemic score, erythrocyte sedimentation rate, ferritin levels, and aspartate transaminase levels. However, serum soluble ST2 levels correlated only with ferritin levels. The numbers of inflammatory cells expressing IL-33 and ST2 were elevated in skin lesions of patients with AOSD compared to HC, but did not differ from those of the skin lesions of eczema or psoriasis. We found significantly higher serum IL-33 and soluble ST2 levels in patients with active AOSD. Results indicate that the IL-33/ST2 signaling pathway may play a role in the pathogenesis of the acute inflammation and skin manifestations associated with AOSD.

  2. High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate beta-lyase activity : application to S-1,2-dichlorovinyl-L-cysteine and S-2-benzothiazolyl-L-cysteine

    NARCIS (Netherlands)

    Stijntjes, G.J.; te Koppele, J.M.; Vermeulen, N P

    1992-01-01

    An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid,

  3. Serum Antibody Biomarkers for ASD

    Science.gov (United States)

    2015-10-01

    typically developing control. US, unaffected sibling control. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a...typically developing (TD) children (e.g., Warren et al., 1990; Singh, 2009). The goal of this study is to identify a serum antibody biomarker for ASD using...50% less IgG1 antibody in ASD boys vs . TD boys (p=0.0096). The level of ASD1 binding to the AM group was the same as to the ASD boys. These data

  4. Assessment of real-time myocardial uptake and enzymatic conversion of hyperpolarized [1-¹³C]pyruvate in pigs using slice selective magnetic resonance spectroscopy

    DEFF Research Database (Denmark)

    Menichetti, Luca; Frijia, Francesca; Flori, Alessandra

    2012-01-01

    . We applied a numerical approach for spectral analysis and kinetic fitting (LSFIT/KIMOfit), making a comparison with a well-known jMRUI/AMARES analysis and γ-variate function, and we estimated the apparent conversion rate of hyperpolarized [1-¹³C]pyruvate into its downstream metabolites [1-¹³C......]lactate, [1-¹³C]alanine and [¹³C]bicarbonate in a 3 T MR scanner. We detected an increase in the apparent kinetic constants (k(PX) ) for bicarbonate and lactate of two-fold during dobutamine infusion. These data correlate with the double product (rate-pressure product), an indirect parameter of cardiac oxygen...

  5. Differentiating inflamed and normal lungs by the apparent reaction rate constants of lactate dehydrogenase probed by hyperpolarized (13)C labeled pyruvate.

    Science.gov (United States)

    Xu, He N; Kadlececk, Stephen; Shaghaghi, Hoora; Zhao, Huaqing; Profka, Harilla; Pourfathi, Mehrdad; Rizi, Rahim; Li, Lin Z

    2016-02-01

    Clinically translatable hyperpolarized (HP) (13)C-NMR can probe in vivo enzymatic reactions, e.g., lactate dehydrogenase (LDH)-catalyzed reaction by injecting HP (13)C-pyruvate into the subject, which is converted to (13)C labeled lactate by the enzyme. Parameters such as (13)C-lactate signals and lactate-to-pyruvate signal ratio are commonly used for analyzing the HP (13)C-NMR data. However, the biochemical/biological meaning of these parameters remains either unclear or dependent on experimental settings. It is preferable to quantify the reaction rate constants with a clearer physical meaning. Here we report the extraction of the kinetic parameters of the LDH reaction from HP (13)C-NMR data and investigate if they can be potential predictors of lung inflammation. Male Sprague-Dawley rats (12 controls, 14 treated) were used. One dose of bleomycin (2.5 U/kg) was administered intratracheally to the treatment group. The lungs were removed, perfused, and observed by the HP-NMR technique, where a HyperSense dynamic nuclear polarization system was used to generate the HP (13)C-pyruvate for injecting into the lungs. A 20 mm (1)H/(13)C dual-tuned coil in a 9.4-T Varian vertical bore NMR spectrometer was employed to acquire the (13)C spectral data every 1 s over a time period of 300 s using a non-selective, 15-degree radiofrequency pulse. The apparent rate constants of the LDH reaction and their ratio were quantified by applying ratiometric fitting analysis to the time series data of (13)C labeled pyruvate and lactate. The apparent forward rate constant kp =(3.67±3.31)×10(-4) s(-1), reverse rate constant kl =(4.95±2.90)×10(-2) s(-1), rate constant ratio kp /kl =(7.53±5.75)×10(-3) for the control lungs; kp =(11.71±4.35)×10(-4) s(-1), kl =(9.89±3.89)×10(-2) s(-1), and kp /kl =(12.39±4.18)×10(-3) for the inflamed lungs at the 7(th) day post treatment. Wilcoxon rank-sum test showed that the medians of these kinetic parameters of the 7-day cohort were significantly

  6. Impact of the Staphylococcus epidermidis LytSR two-component regulatory system on murein hydrolase activity, pyruvate utilization and global transcriptional profile

    Directory of Open Access Journals (Sweden)

    Yu Fangyou

    2010-11-01

    Full Text Available Abstract Background Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown. Results In the present study, we demonstrated that lytSR knock-out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytSR in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart, 1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside. Microarray analysis showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated. Specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457ΔlytSR, which is consistent with the microarray data. Conclusions The preliminary results suggest that in S. epidermidis LytSR two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. Based on the microarray data, it appears that lytSR inactivation induces a stringent response. In addition, LytSR may indirectly enhance biofilm formation by altering the metabolic status of the bacteria.

  7. ¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.

    Directory of Open Access Journals (Sweden)

    Dany J V Beste

    2011-07-01

    Full Text Available Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA. Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

  8. Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

    Science.gov (United States)

    McDonald, Tanya S; Borges, Karin

    2017-07-01

    To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. [U- 13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U- 13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U- 13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from

  9. Enzymological evidence for the function of a plastid-located pyruvate carboxylase in the Haptophyte alga Emiliania huxleyi: a novel pathway for the production of C4 compounds.

    Science.gov (United States)

    Tsuji, Yoshinori; Suzuki, Iwane; Shiraiwa, Yoshihiro

    2012-06-01

    Pyruvate carboxylase (PYC) catalyzes the β-carboxylation of pyruvate to yield oxaloacetate (OAA). We previously isolated a cDNA encoding a putative PYC (EhPYC1) from the haptophyte alga Emiliania huxleyi and then proposed that EhPYC1 contributes to active anaplerotic β-carboxylation during photosynthesis although PYC activity was not detected in the cell extracts. Involvement of PYC in photosynthetic carbon metabolism is unique, since PYC generally functions in non-photosynthetic organisms. In the present study, we demonstrate that EhPYC1 is highly sensitive to endogenous proteases and therefore is easily degraded in cell extracts. By avoiding proteolytic degradation, PYC activity can be detected in the cell extracts of E. huxleyi. The activity of a recombinant His-tagged EhPYC1 expressed in Streptomyces lividans was inhibited by l-malate in a mixed non-competitive manner. Immunofluorescence labeling showed that EhPYC1 is located in the plastid. This result agrees with the prediction that a bipartite plastid-targeting signal is present that functions to deliver proteins into the four-membrane plastid of haptophyte algae. This is the first finding of a plastid-located PYC. These results indicate that E. huxleyi possesses a unique pathway to produce OAA catalyzed by PYC, and the pathway may provide carbon skeletons for amino acid biosynthesis in the plastid. A database search indicates that PYC genes are widespread in green algae, diatoms and brown algae, suggesting the crucial role of PYC in various aquatic phototrophs.

  10. Beneficial effect of feeding a ketogenic diet to mothers on brain development in their progeny with a murine model of pyruvate dehydrogenase complex deficiency.

    Science.gov (United States)

    Pliss, Lioudmila; Jatania, Urvi; Patel, Mulchand S

    2016-06-01

    Pyruvate dehydrogenase complex (PDC) deficiency is a major inborn error of oxidative metabolism of pyruvate in the mitochondria causing congenital lactic acidosis and primarily structural and functional abnormalities of the central nervous system. To provide an alternate source of acetyl-CoA derived from ketone bodies to the developing brain, a formula high in fat content is widely employed as a treatment. In the present study we investigated efficacy of a high-fat diet given to mothers during pregnancy and lactation on lessening of the impact of PDC deficiency on brain development in PDC-deficient female progeny. A murine model of systemic PDC deficiency by interrupting the X-linked Pdha1 gene was employed in this study. Maternal consumption of a high-fat diet during pregnancy and lactation had no effect on number of live-birth, body growth, tissue PDC activity levels, as well as the in vitro rates of glucose oxidation and fatty acid biosynthesis by the developing brain of PDC-deficient female offspring during the postnatal age 35 days, as compared to the PDC-deficient progeny born to dams on a chow diet. Interestingly, brain weight was normalized in PDC-deficient progeny of high fat-fed mothers with improvement in impairment in brain structure deficit whereas brain weight was significantly decreased and was associated with greater cerebral structural defects in progeny of chow-fed mothers as compared to control progeny of mothers fed either a chow or high fat diet. The findings provide for the first time experimental support for beneficial effects of a ketogenic diet during the prenatal and early postnatal periods on the brain development of PDC-deficient mammalian progeny.

  11. Disruption of the pdhB pyruvate dehydrogenase [corrected] gene affects colony morphology, in vitro growth and cell invasiveness of Mycoplasma agalactiae.

    Directory of Open Access Journals (Sweden)

    Shivanand Hegde

    Full Text Available The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.

  12. Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

    Directory of Open Access Journals (Sweden)

    Robert M Gill

    Full Text Available Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-/hydrogen peroxide (H2O2 release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 μM. This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

  13. Hepatoprotective potential of ethanolic extract of Ziziphus oenoplia (L.) Mill roots against antitubercular drugs induced hepatotoxicity in experimental models.

    Science.gov (United States)

    Rao, Ch V; Rawat, A K S; Singh, Anil P; Singh, Arpita; Verma, Neeraj

    2012-04-01

    To evaluate the hepatoprotective potential of ethanolic (50%) extract of Ziziphus oenoplia (L.) Mill (Z. oenoplia) root against isoniazid (INH) and rifampicin (RIF) induced liver damage in animal models. Five groups of six rats each were selected for the study. Ethanolic extract at a dose of 150 and 300 mg/kg as well as silymarin (100 mg/kg) were administered orally once daily for 21 d in INH + RIF treated groups. The serum levels of glutamic oxaloacetic transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase (SALP), and bilirubin were estimated along with activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione peroxidase, and hepatic melondialdehyde formation. Histopathological analysis was carried out to assess injury to the liver. The considerably elevated serum enzymatic activities of glutamic oxaloacetic transaminase, glutamate pyruvate transaminase, alkaline phosphatase and bilirubin due to INH + RIF treatment were restored towards normal in a dose dependent manner after the treatment with ethanolic extract of Z. oenoplia roots. Meanwhile, the decreased activities of superoxide dismutase, catalase, glutathione S-transferase and glutathione peroxidase were also restored towards normal dose dependently. In addition, ethanolic extract also significantly prevented the elevation of hepatic melondialdehyde formation in the liver of INH + RIF intoxicated rats in a dose dependent manner. The biochemical observations were supplemented with histopathological examination of rat liver sections. The results of this study strongly indicate that ethanolic extract of Z. oenoplia has a potent hepatoprotective action against INH + RIF induced hepatic damage in rats. Copyright © 2012 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  14. Effect of different levels of alpha tocopherol on performance traits, serum antioxidant enzymes, and trace elements in Japanese quail ( Coturnix coturnix japonica under low ambient temperature

    Directory of Open Access Journals (Sweden)

    Assar Ali Shah

    Full Text Available ABSTRACT This study was designed to find the effect of vitamin E supplementation on growth, serum antioxidant enzymes, and some trace elements in Japanese quail (Coturnix coturnix japonica under low ambient temperature. A total of 180 day-old Japanese quails were randomly divided into four groups and provided with 0 (group A, 50 (group B, 100 (group C, and 150 IU/kg (group D vitamin E (dl-α-tocopherol acetate under an average temperature of 9±0.5 °C for an experimental period of 42 days. The result showed that feed intake per day, body weight, weight gain per day, and feed conversion ratio did not differ significantly between the groups. Serum concentrations of superoxide and glutathione peroxidase were significantly high in birds supplemented with 150 mg/kg of vitamin E. The concentration of aspartate aminotransferase was not significantly affected between the control and treated groups; however, alanine transaminase concentration significantly reduced in group D. Zinc concentration in the blood increased significantly in group D, with no significant effect on copper and manganese between the control and treated groups. Vitamin E at the level of 150 IU/kg of feed improves the blood antioxidant status and zinc concentration, with no effect on the performance traits of quail reared under low ambient temperature.

  15. Initial Serum Ferritin Predicts Number of Therapeutic Phlebotomies to Iron Depletion in Secondary Iron Overload

    Science.gov (United States)

    Panch, Sandhya R.; Yau, Yu Ying; West, Kamille; Diggs, Karen; Sweigart, Tamsen; Leitman, Susan F.

    2014-01-01

    Background Therapeutic phlebotomy is increasingly used in patients with transfusional siderosis to mitigate organ injury associated with iron overload (IO). Laboratory response parameters and therapy duration are not well characterized in such patients. Methods We retrospectively evaluated 99 consecutive patients undergoing therapeutic phlebotomy for either transfusional IO (TIO, n=88; 76% had undergone hematopoietic transplantation) or non-transfusional indications (hyperferritinemia or erythrocytosis) (n=11). CBC, serum ferritin (SF), transferrin saturation, and transaminases were measured serially. Phlebotomy goal was an SF< 300 mcg/L. Results Mean SF prior to phlebotomy among TIO and nontransfusional subjects was 3,093 and 396 mcg/L, respectively. Transfusion burden in the TIO group was 94 ± 108 (mean ± SD) RBC units; about half completed therapy with 24 ± 23 phlebotomies (range 1–103). One-third was lost to follow-up. Overall, 15% had mild adverse effects, including headache, nausea, and dizziness, mainly during first phlebotomy. Prior transfusion burden correlated poorly with initial ferritin and total number of phlebotomies to target (NPT) in the TIO group. However, NPT was strongly correlated with initial SF (R2=0.8; p<0.0001) in both TIO and nontransfusional groups. ALT decreased significantly with serial phlebotomy in all groups (mean initial and final values, 61 and 39 U/L; p = 0.03). Conclusions Initial SF but not transfusion burden predicted number of phlebotomies to target in patients with TIO. Despite good treatment tolerance, significant losses to follow-up were noted. Providing patients with an estimated phlebotomy number and follow-up duration, and thus a finite endpoint, may improve compliance. Hepatic function improved with iron off-loading. PMID:25209879

  16. Serum lysozyme determinations, April-June 1961

    Energy Technology Data Exchange (ETDEWEB)

    Finch, S C; Lamphere, J P; Jablon, S

    1963-04-18

    Serum lysozyme levels were determined on 670 consecutive subjects seen for regularly scheduled clinic examinations of the Adult Health Study in Hiroshima. Serum lysozyme levels were found to vary significantly with the absolute peripheral granulocyte count, age, sex, and month of study. A high level of correlation also was noted between serum lysozyme and diabetes mellitus. This was at least in part attributable to greater average age in patients with diabetes. Suggestive relationship was established between serum lysozyme levels, respiratory diseases, and tuberculosis. These changes are believed to reflect active inflammation with excessive destruction of granulocytes and parenchymal tissues in those patients with the more acute processes. No relationship was found between serum lysozyme and previous exposure to ionizing radiation. These studies indicate that the serum lysozyme level may be useful in the study of the kinetics of leukopoiesis, the aging process, and in the detection of subtle inflammatory processes. 21 references, 5 tables.

  17. 125I Radioimmunoassay of serum ursodeoxycholyl conjugates

    International Nuclear Information System (INIS)

    Hill, A.; Ross, P.E.; Bouchier, I.A.D.

    1983-01-01

    A radioimmunoassay for serum ursodeoxycholic conjugates using an iodine-125 ligand has been developed. The bile acid was present in normal fasting serum (0.19 +- SD 0.19 μmol/l, n=24) and 2-hour post-prandial serum (0.8 +- SD 0.8 μmol/l, n=16). Gallstone patients undergoing oral ursodeoxycholic acid therapy had significantly higher post-prandial serum levels (21.5 +- SD 14.0 μmol/l, n=15) by radioimmunoassay. Gas liquid chromatography analysis indicated that in normal serum ursodeoxycholic acid was totally conjugated, whereas sera from gallstone patients contained a proportion as the free bile acid (10.2 +- SD 8.1 μmol/l, n=15). Following an oral dose of ursodeoxycholic acid, both unconjugated and conjugated forms of the bile acid appeared in the serum of healthy individuals. (Auth.)

  18. Radioimmunoassay of thyrotropin concentrated from serum

    International Nuclear Information System (INIS)

    Nisula, B.C.; Louvet, J.P.

    1978-01-01

    A method for concentrating human TSH (hTSH) from serum for use in RIAs is described. The method takes advantage of the affinity of the plant lectin, concanavalin A, for the carbohydrate portion of the hTSH molecule. The hTSH from 2.5 ml serum was adsorbed to concanavalin A covalently linked to sepharose and then radioimmunoassayed using the hTSH antiserum and hTSH for iodination distributed by the National Pituitary Agency. For the RIA standard curve, the hTSH reference preparation was concentrated from a serum wwith undetectable hTSH in order to correct for recovery and to control for nonspecific effects. The percentage of serum hTSH extracted from 2.5 ml serum with the concentration procedure was 76.6 +- 3.4% (mean +- SD). The coefficient of correlation between serum hTSH, determined with the concentration procedure, and serum hTSH determined without was 0.979 (P < 0.001). Over 95% of normal adult men and women had detectable levels of serum hTSH, ranging from < 0.56 to 4.0 μU/ml. The mean of detectable serum hTSH levels in normal adult women (n = 11) was 1.54 +- 1.03 μU/ml (mean +- SD) and in normal men (n = 9) was 2.02 +- 1.15 μU/ml (mean +- SD). Clinically hyperthyroid patients with diffuse and nodular toxic goiters (n = 8) and patients with hypothyroidism secondary to pituitary disease (n = 6), four of whom were taking replacement doses of thyroid hormone, had undetectable serum hTSH levels. Serum hTSH in patients with primary hypothyroidism uniformly exceeded the normal range. This hTSH concentrating procedure enhances the effective sensitivity and, therefore, the clinical utility of the RIA for hTSH in serum

  19. Antivenom reversal of biochemical alterations induced by black scorpion Heterometrus fastigiousus Couzijn venom in mice

    Directory of Open Access Journals (Sweden)

    MK Chaubey

    2009-01-01

    Full Text Available In the present study, Heterometrus fastigiousus venom (HFV was employed as antigen to produce species-specific scorpion antivenom (SAV in albino mice (NIH strain. To determine SAV efficacy, it was pre-incubated with 10 LD50 of HFV and then injected subcutaneously into mice. Subsequently, mortality was observed after 24 hours. Minimum effective dose (MED was 12.5 LD50 of HFV/mL of SAV. SAV effectiveness to reverse HFV-induced biochemical alterations in mice was analyzed by challenge method. Simultaneously, mice received subcutaneously 40% of 24-hour-LD50 of HFV and intravenously SAV. After four hours, changes in serum glucose, free amino acids, uric acids, pyruvic acid, cholesterol, total protein, alkaline phosphatase, acid phosphatase, lactic dehydrogenase and glutamate-pyruvate transaminase enzyme level were determined. Treatment with species-specific SAV resulted in the reversal of HFV-induced biochemical alterations.

  20. Serum lipids in hypothyroidism: Our experience

    OpenAIRE

    Prakash, Archana; Lal, Ashok Kumar

    2006-01-01

    In order to determine whether the screening of lipid profile is justified in patients with hypothyroidism we estimated serum lipids in cases having different levels of serum TSH. 60 patients of hypothyroidism in the age group of 20 to 60 yrs were studied for thyroid profile over a period of one year. On the basis of serum TSH level the cases were divided into three groups: In the first group TSH concentration was 8.8±2.99 μlU/ml, 95% confidence interval (Cl) 8.8±1.07, whereas serum total chol...