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Sample records for glucose-6-phosphate dehydrogenase extends

  1. Genetics Home Reference: glucose-6-phosphate dehydrogenase deficiency

    Science.gov (United States)

    ... deficiency Encyclopedia: Glucose-6-phosphate dehydrogenase test Encyclopedia: Hemolytic anemia Encyclopedia: Newborn jaundice Health Topic: Anemia Health Topic: G6PD Deficiency Health Topic: Newborn Screening Genetic and Rare Diseases Information Center (1 link) Glucose-6-phosphate dehydrogenase ...

  2. Screening of Glucose-6-Phosphate Dehydrogenase Deficiency in Cord Blood

    Directory of Open Access Journals (Sweden)

    Can Acipayam

    2014-02-01

    Aim: Glucose-6-phosphate dehydrogenase deficiency is an important factor in etiology of pathologic neonatal jaundice. The aim of this study was to indicate the significance of screening glucose-6-phosphate dehydrogenase deficiency in the cord blood of neonates and the frequency of this deficiency in the etiology of neonatal hyperbilirubinemia. Material and Method: The study was performed consecutive 1015 neonates were included. Five hundred fifty six (54.8% of them were male and 459 (45.2% were female. The following parameters were recorded: Gender, birth weight, birth height, head circumference and gestational age. The glucose-6-phosphate dehydrogenase level of neonates were measured with quantitative method in cord blood. Also, hemoglobine, hematocrite, red blood cell count and blood group were measured. The following parameters were recorded in cases with jaundice: exchange transfusion, phototherapy, physiologic and pathologic jaundice, peak bilirubin day, maximum bilirubin level, total bilirubin level at the first day of jaundice, beginning time of jaundice. Results: Enzyme deficiency was detected in 133 (13.1% of neonates and 76 (57% of them were male, 57 (43% were female. Significant difference was detected in low glucose-6-phosphate dehydrogenase enzyme level with jaundice group for total bilirubin level at the first day of jaundice, maximum total bilirubin level and pathologic jaundice (p<0.05. Discussion: The ratio of glucose-6-phosphate dehydrogenase deficiency was found in Edirne in this study and this ratio was higher than other studies conducted in our country. For this reason, glucose-6-phosphate dehydrogenase enzyme level in cord blood of neonates should be measured routinely and high risk neonates should be followed up for hyperbilirubinemia and parents should be informed in our region.

  3. Prevalence of glucose-6-phosphate dehydrogenase deficiency in ...

    African Journals Online (AJOL)

    Background: Glucose-6-phosphate dehydrogenase (G6PD) is a house keeping enzyme which catalyzes the first step in the hexose monophosphate pathway of glucose metabolism. G6PD deficiency is the commonest hemolytic X-linked genetic disease, which affects approximately 400 million people worldwide.

  4. Identification of glucose 6 phosphate dehydrogenase mutations by ...

    African Journals Online (AJOL)

    Identification of glucose 6 phosphate dehydrogenase mutations by single strand conformation polymorphism and gene sequencing analysis. ... Subject: Six DNA samples from Turkish males confirmed to have G-6-PD deficiency where available for the study. Results: One subject was found to have an abnormal mobility shift ...

  5. Glucose-6-phosphate dehydrogenase deficiency; the single most ...

    African Journals Online (AJOL)

    Introduction: Glucose- 6-phosphate dehydrogenase deficiency is the most common enzymatic disorder of the red cell and an important risk factor for neonatal jaundice. Methodology: The aim of the study was to determine the incidence of G-6-PD deficiency among jaundiced neonates, and describe the associated morbidity ...

  6. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency in patients ...

    African Journals Online (AJOL)

    This is a study of Glucose-6-phosphate dehydrogenase(G6PD) deficiency in sickle cell anaemia patients attending the haematology clinic of the Jos University Teaching Hospital (JUTH), Jos- Nigeria. The prevalence of G6PD deficiency among the 130 sickle cell anaemia patients studied was found to be 18.5%. G6PD ...

  7. Kernicterus by glucose-6-phosphate dehydrogenase deficiency: a case report and review of the literature

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    Cossio de Gurrola Gladys

    2008-05-01

    Full Text Available Abstract Introduction Glucose-6-phosphate dehydrogenase deficiency is an X-linked recessive disease that causes acute or chronic hemolytic anemia and potentially leads to severe jaundice in response to oxidative agents. This deficiency is the most common human innate error of metabolism, affecting more than 400 million people worldwide. Case presentation Here, we present the first documented case of kernicterus in Panama, in a glucose-6-phosphate dehydrogenase-deficient newborn clothed in naphthalene-impregnated garments, resulting in reduced psychomotor development, neurosensory hypoacousia, absence of speech and poor reflex of the pupil to light. Conclusion Mutational analysis revealed the glucose-6-phosphate dehydrogenase Mediterranean polymorphic variant, which explained the development of kernicterus after exposition of naphthalene. As the use of naphthalene in stored clothes is a common practice, glucose-6-phosphate dehydrogenase testing in neonatal screening could prevent severe clinical consequences.

  8. Neonatal jaundice and glucose-6-phosphate dehydrogenase

    OpenAIRE

    Leite, Amauri Antiquera [UNESP

    2010-01-01

    A deficiência de glicose-6-fosfato desidrogenase em neonatos pode ser a responsável pela icterícia neonatal. Este comentário científico é decorrente do relato sobre o tema publicado neste fascículo e que preocupa diversos autores de outros países em relação às complicações em neonatos de hiperbilirrubinemia, existindo inclusive proposições de alguns autores em incluir o teste para identificar a deficiência de glicose-6-fosfato desidrogenase nos recém-nascidos.Glucose-6-phosphate dehydrogenase...

  9. Intravenous immunoglobulin to treat hyperbilirubinemia in neonates with isolated Glucose-6-Phosphate dehydrogenase deficiency

    Directory of Open Access Journals (Sweden)

    Wadah Khriesat

    2017-04-01

    Full Text Available Background Glucose-6-phosphate dehydrogenase deficiency alone or concomitant with ABO isoimmunisation is a widespread indication for neonatal exchange transfusion. Aims To evaluate the effectiveness of Intravenous Immunoglobulin in the treatment of neonatal hyperbilirubinemia due to glucose-6-phosphate dehydrogenase deficiency. Methods A retrospective cohort study was conducted between 2006 and 2014 at the Jordan University of Science and technology. The medical records of 43 infants admitted to the neonatal intensive care unit for isolated glucose-6- phosphate dehydrogenase deficiency hemolytic disease of the newborns were reviewed. Patients were divided into two groups. Group I, a historical cohort, included newborns born between 2006 and 2010, Treatment included phototherapy and exchange transfusion. Group II included newborns born between 2011 and 2014, where, in addition to phototherapy, intravenous immunoglobulin was administered. The duration of phototherapy and number of exchange transfusions were evaluated. Results Of 412 newborns that were admitted with neonatal hyperbilirubinemia, Glucose-6-phosphate dehydrogenase deficiency was present in 43. Of these, 22, did not receive intravenous immunoglobulin and served as a control group. The other 21 newborns received intravenous immunoglobulin. There was no difference in the demographic characteristics between the two groups. Infants in the control group were significantly more likely to receive exchange blood transfusion than infants in the immunoglobulin treatment group, but were significantly less likely to need phototherapy. Conclusion Intravenous immunoglobulin is an effective alternative to exchange transfusion in infants with glucose-6-phosphate dehydrogenase deficiency hemolytic disease of the newborn. It is suggested that intravenous immunoglobulin may be beneficial as a prophylaxis for infants with hyperbilirubinemia.

  10. The Effects of Fenarimol and Methyl Parathion on Glucose 6-Phosphate Dehydrogenase Enzyme Activity in Rats

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    Ferda ARI

    2017-10-01

    Full Text Available Fenarimol and methyl parathion are pesticides that have been used in agriculture for several years. These pesticides have significant effects on environmental and human health. Therefore, we investigated the effects of methyl parathion and fenarimol on glucose 6-phosphate dehydrogenase (EC 1.1.1.49 enzyme activity in rats. The glucose 6- phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway and it is important in detoxifying reactions by NADPH generated. In this study, wistar albino rats administrated with methyl parathion (7 mg kg–1 and fenarimol (200 mg kg−1 by intraperitoneally for different periods (2, 4, 8, 16, 32, 64, and 72 h. The glucose 6-phosphate dehydrogenase enzyme activity was assayed in liver, kidney, brain, and small intestine in male and female rats. The exposure of fenarimol and methyl parathion caused increase of glucose 6-phosphate dehydrogenase enzyme activity in rat tissues, especially at last periods. We suggest that this increment of enzyme activity may be the reason of toxic effects of fenarimol and methyl parathion.

  11. Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells

    NARCIS (Netherlands)

    van Noorden, C. J.; Tas, J.; Vogels, I. M.

    1983-01-01

    With the aid of thin films of polyacrylamide gel containing purified glucose-6-phosphate dehydrogenase subjected to cytochemical procedures for the enzyme using tetranitro blue tetrazolium, arbitrary units of integrated absorbance obtained with a Barr & Stroud GN5 cytophotometer were converted into

  12. D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens

    International Nuclear Information System (INIS)

    Lessmann, D.; Schimz, K.L.; Kurz, G.

    1975-01-01

    The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300μmol NADH formed per min per mg protein, was shown to be homogeneous. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220,000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265,000. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal acivity at pH 8.9. The Entner-Doudoroff enzyme showed specificity for NAD + as well as for NADP + and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of β-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase. (orig.) [de

  13. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages...

  14. Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium

    NARCIS (Netherlands)

    Biagiotti, E.; Bosch, K. S.; Ninfali, P.; Frederiks, W. M.; van Noorden, C. J.

    2000-01-01

    Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown, it may be related;either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized

  15. Glucose-6-phosphate dehydrogenase activity decreases during storage of leukoreduced red blood cells

    NARCIS (Netherlands)

    Peters, Anna L.; van Bruggen, Robin; de Korte, Dirk; van Noorden, Cornelis J. F.; Vlaar, Alexander P. J.

    2016-01-01

    During storage, the activity of the red blood cell (RBC) antioxidant system decreases. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection against oxidative stress by producing NADPH. G6PD function of RBC transfusion products is reported to remain stable during storage, but activity

  16. Biochemical and cytochemical evaluation of heterozygote individuals with glucose-6-phosphate dehydrogenase deficiency

    NARCIS (Netherlands)

    Gurbuz, Nilgun; Aksu, Tevfik Aslan; van Noorden, Cornelis J. F.

    2005-01-01

    The aim of this study was to diagnose heterozygous glucose-6-phosphate dehydrogenase (G6PD) deficient females by an inexpensive cytochemical G6PD staining method that is easy to perform, allowing diagnosis of G6PD deficiency without cumbersome genetic analysis. Three subject groups were included in

  17. Glucose-6-phosphate dehydrogenase protects Escherichia coli from tellurite-mediated oxidative stress.

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    Juan M Sandoval

    Full Text Available The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH, which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH. Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P, suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH, better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress.

  18. Post-irradiation repairing processes of glucose-6-phosphate dehydrogenase and catalase from Hansenula Polymorpha yeast

    International Nuclear Information System (INIS)

    Postolache, Carmen; Postolache, Cristian; Dinu, Diana; Dinischiotu, Anca; Sahini, Victor Emanuel

    2002-01-01

    The post-irradiation repairing mechanisms of two Hansenula Polymorpha yeast enzymes, glucose-6-phosphate dehydrogenase and catalase, were studied. The kinetic parameters of the selected enzymes were investigated over one month since the moment of γ-irradiation with different doses in the presence of oxygen. Dose dependent decrease of initial reaction rates was noticed for both enzymes. Small variation of initial reaction rate was recorded for glucose-6-phosphate dehydrogenase over one month, with a decreasing tendency. No significant electrophoretic changes of molecular forms of this enzyme were observed after irradiation. Continuous strong decrease of catalase activity was evident for the first 20 days after irradiation. Partial recovery process of the catalytic activity was revealed by this study. (authors)

  19. Glucose 6-phosphate dehydrogenase variants in Japan.

    Science.gov (United States)

    Miwa, S

    1980-01-01

    Fifty-four cases of glucose 6-phosphate dehydrogenase (G6PD) deficiency have so far been reported in Japan. Among them, 21 G6PD variants have been characterized. Nineteen out of the 21 variants were characterized in our laboratory and G6PD Heian and "Kyoto" by others. G6PD Tokyo, Tokushima, Ogikubo, Kurume, Fukushima, Yokohama, Yamaguchi, Wakayama, Akita, Heian and "Kyoto" were classified as Class 1, because all these cases showed chronic hemolytic anemia and severe enzyme deficiency. All these variants showed thermal instability. G6PD Mediterranean-like, Ogori, Gifu and Fukuoka were classified as Class 2, whereas G6PD Hofu, B(-) Chinese, Ube, Konan, Kamiube and Kiwa belonged to Class 3. All the 6 Class 3 variants were found as the results of the screening tests. The incidence of the deficiency in Japanese seems to be 0.1-0.5% but that of the cases which may slow drug-induced hemolysis would be much less. G6PD Ube and Konan appear to be relatively common in Japan.

  20. Glucose-6-phosphate dehydrogenase deficiency in Singapore.

    Science.gov (United States)

    Quak, S H; Saha, N; Tay, J S

    1996-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) in man is an X-linked enzyme. The deficiency of this enzyme is one of the most common inherited metabolic disorders in man. In Singapore, three clinical syndromes associated with G6PD deficiency had been described: severe haemolysis in neonates with kernicterus, haemoglobinuria and "viral hepatitis"-like syndrome. The human G6PD monomer consists of 515 amino acids. Only the tetrameric or dimeric forms composed of a single type subunit are catylitically active. The complete amino acid sequence of G6PD had been elucidated in man and various other animals. The region of high homology among the enzymes of various animals is presumably functionally active. Among the Chinese in Singapore, three common molecular variants had been identified: Canton (nt 1376 G --> T), Kaiping (nt 1388 G --> A) and Mediterranean (nt 563 C --> T) in frequencies of 24%, 21% and 10% respectively. In addition, two common mutants (Gaozhou, nt 95 A --> G and Chinese 5, nt 1024 C --> T) have been detected in Singapore Chinese in low frequencies. In Malays, 6 different deficient variants are known in Singapore (3 new, 1 Mahidol, 1 Indonesian and 1 Mediterranean).

  1. Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance

    Czech Academy of Sciences Publication Activity Database

    Šindelář, Luděk; Šindelářová, Milada

    2002-01-01

    Roč. 215, - (2002), s. 862-869 ISSN 0032-0935 R&D Projects: GA ČR GA522/99/1264 Institutional research plan: CEZ:AV0Z5038910 Keywords : Glucose 6 phosphate dehydrogenase * Nicotiana (viral infection) * Plant viruses Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.960, year: 2002

  2. Icterícia neonatal e deficiência de glicose-6-fosfato desidrogenase Neonatal jaundice and glucose-6-phosphate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Amauri Antiquera Leite

    2010-01-01

    Full Text Available A deficiência de glicose-6-fosfato desidrogenase em neonatos pode ser a responsável pela icterícia neonatal. Este comentário científico é decorrente do relato sobre o tema publicado neste fascículo e que preocupa diversos autores de outros países em relação às complicações em neonatos de hiperbilirrubinemia, existindo inclusive proposições de alguns autores em incluir o teste para identificar a deficiência de glicose-6-fosfato desidrogenase nos recém-nascidos.Glucose-6-phosphate dehydrogenase in newborn babies may be responsible for neonatal jaundice. There is a concern of many authors from other countries in respect to complications in neonates with hyperbilirubinemia; some authors even propose screening for glucose-6-phosphate dehydrogenase deficiency in newborn babies. A scientific report on this subject is published in this issue.

  3. Glucose 6 phosphate dehydrogenase deficiency in adults

    International Nuclear Information System (INIS)

    Khan, M.

    2004-01-01

    Objective: To determine the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency in adults presented with anemia. Subjects and Methods: Eighteen months admission data was reviewed for G6PD deficiency as a cause of anemia. Anemia was defined by world health organization (WHO) criteria as haemoglobin less than 11.3 gm%. G6PD activity was measured by Sigma dye decolorisation method. All patients were screened for complications of hemolysis and its possible cause. Patients with more than 13 years of age were included in the study. Results: Out of 3600 patients admitted, 1440 were found anaemic and 49 as G6PD deficient. So the frequency of G6PD deficiency in anaemic patients was 3.4% and the overall frequency is 1.36%. G6PD deficiency among males and females was three and six percent respectively. Antimalarials and antibiotics containing sulphonamide group were the most common precipitating factors for hemolysis. Anemia and jaundice were the most common presentations while malaria was the most common associated disease. Acute renal failure was the most severe complication occurring in five patients with two deaths. Conclusion: G6PD deficiency is a fairly common cause of anemia with medicine as common precipitating factor for hemolysis. Such complications can be avoided with early recognition of the disease and avoiding indiscriminate use of medicine. (author)

  4. Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

    Directory of Open Access Journals (Sweden)

    Barretto O.C. de O.

    2006-01-01

    Full Text Available In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa. The Michaelis-Menten constants (Km: 55 µM for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively. A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

  5. Glucose-6-phosphate dehydrogenase deficiency and Alzheimer's disease: Partners in crime? The hypothesis.

    Science.gov (United States)

    Ulusu, N Nuray

    2015-08-01

    Alzheimer's disease is a multifaceted brain disorder which involves various coupled irreversible, progressive biochemical reactions that significantly reduce quality of life as well as the actual life expectancy. Aging, genetic predispositions, head trauma, diabetes, cardiovascular disease, deficiencies in insulin signaling, dysfunction of mitochondria-associated membranes, cerebrovascular changes, high cholesterol level, increased oxidative stress and free radical formation, DNA damage, disturbed energy metabolism, and synaptic dysfunction, high blood pressure, obesity, dietary habits, exercise, social engagement, and mental stress are noted among the risk factors of this disease. In this hypothesis review I would like to draw the attention on glucose-6-phosphate dehydrogenase deficiency and its relationship with Alzheimer's disease. This enzymopathy is the most common human congenital defect of metabolism and defined by decrease in NADPH+H(+) and reduced form of glutathione concentration and that might in turn, amplify oxidative stress due to essentiality of the enzyme. This most common enzymopathy may manifest itself in severe forms, however most of the individuals with this deficiency are not essentially symptomatic. To understand the sporadic Alzheimer's disease, the writer of this paper thinks that, looking into a crystal ball might not yield much of a benefit but glucose-6-phosphate dehydrogenase deficiency could effortlessly give some clues. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Prevalence of glucose-6-phosphate dehydrogenase deficiency and diagnostic challenges in 1500 immigrants in Denmark examined for haemoglobinopathies

    DEFF Research Database (Denmark)

    Warny, Marie; Klausen, Tobias Wirenfeldt; Petersen, Jesper

    2015-01-01

    Similar to the thalassaemia syndromes, glucose-6-phosphate dehydrogenase (G6PD) deficiency is highly prevalent in areas historically exposed to malaria. In the present study, we used quantitative and molecular methods to determine the prevalence of G6PD deficiency in a population of 1508 immigran...

  7. Effect of Punica granatum fruit peel on glucose-6-phosphate dehydrogenase and malate dehydrogenase in amphistome Gastrothylax indicus.

    Science.gov (United States)

    Aggarwal, Rama; Bagai, Upma

    2017-03-01

    Increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against helminth parasite in sheep. Important lipogenic enzymes like glucose-6-phosphate dehydrogenase (G-6-PDH) and malate dehydrogenase (MDH) show subcellular distribution pattern. Activity of G-6-PDH was largely restricted to cytosolic fraction while MDH was found in both cytosolic and mitochondrial fraction in Gastrothylax indicus. Following in vitro treatment with ethanolic and aqueous extracts of Punica granatum fruit peel and commercial anthelmintic, albendazole G-6-PDH activity was decreased by 19-32 %, whereas MDH was suppressed by 24-41 %, compared to the respective control. Albendazole was quite effective when compared with negative control and both the extracts. The results indicate that phytochemicals of plant may act as potential vermifuge or vermicide.

  8. Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves.

    Science.gov (United States)

    Coban, T Abdül Kadir; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

    2002-05-01

    In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

  9. Rasburicase-induced Hemolytic Anemia in an Adolescent With Unknown Glucose-6-Phosphate Dehydrogenase Deficiency.

    Science.gov (United States)

    Akande, Manzilat; Audino, Anthony N; Tobias, Joseph D

    2017-01-01

    Rasburicase, used in the prevention and treatment of tumor lysis syndrome (TLS), may cause hemolytic anemia and methemoglobinemia in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Although routine screening for G6PD deficiency has been recommended, given the turnaround time for test results and the urgency to treat TLS, such screening may not be feasible. We report a case of rasburicase-induced hemolytic anemia without methemoglobinemia in an adolescent with T-cell lymphoblastic lymphoma, TLS, and previously unrecognized G6PD deficiency. Previous reports of hemolytic anemia with rasburicase are reviewed, mechanisms discussed, and preventative strategies presented.

  10. Glucose-6-phosphate dehydrogenase: the key to sex-related xenobiotic toxicity in hepatocytes of European flounder (Platichthys flesus L.)?

    NARCIS (Netherlands)

    Winzer, Katja; van Noorden, Cornelis J. F.; Köhler, Angela

    2002-01-01

    The role of glucose-6-phosphate dehydrogenase (G6PDH) in oxidative stress responses was investigated in isolated intact living hepatocytes of immature female and male European flounder (Platichthys flesus L.) because it is the major provider of NADPH needed as reducing power for various

  11. Glucose-6-Phosphate Dehydrogenase Deficiency and Adrenal Hemorrhage in a Filipino Neonate with Hyperbilirubinemia

    Directory of Open Access Journals (Sweden)

    Akira Ohishi

    2013-05-01

    Full Text Available We report on a Filipino neonate with early onset and prolonged hyperbilirubinemia who was delivered by a vacuum extraction due to a prolonged labor. Subsequent studies revealed adrenal hemorrhage and glucose-6-phosphate dehydrogenase (G6PD deficiency. It is likely that asphyxia and resultant hypoxia underlie the occurrence of adrenal hemorrhage and the clinical manifestation of G6PD deficiency and that the presence of the two events explains the early onset and prolonged hyperbilirubinemia of this neonate. Our results represent the importance of examining possible underlying factors for the development of severe, early onset, or prolonged hyperbilirubinemia.

  12. [Glucose-6-phosphate dehydrogenase deficiency in children: a case report].

    Science.gov (United States)

    Verdugo L, Patricia; Calvanese T, Marlene; Rodríguez V, Diego; Cárcamo C, Cassandra

    2014-02-01

    Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is the most common red blood cell (RBC) enzyme disorder. The decrease as well as the absence of the enzyme increase RBC vulnerability to oxidative stress caused by exposure to certain medications or intake of fava beans. Among the most common clinical manifestations of this condition, acute hemolysis, chronic hemolysis, neonatal hyperbilirubinemia, and an asymptomatic form are observed. To analyze the case of a child who presented hemolytic crisis due to favism. A 2 year and 7 month old boy with a history of hyperbilirubinemia during the newborn period with no apparent cause, no family history of hemolytic anemia or parental consanguinity. He presented a prolonged neonatal jaundice and severe anemia requiring RBC transfusion. An intake of fava beans 48 h prior to onset of symptoms was reported. G6PD qualitative determination was compatible with this enzyme deficiency. G6PD deficiency can be highly variable in its clinical presentation, so it is necessary to keep it in mind during the diagnosis of hemolytic anemia at any age.

  13. Reaction rate studies of glucose-6-phosphate dehydrogenase activity in sections of rat liver using four tetrazolium salts

    NARCIS (Netherlands)

    Butcher, R. G.; van Noorden, C. J.

    1985-01-01

    The reaction rate of glucose-6-phosphate dehydrogenase activity in liver sections from fed and starved rats has been monitored by the continuous measurement at 37 degrees C of the reaction product as it is formed using scanning and integrating microdensitometry. Control media lacked either substrate

  14. Effect of High-Dose Vitamin C Infusion in a Glucose-6-Phosphate Dehydrogenase-Deficient Patient

    Science.gov (United States)

    Gerber, Bryan; Kenyon, Katharine; Muthukanagaraj, Purushothaman

    2017-01-01

    Vitamin C supplementation is generally regarded as benign. There has been a resurgence of interest in the general medical community regarding the use of vitamin C most notably in the care of sepsis. Nonetheless, caution must be taken if supraphysiologic vitamin C supplementation is being administered as it should be considered a medication just like any other. We present a case of hemolysis in a glucose-6-phosphate dehydrogenase- (G6PD-) deficient patient receiving high-dose vitamin C infusions for his rheumatoid arthritis. PMID:29317868

  15. Glucose-6-Phosphate Dehydrogenase: Update and Analysis of New Mutations around the World

    Science.gov (United States)

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; Ortega-Cuellar, Daniel; González-Valdez, Abigail; Castillo-Rodríguez, Rosa Angélica; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme in the pentose phosphate pathway which produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain an adequate reducing environment in the cells and is especially important in red blood cells (RBC). Given its central role in the regulation of redox state, it is understandable that mutations in the gene encoding G6PD can cause deficiency of the protein activity leading to clinical manifestations such as neonatal jaundice and acute hemolytic anemia. Recently, an extensive review has been published about variants in the g6pd gene; recognizing 186 mutations. In this work, we review the state of the art in G6PD deficiency, describing 217 mutations in the g6pd gene; we also compile information about 31 new mutations, 16 that were not recognized and 15 more that have recently been reported. In order to get a better picture of the effects of new described mutations in g6pd gene, we locate the point mutations in the solved three-dimensional structure of the human G6PD protein. We found that class I mutations have the most deleterious effects on the structure and stability of the protein. PMID:27941691

  16. Loss of peroxisomes causes oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity to detect cancer cells

    NARCIS (Netherlands)

    Frederiks, Wilma M.; Vreeling-Sindelárová, Heleen; van Noorden, Cornelis J. F.

    2007-01-01

    Oxygen insensitivity of carcinoma cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) enables detection of carcinoma cells in unfixed cell smears or cryostat sections of biopsies. The metabolic background of oxygen insensitivity is

  17. Identification of Mutation of Glucose-6-Phosphate Dehy-drogenase (G6PD) in Iran: Meta- analysis Study.

    Science.gov (United States)

    Moosazadeh, Mahmood; Nekoei-Moghadam, Mahmood; Aliram-Zany, Maryam; Amiresmaili, Mohammadreza

    2013-09-01

    Glucose-6-phosphate dehydrogenase is one of the most common genetic deficiencies, which approximately 400 million people in the world suffer from. According to authors' initial search, numerous studies have been carried out in Iran regarding molecular variants of this enzyme. Thus, this meta-analysis presented a reliable estimation about prevalence of different types of molecular mutations of G6PD Enzyme in Iran. Keywords "glucose 6 phosphate dehydrogenase or G6PD, Mediterranean or Chatham or Cosenza and mutation, Iran or Iranian and their Persian equivalents" were searched in different databases. Moreover, reference list of the published studies were examined to increase sensitivity and to select more studies. After studying titles and abstracts of retrieved articles, excluding the repeated and unrelated ones, and evaluating quality of articles, documents were selected. Data was analyzed using STATA. After performing systematic review, 22 papers were entered this meta-analysis and 1698 subjects were examined concerning G6PD molecular mutation. In this meta-analysis, prevalence of Mediterranean mutation, Chatham mutation and Cosenza mutation in Iran was estimated 78.2%, 9.1% and 0.5% respectively. This meta-analysis showed that in spite of prevalence of different types of G6PD molecular mutations in center, north, north-west and west of Iran, the most common molecular mutations in people with G6PD deficiency in Iran, like other Mediterranean countries and countries around Persian Gulf, were Mediterranean mutation, Chatham mutation and Cosenza mutation. It is also recommended that future studies may focus on races and regions which haven't been taken into consideration up to now.

  18. Glucose-6-phosphate dehydrogenase deficiency: an unusual cause of acute jaundice after paracetamol overdose.

    Science.gov (United States)

    Phillpotts, Simon; Tash, Elliot; Sen, Sambit

    2014-11-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest human enzyme defect causing haemolytic anaemia after exposure to specific triggers. Paracetamol-induced haemolysis in G6PD deficiency is a rare complication and mostly reported in children. We report the first case (to the best of our knowledge) of acute jaundice without overt clinical features of a haemolytic crisis, in an otherwise healthy adult female following paracetamol overdose, due to previously undiagnosed G6PD deficiency. It is important that clinicians consider this condition when a patient presents following a paracetamol overdose with significant and disproportionate jaundice, without transaminitis or coagulopathy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1988-10-01

    Full Text Available The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05 for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s., respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01 for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01 before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1

  20. Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films

    NARCIS (Netherlands)

    van Noorden, C. J.; Tas, J.

    1980-01-01

    The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters

  1. Glucose 6 phosphatase dehydrogenase (G6PD) and neurodegenerative disorders: Mapping diagnostic and therapeutic opportunities

    OpenAIRE

    Manju Tiwari

    2017-01-01

    Glucose 6 phosphate dehydrogenase (G6PD) is a key and rate limiting enzyme in the pentose phosphate pathway (PPP). The physiological significance of enzyme is providing reduced energy to specific cells like erythrocyte by maintaining co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH). There are preponderance research findings that demonstrate the enzyme (G6PD) role in the energy balance, and it is associated with blood-related diseases and disorders, primarily the anemia resulted f...

  2. Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetrazolium salts studied in a model system of polyacrylamide films

    NARCIS (Netherlands)

    van Noorden, C. J.; Tas, J.; Sanders, J. A.

    1981-01-01

    The enzyme cytochemical demonstration of glucose-6-phosphate dehydrogenase (G6PDH) with several tetrazolium salts has been studied with an artificial model of polyacrylamide films in corporated with the enzyme, which enabled teh correlation of cytochemical and biochemical data. In the model films no

  3. Glucose-6-Phosphate Dehydrogenase Deficiency A− Variant in Febrile Patients in Haiti

    Science.gov (United States)

    Carter, Tamar E.; Maloy, Halley; von Fricken, Michael; St. Victor, Yves; Romain, Jean R.; Okech, Bernard A.; Mulligan, Connie J.

    2014-01-01

    Haiti is one of two remaining malaria-endemic countries in the Caribbean. To decrease malaria transmission in Haiti, primaquine was recently added to the malaria treatment public health policy. One limitation of primaquine is that, at certain doses, primaquine can cause hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd). In this study, we genotyped two mutations (A376G and G202A), which confer the most common G6PDd variant in West African populations, G6PDd A−. We estimated the frequency of G6PDd A− in a sample of febrile patients enrolled in an on-going malaria study who represent a potential target population for a primaquine mass drug administration. We found that 33 of 168 individuals carried the G6PDd A− allele (includes A− hemizygous males, A− homozygous or heterozygous females) and could experience toxicity if treated with primaquine. These data inform discussions on safe and effective primaquine dosing and future malaria elimination strategies for Haiti. PMID:24891465

  4. Quantitative cytochemical analysis of glucose-6-phosphate dehydrogenase activity in living isolated hepatocytes of European flounder for rapid analysis of xenobiotic effects

    NARCIS (Netherlands)

    Winzer, K.; van Noorden, C. J.; Köhler, A.

    2001-01-01

    There is a great need for rapid but reliable assays to determine quantitatively effects of xenobiotics on biological systems in environmental research. Hepatocytes of European flounder are sensitive to low-dose toxic stress. Glucose-6-phosphate dehydrogenase (G6PDH) is the major source of NADPH in

  5. Glucose 6-phosphate dehydrogenase: isoenzymatic pattern in Oesophagostomum venulosum, Trichuris ovis and T. suis.

    Science.gov (United States)

    Rodriguez, B; Cutillas, C; German, P; Guevara, D

    1991-12-01

    In the present communication we have studied the isoenzymatic pattern activity of the glucose 6-phosphate dehydrogenase (G6PD) in Oesophagostomum venulosum, Trichuris ovis and T. suis, parasites of Capra hircus (goat), Ovis aries (sheep) and Sus scrofa domestica (pig) respectively, by polyacrylamide gel electrophoresis. Different phenotypes have been observed in the G6PD isoenzymatic pattern activity in males and females of Oesophagostomum venulosum. Furthermore, G6PD activity has been assayed in Trichuris ovis collected from Ovis aries and Capra hircus. No differences have been observed in the isoenzymatic patterns attending to the different hosts. All the individuals exhibited one single band or two bands; this suggests a monomeric condition for G6PD in T. ovis. In T. suis the enzyme G6PD appeared as a single electrophoretic band in about 85.7% of the individuals.

  6. Peroxyl radical- and photo-oxidation of glucose 6- phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues

    DEFF Research Database (Denmark)

    Leinisch, Fabian; Mariotti, Michele; Rykær, Martin

    2017-01-01

    indicate that pathophysiological processes and multiple human diseases are associated with the accumulation of damaged proteins. In this study we investigated the mechanisms and consequences of exposure of the key metabolic enzyme glucose-6-phosphate dehydrogenase (G6PDH) to peroxyl radicals (ROO...

  7. Prevalence of Sickle Cell Trait and Glucose 6 Phosphate ...

    African Journals Online (AJOL)

    Blood donation from sickle cell trait (SCT) and glucose-6-phosphate dehydrogenase (G6PD)-deficient donors might alter the quality of the donated blood during processing, storage or in the recipients' circulatory system. The aim of this study was to determine the prevalence of SCT and G6PD deficiency among blood ...

  8. Data on how several physiological parameters of stored red blood cells are similar in glucose 6-phosphate dehydrogenase deficient and sufficient donors

    Directory of Open Access Journals (Sweden)

    Vassilis L. Tzounakas

    2016-09-01

    Full Text Available This article contains data on the variation in several physiological parameters of red blood cells (RBCs donated by eligible glucose-6-phosphate dehydrogenase (G6PD deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD+ cells. Intracellular reactive oxygen species (ROS generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units. Finally, procoagulant activity and protein carbonylation levels were measured in the microparticles population. Further information can be found in “Glucose 6-phosphate dehydrogenase deficient subjects may be better “storers” than donors of red blood cells” [1]. Keywords: G6PD deficiency, Red blood cell storage lesion, Oxidative stress, Cell fragility, Microparticles

  9. Glucose-6-phosphate dehydrogenase deficiency in Nigerian children.

    Directory of Open Access Journals (Sweden)

    Olatundun Williams

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency is the most common human enzymopathy and in Sub-Saharan Africa, is a significant cause of infection- and drug-induced hemolysis and neonatal jaundice. Our goals were to determine the prevalence of G6PD deficiency among Nigerian children of different ethnic backgrounds and to identify predictors of G6PD deficiency by analyzing vital signs and hematocrit and by asking screening questions about symptoms of hemolysis. We studied 1,122 children (561 males and 561 females aged 1 month to 15 years. The mean age was 7.4 ± 3.2 years. Children of Yoruba ethnicity made up the largest group (77.5% followed by those Igbo descent (10.6% and those of Igede (10.2% and Tiv (1.8% ethnicity. G6PD status was determined using the fluorescent spot method. We found that the overall prevalence of G6PD deficiency was 15.3% (24.1% in males, 6.6% in females. Yoruba children had a higher prevalence (16.9% than Igede (10.5%, Igbo (10.1% and Tiv (5.0% children. The odds of G6PD deficiency were 0.38 times as high in Igbo children compared to Yoruba children (p=0.0500. The odds for Igede and Tiv children were not significantly different from Yoruba children (p=0.7528 and 0.9789 respectively. Mean oxygen saturation, heart rate and hematocrit were not significantly different in G6PD deficient and G6PD sufficient children. The odds of being G6PD deficient were 2.1 times higher in children with scleral icterus than those without (p=0.0351. In conclusion, we determined the prevalence of G6PD deficiency in Nigerian sub-populations. The odds of G6PD deficiency were decreased in Igbo children compared to Yoruba children. There was no association between vital parameters or hematocrit and G6PD deficiency. We found that a history of scleral icterus may increase the odds of G6PD deficiency, but we did not exclude other common causes of icterus such as sickle cell disease or malarial infection.

  10. Pedigree analysis of glucose-6 phosphate dehydrogenase (G6PD deficiency of a Javanese Chinese family in Indonesia

    Directory of Open Access Journals (Sweden)

    IDG Ugrasena

    2017-02-01

    Full Text Available The molecular and pedigree analyses in a Javanese Chinese family were carried oul on glucose-6-phosphate dehydrogenase deficiencies. By method of  MPTP scanning without the sequencing steps, those variants could be confirmed. Two out of three sons were clinically jaundiced at birth due to G6PD deficiency and identified to have a G to T nucleotide change al 1376th nucleotide 01 the G6PD gene (GI376T, corresponding to G6PD Canton. Another son was also identified to have a C to T nucleotide change at 1311st nucleotide 01 the G6PD gene (CI311T, corresponding to a Silent mutation. Their father was normal, but their mother obsorved to have the heleromutation 01 G1376T (G6PD Canton and C1311T (a Silent mutation.

  11. Glucose-6-phosphate dehydrogenase in rat lung alveolar epithelial cells. An ultrastructural enzyme-cytochemical study

    Directory of Open Access Journals (Sweden)

    S Matsubara

    2010-01-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is the key enzyme of the pentose phosphate pathway in carbohydrate metabolism, and it plays an important role in cell proliferation and antioxidant regulation within cells in various organs. Although marked cell proliferation and oxidant/antioxidant metabolism occur in lung alveolar epithelial cells, definite data has been lacking as to whether cytochemically detectable G6PD is present in alveolar epithelial cells. The distribution pattern of G6PD within these cells, if it is present, is also unknown. The purpose of the present study was to investigate the subcellular localization of G6PD in alveolar cells in the rat lung using a newly- developed enzyme-cytochemistry (copper-ferrocyanide method. Type I cells and stromal endothelia and fibroblasts showed no activities. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of type II alveolar epithelial cells. The cytochemical controls ensured specific detection of enzyme activity. This enzyme may play a role in airway defense by delivering substances for cell proliferation and antioxidant forces, thus maintaining the airway architecture.

  12. EFFECTS OF PARTIAL HEPATECTOMY, PHENOBARBITAL AND 3-METHYLCHOLANTHRENE ON KINETIC-PARAMETERS OF GLUCOSE-6-PHOSPHATE AND PHOSPHOGLUCONATE DEHYDROGENASE IN-SITU IN PERIPORTAL, INTERMEDIATE AND PERICENTRAL ZONES OF RAT-LIVER LOBULES

    NARCIS (Netherlands)

    Jonges, G. N.; Vogels, I. M. C.; van Noorden, C. J. F.

    1995-01-01

    Glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) are heterogeneously distributed in liver lobules of female rats. The maximum activity of both enzymes is approximately twice higher in intermediate and pericentral zones than in periportal zones. Enzyme activities

  13. Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis▿

    Science.gov (United States)

    Saliola, Michele; Scappucci, Gina; De Maria, Ilaria; Lodi, Tiziana; Mancini, Patrizia; Falcone, Claudio

    2007-01-01

    In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield. PMID:17085636

  14. Radiation target analyses of free and immobilized glucose 6-phosphate dehydrogenase

    International Nuclear Information System (INIS)

    Kempner, E.S.; Miller, J.H.

    2010-01-01

    The sensitivity of the enzyme glucose 6-phosphate dehydrogenase to ionizing radiation was examined under several conditions, including the presence of several free-radical scavengers. The enzyme was also irradiated when covalently bound to polyacrylamide beads whose structure is very similar to the polypeptide backbone of proteins. All the enzyme forms were irradiated in the frozen state with high-energy electrons from a linear accelerator. Surviving enzyme activity and surviving monomers were determined; the data were analyzed by target theory. Free-radical scavengers reduced the radiation target size of both the activity and monomers of the free enzyme, but not that of the immobilized enzyme activity. The target size of the activity of the free enzyme was that of a dimer mass, but in the case of the immobilized enzyme it was equal to the smaller mass of the monomer. Free-radical scavengers reduce the target size by modifying radiation energy transfer. The target size of the polyacrylamide-bound enzyme activity was expected to be very large since the connection between polyacrylamide and protein is a peptide bond which permits transfer of radiation-deposited energy. Several explanations concerning energy transfer are suggested for this result.

  15. Physiological role of glucose-6-phosphate dehydrogenase in cold acclimation of strawberry (Fragaria × ananassa)

    Science.gov (United States)

    Zhang, Yong; Yu, Dingqun; Luo, Ya; Wang, Xiaorong; Chen, Qing; Sun, Bo; Wang, Yan; Liu, Zejing; Tang, Haoru

    2018-04-01

    In recent years, there has been an increasing interest in study of new resistance mechanism in fruit trees. All these regard the climate change and subsequent fruit production. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting step of the oxidative pentose phosphate pathway (OPPP), and the expression of this enzyme is related to different biotic and abiotic stresses. Under accumulation of low temperature stress, the significant increase in G6PDH activity was found to be closely correlated to the levels of antioxidant enzymes, malondialdehyde (MDA) contents, sugar contents as well as changes of superoxide (O2•-). It is suggested that the enhancement of cold resistance of strawberry, which induced by cold acclimation, related to the significant increase in G6PDH activity. On one hand, G6PDH activates NADPH oxidase to generate reactive oxygen species (ROS); on the other hand, it may be involved in the activation of antioxidant enzymes, and accelerates many other important NADPH-dependent enzymatic reactions. Then further result in the elevation of membrane stability and cold resistance of strawberry. Interestingly, even though the plants were placed again under a temperature of 25°C for 1 d, the higher cold resistance, enzyme activities and soluble sugar content acquired.

  16. Hexose-6-phosphate dehydrogenase contributes to skeletal muscle homeostasis independent of 11β-hydroxysteroid dehydrogenase type 1.

    LENUS (Irish Health Repository)

    Semjonous, Nina M

    2011-01-01

    Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11β-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11β-HSD1\\/H6PDH double-KO (DKO) mice, in which 11β-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11β-HSD1KO mice. Critically, in contrast to 11β-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11β-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)\\/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.

  17. Importance of glucose-6-phosphate dehydrogenase (G6PDH) for vanillin tolerance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nguyen, Trinh Thi My; Kitajima, Sakihito; Izawa, Shingo

    2014-09-01

    Vanillin is derived from lignocellulosic biomass and, as one of the major biomass conversion inhibitors, inhibits yeast growth and fermentation. Vanillin was recently shown to induce the mitochondrial fragmentation and formation of mRNP granules such as processing bodies and stress granules in Saccharomyces cerevisiae. Furfural, another major biomass conversion inhibitor, also induces oxidative stress and is reduced in an NAD(P)H-dependent manner to its less toxic alcohol derivative. Therefore, the pentose phosphate pathway (PPP), through which most NADPH is generated, plays a role in tolerance to furfural. Although vanillin also induces oxidative stress and is reduced to vanillyl alcohol in a NADPH-dependent manner, the relationship between vanillin and PPP has not yet been investigated. In the present study, we examined the importance of glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the rate-limiting NADPH-producing step in PPP, for yeast tolerance to vanillin. The growth of the null mutant of G6PDH gene (zwf1Δ) was delayed in the presence of vanillin, and vanillin was efficiently reduced in the culture of wild-type cells but not in the culture of zwf1Δ cells. Furthermore, zwf1Δ cells easily induced the activation of Yap1, an oxidative stress responsive transcription factor, mitochondrial fragmentation, and P-body formation with the vanillin treatment, which indicated that zwf1Δ cells were more susceptible to vanillin than wild type cells. These findings suggest the importance of G6PDH and PPP in the response of yeast to vanillin. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Reduced glutathione and glutathione disulfide in the blood of glucose-6-phosphate dehydrogenase-deficient newborns.

    Science.gov (United States)

    Gong, Zhen-Hua; Tian, Guo-Li; Huang, Qi-Wei; Wang, Yan-Min; Xu, Hong-Ping

    2017-07-20

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is commonly detected during mass screening for neonatal disease. We developed a method to measure reduced glutathione (GSH) and glutathione disulfide (GSSG) using tandem mass spectrometry (MS/MS) for detecting G6PD deficiency. The concentration of GSH and the GSH/GSSG ratio in newborn dry-blood-spot (DBS) screening and in blood plus sodium citrate for test confirmation were examined by MS/MS using labeled glycine as an internal standard. G6PD-deficient newborns had a lower GSH content (242.9 ± 15.9 μmol/L)and GSH/GSSG ratio (14.9 ± 7.2) than neonatal controls (370.0 ± 53.2 μmol/L and 46.7 ± 19.6, respectively). Although the results showed a significance of P blood measured using MS/MS on the first day of sample preparation are consistent with G6PD activity and are helpful for diagnosing G6PD deficiency.

  19. Incorporation of 14C glucose into glycogen and glucose-6-phosphate dehydrogenase activity in rat brain following carbon monoxide intoxication

    International Nuclear Information System (INIS)

    Sikorska, M.; Gorzkowski, B.; Szumanska, G.; Smialek, M.

    1975-01-01

    Incorporation of 14 C glucose into glycogen and glucose-6-phosphate dehydrogenase activity in rat brain following carbon monoxide intoxication was studied. In brains of rats tested on the 20, 30 and 60th minute of exposure to CO and immediately after removal from the chamber the enzyme activity showed no essential deviation from the control level. In the group of rats tested 1 hour after taking them out from the chamber increase of the enzyme activity was noticed, amounting to about 33% of the control value. The brains tested 24 hours after exposure showed the largest increase of the enzyme activity by about 94%. In the next time periods, 48 and 72 hours after intoxication, the enzyme activity was decreasing. The glycogen content in brains of control animals increased 3 hours after CO intoxication by about 69%. The increase of glycogen synthesis was expressed by increase of the total radioactivity, which amounted to 160% of the control value. (Z.M.)

  20. Kinetic and thermodynamic study of the reaction catalyzed by glucose-6-phosphate dehydrogenase with nicotinamide adenine dinucleotide

    International Nuclear Information System (INIS)

    Martin del Campo, Julia S.; Patino, Rodrigo

    2011-01-01

    Research highlights: → The reaction catalyzed by one enzyme of the pentose phosphate pathway was studied. → A spectrophotometric method is proposed for kinetic and thermodynamic analysis. → The pH and the temperature influences are reported on physical chemical properties. → Relative concentrations of substrates are also important in the catalytic process. - Abstract: The enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) from Leuconostoc mesenteroides has a dual coenzyme specificity with oxidized nicotinamide adenine dinucleotide (NAD ox ) and oxidized nicotinamide adenine dinucleotide phosphate as electron acceptors. The G6PD coenzyme selection is determined by the metabolic cellular prevailing conditions. In this study a kinetic and thermodynamic analysis is presented for the reaction catalyzed by G6PD from L. mesenteroides with NAD ox as coenzyme in phosphate buffer. For this work, an in situ spectrophotometric technique was employed based on the detection of one product of the reaction. Substrate and coenzyme concentrations as well as temperature and pH effects were evaluated. The apparent equilibrium constant, the Michaelis constant, and the turnover number were determined as a function of each experimental condition. The standard transformed Gibbs energy of reaction was determined from equilibrium constants at different initial conditions. For the product 6-phospho-D-glucono-1,5-lactone, a value of the standard Gibbs energy of formation is proposed, Δ f G o = -1784 ± 5 kJ mol -1 .

  1. Kinetic and thermodynamic study of the reaction catalyzed by glucose-6-phosphate dehydrogenase with nicotinamide adenine dinucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Martin del Campo, Julia S. [Departamento de Fisica Aplicada, Centro de Investigacion y de Estudios Avanzados - Unidad Merida, Carretera antigua a Progreso Km. 6, A.P. 73 Cordemex, 97310, Merida, Yucatan (Mexico); Patino, Rodrigo, E-mail: rtarkus@mda.cinvestav.mx [Departamento de Fisica Aplicada, Centro de Investigacion y de Estudios Avanzados - Unidad Merida, Carretera antigua a Progreso Km. 6, A.P. 73 Cordemex, 97310, Merida, Yucatan (Mexico)

    2011-04-20

    Research highlights: {yields} The reaction catalyzed by one enzyme of the pentose phosphate pathway was studied. {yields} A spectrophotometric method is proposed for kinetic and thermodynamic analysis. {yields} The pH and the temperature influences are reported on physical chemical properties. {yields} Relative concentrations of substrates are also important in the catalytic process. - Abstract: The enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) from Leuconostoc mesenteroides has a dual coenzyme specificity with oxidized nicotinamide adenine dinucleotide (NAD{sub ox}) and oxidized nicotinamide adenine dinucleotide phosphate as electron acceptors. The G6PD coenzyme selection is determined by the metabolic cellular prevailing conditions. In this study a kinetic and thermodynamic analysis is presented for the reaction catalyzed by G6PD from L. mesenteroides with NAD{sub ox} as coenzyme in phosphate buffer. For this work, an in situ spectrophotometric technique was employed based on the detection of one product of the reaction. Substrate and coenzyme concentrations as well as temperature and pH effects were evaluated. The apparent equilibrium constant, the Michaelis constant, and the turnover number were determined as a function of each experimental condition. The standard transformed Gibbs energy of reaction was determined from equilibrium constants at different initial conditions. For the product 6-phospho-D-glucono-1,5-lactone, a value of the standard Gibbs energy of formation is proposed, {Delta}{sub f}G{sup o} = -1784 {+-} 5 kJ mol{sup -1}.

  2. Data mining and pathway analysis of glucose-6-phosphate dehydrogenase with natural language processing

    Science.gov (United States)

    Chen, Long; Zhang, Chunhua; Wang, Yanling; Li, Yuqian; Han, Qiaoqiao; Yang, Huixin; Zhu, Yuechun

    2017-01-01

    Human glucose-6-phosphate dehydrogenase (G6PD) is a crucial enzyme in the pentose phosphate pathway, and serves an important role in biosynthesis and the redox balance. G6PD deficiency is a major cause of neonatal jaundice and acute hemolyticanemia, and recently, G6PD has been associated with diseases including inflammation and cancer. The aim of the present study was to conduct a search of the National Center for Biotechnology Information PubMed library for articles discussing G6PD. Genes that were identified to be associated with G6PD were recorded, and the frequency at which each gene appeared was calculated. Gene ontology (GO), pathway and network analyses were then performed. A total of 98 G6PD-associated genes and 33 microRNAs (miRNAs) that potentially regulate G6PD were identified. The 98 G6PD-associated genes were then sub-classified into three functional groups by GO analysis, followed by analysis of function, pathway, network, and disease association. Out of the 47 signaling pathways identified, seven were significantly correlated with G6PD-associated genes. At least two out of four independent programs identified the 33 miRNAs that were predicted to target G6PD. miR-1207-5P, miR-1 and miR-125a-5p were predicted by all four software programs to target G6PD. The results of the present study revealed that dysregulation of G6PD was associated with cancer, autoimmune diseases, and oxidative stress-induced disorders. These results revealed the potential roles of G6PD-regulated signaling and metabolic pathways in the etiology of these diseases. PMID:28627690

  3. Neonatal screening for sickle cell disease, Glucose-6-PhosphateDehydrogenase deficiency and Alpha-Thalassemia in Qatif and Al-Hasa

    International Nuclear Information System (INIS)

    Nasserullah, Z.; Srair, Hussain Abu; Al-Jame, A.; Mokhtar, M.; Al-Qatari, G.; Al-Naim, S.; Al-Aqib, A.

    1998-01-01

    Screening programs to determine the frequency of sickle cell,glucose-6-phosphate dehydrogenase deficiency and alpha-thalassemia gene areavailable in Saudi Arabia, although not used frequently. Greater use of theseprograms will decrease the morbidity and mortality of Saudi children affectedby these disorders. Neonatal hemoglobin electrophoresis andglucose-6-dehydrogenase fluorescent spot tests were performed on new bornbabies delivered between December 1992 and December 1993 at the Qatif CentralHospital and at the King Fahd Hospital in Al-Hasa. Cord blood samples werecollected from babies born in these two hospitals. Babies born in otherhospitals had blood collected in their first visit to Qatif primary carecenters at the time of vaccination. All specimens were sent to Dammam CentralLaboratory. The diagnosis of sickle cell and alpha-thalassemia was based oncellulose acetate electrophoresis and confirmed by agar gel electrophoresisand glucose-6-phosphate dehydrgenase was confirmed by fluorescent spot test.A total of 12,220 infants, including 11,313 Saudis (92.6%), were screenedover a 12-month period. The common phenotype detected in these infantsincluded AF, SFA, SFA Bart's, FS and FS Bart's. In Saudi infants, homozygoussickle cell disease was detected in 2.35% and 1.08% in Qatif and Al-Hasa,respectively. The frequencies of sickle cell gene were 0.1545% and 0.1109% inQatif and Al-Hasa. Alpha-thalassemia genes based on an elevated level of HbBart's were 28% and 16.3% in Qatif and Al-Hasa. The screening for G6PDdeficiency revealed a high prevalence of 30.6% and 14.7% in Qatif andAl-Hasa. In the non-Saudi infants the frequencies were low. The outcome ofthis study indicates that the Saudi populations in Qatif and Al-Hasa are atrisk for hemoglobinopathies and G6PD. Neonatal screening programs areessential and cost effective and should be maintained as a routine practice.(author)

  4. Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry

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    S Matsubara

    2009-06-01

    Full Text Available Although various tissue macrophages possess high glucose- 6-phosphate dehydrogenase (G6PD activity, which is reported to be closely associated with their phagocytotic/bactericidal function, the fine subcellular localization of this enzyme in liver resident macrophages (Kupffer cells has not been determined.We have investigated the subcellular localization of G6PD in Kupffer cells in rat liver, using a newly developed enzyme-cytochemical (copper-ferrocyanide method. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Kupffer cells. Cytochemical controls ensured specific detection of the enzymatic activity. Rat Kupffer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Kupffer cell G6PD may play a role in liver defense by delivering NADPH to NADPH-dependent enzymes. G6PD enzyme-cytochemistry may be a useful tool for the study of Kupffer cell functions.

  5. Incorporation of /sup 14/C glucose into glycogen and glucose-6-phosphate dehydrogenase activity in rat brain following carbon monoxide intoxication

    Energy Technology Data Exchange (ETDEWEB)

    Sikorska, M; Gorzkowski, B; Szumanska, G; Smialek, M [Polska Akademia Nauk, Warsaw. Centrum Medycyny Doswiadczalnej i Klinicznej; Panstwowy Zaklad Higieny, Warsaw (Poland))

    1975-01-01

    Incorporation of /sup 14/C glucose into glycogen and glucose-6-phosphate dehydrogenase activity in rat brain following carbon monoxide intoxication was studied. In brains of rats tested on the 20, 30 and 60th minute of exposure to CO and immediately after removal from the chamber the enzyme activity showed no essential deviation from the control level. In the group of rats tested 1 hour after taking them out from the chamber increase of the enzyme activity was noticed, amounting to about 33% of the control value. The brains tested 24 hours after exposure showed the largest increase of the enzyme activity by about 94%. In the next time periods, 48 and 72 hours after intoxication, the enzyme activity was decreasing. The glycogen content in brains of control animals increased 3 hours after CO intoxication by about 69%. The increase of glycogen synthesis was expressed by increase of the total radioactivity, which amounted to 160% of the control value.

  6. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    International Nuclear Information System (INIS)

    Li, Ming V.; Chen, Weiqin; Harmancey, Romain N.; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

    2010-01-01

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

  7. Effect of thoracic x-irradiation on glucose-6-phosphate dehydrogenase activity of the pectoral muscle of guinea pig

    International Nuclear Information System (INIS)

    Bhatavdekar, J.M.; Shah, V.C.

    1981-01-01

    The histochemical distribution of glucose-6-phosphate dehydrogenase (G6PD) was observed in the major pectoral muscle of a guinea pig that had received 240 R thoracic X-irradiation. The irradiation effects were studied at 24, 48 and 72 hrs after X-irradiation. Type I fibres of the pectoral muscle were deeply stained showing high activity whereas type II fibres demonstrated minimum enzyme activity. The intermediate fibres had medium levels of G6PD activity. Type II fibres showed more staining at 24 and 48 hrs as compared with control muscle. However, at 72 hrs all three fibre types showed a marked inhibition of G6PD activity. The significance of these changes suggests that muscle G6PD metabolism generally altered after irradiation, but the specific nature of these changes and their causes still remain unclear. (author)

  8. Five novel glucose-6-phosphate dehydrogenase deficiency haplotypes correlating with disease severity

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    Dallol Ashraf

    2012-09-01

    Full Text Available Abstract Background Glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49 deficiency is caused by one or more mutations in the G6PD gene on chromosome X. An association between enzyme levels and gene haplotypes remains to be established. Methods In this study, we determined G6PD enzyme levels and sequenced the coding region, including the intron-exon boundaries, in a group of individuals (163 males and 86 females who were referred to the clinic with suspected G6PD deficiency. The sequence data were analysed by physical linkage analysis and PHASE haplotype reconstruction. Results All previously reported G6PD missense changes, including the AURES, MEDITERRANEAN, A-, SIBARI, VIANGCHAN and ANANT, were identified in our cohort. The AURES mutation (p.Ile48Thr was the most common variant in the cohort (30% in males patients followed by the Mediterranean variant (p.Ser188Phe detectable in 17.79% in male patients. Variant forms of the A- mutation (p.Val68Met, p.Asn126Asp or a combination of both were detectable in 15.33% of the male patients. However, unique to this study, several of such mutations co-existed in the same patient as shown by physical linkage in males or PHASE haplotype reconstruction in females. Based on 6 non-synonymous variants of G6PD, 13 different haplotypes (13 in males, 8 in females were identified. Five of these were previously unreported (Jeddah A, B, C, D and E and were defined by previously unreported combinations of extant mutations where patients harbouring these haplotypes exhibited severe G6PD deficiency. Conclusions Our findings will help design a focused population screening approach and provide better management for G6PD deficiency patients.

  9. Evaluation of Glucose-6-Phosphate Dehydrogenase stability in stored blood samples.

    Science.gov (United States)

    Jalil, Norunaluwar; Azma, Raja Zahratul; Mohamed, Emida; Ithnin, Azlin; Alauddin, Hafiza; Baya, Siti Noor; Othman, Ainoon

    2016-01-01

    Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the commonest cause of neonatal jaundice in Malaysia. Recently, OSMMR2000-D G6PD Assay Kit has been introduced to quantitate the level of G6PD activity in newborns delivered in Universiti Kebangsaan Malaysia Medical Centre (UKMMC). As duration of sample storage prior to analysis is one of the matters of concern, this study was conducted to identify the stability of G6PD enzyme during storage. A total of 188 cord blood samples from normal term newborns delivered at UKMMC were selected for this study. The cord bloods samples were collected in ethylene-diamine-tetra-acetic acid (EDTA) tubes and refrigerated at 2-8 °C. In addition, 32 out of 188 cord blood samples were spotted on chromatography paper, air-dried and stored at room temperature. G6PD enzyme activities were measured daily for 7 days using the OSMMR2000-D G6PD Assay Kit on both the EDTA blood and dried blood samples. The mean value for G6PD activity was compared between days of analysis using Student Paired T-Test. In this study, 172 out of 188 cord blood samples showed normal enzyme levels while 16 had levels corresponding to severe enzyme deficiency. The daily mean G6PD activity for EDTA blood samples of newborns with normal G6PD activity showed a significant drop on the fourth day of storage (p samples with severely deficient G6PD activity, significant drop was seen on third day of storage (p = 0.002). Analysis of dried cord blood showed a significant reduction in enzyme activity as early as the second day of storage (p = 0.001). It was also noted that mean G6PD activity for spotted blood samples were lower compared to those in EDTA tubes for all days (p = 0.001). Thus, EDTA blood samples stored at 2-8 °C appeared to have better stability in terms of their G6PD enzyme level as compared to dried blood samples on filter paper, giving a storage time of up to 3 days.

  10. Molecular Characterization of Cosenza Mutation among Patients with Glucose-6-Phosphate Dehydrogenase Deficiency in huzestan Province, Southwest Iran

    Science.gov (United States)

    Kazemi Nezhad, Seyed Reza; Fahmi, Fatemeh; Khatami, Saeid Reza; Musaviun, Mohsen

    2011-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common hereditary enzymatic disorders in human, increases the vulnerability of erythrocytes to oxidative stress. It is also characterized by remarkable molecular and biochemical heterogeneity. According to previous investigations, G6PD Cosenza (G1376C) is a common G6PD mutation in some parts of . Therefore in the present study we have characterized mutation among G6PD deficient individuals in Khuzestan province. In order to identify G6PD Cosenza, we analyzed the G6PD gene in 64 samples out of 231 deficient individuals who had not G6PD Mediterranean mutation, using PCR- restriction fragment length polymorphism (RFLP) method. G6PD Cosenza mutation was found in 6 males of 231 samples, resulting in the relative rate of 2.6% and allele frequency of 0.023 among Khuzestanian G6PD deficient subjects. A comparison of these results with previous findings in some parts of suggests that G6PD Cosenza is a common mutation in Khuzestanian G6PD deficient individuals. PMID:23365477

  11. Seizure is a rare presentation for acute hemolysis due to G6PD deficiency. We report a previously healthy boy who presented initially with seizure and cyanosis and subsequently acute hemolysis, due to glucose-6-phosphate dehydrogenase deficiency (G6PD) an

    OpenAIRE

    Afshin FAYYAZI; Ali KHAJEH; Hosein ESFAHANI

    2012-01-01

    Seizure is a rare presentation for acute hemolysis due to G6PD deficiency. We report a previously healthy boy who presented initially with seizure and cyanosis and subsequently acute hemolysis, due to glucose-6-phosphate dehydrogenase deficiency (G6PD) and probably secondary methemoglobinemia, following the ingestion of fava beans.

  12. In Vitro Effects of Imidacloprid and Lambda-cyhalothrin on Capoeta capoeta umbla Kidney Glucose 6-Phosphate Dehydrogenase Enzyme

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    Mahinur KIRICI

    2015-03-01

    Full Text Available Pesticide toxicity causes oxidative damage such as DNA damage, enhanced lipid peroxidation, the oxidation of protein sulfydryl groups and enzyme inactivation in the metabolism. In this study, we investigated the in vitro effects on glucose 6-phosphate dehydrogenase (E.C.1.1.49; G6PD from Capoeta capoeta umbla kidney of imidacloprid and lambda-cyhalothrin. For this purpose, the enzymewas purified from kidney of C. c. umbla with a specific activity of 11.26 EU mg-1 proteins and 22.7% yield using hemolysate preparation, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity gel chromatography methods. In order to control the enzyme purification sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE was done. SDS-PAGE showed a single band for the enzyme. The results of this study suggested that imidacloprid and lambda-cyhalothrin have significant inhibition effect on the activity of G6PD in in vitro. In conclusion, lambda-cyhalothrin inhibits the enzyme activity more than imidacloprid.

  13. Clonal evolution following chemotherapy-induced stem cell depletion in cats heterozygous for glucose-6-phosphate dehydrogenase

    International Nuclear Information System (INIS)

    Abkowitz, J.L.; Ott, R.M.; Holly, R.D.; Adamson, J.W.

    1988-01-01

    The number of hematopoietic stem cells necessary to support normal hematopoiesis is not known but may be small. If so, the depletion or damage of such cells could result in apparent clonal dominance. To test this hypothesis, dimethylbusulfan [2 to 4 mg/kg intravenously (IV) x 3] was given to cats heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G-6-PD). These cats were the daughters of domestic X Geoffroy parents. After the initial drug-induced cytopenias (2 to 4 weeks), peripheral blood counts and the numbers of marrow progenitors detected in culture remained normal, although the percentages of erythroid burst-forming cells (BFU-E) and granulocyte/macrophage colony-forming cells (CFU-GM) in DNA synthesis increased, as determined by the tritiated thymidine suicide technique. In three of six cats treated, a dominance of Geoffroy-type G-6-PD emerged among the progenitor cells, granulocytes, and RBCs. These skewed ratios of domestic to Geoffroy-type G-6-PD have persisted greater than 3 years. No changes in cell cycle kinetics or G-6-PD phenotypes were noted in similar studies in six control cats. These data suggest that clonal evolution may reflect the depletion or damage of normal stem cells and not only the preferential growth and dominance of neoplastic cells

  14. Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension

    Science.gov (United States)

    Chettimada, Sukrutha; Gupte, Rakhee; Rawat, Dhwajbahadur; Gebb, Sarah A.; McMurtry, Ivan F.

    2014-01-01

    Severe pulmonary hypertension is a debilitating disease with an alarmingly low 5-yr life expectancy. Hypoxia, one of the causes of pulmonary hypertension, elicits constriction and remodeling of the pulmonary arteries. We now know that pulmonary arterial remodeling is a consequence of hyperplasia and hypertrophy of pulmonary artery smooth muscle (PASM), endothelial, myofibroblast, and stem cells. However, our knowledge about the mechanisms that cause these cells to proliferate and hypertrophy in response to hypoxic stimuli is still incomplete, and, hence, the treatment for severe pulmonary arterial hypertension is inadequate. Here we demonstrate that the activity and expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, are increased in hypoxic PASM cells and in lungs of chronic hypoxic rats. G6PD overexpression and -activation is stimulated by H2O2. Increased G6PD activity contributes to PASM cell proliferation by increasing Sp1 and hypoxia-inducible factor 1α (HIF-1α), which directs the cells to synthesize less contractile (myocardin and SM22α) and more proliferative (cyclin A and phospho-histone H3) proteins. G6PD inhibition with dehydroepiandrosterone increased myocardin expression in remodeled pulmonary arteries of moderate and severe pulmonary hypertensive rats. These observations suggest that altered glucose metabolism and G6PD overactivation play a key role in switching the PASM cells from the contractile to synthetic phenotype by increasing Sp1 and HIF-1α, which suppresses myocardin, a key cofactor that maintains smooth muscle cell in contractile state, and increasing hypoxia-induced PASM cell growth, and hence contribute to pulmonary arterial remodeling and pathogenesis of pulmonary hypertension. PMID:25480333

  15. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

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    Bo Fu

    2015-12-01

    Full Text Available The in vitro maturation (IVM efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+ oocytes with low glucose-6-phosphate dehydrogenase (G6PDH activity have shown superior quality than BCB negative (− oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9 and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

  16. Kinetic Behaviour of Glucose 6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase in Different Tissues of Rainbow Trout (Oncorhynchus mykiss Exposed to Non-Lethal Concentrations of Cadmium

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    Olcay Hisar

    2009-01-01

    Full Text Available The effects of cadmium (Cd on the enzymatic activities of glucose 6-phosphate dehydrogenase (G6PD and 6-phosphogluconate dehydrogenase (6PGD were investigated in the gill, liver and kidney tissues of rainbow trout (Oncorhynchus mykiss. Three test groups of fish were subjected to increasing concentrations (1, 3 and 5 mg/l of cadmium (Cd in vivo, respectively. The G6PD and 6PGD activities in the gill, liver, and kidney tissues of each group of fish were measured on days 1, 3, 5 and 7. G6PD and 6PGD enzyme activities, measured in gill, liver and kidney homogenates, were stimulated by various concentrations (1, 3, and 5 mg/l of cadmium. Although the dose-response pattern of G6PD enzyme activities in liver and kidney tissue was very similar, that in gill was different from both other tissues. The enzyme activity of G6PD enzyme was significantly stimulated after three days (Day 3 in liver and kidney tissues at a dose of 1 mg/l Cd (p p p p p p < 0.05 in liver and kidney tissues at the doses of 3 and 1 mg/l Cd. The stimulation effect of cadmium on the three tissues studied was also calculated; for both of the enzymes (G6PD and 6PGD, the enzyme activity levels were stimulated by approximately 60% and 38% in gills, 68% and 44% in liver, and 67% and 41% in kidneys, respectively, over the base-line enzyme activity of the control groups during the sevenday experimental period. These findings indicate that tissue G6PD and 6PGD enzymes function to protect against cadmium toxicity.

  17. Molecular Characterization of Cosenza Mutation among Patients with Glucose-6-Phosphate Dehydrogenase Deficiency in Khuzestan Province, Southwest Iran

    Directory of Open Access Journals (Sweden)

    Seyed Reza Kazemi Nezhad

    2011-03-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency is one of the most common hereditary enzymatic disorders in human, increases the vulnerability of erythrocytes to oxidative stress. It is also characterized by remarkable molecular and biochemical heterogeneity. According to previous investigations, G6PD Cosenza (G1376C is a common G6PD mutation in some parts of Iran. Therefore in the present study we have characterized Cosenza mutation among G6PD deficient individuals in Khuzestan province. In order to identify G6PD Cosenza, we analyzed the G6PD gene in 64 samples out of 231 deficient individuals who had not G6PD Mediterranean mutation, using PCR- restriction fragment length polymorphism (RFLP method. G6PD Cosenza mutation was found in 6 males of 231 samples, resulting in the relative rate of 2.6% and allele frequency of 0.023 among Khuzestanian G6PD deficient subjects. A comparison of these results with previous findings in some parts of Iran suggests that G6PD Cosenza is a common mutation in Khuzestanian G6PD deficient individuals

  18. Prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency among malaria patients in Upper Myanmar.

    Science.gov (United States)

    Lee, Jinyoung; Kim, Tae Im; Kang, Jung-Mi; Jun, Hojong; Lê, Hương Giang; Thái, Thị Lam; Sohn, Woon-Mok; Myint, Moe Kyaw; Lin, Khin; Kim, Tong-Soo; Na, Byoung-Kuk

    2018-03-16

    Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is one of the most common X-linked recessive hereditary disorders in the world. Primaquine (PQ) has been used for radical cure of P. vivax to prevent relapse. Recently, it is also used to reduce P. falciparum gametocyte carriage to block transmission. However, PQ metabolites oxidize hemoglobin and generate excessive reactive oxygen species which can trigger acute hemolytic anemia in malaria patients with inherited G6PD deficiency. A total of 252 blood samples collected from malaria patients in Myanmar were used in this study. G6PD variant was analysed by a multiplex allele specific PCR kit, DiaPlexC™ G6PD Genotyping Kit [Asian type]. The accuracy of the multiplex allele specific PCR was confirmed by sequencing analysis. Prevalence and distribution of G6PD variants in 252 malaria patients in Myanmar were analysed. Six different types of G6PD allelic variants were identified in 50 (7 females and 43 males) malaria patients. The predominant variant was Mahidol (68%, 34/50), of which 91.2% (31/34) and 8.8% (3/34) were males and females, respectively. Other G6PD variants including Kaiping (18%, 9/50), Viangchan (6%, 3/50), Mediterranean (4%, 2/50), Union (2%, 1/50) and Canton (2%, 1/50) were also observed. Results of this study suggest that more concern for proper and safe use of PQ as a radical cure of malaria in Myanmar is needed by combining G6PD deficiency test before PQ prescription. Establishment of a follow-up system to monitor potential PQ toxicity in malaria patients who are given PQ is also required.

  19. Glucose and fructose 6-phosphate cycle in humans

    International Nuclear Information System (INIS)

    Karlander, S.; Roovete, A.; Vranic, M.; Efendic, S.

    1986-01-01

    We have determined the rate of glucose cycling by comparing turnovers of [2- 3 H]- and [6- 3 H]glucose under basal conditions and during a glucose infusion. Moreover, the activity of the fructose 6-phosphate cycle was assessed by comparing [3- 3 H]- and [6- 3 H]glucose. The study included eight lean subjects with normal glucose tolerance. They participated in two randomly performed investigations. In one experiment [2- 3 H]- and [6- 3 H]glucose were given simultaneously, while in the other only [3- 3 H]glucose was given. The basal rate of glucose cycling was 0.32 +/- 0.08 mg X kg-1 X min-1 or 17% of basal glucose production (P less than 0.005). During glucose infusion the activity of endogenous glucose cycling did not change but since glucose production was suppressed it amounted to 130% of glucose production. The basal fructose 6-phosphate cycle could be detected only in three subjects and was suppressed during glucose infusion. In conclusion, the glucose cycle is active in healthy humans both in basal conditions and during moderate hyperglycemia. In some subjects, the fructose 6-phosphate cycle also appears to be active. Thus it is preferable to use [6- 3 H]glucose rather than [3- 3 H]glucose when measuring glucose production and particularly when assessing glucose cycle

  20. Two apparent glucose-6-phosphate dehydrogenase variants in normal XY males: G6PD Alabama.

    Science.gov (United States)

    Prchal, J T; Hall, K; Csepreghy, M; Lilly, M; Berkow, R; Scott, C W

    1988-03-01

    A six-year-old black boy who had transient hemolysis after a viral infection was found to have mildly decreased red cell glucose-6-phosphate dehydrogenase (G6PD) activity (1.25 IU/g hemoglobin). Two G6PD bands, both slightly faster than normal G6PD B, were seen on electrophoresis in both the propositus as well as in his maternal grandfather. This is an unexpected finding, since the G6PD gene is located on the long arm of the X chromosome that is subject to X-chromosome inactivation, and available evidence indicates that it is present as a single functional copy in the human genome. The obvious possibility of duplication of the X chromosome was eliminated by cytogenetic analysis with G-banding. G6PD duplication is unlikely, since peripheral blood granulocytes, platelets, and lymphocytes; cultured skin and bone marrow fibroblasts; and Epstein-Barr virus-stimulated lymphocytes yielded only a single electrophoretic band with mobility identical to the slower band seen in crude red blood cell hemolysate. Study of partially purified red blood cell hemolysate G6PD also yielded a single band with identical mobility. Kinetic studies of the enzyme in the propositus and in three generations of his family identified a unique, previously unpublished G6PD mutant that is herein designated G6PD Alabama. Red blood cells were separated by density gradient into a reticulocyte-enriched, an intermediate, and a dense, older portion. Two distinct enzyme bands were identified on electrophoresis of hemolysate from the reticulocyte-enriched portion, but not from the other two portions. It is postulated that two transcriptional products of the mutant G6PD gene exist; one with a short half-life and detectable only in young red blood cells, and another with a longer half-life present in all cells. The existence of two distinct mutant genes in the genome or a unique post-translational form of the mutant G6PD detected only in reticulocytes cannot be excluded.

  1. Simultaneous demonstration of acid phosphatase and glucose-6-phosphate dehydrogenase in mouse hepatocytes. A novel electron-microscopic dual staining enzyme-cytochemistry

    Directory of Open Access Journals (Sweden)

    S Matsubara

    2010-01-01

    Full Text Available Acid phosphatase (ACPase and glucose-6-phosphate dehydrogenase (G6PD play important roles in cell biology/disease pathophysiology in various organs including the liver. The purpose of the present report is to introduce a new enzymecytochemical method to simultaneously demonstrate the subcellular localization of ACPase and G6PD within the same hepatocyte in the mouse liver. The ultrastructural localization of ACPase and G6PD were demonstrated, with concomitant use of the cerium method and the copper-ferrocyanide method, respectively. ACPase labelings were localized in the lysosomes, and G6PD labelings were visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of the hepatocyte. This novel double staining procedure may be a useful histochemical tool for the study of liver functions in both physiological and pathological conditions.

  2. Prevalence of thalassaemia, iron-deficiency anaemia and glucose-6-phosphate dehydrogenase deficiency among Arab migrating nomad children, southern Islamic Republic of Iran.

    Science.gov (United States)

    Pasalar, M; Mehrabani, D; Afrasiabi, A; Mehravar, Z; Reyhani, I; Hamidi, R; Karimi, M

    2014-12-17

    This study investigated the prevalence of iron-deficiency anaemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency and β-thalassaemia trait among Arab migrating nomad children in southern Islamic Republic of Iran. Blood samples were analysed from 134 schoolchildren aged child had G6PD deficiency. A total of 9.7% of children had HbA2 ≥ 3.5 g/dL, indicating β-thalassaemia trait (10.8% in females and 7.8% in males). Mean serum iron, serum ferritin and total iron binding capacity were similar in males and females. Serum ferritin index was as accurate as Hb index in the diagnosis of iron-deficiency anaemia. A high prevalence of β-thalassaemia trait was the major potential risk factor in this population.

  3. Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1991-06-01

    Full Text Available The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females aged 17 to 58 years. Twenty one (53.84% of the patients presented a slow acetylatingphenotype and 18(46.16% a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD acti vity was decreased in 5(23.80% slow acetylators and in 4(22.22% fast acetylators. Glutathione reductase activity was decreased in 14 (66.66% slow acetylators and in 12 (66.66% fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p Os autores avaliaram o fenótipo acetilador da isoniazida, hematócrito, hemoglobina, atividade da glicose-6- fosfato desidrogenase, glutationa redutase e os níveis séricos de sulfadoxina de 39 doentes com paracoccidíoidomicose, senão 33 do sexo masculino e 6 do feminino, com idades compreendidas entre 17 e 58 anos. Vinte e um (53,84% doentes apresentaram fenótipo acetilador lento e 18 (46,16% rápido. A atividade da glicose-6-fosfato desidrogenase (G6PD esteve diminuída em 5 (23,80% acetiladores lentos e 4 (22,22% rápidos. A atividade da glutationa redutase esteve diminuída em 14 (66,66% acetiladores lentos e 12 (66,66% rápidos. Os níveis séricos de sulfadoxina livre e total foram maiores nos acetiladores lentos (p < 0,02. A análise dos resultados permite concluir que os níveis séricos de sulfadoxina relaciona-se com o fenótipo acetilador. Além disso, os níveis estiveram sempre acima de 50 µg/ml, níveis estes considerados terapêuticos. Por outro lado, a deficiência de glutationa redutase pode estar relacionada com a má absorção intestinal de nutrientes, entre eles riboflavina, vitamina precursora de FAD.

  4. High prevalence of Dapsone-induced oxidant hemolysis in North American SCT recipients without glucose-6-phosphate-dehydrogenase deficiency.

    Science.gov (United States)

    Olteanu, H; Harrington, A M; George, B; Hari, P N; Bredeson, C; Kroft, S H

    2012-03-01

    Dapsone (4-4'-diaminodiphenylsulfone) is commonly used for Pneumocystis jirovecii pneumonia (PCP) prophylaxis in immunocompromised patients. Oxidant hemolysis is a known complication of dapsone, but its frequency in adult patients who have undergone a SCT for hematological malignancies is not well established. We studied the presence of oxidant hemolysis, by combining examination of RBC morphology and laboratory data, in 30 patients who underwent a SCT and received dapsone for PCP prophylaxis, and compared this group with 26 patients who underwent a SCT and received trimethoprim-sulfamethoxazole (TMP-SMX) for PCP prophylaxis. All patients had normal glucose-6-phosphate dehydrogenase (G6PDH) enzymatic activity. In SCT patients, dapsone compared with TMP-SMX for PCP prophylaxis was associated with a high incidence of oxidant hemolysis (87 vs 0%, PSCT patients is 20-fold higher than the reported rate in the population of HIV-infected patients, and thus much higher than the prevalence of G6PDH variants in the general population. In our patients, it manifested clinically as a lower Hb that was not significant enough to result in increased packed RBC transfusions.

  5. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

    Science.gov (United States)

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; González-Valdez, Abigail; Martínez-Rosas, Víctor; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa Angélica; Cuevas-Cruz, Miguel; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site. PMID:27213370

  6. Glucose-6-phosphate dehydrogenase deficiency in people living in malaria endemic districts of Nepal.

    Science.gov (United States)

    Ghimire, Prakash; Singh, Nihal; Ortega, Leonard; Rijal, Komal Raj; Adhikari, Bipin; Thakur, Garib Das; Marasini, Baburam

    2017-05-23

    Glucose-6-phosphate dehydrogenase (G6PD) is a rate limiting enzyme of the pentose phosphate pathway and is closely associated with the haemolytic disorders among patients receiving anti-malarial drugs, such as primaquine. G6PD deficiency (G6PDd) is an impending factor for radical treatment of malaria which affects the clearance of gametocytes from the blood and subsequent delay in the achievement of malaria elimination. The main objective of this study was to assess the prevalence of G6PD deficiency in six malaria endemic districts in Southern Nepal. A cross-sectional population based prevalence survey was conducted in six malaria endemic districts of Nepal, during April-Dec 2013. A total of 1341 blood samples were tested for G6PDd using two different rapid diagnostic test kits (Binax-Now ® and Care Start™). Equal proportions of participants from each district (n ≥ 200) were enrolled considering ethnic and demographic representation of the population groups. Out of total 1341 blood specimens collected from six districts, the overall prevalence of G6PDd was 97/1341; 7.23% on Binax Now and 81/1341; 6.0% on Care Start test. Higher prevalence was observed in male than females [Binax Now: male 10.2%; 53/521 versus female 5.4%; 44/820 (p = 0.003) and Care Start: male 8.4%; 44/521 versus female 4.5%; 37/820 (p = 0.003)]. G6PDd was higher in ethnic groups Rajbanshi (11.7%; 19/162) and Tharu (5.6%; 56/1005) (p = 0.006), major inhabitant of the endemic districts. Higher prevalence of G6PDd was found in Jhapa (22/224; 9.8%) and Morang districts (18/225; 8%) (p = 0.031). In a multivariate analysis, male were found at more risk for G6PDd than females, on Binax test (aOR = 1.97; CI 1.28-3.03; p = 0.002) and Care Start test (aOR = 1.86; CI 1.16-2.97; p = 0.009). The higher prevalence of G6PDd in certain ethnic group, gender and geographical region clearly demonstrates clustering of the cases and ascertained the risk groups within the population. This is the

  7. Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling

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    Yi-Hsuan Wu

    2015-12-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α and GTPase myxovirus resistance 1 (MX1—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E and enterovirus 71 (EV71 infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.

  8. Inhibition of catalase by aminotriazole in vivo results in reduction of glucose-6-phosphate dehydrogenase activity in Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Bayliak, M; Gospodaryov, D; Semchyshyn, H; Lushchak, V

    2008-04-01

    The inhibitor of catalase 3-amino-1,2,4-triazole (AMT) was used to study the physiological role of catalase in the yeast Saccharomyces cerevisiae under starvation. It was shown that AMT at the concentration of 10 mM did not affect the growth of the yeast. In vivo and in vitro the degree of catalase inhibition by AMT was concentration- and time-dependent. Peroxisomal catalase in bakers' yeast was more sensitive to AMT than the cytosolic one. In vivo inhibition of catalase by AMT in S. cerevisiae caused a simultaneous decrease in glucose-6-phosphate dehydrogenase activity and an increase in glutathione reductase activity. At the same time, the level of protein carbonyls, a marker of oxidative modification, was not affected. Possible mechanisms compensating the negative effects caused by AMT inhibition of catalase are discussed.

  9. Prevalence of glucose-6-phosphate dehydrogenase deficiency and sickle cell trait among blood donors in Riyadh

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    Alabdulaali Mohammed

    2010-01-01

    Full Text Available Background and Aims: Blood donation from glucose-6-phosphate dehydrogenase (G6PD-deficient and sickle cell trait (SCT donors might alter the quality of the donated blood during processing, storage or in the recipient′s circulatory system. The aim of this study was to determine the prevalence of G6PD deficiency and SCT among blood donors coming to King Khalid University Hospital (KKUH in Riyadh. It was also reviewed the benefits and risks of transfusing blood from these blood donors. Materials and Methods: This cross-sectional study was conducted on 1150 blood samples obtained from blood donors that presented to KKUH blood bank during the period April 2006 to May 2006. All samples were tested for Hb-S by solubility test, alkaline gel electrophoresis; and for G6PD deficiency, by fluorescent spot test. Results: Out of the 1150 donors, 23 (2% were diagnosed for SCT, 9 (0.78% for G6PD deficiency and 4 (0.35% for both conditions. Our prevalence of SCT and G6PD deficiency is higher than that of the general population of Riyadh. Conclusion: We recommend to screen all units for G6PD deficiency and sickle cell trait and to defer donations from donors with either of these conditions, unless if needed for special blood group compatibility, platelet apheresis or if these are likely to affect the blood bank inventory. If such blood is to be used, special precautions need to be undertaken to avoid complications in high-risk recipients.

  10. Glucose-6-phosphate dehydrogenase deficiency in neonatal hyperbilirubinaemia: Hacettepe experıence.

    Science.gov (United States)

    Celik, H Tolga; Günbey, Ceren; Unal, Sule; Gümrük, Fatma; Yurdakök, Murat

    2013-05-01

    The aim of this study was to investigate the prevalence of glucose-6-phospate dehydrogenase (G6PD) deficiency in newborn infants with neonatal hyperbilirubinaemia and to compare the clinical features of G6PD-deficient and G6PD-normal newborn infants. A total of 4906 term and preterm neonates with indirect hyperbilirubinaemia were retrospectively evaluated according to demographic, neonatal features, bilirubin levels, erythrocyte G6PD levels, other risk factors and treatments. Among 4906 newborn infants with indirect hyperbilirubinaemia, 55 (1.12%) neonates were G6PD-deficient. In our study, no statistically significant difference was detected between G6PD-deficient and G6PD-normal infants in relation to the time of onset of jaundice, bilirubin levels and duration of phototherapy. However, the incidence of exchange transfusion in G6PD-deficient infants was 16.4% while it was only 3.3% in G6PD normal infants (P G6PD must be ordered to all newborns who are receiving phototherapy and especially to those who are coming from the high incident geographical regions and less responsive to phototherapy. © 2013 The Authors. Journal of Paediatrics and Child Health © 2013 Paediatrics and Child Health Division (Royal Australasian College of Physicians).

  11. Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

    DEFF Research Database (Denmark)

    Olavarria, K.; De Ingeniis, J.; Zielinski, D. C.

    2014-01-01

    In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work......PDH(R46E,Q47E). Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDH(R46E,Q47E). Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect...

  12. Glucose 6 phosphatase dehydrogenase (G6PD and neurodegenerative disorders: Mapping diagnostic and therapeutic opportunities

    Directory of Open Access Journals (Sweden)

    Manju Tiwari

    2017-12-01

    Full Text Available Glucose 6 phosphate dehydrogenase (G6PD is a key and rate limiting enzyme in the pentose phosphate pathway (PPP. The physiological significance of enzyme is providing reduced energy to specific cells like erythrocyte by maintaining co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH. There are preponderance research findings that demonstrate the enzyme (G6PD role in the energy balance, and it is associated with blood-related diseases and disorders, primarily the anemia resulted from G6PD deficiency. The X-linked genetic deficiency of G6PD and associated non-immune hemolytic anemia have been studied widely across the globe. Recent advancement in biology, more precisely neuroscience has revealed that G6PD is centrally involved in many neurological and neurodegenerative disorders. The neuroprotective role of the enzyme (G6PD has also been established, as well as the potential of G6PD in oxidative damage and the Reactive Oxygen Species (ROS produced in cerebral ischemia. Though G6PD deficiency remains a global health issue, however, a paradigm shift in research focusing the potential of the enzyme in neurological and neurodegenerative disorders will surely open a new avenue in diagnostics and enzyme therapeutics. Here, in this study, more emphasis was made on exploring the role of G6PD in neurological and inflammatory disorders as well as non-immune hemolytic anemia, thus providing diagnostic and therapeutic opportunities.

  13. Two new glucose 6-phosphate dehydrogenase variants associated with congenital nonspherocytic hemolytic anemia found in Japan: GD(-) Tokushima and GD(-) Tokyo.

    Science.gov (United States)

    Miwa, S; Ono, J; Nakashima, K; Abe, S; Kageoka, T

    1976-01-01

    Two new variants of glucose 6-phosphate dehydrogenase (G6PD) deficiency associated with chronic nonspherocytic hemolytic anemia were discovered in Japan. Gd(-) Tokushima was found in a 17-years-old male whose erythrocytes contained 4.4% of normal enzyme activity. Partially purified enzyme revealed a main band of normal electrophoretic mobility with additional two minor bands of different mobility; normal Km G6P, and Km NADP five-to sixfold higher than normal; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; marked thermal instability; a normal pH curve; and normal Ki NADPH. The hemolytic anemia was moderate to severe. Gd(-) Tokyo was characterized from a 15-year-old male who had chronic nonspherocytic hemolytic anemia of mild degree. The erythrocytes contained 3% of normal enzyme activity, and partially purified enzyme revealed slow electrophoretic mobility (90% of normal for both a tris-hydrochloride buffer system and a tris-EDTA-borate buffer system, and 70% of normal for a phosphate buffer system); normal Km G6P and Km NADP; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; greatly increased thermal instability; a normal pH curve; and normal Ki NADPH. These two variants are clearly different from hitherto described G6PD variants, including the Japanese variants Gd(-) Heian and Gd(-) Kyoto. The mothers of both Gd(-) Tokushima and Gd(-) Tokoyo were found to be heterozygote by an ascorbate-cyanide test.

  14. Prevalence of anemia, iron deficiency, thalassemia and glucose-6-phosphate dehydrogenase deficiency among hill-tribe school children in Omkoi District, Chiang Mai Province, Thailand.

    Science.gov (United States)

    Yanola, Jintana; Kongpan, Chatpat; Pornprasert, Sakorn

    2014-07-01

    The prevalaence of anemia, iron deficiency, thalassemia and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency were examined among 265 hill-tribe school children, 8-14 years of age, from Omkoi District, Chiang Mai Province, Thailand. Anemia was observed in 20 school children, of whom 3 had iron deficiency anemia. The prevalence of G-6-PD deficiency and β-thalassemia trait [codon 17 (A>T), IVSI-nt1 (G>T) and codons 71/72 (+A) mutations] was 4% and 8%, respectively. There was one Hb E trait, and no α-thalassemia-1 SEA or Thai type deletion. Furthermore, anemia was found to be associated with β-thalassemia trait in 11 children. These data can be useful for providing appropriate prevention and control of anemia in this region of Thailand.

  15. Triiodothyronine (T3)-associated upregulation and downregulation of nuclear T3 binding in the human fibroblast cell (MRC-5)--stimulation of malic enzyme, glucose-6-phosphate-dehydrogenase, and 6-phosphogluconate-dehydrogenase by insulin, but not by T3

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The specific nuclear binding of triiodothyronine (T3) (NBT3) and the activity of malic enzyme (ME), glucose-6-phosphate-dehydrogenase (G6PD), and 6-phosphogluconate-dehydrogenase (6PGD) were studied in the human fibroblast cell (MRC-5). The overall apparent binding affinity (Ka) was 2.7 x 10(9) L.......mol-1 estimated from kinetic studies of nuclear T3 binding, and 2.5 x 10(9) L.mol-1 estimated from equilibrium studies. The scatchard plots were curvilinear and composed of a high-affinity binding site with Ka1 3.4 +/- 0.7 x 10(9) L.mol-1 and maximal binding capacity (MBC) MBC1 57.0 +/- 11.9 fmol/mg DNA...... and a low-affinity binding site with Ka2 2.9 +/- 1.1 x 10(8) L.mol-1 and MBC2 124.7 +/- 22.1 fmol/mg DNA (n = 6). Incubation of cells with 6 nmol/L T3 for 20 hours reduced NBT3 to 62.2% +/- 15.7% (P less than .01, n = 11). The Ka estimated from kinetic studies was reduced to 6.7 x 10(7) L.mol-1...

  16. Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high ...

  17. Glucose-6-phosphate dehydrogenase Lodi844C: a study on its expression in blood cells and muscle.

    Science.gov (United States)

    Ninfali, P; Bresolin, N; Baronciani, L; Fortunato, F; Comi, G; Magnani, M; Scarlato, G

    1991-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in erythrocytes, lymphocytes and muscle of an Italian male, whose family has lived for at least three generations in Lodi (Lombardy, northern Italy). The subject was hospitalized for myalgia and dark urine after intense physical exercise, but no sign of anemia and chronic hemolysis were present at rest. Family studies revealed that the mother and the maternal aunt had the same enzymopathy. The enzyme-specific activity in red blood cells was 15% of control and the kinetic properties were the following: slower electrophoretic mobility; biphasic pH activity curve; slightly reduced thermal stability, and increased utilization of the substrate analogs. The analysis of our patient's DNA showed a G----C mutation at nucleotide 844 which causes an Asp----His amino acid change in position 282. This is the same mutation found by De Vita et al. in the G6PD Seattle-like variant. However, by following a new convention, we labelled our variant as G6PD Lodi844C. As far as the muscle is concerned, we found that the enzyme-specific activity in this tissue was 14% of control values, but cultured myotubes and myoblasts revealed a normal level of G6PD as well as skin fibroblasts. On the contrary in the same type of cultured cells obtained from G6PD Mediterranean subjects, the G6PD activity was about 20% of normal. Our results complete the characterization of this mutant enzyme, demonstrate the expression of the deficit in muscle and describe the enzyme behaviour in cultured cells.

  18. Glucose-6-phosphate dehydrogenase deficiency does not increase the susceptibility of sperm to oxidative stress induced by H2O2.

    Science.gov (United States)

    Roshankhah, Shiva; Rostami-Far, Zahra; Shaveisi-Zadeh, Farhad; Movafagh, Abolfazl; Bakhtiari, Mitra; Shaveisi-Zadeh, Jila

    2016-12-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. G6PD plays a key role in the pentose phosphate pathway, which is a major source of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH provides the reducing equivalents for oxidation-reduction reductions involved in protecting against the toxicity of reactive oxygen species such as H 2 O 2 . We hypothesized that G6PD deficiency may reduce the amount of NADPH in sperms, thereby inhibiting the detoxification of H 2 O 2 , which could potentially affect their motility and viability, resulting in an increased susceptibility to infertility. Semen samples were obtained from four males with G6PD deficiency and eight healthy males as a control. In both groups, motile sperms were isolated from the seminal fluid and incubated with 0, 10, 20, 40, 60, 80, and 120 µM concentrations of H 2 O 2 . After 1 hour incubation at 37℃, sperms were evaluated for motility and viability. Incubation of sperms with 10 and 20 µM H 2 O 2 led to very little decrease in motility and viability, but motility decreased notably in both groups in 40, 60, and 80 µM H 2 O 2 , and viability decreased in both groups in 40, 60, 80, and 120 µM H 2 O 2 . However, no statistically significant differences were found between the G6PD-deficient group and controls. G6PD deficiency does not increase the susceptibility of sperm to oxidative stress induced by H 2 O 2 , and the reducing equivalents necessary for protection against H 2 O 2 are most likely produced by other pathways. Therefore, G6PD deficiency cannot be considered as major risk factor for male infertility.

  19. Prevalence of glucose-6-phosphate dehydrogenase (G6PD deficiency in neonates in Bunda Women's and Children's Hospital, Jakarta, Indonesia

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    Risma Kerina Kaban

    2011-02-01

    Full Text Available Background Glucose-6-phosphate dehydrogenase (G6PD deficiency is the most connnon enzyme deficiency in the world. Itis a risk factor for hyperbilirubinemia in neonates, which can cause serious complications such as bilirubininduced encephalopathy or kernicterus. WHO recommends universal neonatal screening for G6PD deficiency when the frequency exceeds 35% of male newborns. Objective To assess the prevalence of G6PD deficiency among neonates in Bunda Women and C hildren Hospital (Bunda WCH, Jakarta, in order to detennine if there is a need for routine G6PD neonatal screening. Methods This is a cross-sectional and retrospective study; infants' data were obtained from medical records. From January 2009 to May 2010, all neonates in Bunda WCH were screened for G6PD deficiency on the yd day of life. Blood samples were collected using filter papers. We considered a result to be nonnal if it exceeded 3.6 U/g Hb. Results A total 1802 neonates were screened. We found 94 neonates (5.2% with G6PD deficiency. Out of 943 males, 59 (6.26% were G6PD deficient, and out of 859 females, 35 (4.07% were G6PD deficient. We observed that prevalence of G6PD deficiency according to sex distribution was significantly higher in males than females (6.26% vs. 4.07%, P=0.037. There was no significant difference in the risk for severe hyperbilirubinemia between the G6PD deficient infants and the nonnal infants (P=0.804. Conclusions The frequencies of G6PD deficiency were 6.26% of male neonates and 4.07% of female neonates. We recommend universal neonatal screening for G6PD deficiencies in Jakarta since our findings exceed the WHO recommendation for routine testing.

  20. INFLUENCE OF pH, TEMPERATURE AND DISSOLVED OXYGEN CONCENTRATION ON THE PRODUCTION OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE AND INVERTASE BY Saccharomyces cerevisiae

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    J. Abrahão-Neto

    1997-03-01

    Full Text Available The effect of pH (from 4.0 to 5.0, temperature (T (from 30 oC to 40 oC and dissolved oxygen concentration (DO (from 0.2 to 6.0 mg O2/L on glucose 6-phosphate dehydrogenase (G6PDH (EC 1.1.1.49 and Invertase (EC 3.2.1.26 formation by S. cerevisiae were studied. The best culture conditions for G6PDH and Invertase formation were: 2.55 L culture medium (yeast extract, 3.0 g/L; 5peptone, 5.0 g/L; glucose, 2.0 g/L; sucrose, 15.0 g/L; Na2HPO4.12 H2O, 2.4 g/L; (NH42SO4, 5.1 g/L and MgSO4. 7H2O, 0.075 g/L; 0.45 L inoculum (0.70 g dry cell/L; pH = 4.5; T = 35 oC and DO = 4.0 mg/L. G6PDH was highly sensitive to pH, T and DO variation. The increase in G6PDH production was about three times when the DO ranged from 0.2 to 4.0 mg O2/L. Moreover, by shifting pH from 5.0 to 4.5 and temperature from 30 oC to 35 oC, G6PDH formation increased by 57% and 70%, respectively. Invertase activity (IA of whole cells decreased at least 50% at extremes values of DO (2.0 and 6.0 mg O2/L and pH (4.0 and 5.0. Furthermore, IA oscillated during the fermentation due to the glucose repression/derepression mechanism

  1. Screening for glucose-6-phosphate dehydrogenase deficiency in neonates: a comparison between cord and peripheral blood samples.

    Science.gov (United States)

    AlSaif, Saif; Ponferrada, Ma Bella; AlKhairy, Khalid; AlTawil, Khalil; Sallam, Adel; Ahmed, Ibrahim; Khawaji, Mohammed; AlHathlol, Khalid; Baylon, Beverly; AlSuhaibani, Ahmed; AlBalwi, Mohammed

    2017-07-11

    The use of cord blood in the neonatal screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency is being done with increasing frequency but has yet to be adequately evaluated against the use of peripheral blood sample which is usually employed for confirmation. We sought to determine the incidence and gender distribution of G6PD deficiency, and compare the results of cord against peripheral blood in identifying G6PD DEFICIENCY neonates using quantitative enzyme activity assay. We carried out a retrospective and cross-sectional study employing review of primary hospital data of neonates born in a tertiary care center from January to December 2008. Among the 8139 neonates with cord blood G6PD assays, an overall incidence of 2% for G6PD deficiency was computed. 79% of these were males and 21% were females with significantly more deficient males (p blood samples (n = 1253) showed a significantly higher mean G6PD value for peripheral than cord blood (15.12 ± 4.52 U/g and 14.52 ± 4.43 U/g, respectively, p = 0.0008). However, the proportion of G6PD deficient neonates did not significantly differ in the two groups (p = 0.79). Sensitivity of cord blood in screening for G6PD deficiency, using peripheral G6PD assay as a gold standard was 98.6% with a NPV of 99.5%. There was no difference between cord and peripheral blood samples in discriminating between G6PD deficient and non-deficient neonates. A significantly higher mean peripheral G6PD assay reinforces the use of cord blood for neonatal screening since it has substantially low false negative results.

  2. Combined Effect of L-Cysteine and Vitamin E Injected Pre-Irradiation on Glucose-6-Phosphate Dehydrogenase Activity and Certain products of Glycolysis in Blood of Female Rats

    International Nuclear Information System (INIS)

    Abdel-Fattah, K.I.; Abou-Safi, H.M.; Kafafy, Y.A.; Ashry, O.M.

    1999-01-01

    The present work aims to evaluate the protective limits of L-cysteine and vitamin E combination against deleterious effects of gamma radiation on glucose-6-phosphate dehydrogenase activity, liver glycogen, blood glucose, pyruvic and lactic acids and their correlations in adult female rats. Mature female white rats were divided into four groups: 1- Control group. 2- Whole body gamma irradiated group at a dose level two Gy. 3-Group injected with 120 mg/100 g b.wt. L-cysteine+10 mg/100 g b.wt. vitamin E. 4- Group injected with cysteine+ vitamin E one hour before irradiation at 2 Gy dose level. Results revealed that combined administration of cysteine and vitamin E before gamma-irradiation have accelerated the radiation injury on liver glycogen, plasma glucose and G 6 Pd activity, while they showed a protective effect on lactic and pyruvic acids. This could be due to different mechanisms or a biphasic mechanism related to hormonal (like E 2 , T 3 and insulin), enzymatic or metabolic (e.g. oxidation/reduction, catabolic, anabolic factors) control

  3. Co-immobilization of cyclohexanone monooxygenase and glucose-6-phosphate dehydrogenase onto polyethylenimine-porous agarose polymeric composite using γ irradiation to use in biotechnological processes

    International Nuclear Information System (INIS)

    Atia, K.S.

    2005-01-01

    The co-immobilization of cyclohexanone monooxygenase (CHMO) and glucose-6-phosphate dehydrogenase (G6PDH) was optimized by completely coating, via covalent immobilization, the surface aldehyde groups of porous agarose (glyoxyl-agarose) with amine groups of polyethylenimine (PEI). The highest immobilization efficiency (∼87%) (activity of enzyme per amount of immobilized enzyme) was obtained with a CHMO/G6PDH ratio 2:1. The effects of different ratios of the support to the amount of enzymes (CHMO:G6PDH=2:1), the optimum incubation pH and the incubation time on the enzymatic activity of the enzymes were determined and found to be 5:1, 8.5 and 30 min, respectively. Subjecting the co-immobilized enzymes to doses of γ-radiation (5-100 kGy) resulted in complete loss in the activity of the free enzymes at a dose of 40 kGy, while the co-immobilized ones showed relatively high resistance to γ-radiation up to a dose of 50 kGy

  4. Fluorometric determination of free glucose and glucose 6-phosphate in cows' milk and other opaque matrices

    DEFF Research Database (Denmark)

    Larsen, Torben

    2015-01-01

    Analyses of free glucose and glucose 6-phosphate in milk have until now been dependent upon several time consuming and troublesome procedures. This has limited investigations in the area. The present article presents a new, reliable, analytical procedure, based on enzymatic degradation and fluoro......Analyses of free glucose and glucose 6-phosphate in milk have until now been dependent upon several time consuming and troublesome procedures. This has limited investigations in the area. The present article presents a new, reliable, analytical procedure, based on enzymatic degradation...... and fluorometric detection. Standards and control materials were based on milk that was stripped of intrinsic glucose and glucose 6-phosphate in order to obtain standards and samples based on the same matrix. The analysis works without pre-treatment of the samples, e.g. without centrifugation and precipitation...

  5. SLC37A1 and SLC37A2 are phosphate-linked, glucose-6-phosphate antiporters.

    Directory of Open Access Journals (Sweden)

    Chi-Jiunn Pan

    Full Text Available Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P into glucose and phosphate (P(i. This reaction depends on coupling the G6P transporter (G6PT with glucose-6-phosphatase-α (G6Pase-α. Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a P(i-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:P(i exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, P(i-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.

  6. What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

    Science.gov (United States)

    Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M S; Engel, Paul C

    2008-08-01

    Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

  7. Glucose-6-phosphate reduces calcium accumulation in rat brain endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Jeffrey Thomas Cole

    2012-04-01

    Full Text Available Brain cells expend large amounts of energy sequestering calcium (Ca2+, while loss of Ca2+ compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P, a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum to sequester Ca2+. This led to the hypothesis that G6P regulates Ca2+ accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA. Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, 45Ca2+ accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi coupled with Ca2+ accumulation was quantified. Addition of G6P significantly and decreased Ca2+ accumulation in a dose-dependent fashion (1-10 mM. The reduction in Ca2+ accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca2+ accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca2+ uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca2+ dystasis caused by altered G6P regulation of SERCA activity

  8. Comparison of Spectrophotometry, Chromate Inhibition, and Cytofluorometry Versus Gene Sequencing for Detection of Heterozygously Glucose-6-Phosphate Dehydrogenase-Deficient Females.

    Science.gov (United States)

    Peters, Anna L; Veldthuis, Martijn; van Leeuwen, Karin; Bossuyt, Patrick M M; Vlaar, Alexander P J; van Bruggen, Robin; de Korte, Dirk; Van Noorden, Cornelis J F; van Zwieten, Rob

    2017-11-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide. Detection of heterozygously deficient females can be difficult as residual activity in G6PD-sufficient red blood cells (RBCs) can mask deficiency. In this study, we compared accuracy of 4 methods for detection of G6PD deficiency in females. Blood samples from females more than 3 months of age were used for spectrophotometric measurement of G6PD activity and for determination of the percentage G6PD-negative RBCs by cytofluorometry. An additional sample from females suspected to have G6PD deficiency based on the spectrophotometric G6PD activity was used for measuring chromate inhibition and sequencing of the G6PD gene. Of 165 included females, 114 were suspected to have heterozygous deficiency. From 75 females, an extra sample was obtained. In this group, mutation analysis detected 27 heterozygously deficient females. The sensitivity of spectrophotometry, cytofluorometry, and chromate inhibition was calculated to be 0.52 (confidence interval [CI]: 0.32-0.71), 0.85 (CI: 0.66-0.96), and 0.96 (CI: 0.71-1.00, respectively, and the specificity was 1.00 (CI: 0.93-1.00), 0.88 (CI: 0.75-0.95), and 0.98 (CI: 0.89-1.00), respectively. Heterozygously G6PD-deficient females with a larger percentage of G6PD-sufficient RBCs are missed by routine methods measuring total G6PD activity. However, the majority of these females can be detected with both chromate inhibition and cytofluorometry.

  9. 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

  10. The transcriptional regulator NtrC controls glucose-6-phosphate dehydrogenase expression and polyhydroxybutyrate synthesis through NADPH availability in Herbaspirillum seropedicae.

    Science.gov (United States)

    Sacomboio, Euclides Nenga Manuel; Kim, Edson Yu Sin; Correa, Henrique Leonardo Ruchaud; Bonato, Paloma; Pedrosa, Fabio de Oliveira; de Souza, Emanuel Maltempi; Chubatsu, Leda Satie; Müller-Santos, Marcelo

    2017-10-19

    The NTR system is the major regulator of nitrogen metabolism in Bacteria. Despite its broad and well-known role in the assimilation, biosynthesis and recycling of nitrogenous molecules, little is known about its role in carbon metabolism. In this work, we present a new facet of the NTR system in the control of NADPH concentration and the biosynthesis of molecules dependent on reduced coenzyme in Herbaspirillum seropedicae SmR1. We demonstrated that a ntrC mutant strain accumulated high levels of polyhydroxybutyrate (PHB), reaching levels up to 2-fold higher than the parental strain. In the absence of NtrC, the activity of glucose-6-phosphate dehydrogenase (encoded by zwf) increased by 2.8-fold, consequently leading to a 2.1-fold increase in the NADPH/NADP + ratio. A GFP fusion showed that expression of zwf is likewise controlled by NtrC. The increase in NADPH availability stimulated the production of polyhydroxybutyrate regardless the C/N ratio in the medium. The mutant ntrC was more resistant to H 2 O 2 exposure and controlled the propagation of ROS when facing the oxidative condition, a phenotype associated with the increase in PHB content.

  11. The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glucose 6-phosphate dehydrogenase activity

    International Nuclear Information System (INIS)

    Sahin, Ali; Senturk, Murat; Ciftci, Mehmet; Varoglu, Erhan; Kufrevioglu, Omer Irfan

    2010-01-01

    Aim: The inhibitory effects of thallium-201 ( 201 Tl) solution on human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) activity were investigated. Methods: For this purpose, erythrocyte G6PD was initially purified 835-fold at a yield of 41.7% using 2',5'-Adenosine diphosphate sepharose 4B affinity gel chromatography. The purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed a single band for the final enzyme preparation. The in vitro and in vivo effects of the 201 Tl solution including Tl + , Fe +3 and Cu +2 metals and the in vitro effects of the radiation effect of the 201 Tl solution and non-radioactive Tl + , Fe +3 and Cu +2 metals on human erythrocyte G6PD enzyme were studied. Enzyme activity was determined with the Beutler method at 340 nm using a spectrophotometer. All purification procedures were carried out at +4 deg. C. Results: 201 Tl solution and radiation exposure had inhibitory effects on the enzyme activity. IC 50 value of 201 Tl solution was 36.86 μl ([Tl + ]: 0.0036 μM, [Cu +2 ]: 0.0116 μM, [Fe +3 ]: 0.0132 μM), of human erythrocytes G6PD. Seven human patients were also used for in vivo studies of 201 Tl solution. Furthermore, non-radioactive Tl + , Fe +3 and Cu +2 were found not to have influenced the enzyme in vitro. Conclusion: Human erythrocyte G6PD activity was inhibited by exposure for up to 10 minutes to 0.057 mCi/kg 201 Tl solution. It was detected in in vitro and in vivo studies that the human erythrocyte G6PD enzyme is inhibited due to the radiation effect of 201 Tl solution.

  12. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

    Science.gov (United States)

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Enríquez-Flores, Sergio; De la Mora-De la Mora, Ignacio; González-Valdez, Abigail; García-Torres, Itzhel; Martínez-Rosas, Víctor; Sierra-Palacios, Edgar; Lazcano-Pérez, Fernando; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients. PMID:26633385

  13. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

    Directory of Open Access Journals (Sweden)

    Saúl Gómez-Manzo

    2015-12-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency, and the G6PD Santa Maria and A+ (less severe deficiency (Class I, II and III, respectively affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients.

  14. Glucose-6-phosphate dehydrogenase is required for hpa1xoo (harpin protein fragment)-mediated salt stress tolerance in transgenic arabidopsis thaliana

    International Nuclear Information System (INIS)

    Sang, S.L.; Xie, L.L.; Cui, X.W.; Wang, Z.Y.

    2018-01-01

    Harpin induces salicylic acid and abscisic acid signaling in plants under biotic and abiotic stress, respectively. Our previous report showed that the effective harpin fragment Hpa1xoo enhanced H2O2 production and pathogen resistance in a transgenic Arabidopsis mutant. In this study, we examined contents of thiobarbituric acid reactive substance (TBARS), H2O2 and glutathione, and glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR) and glutathione peroxidase (GPX) enzyme activity in Hpa1xoo-expressing Arabidopsis under salt stress. The results revealed increased amounts of TBARS and H2O2 in wild-type (WT) compared to mutant plants under salt stress conditions. In contrast, increased levels were observed in the mutant under stress-free conditions. Moreover, a higher reduced glutathione (GSH) content and ratio of GSH/oxidized glutathione (GSSG) was observed in mutant compared to WT plants under both stress-free and salt stress conditions. In addition, mutant plants exhibited significantly higher G6PDH, GR and GPX activity than WT plants under salt stress. Suppression of G6PDH activity via 6-aminonicotinamide (6-AN, a specific inhibitor of G6PDH) was partly reversed by L-buthionine-sulfoximine (BSO, a specific inhibitor of GSH regeneration) and aggravated by GSH. Combined with previous reports, these findings suggest that the G6PDH enzyme plays a key role in harpin fragment (Hpa1xoo)-mediated salt stress tolerance in transgenic Arabidopsis. (author)

  15. [Evaluations of newborn screening program performance and enzymatic diagnosis of glucose-6-phosphate dehydrogenase deficiency in Guangzhou].

    Science.gov (United States)

    Tang, F; Huang, Y L; Jiang, X; Jia, X F; Li, B; Feng, Y; Chen, Q Y; Tang, C F

    2018-05-02

    Objective: To reveal the molecular epidemiologic characteristics of glucose-6-phosphate dehydrogenase (G6PD) gene and to evaluate based on the genetic analysis the newborn screening program performance and enzymatic diagnosis of G6PD deficiency in Guangzhou. Methods: G6PD enzyme activities were measured by quantitative fluorescence assay in dry blood spots of 16 319 newborns(8 725 males, 7 594 females) 3-7 days after birth in Guangzhou Newborn Center. They were born in Guangzhou form Oct. 1 to 20, 2016. The cutoff value of G6PD was less than 2.6 U/g Hb in dry blood spots. G6PD deficiency was diagnosed when G6PDblood cells. Genetic analysis of G6PD gene was performed on the dry blood spot samples of 823 newborns (including positive 346, negative 477)with various levels of G6PD enzyme activities through fluorescence PCR melting curve analysis(FMCA) to detect 15 kinds of mutations reported to be common among Chinese.G6PD gene Sanger sequency was performed in seven highly suspicious patients with negative results by FMCA. Results: (1) Using the cutoff value of G6PDT, c.551C>T, c.835A>T hemizygote were found in 3 male's samples, respectively. (3) The estimated prevalence of harboring mutation was 6.0% in males and 13.5% in females according to rates of mutation in samples with various levels of G6PD enzyme activities. Six common mutations were c.1388G>A、c.1376G>T, c.95A> G, c.871G>A, c.1024C>T, c.392G>T, accounting for 95.5% of detected alleles .(4) based on results of G6PD gene analysis, the newborn scereening of G6PD deficiency with cutoff value G6PDblood cells were 95.5%, 97.2%, respectively. Conclusions: The prevalence of G6PD deficiency in males was 6.0% in Guangzhou. Six mutations c.1388G>A, c.1376G>T, c.95A>G, c.871G>A, c.1024C>T, c.392G>T accounted for 95.5%. The cutoff value of G6PD<2.6 U/g Hb innewborn screening program and the criteria of biochemical diagnosis could accurately identify G6PD deficiency . Combined with biochemical and molecular analysis will

  16. Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt

    Science.gov (United States)

    Marini, Cecilia; Ravera, Silvia; Buschiazzo, Ambra; Bianchi, Giovanna; Orengo, Anna Maria; Bruno, Silvia; Bottoni, Gianluca; Emionite, Laura; Pastorino, Fabio; Monteverde, Elena; Garaboldi, Lucia; Martella, Roberto; Salani, Barbara; Maggi, Davide; Ponzoni, Mirco; Fais, Franco; Raffaghello, Lizzia; Sambuceti, Gianmario

    2016-01-01

    Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This “Warburg effect” represents a standard to diagnose and monitor tumor aggressiveness with 18F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that 18F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy. PMID:27121192

  17. Glucose-6-phosphate dehydrogenase (G6PD)-deficient infants: Enzyme activity and gene variants as risk factors for phototherapy in the first week of life.

    Science.gov (United States)

    Wong, Fei-Liang; Ithnin, Azlin; Othman, Ainoon; Cheah, Fook-Choe

    2017-07-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a recognised cause of severe neonatal hyperbilirubinaemia, and identifying which infants are at risk could optimise care and resources. In this study, we determined if G6PD enzyme activity (EA) and certain gene variants were associated with neonatal hyperbilirubinaemia requiring phototherapy during the first week after birth. Newborn infants with G6PD deficiency and a group with normal results obtained by the fluorescent spot test were selected for analyses of G6PD EA and the 10 commonly encountered G6PD mutations in this region, relating these with whether the infants required phototherapy before discharge from the hospital in the first week. A total of 222 infants with mean gestation and birth weight of 38.3 ± 1.8 weeks and 3.02 ± 0.48 kg, respectively, were enrolled. Of these, n = 121 were deficient with EA ≤6.76 U/g Hb, and approximately half (43%) received phototherapy in the first week after birth. The mean EA level was 3.7 U/g Hb. The EA had good accuracy in predicting phototherapy use, with area under the receiver-operating-characteristic curve of 0.81 ± 0.05. Infants on phototherapy more commonly displayed World Health Organization Class II mutations (deficiency in EA and mutation at c.1388G>A (adjusted odds ratio, 1.5 and 5.7; 95% confidence interval: 1.31-1.76 and 1.30-25.0, respectively) were independent risk factors for phototherapy. Low G6PD EA (G6PD gene variant, c.1388G>A, are risk factors for the need of phototherapy in newborn infants during the first week after birth. © 2017 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).

  18. Immune Thrombocytopenia Resolved by Eltrombopag in a Carrier of Glucose-6-Phosphate Dehydrogenase Deficiency

    Directory of Open Access Journals (Sweden)

    Laura Scaramucci

    2016-03-01

    Full Text Available Eltrombopag, a thrombopoietin mimetic peptide, may provide excellent clinical efficacy in steroid-refractory patients with immune thrombocytopenic purpura (ITP [1,2]. Eltrombopag is generally well tolerated. However, its use in the particular setting of glucose-6-phosphate dehydrogenase (G6PD and history of acute hemolytic anemia (AHA has not been reported so far. A 51-year-old female was diagnosed as having ITP in September 2014. She was not taking any medication and her past history was negative, apart from having been diagnosed a carrier (heterozygous of G6PD deficiency (Mediterranean variant after a familial screening by molecular and biochemical methods. She presented with only slightly reduced (about 50% enzyme level, belonging to World Health Organization-defined class 3 [3,4]. In the following years, the patient experienced some episodes of AHA, which were managed at outside institutions; in particular, a severe episode of AHA, probably triggered by urinary infection and antibiotics [5], had complicated her second and last delivery. The hemolytic episodes were selflimiting and resolved without sequelae. No other causes of hemolysis were documented. When the case came to our attention, a diagnosis of ITP was made; hemolytic parameters were normal, although the G6PD enzyme concentration was not measured. Oral prednisone (1 mg/kg was given with only a transient benefit. The patient was then a candidate for elective splenectomy. However, given her extremely low platelet count, she was started in October 2014 on eltrombopag at 50 mg/day as a bridge to splenectomy. Given that, to the best of our knowledge, the use of this drug has never been reported in the particular setting of G6PD deficiency, the patient was constantly monitored. A prompt platelet increase (178x109/L was observed 1 week after the start of treatment. After she achieved the target platelet count, the dose of eltrombopag was tapered to the lowest effective dose. The patient

  19. Efficient regeneration of NADPH in a 3-enzyme cascade reaction by in situ generation of glucose 6-phosphate from glucose and pyrophosphate

    NARCIS (Netherlands)

    Hartog, A.F.; van Herk, T.; Wever, R.

    2011-01-01

    We report here a promising method to regenerate NADPH (nicotinamide adenine dinucleotide phosphate) using the intermediate formation of glucose 6-phosphate (G6P) from glucose and pyrophosphate (PPi) catalyzed by the acid phosphatase from Shigella flexneri (PhoN-Sf). The G6P formed is used in turn by

  20. Protective effects of glucose-6-phosphate dehydrogenase on neurotoxicity of aluminium applied into the CA1 sector of rat hippocampus

    Directory of Open Access Journals (Sweden)

    Marina D Jovanovic

    2014-01-01

    Full Text Available Background & objectives: Aluminum (Al toxicity is closely linked to the pathogenesis of Alzheimer′s disease (AD. This experimental study was aimed to investigate the active avoidance behaviour of rats after intrahippocampal injection of Al, and biochemical and immunohistochemical changes in three bilateral brain structures namely, forebrain cortex (FBCx, hippocampus and basal forebrain (BF. Methods: Seven days after intra-hippocampal (CA1 sector injection of AlCl 3 into adult male Wistar rats they were subjected to two-way active avoidance (AA tests over five consecutive days. Control rats were treated with 0.9% w/v saline. The animals were decapitated on the day 12 post-injection. The activities of acetylcholinesterase (AChE and glucose-6-phosphate dehydrogenase (G6PDH were measured in the FBCx, hippocampus and BF. Immunohistochemical staining was performed for transferrin receptors, amyloid β and tau protein. Results: The activities of both AChE and G6PDH were found to be decreased bilaterally in the FBCx, hippocampus and basal forebrain compared to those of control rats. The number of correct AA responses was reduced by AlCl 3 treatment. G6PDH administered prior to AlCl 3 resulted in a reversal of the effects of AlCl 3 on both biochemical and behavioural parameters. Strong immunohistochemical staining of transferrin receptors was found bilaterally in the FBCx and the hippocampus in all three study groups. In addition, very strong amyloid β staining was detected bilaterally in all structures in AlCl 3 -treated rats but was moderate in G6PDH/AlCl 3 -treated rats. Strong tau staining was noted bilaterally in AlCl 3 -treated rats. In contrast, tau staining was only moderate in G6PDH/AlCl 3 -treated rats. Interpretation & conclusions: Our findings indicated that the G6PDH alleviated the signs of behavioural and biochemical effects of AlCl 3 -treatment suggesting its involvement in the pathogenesis of Al neurotoxicity and its potential

  1. Population screening for glucose-6-phosphate dehydrogenase deficiencies in Isabel Province, Solomon Islands, using a modified enzyme assay on filter paper dried bloodspots

    Directory of Open Access Journals (Sweden)

    Landry Losi

    2010-08-01

    Full Text Available Abstract Background Glucose-6-phosphate dehydrogenase deficiency poses a significant impediment to primaquine use for the elimination of liver stage infection with Plasmodium vivax and for gametocyte clearance, because of the risk of life-threatening haemolytic anaemia that can occur in G6PD deficient patients. Although a range of methods for screening G6PD deficiency have been described, almost all require skilled personnel, expensive laboratory equipment, freshly collected blood, and are time consuming; factors that render them unsuitable for mass-screening purposes. Methods A published WST8/1-methoxy PMS method was adapted to assay G6PD activity in a 96-well format using dried blood spots, and used it to undertake population screening within a malaria survey undertaken in Isabel Province, Solomon Islands. The assay results were compared to a biochemical test and a recently marketed rapid diagnostic test. Results Comparative testing with biochemical and rapid diagnostic test indicated that results obtained by filter paper assay were accurate providing that blood spots were assayed within 5 days when stored at ambient temperature and 10 days when stored at 4 degrees. Screening of 8541 people from 41 villages in Isabel Province, Solomon Islands revealed the prevalence of G6PD deficiency as defined by enzyme activity Conclusions The assay enabled simple and quick semi-quantitative population screening in a malaria-endemic region. The study indicated a high prevalence of G6PD deficiency in Isabel Province and highlights the critical need to consider G6PD deficiency in the context of P. vivax malaria elimination strategies in Solomon Islands, particularly in light of the potential role of primaquine mass drug administration.

  2. Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots.

    Science.gov (United States)

    Liu, Yinggao; Wu, Ruru; Wan, Qi; Xie, Gengqiang; Bi, Yurong

    2007-03-01

    The pivotal role of glucose-6-phosphate dehydrogenase (G-6-PDH)-mediated nitric oxide (NO) production in the tolerance to oxidative stress induced by 100 mM NaCl in red kidney bean (Phaseolus vulgaris) roots was investigated. The results show that the G-6-PDH activity was enhanced rapidly in the presence of NaCl and reached a maximum at 100 mM. Western blot analysis indicated that the increase of G-6-PDH activity in the red kidney bean roots under 100 mM NaCl was mainly due to the increased content of the G-6-PDH protein. NO production and nitrate reductase (NR) activity were also induced by 100 mM NaCl. The NO production was reduced by NaN(3) (an NR inhibitor), but not affected by N(omega)-nitro-L-arginine (L-NNA) (an NOS inhibitor). Application of 2.5 mM Na(3)PO(4), an inhibitor of G-6-PDH, blocked the increase of G-6-PDH and NR activity, as well as NO production in red kidney bean roots under 100 mM NaCl. The activities of antioxidant enzymes in red kidney bean roots increased in the presence of 100 mM NaCl or sodium nitroprusside (SNP), an NO donor. The increased activities of all antioxidant enzymes tested at 100 mM NaCl were completely inhibited by 2.5 mM Na(3)PO(4). Based on these results, we conclude that G-6-PDH plays a pivotal role in NR-dependent NO production, and in establishing tolerance of red kidney bean roots to salt stress.

  3. Astroglial Pentose Phosphate Pathway Rates in Response to High-Glucose Environments

    Directory of Open Access Journals (Sweden)

    Shinichi Takahashi

    2012-02-01

    Full Text Available ROS (reactive oxygen species play an essential role in the pathophysiology of diabetes, stroke and neurodegenerative disorders. Hyperglycaemia associated with diabetes enhances ROS production and causes oxidative stress in vascular endothelial cells, but adverse effects of either acute or chronic high-glucose environments on brain parenchymal cells remain unclear. The PPP (pentose phosphate pathway and GSH participate in a major defence mechanism against ROS in brain, and we explored the role and regulation of the astroglial PPP in response to acute and chronic high-glucose environments. PPP activity was measured in cultured neurons and astroglia by determining the difference in rate of 14CO2 production from [1-14C]glucose and [6-14C]glucose. ROS production, mainly H2O2, and GSH were also assessed. Acutely elevated glucose concentrations in the culture media increased PPP activity and GSH level in astroglia, decreasing ROS production. Chronically elevated glucose environments also induced PPP activation. Immunohistochemical analyses revealed that chronic high-glucose environments induced ER (endoplasmic reticulum stress (presumably through increased hexosamine biosynthetic pathway flux. Nuclear translocation of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2, which regulates G6PDH (glyceraldehyde-6-phosphate dehydrogenase by enhancing transcription, was also observed in association with BiP (immunoglobulin heavy-chain-binding protein expression. Acute and chronic high-glucose environments activated the PPP in astroglia, preventing ROS elevation. Therefore a rapid decrease in glucose level seems to enhance ROS toxicity, perhaps contributing to neural damage when insulin levels given to diabetic patients are not properly calibrated and plasma glucose levels are not adequately maintained. These findings may also explain the lack of evidence for clinical benefits from strict glycaemic control during the acute phase of stroke.

  4. Astroglial pentose phosphate pathway rates in response to high-glucose environments

    Science.gov (United States)

    Takahashi, Shinichi; Izawa, Yoshikane; Suzuki, Norihiro

    2012-01-01

    ROS (reactive oxygen species) play an essential role in the pathophysiology of diabetes, stroke and neurodegenerative disorders. Hyperglycaemia associated with diabetes enhances ROS production and causes oxidative stress in vascular endothelial cells, but adverse effects of either acute or chronic high-glucose environments on brain parenchymal cells remain unclear. The PPP (pentose phosphate pathway) and GSH participate in a major defence mechanism against ROS in brain, and we explored the role and regulation of the astroglial PPP in response to acute and chronic high-glucose environments. PPP activity was measured in cultured neurons and astroglia by determining the difference in rate of 14CO2 production from [1-14C]glucose and [6-14C]glucose. ROS production, mainly H2O2, and GSH were also assessed. Acutely elevated glucose concentrations in the culture media increased PPP activity and GSH level in astroglia, decreasing ROS production. Chronically elevated glucose environments also induced PPP activation. Immunohistochemical analyses revealed that chronic high-glucose environments induced ER (endoplasmic reticulum) stress (presumably through increased hexosamine biosynthetic pathway flux). Nuclear translocation of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), which regulates G6PDH (glyceraldehyde-6-phosphate dehydrogenase) by enhancing transcription, was also observed in association with BiP (immunoglobulin heavy-chain-binding protein) expression. Acute and chronic high-glucose environments activated the PPP in astroglia, preventing ROS elevation. Therefore a rapid decrease in glucose level seems to enhance ROS toxicity, perhaps contributing to neural damage when insulin levels given to diabetic patients are not properly calibrated and plasma glucose levels are not adequately maintained. These findings may also explain the lack of evidence for clinical benefits from strict glycaemic control during the acute phase of stroke. PMID:22300409

  5. Process Integration for the Disruption of Candida guilliermondii Cultivated in Rice Straw Hydrolysate and Recovery of Glucose-6-Phosphate Dehydrogenase by Aqueous Two-Phase Systems.

    Science.gov (United States)

    Gurpilhares, Daniela B; Pessoa, Adalberto; Roberto, Inês C

    2015-07-01

    Remaining cells of Candida guilliermondii cultivated in hemicellulose-based fermentation medium were used as intracellular protein source. Recovery of glucose-6-phosphate dehydrogenase (G6PD) was attained in conventional aqueous two-phase systems (ATPS) was compared with integrated process involving mechanical disruption of cells followed by ATPS. Influences of polyethylene glycol molar mass (M PEG) and tie line lengths (TLL) on purification factor (PF), yields in top (Y T ) and bottom (Y B ) phases and partition coefficient (K) were evaluated. First scheme resulted in 65.9 % enzyme yield and PF of 2.16 in salt-enriched phase with clarified homogenate (M PEG 1500 g mol(-1), TLL 40 %); Y B of 75.2 % and PF B of 2.9 with unclarified homogenate (M PEG 1000 g mol(-1), TLL 35 %). The highest PF value of integrated process was 2.26 in bottom phase (M PEG 1500 g mol(-1), TLL 40 %). In order to optimize this response, a quadratic model was predicted for the response PFB for process integration. Maximum response achieved was PFB = 3.3 (M PEG 1500 g mol(-1), TLL 40 %). Enzyme characterization showed G6P Michaelis-Menten constant (K M ) equal 0.07-0.05, NADP(+) K M 0.02-1.98 and optimum temperature 70 °C, before and after recovery. Overall, our data confirmed feasibility of disruption/extraction integration for single-step purification of intracellular proteins from remaining yeast cells.

  6. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP+-dependent dehydrogenases of the pentose phosphate pathway

    International Nuclear Information System (INIS)

    Rodrigues, Juan; Branco, Vasco; Lu, Jun; Holmgren, Arne; Carvalho, Cristina

    2015-01-01

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP + -dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI 50 : 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg 2+ > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates the

  7. Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6.

    Science.gov (United States)

    Papapetridis, Ioannis; van Dijk, Marlous; Dobbe, Arthur P A; Metz, Benjamin; Pronk, Jack T; van Maris, Antonius J A

    2016-04-26

    Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption. Replacement of the native NADP(+)-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD(+)-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15% increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP(+)-dependent acetaldehyde dehydrogenase, led to a 39% increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44% increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13% increase in the ethanol yield on glucose. The combination of NAD(+)-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor

  8. Peculiarities of glucose and glycerol metabolism in Nocardia vaccinii IMB B-7405

    Directory of Open Access Journals (Sweden)

    T. P. Pirog

    2015-04-01

    Full Text Available It has been established that in cells of Nocardia vaccinii IMB B-7405 (surfactant producer glucose catabolism is performed through pentose phosphate cycle as well as through gluconate (activi­ty of NAD+-dependent glucose-6- phosphate dehydrogenase and FAD+-dependent glucose dehydrogenase 835 ± 41 and 698 ± 35 nmol∙min-1∙mg-1 of protein respectively. 6-Phosphogluconate formed in the gluconokinase reaction is involved in the pentose phosphate cycle (activity of constitutive NADP+-dependent 6-phosphogluconate dehydrogenase 357 ± 17 nmol∙min-1∙mg-1 of protein. Glyce­rol catabolism to dihydroxyacetonephosphate (the intermediate of glycolysis may be performed in two ways: through glycerol-3-phosphate (glycerol kinase activity 244 ± 12 nmol∙min-1∙mg-1 of protein and through dihydroxyacetone. Replenishment of the C4-dicarboxylic acids pool in N. vaccinii IMV B-7405 grown on glucose and glycerol occurs in the phosphoenolpyruvate(PEPcarboxylase reaction (714–803 nmol∙min-1∙mg-1 of protein. 2-Oxoglutara­te was involved in tricarboxylic acid cycle by alternate pathway with the participation of 2-oxoglutarate synthase. The observed activity of both key enzymes of gluconeogenesis (PEP- carboxykinase and PEP-synthase, trehalose phosphate synthase and NADP+-dependent glutamate dehydrogenase confirmed the ability of IMV B-7405 strain to the synthesis of surface active glyco- and aminolipids, respectively.

  9. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, Juan, E-mail: juanricardorodrigues@gmail.com [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, Central University of Venezuela (Venezuela, Bolivarian Republic of); Branco, Vasco [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal); Lu, Jun; Holmgren, Arne [Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet (Sweden); Carvalho, Cristina, E-mail: cristina.carvalho@ff.ulisboa.pt [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal)

    2015-08-01

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI{sub 50}: 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg{sup 2+} > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates

  10. Oxidatively modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Alzheimer's disease: many pathways to neurodegeneration.

    Science.gov (United States)

    Butterfield, D Allan; Hardas, Sarita S; Lange, Miranda L Bader

    2010-01-01

    Recently, the oxidoreductase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has become a subject of interest as more and more studies reveal a surfeit of diverse GAPDH functions, extending beyond traditional aerobic metabolism of glucose. As a result of multiple isoforms and cellular locales, GAPDH is able to come in contact with a variety of small molecules, proteins, membranes, etc., that play important roles in normal and pathologic cell function. Specifically, GAPDH has been shown to interact with neurodegenerative disease-associated proteins, including the amyloid-beta protein precursor (AbetaPP). Studies from our laboratory have shown significant inhibition of GAPDH dehydrogenase activity in Alzheimer's disease (AD) brain due to oxidative modification. Although oxidative stress and damage is a common phenomenon in the AD brain, it would seem that inhibition of glycolytic enzyme activity is merely one avenue in which AD pathology affects neuronal cell development and survival, as oxidative modification can also impart a toxic gain-of-function to many proteins, including GAPDH. In this review, we examine the many functions of GAPDH with respect to AD brain; in particular, the apparent role(s) of GAPDH in AD-related apoptotic cell death is emphasized.

  11. The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae.

    Science.gov (United States)

    Asención Diez, Matías D; Miah, Farzana; Stevenson, Clare E M; Lawson, David M; Iglesias, Alberto A; Bornemann, Stephen

    2017-01-20

    Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae*

    Science.gov (United States)

    Asención Diez, Matías D.; Miah, Farzana; Stevenson, Clare E. M.; Lawson, David M.; Iglesias, Alberto A.; Bornemann, Stephen

    2017-01-01

    Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli. However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae. The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate. PMID:27903647

  13. Review of key knowledge gaps in glucose-6-phosphate dehydrogenase deficiency detection with regard to the safe clinical deployment of 8-aminoquinoline treatment regimens: a workshop report.

    Science.gov (United States)

    von Seidlein, Lorenz; Auburn, Sarah; Espino, Fe; Shanks, Dennis; Cheng, Qin; McCarthy, James; Baird, Kevin; Moyes, Catherine; Howes, Rosalind; Ménard, Didier; Bancone, Germana; Winasti-Satyahraha, Ari; Vestergaard, Lasse S; Green, Justin; Domingo, Gonzalo; Yeung, Shunmay; Price, Ric

    2013-03-27

    The diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency is a crucial aspect in the current phases of malaria control and elimination, which will require the wider use of 8-aminoquinolines for both reducing Plasmodium falciparum transmission and achieving the radical cure of Plasmodium vivax. 8-aminoquinolines, such as primaquine, can induce severe haemolysis in G6PD-deficient individuals, potentially creating significant morbidity and undermining confidence in 8-aminoquinoline prescription. On the other hand, erring on the side of safety and excluding large numbers of people with unconfirmed G6PD deficiency from treatment with 8-aminoquinolines will diminish the impact of these drugs. Estimating the remaining G6PD enzyme activity is the most direct, accessible, and reliable assessment of the phenotype and remains the gold standard for the diagnosis of patients who could be harmed by the administration of primaquine. Genotyping seems an unambiguous technique, but its use is limited by cost and the large range of recognized G6PD genotypes. A number of enzyme activity assays diagnose G6PD deficiency, but they require a cold chain, specialized equipment, and laboratory skills. These assays are impractical for care delivery where most patients with malaria live. Improvements to the diagnosis of G6PD deficiency are required for the broader and safer use of 8-aminoquinolines to kill hypnozoites, while lower doses of primaquine may be safely used to kill gametocytes without testing. The discussions and conclusions of a workshop conducted in Incheon, Korea in May 2012 to review key knowledge gaps in G6PD deficiency are reported here.

  14. Brain glucose metabolism in an animal model of depression.

    Science.gov (United States)

    Detka, J; Kurek, A; Kucharczyk, M; Głombik, K; Basta-Kaim, A; Kubera, M; Lasoń, W; Budziszewska, B

    2015-06-04

    An increasing number of data support the involvement of disturbances in glucose metabolism in the pathogenesis of depression. We previously reported that glucose and glycogen concentrations in brain structures important for depression are higher in a prenatal stress model of depression when compared with control animals. A marked rise in the concentrations of these carbohydrates and glucose transporters were evident in prenatally stressed animals subjected to acute stress and glucose loading in adulthood. To determine whether elevated levels of brain glucose are associated with a change in its metabolism in this model, we assessed key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase), products of glycolysis, i.e., pyruvate and lactate, and two selected enzymes of the tricarboxylic acid cycle (pyruvate dehydrogenase and α-ketoglutarate dehydrogenase) in the hippocampus and frontal cortex. Additionally, we assessed glucose-6-phosphate dehydrogenase activity, a key enzyme in the pentose phosphate pathway (PPP). Prenatal stress increased the levels of phosphofructokinase, an important glycolytic enzyme, in the hippocampus and frontal cortex. However, prenatal stress had no effect on hexokinase or pyruvate kinase levels. The lactate concentration was elevated in prenatally stressed rats in the frontal cortex, and pyruvate levels remained unchanged. Among the tricarboxylic acid cycle enzymes, prenatal stress decreased the level of pyruvate dehydrogenase in the hippocampus, but it had no effect on α-ketoglutarate dehydrogenase. Like in the case of glucose and its transporters, also in the present study, differences in markers of glucose metabolism between control animals and those subjected to prenatal stress were not observed under basal conditions but in rats subjected to acute stress and glucose load in adulthood. Glucose-6-phosphate dehydrogenase activity was not reduced by prenatal stress but was found to be even higher in animals exposed to

  15. Effects of whole body x-ray irradiation on induction by phenobarbital of rat liver glucose-6-phosphate dehydrogenase and glutathione reductase

    Energy Technology Data Exchange (ETDEWEB)

    Bitny-Szlachto, S.; Szyszko, A. (Wojskowy Inst. Higieny i Epidemiologii, Warsaw (Poland))

    1979-01-01

    In rats treated with phenobarbital (3x100 mg/kg, i.p.), liver G-6-P dehydrogenase activity increased by 70% in the cytosol and in the 9.000xg supernatant, and only by 20% in microsomes. Moreover, the phenobarbital treatment increased rat liver GSSG reductase activity by 30%. On the other hand, activity of the liver microsomal G-6-P dehydrogenase was found to increase by some 20% in whole body irradiated, both control and phenobarbital treated rats. In rats irradiated with 600 R prior to the first dose of the inducer there was not noted any increase in G-6-P dehydrogenase of the 9.000xg supernatant, and increase in the cytosol activity dropped to 38%. Thus, induction of the soluble liver G-6-P dehydrogenase by phenobarbital has turned out to be radiosensitive, whereas phenobarbital induction of GSSG reductase was unaffected by irradiation.

  16. Genetics Home Reference: glucose phosphate isomerase deficiency

    Science.gov (United States)

    ... glycolytic pathway; in this step, a molecule called glucose-6-phosphate is converted to another molecule called fructose-6-phosphate. When GPI remains a single molecule (a monomer) it is involved in the development and maintenance of nerve cells ( neurons ). In this context, it is often known as ...

  17. Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2006-02-01

    Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B{sub 6} (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V{sub M}) of 3.64 Å{sup 3} Da{sup −1} and a solvent content of 66%.

  18. Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-01-01

    Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B 6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M ) of 3.64 Å 3 Da −1 and a solvent content of 66%

  19. Use of a simplified spectrophotometric method for quantitative determination of glucose-6-phosphate dehydrogenase activity in normal children from two day-care centers of the city of São Paulo

    Directory of Open Access Journals (Sweden)

    Roberto Muller

    2003-06-01

    Full Text Available Objective: To evaluate the applicability of a simplified method forquantitative determination of glucose-6-phosphate dehydrogenaseactivity in normal children; to determine the mean, standarddeviation and threshold value under which the enzyme activity isconsidered deficient. Methods: Blood samples were collected from201 children from two day-care centers in the city of São Paulo.The subjects were considered normal based on physicalexamination and laboratory tests. The enzyme activity wasdetermined in red blood cells of normal children using the “TestCombination G-6-PDH®” kit. The following statistical analyses werecarried out: the results were submitted to Student’s t test,Kolmogorov-Smirnov test, lower confidence interval (one-tailedtest and Spearman’s correlation coefficient. Results: The meanhemoglobin value for girls was slightly higher than the mean valuefor boys, but this difference was not statistically significant. Therewas no statistical difference in mean enzyme activities for Caucasianand non-Caucasian children. There was no significant correlation amongenzyme activity levels, red blood cells, hemoglobin levels,hematocrit, reticulocytes, white blood cells and age of patients.The mean enzyme activity for boys was 4.448 U/g Hb, standarddeviation = 1.380 U/g Hb. For girls, the mean enzyme activity was4.531 U/g Hb, standard deviation = 1.386 U/g Hb, and the differencewas not statistically significant. Therefore, the two populationgroups were considered as one single population, presenting amean enzyme activity of 4.490 U/g Hb, standard deviation = 1.380 U/g Hb.Since the distribution curve of enzyme activity values was normal,a lower confidence interval was determined (one-tailed test, witha cutoff point of 2.227 U/g Hb. Conclusion: The method used bySolem proved to be simple, fast, very accurate and useful to detectglucose-6-phosphate dehydrogenase activity and to identifychildren with enzyme deficiency.

  20. Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-02-01

    The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-alpha-ketobutyrate. It belongs to the D-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 A from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 A. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.64 A3 Da(-1) and a solvent content of 66%.

  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. Time course of radiolabeled 2-deoxy-D-glucose 6-phosphate turnover in cerebral cortex of goats

    International Nuclear Information System (INIS)

    Pelligrino, D.A.; Miletich, D.J.; Albrecht, R.F.

    1987-01-01

    The vivo dephosphorylation rate of 2-deoxy-D-glucose 6-phosphate (DGP) in the cerebral cortex of goats injected intravenously with radiolabeled 2-deoxy-D-glucose (DG) was investigated. Serial rapidly frozen samples of parietal cortical gray tissue were obtained at regular intervals over time periods from 45 min to 3 h in awake goats or in paralyzed and artificially ventilated goats maintained under 70% N 2 O or pentobarbital sodium anesthesia. The samples were analyzed for glucose content and separate DG and DGP activities. The rate parameters for phosphorylation (k/sup */ 4 ) and dephosphorylation (k/sup */ 4 ) were estimated in each animal. The glucose phosphorylation rate (PR) was calculated over the intervals 3-5 (or 6), 3-10, 3-20, 3-30, and 3-45 min, assuming k/sup */ 4 = O. As the evaluation period was extended beyond 10 min, the calculated PR became increasingly less when compared with that calculated over the 3- to 5- (or 6) min interval (PR/sub i/). Furthermore, as metabolic activity decreased, the magnitude of the error increased such that at 45 min pentobarbital-anesthetize goats underestimated the PR/sub i/ by 46.5% compared with only 23.1 in N 2 O-anesthetized goats. This was also reflected in the >twofold higher k/sup */ 4 /k/sup */ 3 ratio in the pentobarbital vs. N 2 O-anesthetized group. It is concluded that when using the DG method in the goat, DGP dephosphorylation cannot be ignored when employing >10-min evaluation periods

  3. Complex formation of uranium(VI) with fructose and glucose phosphates

    International Nuclear Information System (INIS)

    Koban, A.; Geipel, G.; Bernhard, G.; Fanghaenel, T.

    2002-01-01

    The uptake of heavy metals into plants is commonly quantified by the soil-plant transfer factor. Up to now little is known about the chemical speciation of actinides in plants. To compare the obtained spectroscopic data of uranium complexes in plants with model compounds, we investigate the complexation of uranium with relevant bioligands of various functionalities. A very important class of ligands consists of phosphate esters, which serve as phosphate group and energy transmitters as well as energy storage media in biological systems. Heavy metal ions bound to the phosphate esters can be transported into living cells and then deposited. Therefore, in our study we present the results of uranium complexation with glucose-6-phosphate (G6P), and fructose-6-phosphate (F6P) obtained by time-resolved laser-induced fluorescence spectroscopy (TRLFS). The experiments were performed at a fixed uranyl concentration (10 -5 M) as a function of the ligand concentrations (10 -5 to 10 -3 M) in a pH range from 2 to 4.5. For the glucose phosphate system we observed, using increasing ligand concentrations, a decrease in the fluorescence intensity and a small red shift of the emission bands. From this we conclude that the complexed uranyl glucose phosphate species show only minor or no fluorescence properties. The TRLFS spectra of the glucose phosphate samples indicated the presence of a single species with fluorescence properties. This species has a lifetime of approximately 1.5 μs and was identified as the free uranyl ion. An opposite phenomenon was observed for the fructose phosphate system: there was no decrease in fluorescence intensity. However, a strong red shift of the spectra was observed, illustrating the fluorescence properties of the uranyl fructose phosphate complex. The TRLFS spectra of the fructose phosphate system showed a second lifetime ( 2 2+ UO 2 (lig) x (2-y)+ + y H + (lig = sugar phosphate). Applying the mass action law and transformation to the logarithmic

  4. Prevalence of glucose-6-phosphate dehydrogenase deficiency in ...

    African Journals Online (AJOL)

    Pradeep Kumar

    2016-02-06

    Feb 6, 2016 ... Hemolytic anemia; ... G6PD deficiency is the commonest hemolytic X-linked genetic disease, which affects .... tain drugs or infection, can elicit acute hemolysis. ..... down syndrome risk: a meta-analysis from 34 studies.

  5. High glucose impairs superoxide production from isolated blood neutrophils

    DEFF Research Database (Denmark)

    Perner, A; Nielsen, S E; Rask-Madsen, J

    2003-01-01

    Superoxide (O(2)(-)), a key antimicrobial agent in phagocytes, is produced by the activity of NADPH oxidase. High glucose concentrations may, however, impair the production of O(2)(-) through inhibition of glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the formation of NADPH. This study...... measured the acute effects of high glucose or the G6PD inhibitor dehydroepiandrosterone (DHEA) on the production of O(2)(-) from isolated human neutrophils....

  6. Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

    Science.gov (United States)

    Wisselink, H Wouter; Mars, Astrid E; van der Meer, Pieter; Eggink, Gerrit; Hugenholtz, Jeroen

    2004-07-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

  7. Overexpression, crystallization and preliminary X-­ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

    Science.gov (United States)

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-01-01

    The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-­phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M) of 3.64 Å3 Da−1 and a solvent content of 66%. PMID:16511285

  8. Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, Department of Biochemistry, University of Leicester, Leicester LE1 9HN (United Kingdom)

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2{sub 1}; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3{sub 2}. The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.

  9. Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    International Nuclear Information System (INIS)

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E.

    2008-01-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2 1 ; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3 2 . The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP

  10. [Intensity of pentose phosphate metabolism of carbohydrates in various brain areas in normal and starved animals].

    Science.gov (United States)

    Kerimov, B F

    2002-01-01

    The activities of key enzymes of pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G-6 PD) and 6-phosphogluconate dehydrogenase (6-PGD), were studied in cytoplasmatic fractions of brain cortical (limbic, orbital, sensorimotor cortex) and subcortical (myelencefalon, mesencefalon, hypothalamus) structures of rats subjected to starvation for 1, 2, 3, 5 and 7 days. Short-term starvation (1-3 days) caused activation of 6-GPD and 6-PGD both in cortical and subcortical structures. Long-term starvation for 5-7 days caused a decrease of activities of the pentose phosphate pathway enzymes in all studied structures. It is suggested that enzymes of pentose phosphate pathway in nervous tissues are functionally and metabolically related to glutathione system and during starvation they indirectly participate in the regulation lipid peroxidation processes.

  11. Cannabidiol attenuates OGD/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Shanshan Sun

    2017-04-01

    Full Text Available Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD, a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen–glucose-deprivation/reperfusion (OGD/R model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XFe24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP+ ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance.

  12. Cannabidiol attenuates OGD/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons.

    Science.gov (United States)

    Sun, Shanshan; Hu, Fangyuan; Wu, Jihong; Zhang, Shenghai

    2017-04-01

    Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD), a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen-glucose-deprivation/reperfusion (OGD/R) model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XF e 24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP + ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

    Science.gov (United States)

    Guadalupe-Medina, Víctor; Metz, Benjamin; Oud, Bart; van Der Graaf, Charlotte M; Mans, Robert; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h−1. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l−1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth. PMID:24004455

  14. Prevalence of glucose-6-phosphate dehydrogenase deficiency and ...

    African Journals Online (AJOL)

    . ... while statistical analysis was done using STATA soft- ware version 8 (STATA Corp., College station, TX). Prevalence of G6PD and HbAS in bivariate variables ... Multivariate logis- .... technique (Enevold et al., 2007) found prevalence of.

  15. Noninferiority of glucose-6-phosphate dehydrogenase deficiency diagnosis by a point-of-care rapid test vs the laboratory fluorescent spot test demonstrated by copper inhibition in normal human red blood cells.

    Science.gov (United States)

    Baird, J Kevin; Dewi, Mewahyu; Subekti, Decy; Elyazar, Iqbal; Satyagraha, Ari W

    2015-06-01

    Tens of millions of patients diagnosed with vivax malaria cannot safely receive primaquine therapy against repeated attacks caused by activation of dormant liver stages called hypnozoites. Most of these patients lack access to screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency, a highly prevalent disorder causing serious acute hemolytic anemia with primaquine therapy. We optimized CuCl inhibition of G6PD in normal red blood cells (RBCs) to assess G6PD diagnostic technologies suited to point of care in the impoverished rural tropics. The most widely applied technology for G6PD screening-the fluorescent spot test (FST)-is impractical in that setting. We evaluated a new point-of-care G6PD screening kit (CareStart G6PD, CSG) against FST using graded CuCl treatments to simulate variable hemizygous states, and varying proportions of CuCl-treated RBC suspensions to simulate variable heterozygous states of G6PD deficiency. In experiments double-blinded to CuCl treatment, technicians reading FST and CSG test (n = 269) classified results as positive or negative for deficiency. At G6PD activity ≤40% of normal (n = 112), CSG test was not inferior to FST in detecting G6PD deficiency (P = 0.003), with 96% vs 90% (P = 0.19) sensitivity and 75% and 87% (P = 0.01) specificity, respectively. The CSG test costs less, requires no specialized equipment, laboratory skills, or cold chain for successful application, and performs as well as the FST standard of care for G6PD screening. Such a device may vastly expand access to primaquine therapy and aid in mitigating the very substantial burden of morbidity and mortality imposed by the hypnozoite reservoir of vivax malaria. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Glucose 6-phosphate dehydrogenase deficiency and cystic fibrosis

    OpenAIRE

    Congdon, P. J.; Aggarwal, R. K.; Littlewood, J. M.; Shapiro, H.

    1981-01-01

    A child born to Pakistani parents is described. He had both cystic fibrosis and G-6PD-deficiency. So far as can be ascertained, the occurrence of both these conditions in the same individual has not previously been reported.

  17. Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase

    International Nuclear Information System (INIS)

    Viana, Renato Marcio Ribeiro; Prado, Maria Auxiliadora Fontes; Alves, Ricardo Jose

    2008-01-01

    The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(d-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from d-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethyl phosphoryl chloride. The resulting 5-[d-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase. (author)

  18. Glucose-6-phosphate dehydrogenase deficiency in northern Mexico ...

    Indian Academy of Sciences (India)

    screening, in which the haplotype analysis was performed. Group B .... Thr65 in the native structure of human G6PD, the same protein .... this mutation has a very low frequency in the Mexican popu- lation, we can predict a significant health impact in the males .... genase deficiency: a systematic review and meta-analysis.

  19. ONLINE MONITORING OF EXTRACELLULAR BRAIN GLUCOSE USING MICRODIALYSIS AND A NADPH-LINKED ENZYMATIC ASSAY

    NARCIS (Netherlands)

    VANDERKUIL, JHF; KORF, J

    A method to monitor extracellular glucose in freely moving rats, based on intracerebral microdialysis coupled to an enzyme reactor is described. The dialysate is continuously mixed with a solution containing the enzymes hexokinase and glucose-6-phosphate dehydrogenase, and the fluorescence of NADPH

  20. G6PD Deficiency (Glucose-6-Phosphate Dehydrogenase) (For Parents)

    Science.gov (United States)

    ... genes from one or both parents to a child. The gene responsible for this deficiency is on the X chromosome. G6PD deficiency is most common in males of African heritage. Many females of African heritage are carriers ...

  1. Modulation of low dose radiation effect on pentose phosphate pathway enzymes by B-multivitamin deficiency

    International Nuclear Information System (INIS)

    Zimatkina, T.I.; Lashak, L.K.; Moiseenok, A.G.

    1997-01-01

    Blood, liver, thymus and spleen of albino rats injected subcutaneously with antivitamins (othythiamine and methotrexate) and subjected to prolonger γ-irradiation in the overall dose of 0.75 Gy were assayed for transketolase and glucose-6-phosphate dehydrogenase after 12h, 1, 2, 5 and 40 days from the last radiation dose. High transketolase sensitivity was found both to radiation (activation) and the combined effects of vitamin deficiency and radiation (potentiation of antivitamin inhibitory action) in all the tissues studied. The activity of glucose-6-phosphate dehydrogenase was little changed under the given experimental manipulations, but the combined effect of the factors considerably inhibited the enzyme activities in the organs of the immune system. Consequently, in B-multivitamin deficiency the effect of low radiation doses was subjected to a considerable modulation resulting in profound inhibition of the oxidation and nonoxidative branches of the pentose phosphate pathway. (author). 9 refs, 2 tabs

  2. Polimorfisme Enzim Glucose-6-Phosphate Isomerase pada Tiga Populasi Tuna Sirip Kuning (Thunnus albacares)

    OpenAIRE

    Permana, Gusti Ngurah; Hutapea, Jhon H.; Moria, Sari Budi; Haryanti, Haryanti

    2006-01-01

    Samples of yellowfin tuna (Thunnus albacares) were taken from three locations Bali, North Sulawesi and North Maluku. The glucose-6-phosphate isomerase (GPI) was analyzed from liver using allozyme electrophoresis method. Polymorphism of GPI enzyme was observed and four alleles (A, B ,C, D) were found in Bali population, three alleles (A,B,C) were found in North Maluku and North Sulawesi populations. Heterozygosity values, from Bali, North Maluku and North Sulawesi were 0.419; 0.417; 0.143 resp...

  3. LDH and G-6PDH activities in the ovaries of adult female Wistar rats ...

    African Journals Online (AJOL)

    The present study was designed to evaluate the effects of aqueous extracts of neem (Azadirachta Indica) leaves (which have been documented for its antifertility effect on experimental animals) on glucose-6-phosphate dehydrogenase (G-6PDH) and lactate dehydrogenase (LDH) levels in the ovaries of adult female wistar ...

  4. Dephosphorylation of 2-deoxyglucose 6-phosphate and 2-deoxyglucose export from cultured astrocytes.

    Science.gov (United States)

    Forsyth, R J; Bartlett, K; Eyre, J

    1996-03-01

    Neurotransmitter-stimulated mobilization of astrocyte glycogen has been proposed as a basis for local energy homeostasis in brain. However, uncertainty remains over the fate of astrocyte glycogen. Upon transfer of cultured astrocytes pre-loaded with [2-3H]2-deoxyglucose 6-phosphate at non-tracer concentrations to a glucose-free, 2-deoxyglucose-free medium, rapid dephosphorylation of a proportion of the intracellular 2-deoxyglucose 6-phosphate pool and export of 2-deoxyglucose to the extracellular fluid occurs. Astrocytes show very low, basal rates of gluconeogenesis from pyruvate (approx. 1 nmol mg protein-1 h-1). Astrocytes in vivo may be capable of physiologically significant glucose export from glucose-6-phosphate. The low gluconeogenic activity in astrocytes suggests that the most likely source of glucose-6-phosphate may be glycogen. These findings support the hypothesis that export, as glucose, to adjacent neurons may be one of the possible fate(s) of astrocytic glycogen. Such export of glycogen as glucose occurring in response to increases in neuronal activity could contribute to energy homeostasis on a paracrine scale within brain.

  5. Molecular Identification of G6PD Chatham (G1003A) in Khuzestan ...

    Indian Academy of Sciences (India)

    Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in pentose phosphate pathway and the main intracel- lular source of NADPH. Since G6PD is the only source of. NADPH in red blood cells, defense against oxidative damage strongly depends on its activity (Mehta et al. 2000). Defi- ciency of G6PD enzyme in ...

  6. Electron-transfer mediator for a NAD-glucose dehydrogenase-based glucose sensor.

    Science.gov (United States)

    Kim, Dong-Min; Kim, Min-yeong; Reddy, Sanapalli S; Cho, Jaegeol; Cho, Chul-ho; Jung, Suntae; Shim, Yoon-Bo

    2013-12-03

    A new electron-transfer mediator, 5-[2,5-di (thiophen-2-yl)-1H-pyrrol-1-yl]-1,10-phenanthroline iron(III) chloride (FePhenTPy) oriented to the nicotinamide adenine dinucleotide-dependent-glucose dehydrogenase (NAD-GDH) system was synthesized through a Paal-Knorr condensation reaction. The structure of the mediator was confirmed by Fourier-transform infrared spectroscopy, proton and carbon nucler magnetic resonance spectroscopy, and mass spectroscopy, and its electron-transfer characteristic for a glucose sensor was investigated using voltammetry and impedance spectroscopy. A disposable amperometric glucose sensor with NAD-GDH was constructed with FePhenTPy as an electron-transfer mediator on a screen printed carbon electrode (SPCE) and its performance was evaluated, where the addition of reduces graphene oxide (RGO) to the mediator showed the enhanced sensor performance. The experimental parameters to affect the analytical performance and the stability of the proposed glucose sensor were optimized, and the sensor exhibited a dynamic range between 30 mg/dL and 600 mg/dL with the detection limit of 12.02 ± 0.6 mg/dL. In the real sample experiments, the interference effects by acetaminophen, ascorbic acid, dopamine, uric acid, caffeine, and other monosaccharides (fructose, lactose, mannose, and xylose) were completely avoided through coating the sensor surface with the Nafion film containing lead(IV) acetate. The reliability of proposed glucose sensor was evaluated by the determination of glucose in artificial blood and human whole blood samples.

  7. InterProScan Result: FS765596 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 001282 Glucose-6-phosphate dehydrogenase Molecular Function: glucose-6-phosphate dehydrogenase activity (GO:0004345)|Biological... Process: glucose metabolic process (GO:0006006)|Biological Process: oxidation reduction (GO:0055114) ...

  8. [Influence of an infusion of 5- or 20% glucose solution on blood glucose and inorganic phosphate concentrations in dairy cows].

    Science.gov (United States)

    Aldaek, T A A; Failing, K; Wehrend, A; Klymiuk, M C

    2011-01-01

    The study was performed to evaluate the influence of an intravenous infusion of 5% and 20% dextrose solution on the plasma concentration of glucose and inorganic phosphate in healthy, dairy cows. Ten healthy, lactating, nonpregnant 3 to 6 year-old Holstein-Friesian cows were included in this investigation. The daily milk yield was 20.3±2.7 liters. Blood plasma concentrations of inorganic phosphate and glucose were determined before, during, immediately and 60 minutes after infusion of 0.9% physiological saline, 5% or 20% dextrose solution. A statistically significant influence of dextrose infusion on blood glucose concentration was observed. After 20% dextrose infusion (200 g dextrose) the blood glucose concentration increased by approximately 13.26 mmol/l. The administration of 5% dextrose solution (50 g dextrose) yielded an increase of blood glucose concentration by 3.31 mmol/l. There was no significant correlation between plasma inorganic phosphate concentrations and infusion of 0.9% saline, 5% or 20% dextrose solution. Intravenous administration of 1000 ml of 5% or 20% dextrose solution does not induce a lasting plasma phosphate reduction and is suitable for elevating the blood glucose concentration.

  9. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  10. Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, University of Leicester, Leicester LE1 9HN (United Kingdom)

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

  11. Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

    Science.gov (United States)

    Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

    2018-04-03

    While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

  12. (G6PD) in stored blood

    African Journals Online (AJOL)

    Red blood cell viability in stored blood determines successful transfusion. Glucose-6-phosphate dehydrogenase (G6PD) activity has been shown to maintain red blood cell membrane integrity. This study was, therefore, aimed at estimating the G6PD activity in stored blood bags at the blood bank of the University of Nigeria ...

  13. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

    Science.gov (United States)

    Cross, Megan; Biberacher, Sonja; Park, Suk-Youl; Rajan, Siji; Korhonen, Pasi; Gasser, Robin B; Kim, Jeong-Sun; Coster, Mark J; Hofmann, Andreas

    2018-04-24

    The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

  14. Boosting the pentose phosphate pathway restores cardiac progenitor cell availability in diabetes.

    Science.gov (United States)

    Katare, Rajesh; Oikawa, Atsuhiko; Cesselli, Daniela; Beltrami, Antonio P; Avolio, Elisa; Muthukrishnan, Deepti; Munasinghe, Pujika Emani; Angelini, Gianni; Emanueli, Costanza; Madeddu, Paolo

    2013-01-01

    Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway. Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1(pos) CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105(pos) progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt. We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity.

  15. Molecular Characterization of Glucose-6-Phosphate ...

    African Journals Online (AJOL)

    Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, ... United Arab Emirates (UAE), 3School of Pharmacy, Pharmacology Department, University Sains Malaysia ... International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index ..... dehydrogenase deficiency in a student population.

  16. The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

    International Nuclear Information System (INIS)

    Sato, Tomoki; Morita, Akihito; Mori, Nobuko; Miura, Shinji

    2014-01-01

    Highlights: • Ethanol administration increased GPD1 mRNA expression. • Ethanol administration increased glucose incorporation into TG glycerol moieties. • No increase in hepatic TG levels was observed in ethanol-injected GPD1 null mice. • We propose that GPD1 is required for ethanol-induced TG accumulation in the liver. - Abstract: Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of 14 C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation

  17. The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Tomoki, E-mail: s13220@u-shizuoka-ken.ac.jp [Laboratory of Nutritional Biochemistry, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Morita, Akihito, E-mail: moritaa@u-shizuoka-ken.ac.jp [Laboratory of Nutritional Biochemistry, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Mori, Nobuko, E-mail: morin@b.s.osakafu-u.ac.jp [Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai 599-8570 (Japan); Miura, Shinji, E-mail: miura@u-shizuoka-ken.ac.jp [Laboratory of Nutritional Biochemistry, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

    2014-02-21

    Highlights: • Ethanol administration increased GPD1 mRNA expression. • Ethanol administration increased glucose incorporation into TG glycerol moieties. • No increase in hepatic TG levels was observed in ethanol-injected GPD1 null mice. • We propose that GPD1 is required for ethanol-induced TG accumulation in the liver. - Abstract: Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of {sup 14}C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.

  18. Deficiencia de glucosa 6-fostato deshidrogenasa en hombres sanos y en pacientes maláricos; Turbo (Antioquia, Colombia Deficiency of glucose-6-phosphate dehydrogenase in healthy men and malaria patients; Turbo (Antioquia, Colombia

    Directory of Open Access Journals (Sweden)

    Jaime Carmona-Fonseca

    2008-06-01

    Full Text Available INTRODUCCIÓN: En América Latina la deficiencia de glucosa 6-fosfato deshidrogenasa (d-G6PD ha sido poco estudiada y en Colombia solo conocemos tres publicaciones antiguas. Urge conocer más la prevalencia de d-G6PD, sobre todo ahora que el tratamiento de la malaria vivax plantea aumentar la dosis diaria o total de primaquina. OBJETIVO: Medir la prevalencia de d-G6PD en poblaciones masculina sana y de enfermos con malaria por Plasmodium vivax, en Turbo (Urabá, departamento de Antioquia, Colombia. METODOLOGÍA: Encuestas de prevalencia, para evaluar la G6PD en dos poblaciones de Turbo (Antioquia: hombres sanos; hombres y mujeres con malaria vivax. Se trabajó con muestras diseñadas con criterios estadístico-epidemiológicos. La actividad enzimática se midió con el método normalizado de Beutler para valorar la G6PD en hemolizados. RESULTADOS: Entre los hombres sanos (n = 508, el intervalo de confianza 95% para el promedio (IC95% estuvo entre 4,15 y 4,51 UI/g hemoglobina y 14,8% presentaron valores por debajo del "límite normal" de INTRODUCTION: Glucose-6-phosphate dehydrogenase (G6PD deficiency in Latin America has not been fully studied and in Colombia only three outdated publications are known. Recent information on the prevalence of G6PD deficiency is required now, because the recommended treatment of vivax malaria requires higher daily or total doses of primaquine. OBJECTIVE: To measure the prevalence of G6PD in a healthy male population and in a Plasmodium vivax infected population in Turbo (Urabá, Antioquia Department, Colombia. METHOD: Prevalence survey to evaluate G6PD in two populations of Turbo (Antioquia: healthy male; male and female with vivax malaria. The work was carried out on population samples selected using statistical and epidemiological criteria. Enzyme activity was measured using Beutler's normalized method to evaluate G6PD after hemolysis. RESULTS: For the healthy male group (n = 508, and with a 95% confidence

  19. Metabolism of tritiated D-glucose in rat erythrocytes

    International Nuclear Information System (INIS)

    Manuel y Keenoy, B.; Malaisse-Lagae, F.; Malaisse, W.J.

    1991-01-01

    The metabolism of D-[U-14C]glucose, D-[1-14C]glucose, D-[6-14C]glucose, D-[1-3H]glucose, D-[2-3H]glucose, D-[3-3H]glucose, D-[3,4-3H]glucose, D-[5-3H]glucose, and D-[6-3H]glucose was examined in rat erythrocytes. There was a fair agreement between the rate of 3HOH production from either D-[3-3H]glucose and D-[5-3H]glucose, the decrease in the 2,3-diphosphoglycerate pool, its fractional turnover rate, the production of 14C-labeled lactate from D-[U-14C]glucose, and the total lactate output. The generation of both 3HOH and tritiated acidic metabolites from D-[3,4-3H]glucose indicated incomplete detritiation of the C4 during interconversion of fructose-1,6-bisphosphate and triose phosphates. Erythrocytes unexpectedly generated 3HOH from D-[6-3H]glucose, a phenomenon possibly attributable to the detritiation of [3-3H]pyruvate in the reaction catalyzed by glutamate pyruvate transaminase. The production of 3HOH from D-[2-3H]glucose was lower than that from D-[5-3H]glucose, suggesting enzyme-to-enzyme tunneling of glycolytic intermediates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. The production of 3HOH from D-[1-3H]glucose largely exceeded that of 14CO2 from D-[1-14C]glucose, a situation tentatively ascribed to the generation of 3HOH in the phosphomannoisomerase reaction. It is further speculated that the adjustment in specific radioactivity of D-[1-3H]glucose-6-phosphate cannot simultaneously match the vastly different degrees of isotopic discrimination in velocity at the levels of the reactions catalyzed by either glucose-6-phosphate dehydrogenase or phosphoglucoisomerase. The interpretation of the present findings thus raises a number of questions, which are proposed as a scope for further investigations

  20. Trehalose-6-Phosphate: connecting plant metabolism and development

    Directory of Open Access Journals (Sweden)

    Jathish ePonnu

    2011-11-01

    Full Text Available Beyond their metabolic roles, sugars can also act as messengers in signal transduction. Trehalose, a sugar found in many species of plants and animals, is a non-reducing disaccharide composed of two glucose moieties. Its synthesis in plants is a two-step process, involving the production of trehalose-6-phosphate (T6P catalyzed by TREHALOSE-6-PHOSPHATE SYNTHASE (TPS and its consecutive dephosphorylation to trehalose, catalyzed by TREHALOSE-6-PHOSPHATE PHOSPHATASE (TPP. T6P has recently emerged as an important signaling metabolite, regulating carbon assimilation and sugar status in plants. In addition, T6P has also been demonstrated to play an essential role in plant development. This review recapitulates the recent advances in our understanding the role of T6P in coordinating diverse metabolic and developmental processes.

  1. A role for glucose-6-phosphate dehydrogenase

    African Journals Online (AJOL)

    STORAGESEVER

    2009-01-19

    Jan 19, 2009 ... mice caused a decrease in the nucleated cell counts in the peripheral blood, the .... Society of hematology, annual meeting abstracts; abstract 1049: p. 106. Salman 141. Leopold J ... Trans Res. Soc. London B. Biol. Sci. 354:.

  2. Subversion of Schwann Cell Glucose Metabolism by Mycobacterium leprae*

    Science.gov (United States)

    Medeiros, Rychelle Clayde Affonso; Girardi, Karina do Carmo de Vasconcelos; Cardoso, Fernanda Karlla Luz; Mietto, Bruno de Siqueira; Pinto, Thiago Gomes de Toledo; Gomez, Lilian Sales; Rodrigues, Luciana Silva; Gandini, Mariana; Amaral, Julio Jablonski; Antunes, Sérgio Luiz Gomes; Corte-Real, Suzana; Rosa, Patricia Sammarco; Pessolani, Maria Cristina Vidal; Nery, José Augusto da Costa; Sarno, Euzenir Nunes; Batista-Silva, Leonardo Ribeiro; Sola-Penna, Mauro; Oliveira, Marcus Fernandes; Moraes, Milton Ozório; Lara, Flavio Alves

    2016-01-01

    Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed. PMID:27555322

  3. Construction of mutant glucose oxidases with increased dye-mediated dehydrogenase activity.

    Science.gov (United States)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-11-02

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

  4. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    Science.gov (United States)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  5. Erroneous glucose recordings while using mutant variant of quinoprotein glucose dehydrogenase glucometer in a child with galactosemia

    Directory of Open Access Journals (Sweden)

    Vivek Mathew

    2013-01-01

    Full Text Available We report a 2-month-old child with galactosemia and falsely high glucose readings with a glucometer using mutant variant of quinoprotein glucose dehydrogenase (MutQ-GDH chemistry. Potentially fatal hypoglycemia could have been induced in the child if insulin infusion had been initiated as per glycemic management protocol. Even though, the product information with the glucometer carries warning regarding interference by high galactose levels, the awareness regarding this interaction is generally poor in many practice settings. Although, false readings have been reported with glucose dehydrogenase pyrroloquinoline quinone (GDH-PQQ glucometers, to our knowledge this is the first case report of a falsely high glucose reading due to high galactose in a proven case of galactosemia with a glucometer using the MutQ-GDH chemistry (a modified GDH-PQQ chemistry. Our experience has prompted us to write this case report and we suggest avoiding these glucometers in neonates and infants when a metabolic disease is suspected.

  6. Infleunce of pH on the partition of glucose-6-phosphate dehydrogenase and hexokinase in aqueous two-phase system Influência do pH na partição da glicose 6-fosfato desidrogenase e hexoquinase em sistema de duas fases aquosas

    Directory of Open Access Journals (Sweden)

    Daniel Pereira da Silva

    2002-09-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PDH and hexokinase (HK are important enzymes used in biochemical and medical studies and in several analytical methods. Aqueous two-phase system (ATPS formed by a polymer solution and an electrolyte solution provides a method for the separation and purification of enzymes with several advantages, including biocompatibility and easy scale up of the process. In this work, the effects of different pH values on the storage stability and partitioning behavior (K, partition coefficient of the enzymes G6PDH and HK from baker's yeast extract were investigated in ATPS. The results, obtained from the 17.5% PEG 400 : 15.0% phosphate system, showed that when the pH was increased from 5.0 to 8.8, the K HK increased 26-fold and the K G6PDH 2.2-fold. In the 20.0% PEG 1500 : 17.5% phosphate system, the K HK and K G6PDH increased 13 and 1.2-fold, when the pH value was increased from 3.8 to 8.8, respectively. This leads to the conclusion that the partition coefficient for both enzymes is favored by high pH values. A statistical analysis of the results was conducted to confirm this conclusion.Glicose-6-fosfato desidrogenase (G6PDH e hexoquinase (HK são importantes enzimas usadas em estudos bioquímicos e médicos e em diversos métodos analíticos. Sistema de duas fases aquosas (SDFA formado por uma solução polimérica e uma solução eletrolítica proporciona um método para separação e purificação de enzimas com diversas vantagens, incluindo biocompatibilidade, que pode ser facilmente escalonado para nível industrial. Neste trabalho, os efeitos de diferentes valores de pH na estabilidade e na partição (K, coeficiente de partição por SDFA das enzimas G6PDH e HK, obtidas através de levedura de panificação, foram investigados. Os resultados, obtidos do sistema constituído por 17,5% de PEG 400 e 15,0% de fosfato, mostraram que com a elevação do pH de 5,0 para 8,8, o K HK aumentou 26 vezes e o K G6PDH 2,2 vezes

  7. Construction of an integrated enzyme system consisting azoreductase and glucose 1-dehydrogenase for dye removal.

    Science.gov (United States)

    Yang, Yuyi; Wei, Buqing; Zhao, Yuhua; Wang, Jun

    2013-02-01

    Azo dyes are toxic and carcinogenic and are often present in industrial effluents. In this research, azoreductase and glucose 1-dehydrogenase were coupled for both continuous generation of the cofactor NADH and azo dye removal. The results show that 85% maximum relative activity of azoreductase in an integrated enzyme system was obtained at the conditions: 1U azoreductase:10U glucose 1-dehydrogenase, 250mM glucose, 1.0mM NAD(+) and 150μM methyl red. Sensitivity analysis of the factors in the enzyme system affecting dye removal examined by an artificial neural network model shows that the relative importance of enzyme ratio between azoreductase and glucose 1-dehydrogenase was 22%, followed by dye concentration (27%), NAD(+) concentration (23%) and glucose concentration (22%), indicating none of the variables could be ignored in the enzyme system. Batch results show that the enzyme system has application potential for dye removal. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2012-11-01

    Full Text Available Mutagenesis studies on glucose oxidases (GOxs were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe and Aspergillus niger GOx (PDB ID; 1cf3. We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

  9. Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions

    Science.gov (United States)

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

    2012-01-01

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

  10. Diurnal fluctuation of leukocyte G6PD activity. A possible explanation for the normal neutrophil bactericidal activity and the low incidence of pyogenic infections in patients with severe G6PD deficiency in Israel

    NARCIS (Netherlands)

    Wolach, Baruch; Ashkenazi, Meir; Grossmann, Rami; Gavrieli, Ronit; Friedman, Ziva; Bashan, Nava; Roos, Dirk

    2004-01-01

    Acute hemolytic anemia associated with red blood cell (RBC) glucose-6-phosphate dehydrogenase (G6PD) deficiency is commonly encountered in the Mediterranean basin. Nevertheless, concomitant clinical evidence of white blood cell G6PD deficiency is extremely rare in Israel. This study sought to assess

  11. Proteomic and biochemical basis for enhanced growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium.

    Science.gov (United States)

    Kumar, Arvind; Rai, Lal Chand

    2015-01-01

    Proteomics and biochemical analyses were used to unravel the basis for higher growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium compared to soluble. Proteomic analysis using 2-DE, MALDI-TOF/MS and LC-MS revealed the involvement of nine proteins. Down-regulation of fructose bisphosphate aldolase with decreased concentrations of glucose-6-phosphate and fructose-6-phosphate indicated diminished glycolysis. However, up-regulation of phosphoglycerate mutase, increase in the activities of 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrogenase suggested induction of Entner-Doudoroff and pentose phosphate pathways. These pathways generate sufficient energy from gluconic acid, which is also used for biosynthesis as indicated by up-regulation of elongation factor Tu, elongation factor G and protein disulfide isomerase. Increased reactive oxygen species (ROS) formation resulting from organic acid oxidation leads to overexpressed manganese superoxide dismutase and increased activities of catalase and ascorbate peroxidase. Thus the organism uses gluconate instead of glucose for energy, while alleviating extra ROS formation by oxidative defense enzymes. Copyright © 2014 Elsevier GmbH. All rights reserved.

  12. Experimentally Induced Bleaching in the Sea Anemone Exaiptasia Supports Glucose as a Main Metabolite Associated with Its Symbiosis

    Directory of Open Access Journals (Sweden)

    Víctor Hugo Molina

    2017-01-01

    Full Text Available Our current understanding of carbon exchange between partners in the Symbiodinium-cnidarian symbioses is still limited, even though studies employing carbon isotopes have made us aware of the metabolic complexity of this exchange. We examined glycerol and glucose metabolism to better understand how photosynthates are exchanged between host and symbiont. The levels of these metabolites were compared between symbiotic and bleached Exaiptasia pallida anemones, assaying enzymes directly involved in their metabolism. We measured a significant decrease of glucose levels in bleached animals but a significant increase in glycerol and G3P pools, suggesting that bleached animals degrade lipids to compensate for the loss of symbionts and seem to rely on symbiotic glucose. The lower glycerol 3-phosphate dehydrogenase but higher glucose 6-phosphate dehydrogenase specific activities measured in bleached animals agree with a metabolic deficit mainly due to the loss of glucose from the ruptured symbiosis. These results corroborate previous observations on carbon translocation from symbiont to host in the sea anemone Exaiptasia, where glucose was proposed as a main translocated metabolite. To better understand photosynthate translocation and its regulation, additional research with other symbiotic cnidarians is needed, in particular, those with calcium carbonate skeletons.

  13. Mediatorless electron transfer in glucose dehydrogenase/laccase system adsorbed on carbon nanotubes

    International Nuclear Information System (INIS)

    Ratautas, D.; Marcinkevičienė, L.; Meškys, R.; Kulys, J.

    2015-01-01

    Highlights: • Glucose dehydrogenase from Ewingella americana (GDH) demonstrated an effective mediatorless oxidation of glucose on single-walled carbon nanotubes (SWCNT). • Laccase from Trichaptum abietinum (LAC) exhibited mediatorless oxygen reduction when the enzyme was adsorbed on SWCNT. • Simultaneous adsorption of GDH and LAC on SWCNT formed an electron transfer chain in which glucose and lactose were oxidized by oxygen in mediatorless manner. - Abstract: A mediatorless electron transfer in the chain of glucose dehydrogenase (GDH) and laccase (LAC) catalysing the oxidation of glucose by molecular oxygen was studied. To demonstrate mediatorless processes, the GDH from Ewingella americana was adsorbed on single-walled carbon nanotubes (SWCNT). The effective mediatorless oxidation of glucose proceeded at 0.2–0.4 V vs. SCE. The electrode was most active at pH 6.1, and generated 0.8 mA cm −2 biocatalytic current in the presence of 50 mM glucose. The electrode showed a bell-shaped pH dependence with pK a values of 4.1 and 7.5. LAC from Trichaptum abietinum adsorbed on SWCNT exhibited mediatorless oxygen reduction at electrode potential less than 0.65 V. The electrode was most active at pH 3.0–4.0 and generated 1.1 mA cm −2 biocatalytic current in the presence of 0.254 mM oxygen, with an apparent pK a of 1.0 and 5.4. The electrodes prepared by simultaneous adsorption of GDH and LAC on SWCNT exhibited glucose oxidation at a potential higher than 0.25 V. The oxygen consumption in the chain was demonstrated using a Clark-type oxygen electrode. The dependence of oxygen consumption on glucose and lactose concentrations as well as activity of the system on pH were measured. A model of the pH dependence as well as mediatorless consecutive glucose oxidation with oxygen catalysed by LAC/GDH system is presented. This work provides a novel approach towards the synthesis of artificial multi enzyme systems by wiring oxidoreductases with SWCNT, and offers a better

  14. Evidence for catabolite degradation in the glucose-dependent inactivation of yeast cytoplasmic malate dehydrogenase

    International Nuclear Information System (INIS)

    Neeff, J.; Haegele, E.; Nauhaus, J.; Heer, U.; Mecke, D.

    1978-01-01

    The cytoplasmic malate dehydrogenase of Saccharomyces cerevisiae was radioactively labeled during its synthesis on a glucose-free derepression medium. After purification a sensitive radioimmunoassay for this enzyme could be developed. The assay showed that after the physiological, glucose-dependent 'catabolite inactivation' of cytoplasmic malate dehydrogenase an inactive enzyme protein is immunologically not detectable. Together with the irreversibility of this reaction in vivo this finding strongly suggests a proteolytic mechanism of enzyme inactivation. For this process the term 'catabolite degradation' is used. (orig.) [de

  15. The chemopreventive properties of chlorogenic acid reveal a potential new role for the microsomal glucose-6-phosphate translocase in brain tumor progression

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    Desgagnés Julie

    2006-03-01

    Full Text Available Abstract Background Chlorogenic acid (CHL, the most potent functional inhibitor of the microsomal glucose-6-phosphate translocase (G6PT, is thought to possess cancer chemopreventive properties. It is not known, however, whether any G6PT functions are involved in tumorigenesis. We investigated the effects of CHL and the potential role of G6PT in regulating the invasive phenotype of brain tumor-derived glioma cells. Results RT-PCR was used to show that, among the adult and pediatric brain tumor-derived cells tested, U-87 glioma cells expressed the highest levels of G6PT mRNA. U-87 cells lacked the microsomal catalytic subunit glucose-6-phosphatase (G6Pase-α but expressed G6Pase-β which, when coupled to G6PT, allows G6P hydrolysis into glucose to occur in non-glyconeogenic tissues such as brain. CHL inhibited U-87 cell migration and matrix metalloproteinase (MMP-2 secretion, two prerequisites for tumor cell invasion. Moreover, CHL also inhibited cell migration induced by sphingosine-1-phosphate (S1P, a potent mitogen for glioblastoma multiform cells, as well as the rapid, S1P-induced extracellular signal-regulated protein kinase phosphorylation potentially mediated through intracellular calcium mobilization, suggesting that G6PT may also perform crucial functions in regulating intracellular signalling. Overexpression of the recombinant G6PT protein induced U-87 glioma cell migration that was, in turn, antagonized by CHL. MMP-2 secretion was also inhibited by the adenosine triphosphate (ATP-depleting agents 2-deoxyglucose and 5-thioglucose, a mechanism that may inhibit ATP-mediated calcium sequestration by G6PT. Conclusion We illustrate a new G6PT function in glioma cells that could regulate the intracellular signalling and invasive phenotype of brain tumor cells, and that can be targeted by the anticancer properties of CHL.

  16. The crystal structure of galactitol-1-phosphate 5-dehydrogenase from Escherichia coli K12 provides insights into its anomalous behavior on IMAC processes.

    Science.gov (United States)

    Esteban-Torres, María; Alvarez, Yanaisis; Acebrón, Iván; de las Rivas, Blanca; Muñoz, Rosario; Kohring, Gert-Wieland; Roa, Ana María; Sobrino, Mónica; Mancheño, José M

    2012-09-21

    Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni(2+)-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn(2+)- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Hepatic glucose-6-phosphatase-α deficiency leads to metabolic reprogramming in glycogen storage disease type Ia.

    Science.gov (United States)

    Cho, Jun-Ho; Kim, Goo-Young; Mansfield, Brian C; Chou, Janice Y

    2018-04-15

    Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in endogenous glucose production. This autosomal recessive disorder is characterized by impaired glucose homeostasis and long-term complications of hepatocellular adenoma/carcinoma (HCA/HCC). We have shown that hepatic G6Pase-α deficiency-mediated steatosis leads to defective autophagy that is frequently associated with carcinogenesis. We now show that hepatic G6Pase-α deficiency also leads to enhancement of hepatic glycolysis and hexose monophosphate shunt (HMS) that can contribute to hepatocarcinogenesis. The enhanced hepatic glycolysis is reflected by increased lactate accumulation, increased expression of many glycolytic enzymes, and elevated expression of c-Myc that stimulates glycolysis. The increased HMS is reflected by increased glucose-6-phosphate dehydrogenase activity and elevated production of NADPH and the reduced glutathione. We have previously shown that restoration of hepatic G6Pase-α expression in G6Pase-α-deficient liver corrects metabolic abnormalities, normalizes autophagy, and prevents HCA/HCC development in GSD-Ia. We now show that restoration of hepatic G6Pase-α expression normalizes both glycolysis and HMS in GSD-Ia. Moreover, the HCA/HCC lesions in L-G6pc-/- mice exhibit elevated levels of hexokinase 2 (HK2) and the M2 isoform of pyruvate kinase (PKM2) which play an important role in aerobic glycolysis and cancer cell proliferation. Taken together, hepatic G6Pase-α deficiency causes metabolic reprogramming, leading to enhanced glycolysis and elevated HMS that along with impaired autophagy can contribute to HCA/HCC development in GSD-Ia. Published by Elsevier Inc.

  18. Glucose uptake and its effect on gene expression in prochlorococcus.

    Directory of Open Access Journals (Sweden)

    Guadalupe Gómez-Baena

    Full Text Available The marine cyanobacteria Prochlorococcus have been considered photoautotrophic microorganisms, although the utilization of exogenous sugars has never been specifically addressed in them. We studied glucose uptake in different high irradiance- and low irradiance-adapted Prochlorococcus strains, as well as the effect of glucose addition on the expression of several glucose-related genes. Glucose uptake was measured by adding radiolabelled glucose to Prochlorococcus cultures, followed by flow cytometry coupled with cell sorting in order to separate Prochlorococcus cells from bacterial contaminants. Sorted cells were recovered by filtration and their radioactivity measured. The expression, after glucose addition, of several genes (involved in glucose metabolism, and in nitrogen assimilation and its regulation was determined in the low irradiance-adapted Prochlorococcus SS120 strain by semi-quantitative real time RT-PCR, using the rnpB gene as internal control. Our results demonstrate for the first time that the Prochlorococcus strains studied in this work take up glucose at significant rates even at concentrations close to those found in the oceans, and also exclude the possibility of this uptake being carried out by eventual bacterial contaminants, since only Prochlorococcus cells were used for radioactivity measurements. Besides, we show that the expression of a number of genes involved in glucose utilization (namely zwf, gnd and dld, encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and lactate dehydrogenase, respectively is strongly increased upon glucose addition to cultures of the SS120 strain. This fact, taken together with the magnitude of the glucose uptake, clearly indicates the physiological importance of the phenomenon. Given the significant contribution of Prochlorococcus to the global primary production, these findings have strong implications for the understanding of the phytoplankton role in the carbon

  19. Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

    Science.gov (United States)

    Besson, Marie Thérèse; Alegría, Karin; Garrido-Gerter, Pamela; Barros, Luis Felipe; Liévens, Jean-Charles

    2015-01-01

    Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93). We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP) impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK) which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3

  20. Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

    Directory of Open Access Journals (Sweden)

    Marie Thérèse Besson

    Full Text Available Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93. We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD, the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to

  1. Hyperbilirubinaemia and erythrocytic glucose 6 phosphate dehydrogenase deficiency in Malaysian children.

    Science.gov (United States)

    Hon, A T; Balakrishnan, S; Ahmad, Z

    1989-03-01

    Cord blood from 8,975 babies delivered in Hospital Sultanah Aminah Johor Bahru over a period of eight months (1st August 1985 to 31st March 1986) were screened for G6PD deficiency. The overall incidence was 4.5% in Chinese, 3.5% in Malays and 1.5% in Indian babies. One hundred of these babies were observed in the nursery for seven days and their daily serum bilirubin recorded. The serum bilirubin peaked at 96 hours to a value of 12mg%. None of the babies in the nursery developed a serum bilirubin level of more than 15mg%. Six of the babies with G6PD deficiency that were sent home were readmitted with hyperbilirubinaemia that needed exchange transfusion.

  2. Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Durand, Fabien [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France); Stines-Chaumeil, Claire [Universite de Bordeaux, CNRS, Institut de Biochimie et de Genetique Cellulaires, 1 rue Camille Saint Saens, 33077 Bordeaux Cedex (France); Flexer, Victoria [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France); Andre, Isabelle [Universite de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France); CNRS, UMR5504, F-31400 Toulouse (France); INRA, UMR 792 Ingenierie des Systemes Biologiques et des Procedes, F-31400 Toulouse (France); Mano, Nicolas, E-mail: mano@crpp-bordeaux.cnrs.fr [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France)

    2010-11-26

    Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

  3. Effects of dehydroepiandrosterone (DHEA) on glucose metabolism in isolated hepatocytes from Zucker rats

    International Nuclear Information System (INIS)

    Finan, A.; Cleary, M.P.

    1986-01-01

    DHEA has been shown to competitively inhibit the pentose phosphate shunt (PPS) enzyme glucose-6-phosphate dehydrogenase (G6PD) when added in vitro to supernatants or homogenates prepared from mammalian tissues. However, no consistent effect on G6PD activity has been determined in tissue removed from DHEA-treated rats. To explore the effects of DHEA on PPS, glucose utilization was measured in hepatocytes from lean and obese male Zucker rats (8 wks of age) following 1 wk of DHEA treatment (0.6% in diet). Incubation of isolated hepatocytes from treated lean Zucker rats with either [1- 14 C] glucose or [6- 14 C] glucose resulted in significant decreases in CO 2 production and total glucose utilization. DHEA-lean rats also had lowered fat pad weights. In obese rats, there was no effect of 1 wk of treatment on either glucose metabolism or fat pad weight. The calculated percent contribution of the PPS to glucose metabolism in hepatocytes was not changed for either DHEA-lean or obese rats when compared to control rats. In conclusion, 1 wk of DHEA treatment lowered overall glucose metabolism in hepatocytes of lean Zucker rats, but did not selectively affect the PPS. The lack of an effect of short-term treatment in obese rats may be due to differences in their metabolism or storage/release of DHEA in tissues in comparison to lean rats

  4. Reduced prevalence of Plasmodium falciparum infection and of concomitant anaemia in pregnant women with heterozygous G6PD deficiency

    NARCIS (Netherlands)

    Mockenhaupt, Frank P.; Mandelkow, Jantina; Till, Holger; Ehrhardt, Stephan; Eggelte, Teunis A.; Bienzle, Ulrich

    2003-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency confers protection against malaria in children, yet its role in malaria in pregnancy is unknown. In a cross-sectional study among 529 pregnant Ghanaian women, Plasmodium falciparum infection, anaemia and G6PD genotypes were assessed. Of these,

  5. Deficiencia de glucosa-6-fosfato deshidrogenasa en un paciente con síndrome de Down

    Directory of Open Access Journals (Sweden)

    Francisco R. Cammarata Scalisi

    2012-07-01

    Full Text Available El síndrome de Down, es una alteración genética que ocurre cuando un individuo exhibe todo o una parte específica adicional del cromosoma 21 y es la entidad más frecuentemente asociada a retardo mental. La deficiencia de glucosa-6-fosfato deshidrogenasa, es el defecto enzimático más común en humanos y presenta patrón de herencia ligado al cromosoma X recesivo. Se debe a la mutación del gen G6PD, el cual causa diversos fenotipos bioquímicos y clínicos. Reportamos un caso de lactante menor masculino, evaluado en la Unidad de Genética Médica de la Universidad de Los Andes, con el diagnóstico de deficiencia de glucosa-6-fosfato deshidrogenasa con doble mutación A376G y G202A y síndrome de Down con estudio citogenético 47, XY, +21. Palabras clave:Síndrome de Down; deficiencia de glucosa-6-fosfato deshidrogenasa; G6PD; A37G6; G202A. Glucose-6-phosphate dehydrogenase deficiency in a patient with Down syndrome Abstract Down syndrome, is a genetic disorder that occurring when an individual exhibits all or part of an extra copy of chromosome 21 and the most common entity associated mental retardation. Glucose-6-phosphate dehydrogenase deficiency, is the most common human enzyme defect and has a X-linked recessive inheritance. Due to mutations in the G6PD gene, which cause many biochemical and clinical phenotypes. We reported a case of child male, evaluated in the Unit of Medical Genetics of the University of The Andes, with diagnosis of glucose-6-phosphate dehydrogenase deficiency with double mutation A376G and G202A and Down syndrome with cytogenetic study 47, XY, + 21.

  6. Glucose-6-phosphate dehydrogenase (G6PD mutations and haemoglobinuria syndrome in the Vietnamese population

    Directory of Open Access Journals (Sweden)

    Day Nick

    2009-07-01

    Full Text Available Abstract Background In Vietnam the blackwater fever syndrome (BWF has been associated with malaria infection, quinine ingestion and G6PD deficiency. The G6PD variants within the Vietnamese Kinh contributing to the disease risk in this population, and more generally to haemoglobinuria, are currently unknown. Method Eighty-two haemoglobinuria patients and 524 healthy controls were screened for G6PD deficiency using either the methylene blue reduction test, the G-6-PDH kit or the micro-methaemoglobin reduction test. The G6PD gene variants were screened using SSCP combined with DNA sequencing in 82 patients with haemoglobinuria, and in 59 healthy controls found to be G6PD deficient. Results This study confirmed that G6PD deficiency is strongly associated with haemoglobinuria (OR = 15, 95% CI [7.7 to 28.9], P G6PD variants were identified in the Vietnamese population, of which two are novel (Vietnam1 [Glu3Lys] and Vietnam2 [Phe66Cys]. G6PD Viangchan [Val291Met], common throughout south-east Asia, accounted for 77% of the variants detected and was significantly associated with haemoglobinuria within G6PD-deficient ethnic Kinh Vietnamese (OR = 5.8 95% CI [114-55.4], P = 0.022. Conclusion The primary frequency of several G6PD mutations, including novel mutations, in the Vietnamese Kinh population are reported and the contribution of G6PD mutations to the development of haemoglobinuria are investigated.

  7. F-19 MR imaging of glucose metabolism in the rat and rabbit

    International Nuclear Information System (INIS)

    Nakada, T.; Kwee, I.L.; Card, P.J.; Matwiyoff, N.A.; Griffey, B.V.; Griffey, R.H.

    1987-01-01

    MR imaging reflecting regional pathway specific glucose metabolism was performed utilizing F-19 as the MR signal probe and two fluorinated glucose analogues, 2-fluoro-2-deoxy-D-glucose (2-FDG) and 3-fluoro-3-deoxy-D-glucose (3-FDG) as the metabolic probe. 2-FDG-6-phosphate images provides regional quantitative information regarding glycolytic activities, while 2-FDG-6-phosphoglyconate images provide information on the pentose monophosphate shunt activities. 3-FDG-sorbitol and 3-FDG-fructose indicate regional aldose reductase and sorbitol dehydrogenase activities of the aldose reductase sorbitol pathway, respectively. The potential toxicity of 2-FDG in high doses precludes the immediate application of the 2-FDG MR imaging method to humans. The extremely low toxicity of 3-FDG, however, indicates promise for clinical application of 3-FDG MR imaging

  8. Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.

    Science.gov (United States)

    Timm, Kerstin N; Hartl, Johannes; Keller, Markus A; Hu, De-En; Kettunen, Mikko I; Rodrigues, Tiago B; Ralser, Markus; Brindle, Kevin M

    2015-12-01

    A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells. © 2014 Wiley Periodicals, Inc.

  9. Atividade da 6-fosfogliconato desidrogenase em deficientes de glicose-6-fosfato desidrogenase Activity of 6-phosphogluconate dehydrogenase in glucose-6-phosphate dehydrogenase deficiency

    Directory of Open Access Journals (Sweden)

    Daniela B. Nicolielo

    2006-06-01

    Full Text Available As enzimas G6PD e 6PGD são responsáveis pela geração do aporte de NADPH, necessário para a detoxificação dos agentes oxidantes produzidos pelo estresse oxidativo metabólico nos eritrócitos. Devido à alta prevalência de deficiência de G6PD na população mundial, principalmente de origem negróide africana, muitos estudos têm sido realizados na tentativa de conhecer melhor a atuação destas enzimas. O objetivo deste estudo foi avaliar a atividade enzimática da 6PGD, nos deficientes de G6PD, para verificar a existência de aumento da atividade desta enzima, correlacionando com um possível aumento do número de reticulócitos ou presença de alterações da série vermelha. A pesquisa em 2.657 indivíduos do sexo masculino resultou em 97 deficientes de G6PD, determinando uma prevalência de 3,65% para a região de Bauru (SP, com atividade enzimática média de G6PD de 1,74 UI.g Hb-1. min-1 a 37ºC, 14,4% da atividade da G6PD normal. A atividade enzimática média da 6PGD foi de 9,5 UI.g Hb-1. min-1 a 37ºC, estando aumentada em 47,4% dos deficientes de G6PD. Os resultados não confirmaram que a hipótese do aumento da atividade enzimática da 6PGD, em deficientes de G6PD, seja decorrente da presença de um número aumentado de reticulócitos na corrente circulatória, faixa etária ou alterações eritrocitométricas que denotem anemia. O mais provável é que a hemólise autolimitada, imposta pelos processos oxidativos, preserve os eritrócitos mais jovens, que possuem atividade enzimática mais elevada, uma vez que naturalmente ocorre diminuição da atividade destas enzimas com o envelhecimento celular.The G6PD and 6PGD enzymes are responsible for the generation of NADPH supply necessary for the detoxification of the oxidant agents produced during the oxidative metabolic stress on erythrocytes. Due to the high prevalence of the deficiency of G6PD on world population, especially on Afro descents, many studies have been done trying

  10. Control of Glycolysis by Glyceraldehyde-3-Phosphate Dehydrogenase in Streptococcus cremoris and Streptococcus lactis

    NARCIS (Netherlands)

    POOLMAN, B; BOSMAN, B; KONINGS, WN

    1987-01-01

    The decreased response of the energy metabolism of lactose-starved Streptococcus cremoris upon readdition of lactose is caused by a decrease of the glycolytic activity. The decrease in glycolysis is accompanied by a decrease in the activities of glyceraldehyde-3-phosphate dehydrogenase and

  11. Determination of dehydrogenase activities involved in D-glucose oxidation in Gluconobacter and Acetobacter strains

    Directory of Open Access Journals (Sweden)

    Florencia Sainz

    2016-08-01

    Full Text Available Acetic acid bacteria (AAB are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane bound dehydrogenases. In the present study, the enzyme activity of the membrane bound dehydrogenases (membrane-bound PQQ-glucose dehydrogenase (mGDH, D-gluconate dehydrogenase (GADH and membrane-bound glycerol dehydrogenase (GLDH involved in the oxidation of D-glucose and D-gluconic acid (GA was determined in six strains of three different species of AAB (three natural and three type strains. Moreover, the effect of these activities on the production of related metabolites (GA, 2-keto-D-gluconic acid (2KGA and 5-keto-D-gluconic acid (5KGA was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the A. malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h, which coincided with glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of G. oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition.Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter were

  12. Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI by high-throughput screening of existing drugs

    Directory of Open Access Journals (Sweden)

    Rana Eltahan

    2018-04-01

    Full Text Available Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI and determined its Michaelis constant towards fructose-6-phosphate (Km = 0.309 mM, Vmax = 31.72 nmol/μg/min. We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC50 = 8.33 μM; Ki = 36.33 μM, while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC50 = 165 μM at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC50 on HCT-8 cells = 700 μM. Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. Keywords: Apicomplexan, Cryptosporidium parvum, Glucose-6-phosphate isomerase (GPI, Ebselen

  13. Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

    NARCIS (Netherlands)

    Wisselink, H.W.; Mars, A.E.; Meer, van der P.; Eggink, G.; Hugenholtz, J.

    2004-01-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance

  14. ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Mráček, Tomáš; Holzerová, Eliška; Drahota, Zdeněk; Kovářová, Nikola; Vrbacký, Marek; Ješina, Pavel; Houštěk, Josef

    2014-01-01

    Roč. 1837, č. 1 (2014), s. 98-111 ISSN 0005-2728 R&D Projects: GA ČR(CZ) GPP303/10/P227; GA MŠk(CZ) LL1204 Grant - others:Univerzita Karlova(CZ) 750213 Institutional support: RVO:67985823 Keywords : mitochondrial glycerol-3-phosphate dehydrogenase * ROS production * supercomplex * in-gel ROS detection Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.353, year: 2014

  15. TLQP-21 protects human umbilical vein endothelial cells against high-glucose-induced apoptosis by increasing G6PD expression.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available Hyperglycemia causes oxidative stress that could damage vascular endothelial cells, leading to cardiovascular complications. The Vgf gene was identified as a nerve growth factor-responsive gene, and its protein product, VGF, is characterized by the presence of partially cleaved products. One of the VGF-derived peptides is TLQP-21, which is composed of 21 amino acids (residues 556-576. Past studies have reported that TLQP-21 could stimulate insulin secretion in pancreatic cells and protect these cells from apoptosis, which suggests that TLQP-21 has a potential function in diabetes therapy. Here, we explore the protective role of TLQP-21 against the high glucose-mediated injury of vascular endothelial cells. Using human umbilical vascular endothelial cells (HUVECs, we demonstrated that TLQP-21 (10 or 50 nM dose-dependently prevented apoptosis under high-glucose (30 mmol/L conditions (the normal glucose concentration is 5.6 mmol/L. TLQP-21 enhanced the expression of NAPDH, resulting in upregulation of glutathione (GSH and a reduction in the levels of reactive oxygen species (ROS. TLQP-21 also upregulated the expression of glucose-6-phosphate dehydrogenase (G6PD, which is known as the main source of NADPH. Knockdown of G6PD almost completely blocked the increase of NADPH induced by TLQP-21, indicating that TLQP-21 functions mainly through G6PD to promote NADPH generation. In conclusion, TLQP-21 could increase G6PD expression, which in turn may increase the synthesis of NADPH and GSH, thereby partially restoring the redox status of vascular endothelial cells under high glucose injury. We propose that TLQP-21 is a promising drug for diabetes therapy.

  16. Effect of tamoxifen pre-treatment on the retention of tritiated oestradiol and 5. cap alpha. -dihydrotestosterone and on glucose metabolism in human breast carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Deshpande, N; Mitchell, I [Imperial Cancer Research Fund, London (UK). Labs.; Hughes, D

    1978-05-01

    The effect of pre-treatment with tamoxifen on glucose metabolism and retention of injected oestradiol-17B and 5..cap alpha..-dihydrotestosterone by human breast carcinomas were studied in patients undergoing mastectomy. The following effects were observed: the pretreatment reduced retention of oestradiol-17B whereas a small but statistically significant rise in 5..cap alpha..-dihydrotestosterone accumulation was observed. There was an increase in both phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase (G6PDH) activities in tumours from treated patients whereas ..cap alpha..-glycerolphosphate dehydrogenase (..cap alpha..-GPDH) activity was significantly reduced in the same tumours. The significance of these findings is discussed and it is argued that these changes in carbohydrate metabolism may not be due to the blocking of hormone receptors.

  17. Detection of transketolase in bone marrow-derived insulin-producing cells: benfotiamine enhances insulin synthesis and glucose metabolism.

    Science.gov (United States)

    Oh, Seh-Hoon; Witek, Rafal P; Bae, Si-Hyun; Darwiche, Houda; Jung, Youngmi; Pi, Liya; Brown, Alicia; Petersen, Bryon E

    2009-01-01

    Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus.

  18. Detection of Transketolase in Bone Marrow—Derived Insulin-Producing Cells: Benfotiamine Enhances Insulin Synthesis and Glucose Metabolism

    Science.gov (United States)

    Witek, Rafal P.; Bae, Si-Hyun; Darwiche, Houda; Jung, Youngmi; Pi, Liya; Brown, Alicia; Petersen, Bryon E.

    2009-01-01

    Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus. PMID:18393672

  19. A pyrroloquinolinequinone-dependent glucose dehydrogenase (PQQ-GDH)-electrode with direct electron transfer based on polyaniline modified carbon nanotubes for biofuel cell application

    International Nuclear Information System (INIS)

    Schubart, Ivo W.; Göbel, Gero; Lisdat, Fred

    2012-01-01

    Graphical abstract: - Abstract: In this study we present a pyrroloquinolinequinone-dependent glucose dehydrogenase [(PQQ)-GDH] electrode with direct electron transfer between the enzyme and electrode. Soluble pyrroloquinolinequinone-dependent glucose dehydrogenase from Acinetobacter calcoaceticus is covalently bound to an electropolymerized polyaniline copolymer film on a multi-walled carbon nanotube (MWCNT)-modified gold electrode. The pulsed electropolymerization of 2-methoxyaniline-5-sulfonic acid (MASA) and m-aminobenzoic acid (ABA) is optimized with respect to the efficiency of the bioelectrocatalytic conversion of glucose. The glucose oxidation starts at −0.1 V vs. Ag/AgCl and current densities up to 500 μA/cm 2 at low potential of +0.1 V vs. Ag/AgCl can be achieved. The electrode shows a glucose sensitivity in the range from 0.1 mM to 5 mM at a potential of +0.1 V vs. Ag/Ag/Cl. The dynamic range is extended to 100 mM at +0.4 V vs. Ag/AgCl. The electron transfer mechanism is studied and buffer effects are investigated. The developed enzyme electrode is examined for bioenergetic application by assembling of a membrane-less biofuel cell. For the cathode a bilirubin oxidase (BOD) based MWCNT-modified gold electrode with direct electron transfer (DET) is used. The biofuel cell exhibits a cell potential of 680 ± 20 mV and a maximum power density of up to 65 μW/cm 2 at 350 mV vs. Ag/AgCl.

  20. Improved Xylitol Production from D-Arabitol by Enhancing the Coenzyme Regeneration Efficiency of the Pentose Phosphate Pathway in Gluconobacter oxydans.

    Science.gov (United States)

    Li, Sha; Zhang, Jinliang; Xu, Hong; Feng, Xiaohai

    2016-02-10

    Gluconobacter oxydans is used to produce xylitol from D-arabitol. This study aims to improve xylitol production by increasing the coenzyme regeneration efficiency of the pentose phosphate pathway in G. oxydans. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were overexpressed in G. oxydans. Real-time PCR and enzyme activity assays revealed that G6PDH/6PGDH activity and coenzyme regeneration efficiency increased in the recombinant G. oxydans strains. Approximately 29.3 g/L xylitol was obtained, with a yield of 73.2%, from 40 g/L d-arabitol in the batch biotransformation with the G. oxydans PZ strain. Moreover, the xylitol productivity (0.62 g/L/h) was 3.26-fold of the wild type strain (0.19 g/L/h). In repetitive batch biotransformation, the G. oxydans PZ cells were used for five cycles without incurring a significant loss in productivity. These results indicate that the recombinant G. oxydans PZ strain is economically feasible for xylitol production in industrial bioconversion.

  1. Metabolism of excised embryos of Lupinus luteus L. VI. An electrophoretic analysis of some dehydrogenases in cultured embryos as compared with the normal seedling axes

    Directory of Open Access Journals (Sweden)

    J. Czosnowski

    2015-01-01

    Full Text Available The electrophoretic patterns (disc electrophoresis of the studied dehydrogenases: glucose-6-phosphate - (A, malate - (B, glutamate - (C, alcohol - (D and lactate dehydrogenase (E, in the axial organs of isolated Lupinus luteus embryos and seedlings cultivated over 12 days are characterized by great similarities. With time, after the third day of cultivation the patterns begin to become less deyeloped. Analyses performed during the first 10 hours of imbibition of seed parts indicate that the maximal development of isozyme patterns occurs during the third hour after which the patterns become poorer. The most uniform type of pattern. and the lowest number of isozymes was shown by glutamate dehydrogenase, the richest pattern was shown by malate dehydrogenase. No band common for a 11 the 27 experimental elements was found.

  2. Gaseous environment of plants and activity of enzymes of carbohydrate catabolism

    International Nuclear Information System (INIS)

    Ivanov, B.F.; Zemlyanukhin, A.A.; Igamberdiev, A.U.; Salam, A.M.M.

    1989-01-01

    The authors investigated the action of hypoxia and high CO 2 concentration in the atmosphere on activity of phosphofructokinase, aldolase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, and isocitrate lyase in pea seedlings (Pisum sativum L.), corn scutella (Zea mays L.), and hemp cotyledons (Cannabis sativa L.). The first 4-12h of hypoxia witnessed suppression of enzymes of the initial stages of glycolysis (glucose-6-phosphate isomerase, phosphofructokinase)and activation of enzymes of its final stages (alcohol dehydrogenase and lactate dehydrogenase) and enzymes linking glycolysis and the pentose phosphate pathway (aldolase and glucose-6-phosphate dehydrogenase). An excess of CO 2 in the environment accelerated and amplified this effect. At the end of a 24-h period of anaerobic incubation, deviations of enzyme activity from the control were leveled in both gaseous environments. An exception was observed in the case of phosphofructokinase, whose activity increased markedly at this time in plants exposed to CO 2 . Changes in activity of the enzymes were coupled with changes in their kinetic parameters (apparent K m and V max values). The activity of isocitrate lyase was suppressed in both variants of hypoxic gaseous environments, a finding that does not agree with the hypothesis as to participation of the glyoxylate cycle in the metabolic response of plants to oxygen stress. Thus, temporary inhibition of the system of glycolysis and activation of the pentose phosphate pathway constituted the initial response of the plants to O 2 stress, and CO 2 intensified this metabolic response

  3. [Enzyme kinetic glucose determination by the glucose dehydrogenase method. Enzyme kinetic substrate determination using competitive inhibitors, II (author's transl)].

    Science.gov (United States)

    Müller-Matthesius, R

    1975-05-01

    The sensitivity of enzyme kinetic substrate determinations can be improved with the aid of competitive inhibitors. As an example, the determination of glucose dehydrogenase in the presence of potassium thiocyanate is described. The method has the advantage of rapid operation with satisfactory precision.

  4. Glucose metabolic alterations in hippocampus of diabetes mellitus rats and the regulation of aerobic exercise.

    Science.gov (United States)

    Li, Jingjing; Liu, Beibei; Cai, Ming; Lin, Xiaojing; Lou, Shujie

    2017-11-04

    Diabetes could negatively affect the structures and functions of the brain, especially could cause the hippocampal dysfunction, however, the potential metabolic mechanism is unclear. The aim of this study was to investigate the changes of glucose metabolism in hippocampus of diabetes mellitus rats and the regulation of aerobic exercise, and to analyze the possible mechanisms. A rat model of type 2 diabetes mellitus was established by high-fat diet feeding in combination with STZ intraperitoneal injection, then 4 weeks of aerobic exercise was conducted. The glucose metabolites and key enzymes involved in glucose metabolism in hippocampus were respectively detected by GC/MS based metabolomics and western blot. Metabolomics results showed that compared with control rats, the level of citric acid was significantly decreased, while the levels of lactic acid, ribose 5-phosphate, xylulose 5-phosphate and glucitol were significantly increased in the diabetic rat. Compared with diabetic rats, the level of citric acid was significantly increased, while the lactic acid, ribose 5-phosphate and xylulose 5-phosphate were significantly decreased in the diabetic exercise rats. Western blot results showed that lower level of citrate synthase and oxoglutarate dehydrogenase, higher level of aldose reductase and glucose 6-phosphatedehydrogenase were found in the diabetic rats when compared to control rats. After 4 weeks of aerobic exercise, citrate synthase was upregulated and glucose 6-phosphatedehydrogenase was downregulated in the diabetic rats. These results suggest that diabetes could cause abnormal glucose metabolism, and aerobic exercise plays an important role in regulating diabetes-induced disorder of glucose metabolism in the hippocampus. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Final Report for research on The Glucose 6-Phosphate Shunt Around the Calvin-Benson Cycle

    Energy Technology Data Exchange (ETDEWEB)

    Sharkey, Thomas D. [Michigan State Univ., East Lansing, MI (United States)

    2017-10-30

    In this research, photosynthetic carbon metabolism was studied to identify mechanisms by which plants store energy from sunlight as carbon compounds, especially sugars. Conditions were identified in which carbon appeared to flow backwards from outside the photosynthetic compartment (chloroplast) back into it. A specific gene product was manipulated to make the flow bigger or smaller. Preventing the flow (by eliminating the gene) had little effect on plant growth but increasing the flow, by overexpressing the gene, caused the plants to become extremely sensitive to changes in light. Plants with the gene overexpressed had high rates of cyclic electron flow, the photosynthetic electron transport pathway that occurs when plants need more of the energy molecule ATP. These and other observations led us to conclude that a metabolic pathway that is normally turned off because it is counter-productive during photosynthesis, in fact occurs at about 10% of the rate of normal photosynthesis. This creates an inefficiency but may stabilize photosynthesis allowing it to cope with the very large and rapid changes that leaves experience such as the hundred-fold changes in light intensity that can occur in seconds on a partly cloudy day. We also concluded that the back flow of carbon into chloroplasts could be important at high rates of photosynthesis allowing increased rates of starch synthesis. Starch synthesis allows plants to store sugars during the day for use at night. At high rates of photosynthesis starch synthesis becomes very important to protect against end-product inhibition of photosynthesis. This research identified two metabolic pathways that extend the primary carbon fixation pathway called the Calvin-Benson cycle. These pathway extensions are now called the cytosolic bypass and the glucose 6-phosphate shunt. This improvement in our understanding of carbon metabolism of photosynthesis will guide efforts to increase photosynthesis to increase production of food, fuel

  6. Modulation of NADP(+)-dependent isocitrate dehydrogenase in aging.

    Science.gov (United States)

    Kil, In Sup; Lee, Young Sup; Bae, Young Seuk; Huh, Tae Lin; Park, Jeen-Woo

    2004-01-01

    NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP(+)-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46-48 population doubling level (PDL) and then gradually decreased at later PDL. 2',7'-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.

  7. Novel fungal FAD glucose dehydrogenase derived from Aspergillus niger for glucose enzyme sensor strips.

    Science.gov (United States)

    Sode, Koji; Loew, Noya; Ohnishi, Yosuke; Tsuruta, Hayato; Mori, Kazushige; Kojima, Katsuhiro; Tsugawa, Wakako; LaBelle, Jeffrey T; Klonoff, David C

    2017-01-15

    In this study, a novel fungus FAD dependent glucose dehydrogenase, derived from Aspergillus niger (AnGDH), was characterized. This enzyme's potential for the use as the enzyme for blood glucose monitor enzyme sensor strips was evaluated, especially by investigating the effect of the presence of xylose during glucose measurements. The substrate specificity of AnGDH towards glucose was investigated, and only xylose was found as a competing substrate. The specific catalytic efficiency for xylose compared to glucose was 1.8%. The specific activity of AnGDH for xylose at 5mM concentration compared to glucose was 3.5%. No other sugars were used as substrate by this enzyme. The superior substrate specificity of AnGDH was also demonstrated in the performance of enzyme sensor strips. The impact of spiking xylose in a sample with physiological glucose concentrations on the sensor signals was investigated, and it was found that enzyme sensor strips using AnGDH were not affected at all by 5mM (75mg/dL) xylose. This is the first report of an enzyme sensor strip using a fungus derived FADGDH, which did not show any positive bias at a therapeutic level xylose concentration on the signal for a glucose sample. This clearly indicates the superiority of AnGDH over other conventionally used fungi derived FADGDHs in the application for SMBG sensor strips. The negligible activity of AnGDH towards xylose was also explained on the basis of a 3D structural model, which was compared to the 3D structures of A. flavus derived FADGDH and of two glucose oxidases. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

    OpenAIRE

    Sygmund, Christoph; Staudigl, Petra; Klausberger, Miriam; Pinotsis, Nikos; Djinovic-Carugo, Kristina; Gorton, Lo; Haltrich, Dietmar; Ludwig, Roland

    2011-01-01

    Abstract Background FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spr...

  9. Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

    OpenAIRE

    Wisselink, H. Wouter; Mars, Astrid E.; van der Meer, Pieter; Eggink, Gerrit; Jeroen Hugenholtz

    2004-01-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and 13C nuclear magnetic resonance analysis revealed that small amounts (

  10. Analysis of trehalose-6-phosphate control over carbon allocation and growth in plants

    NARCIS (Netherlands)

    Aghdasi, M.

    2007-01-01

    Trehalose is the non-reducing alpha-alpha-1, 1-linked glucose disaccharide. The biosynthesic precursor of trehalose, trehalose-6-phosphate (T6P), is essential for plant development, growth, carbon utilization and alters photosynthetic capacity but its mode of action is not underestood. This thesis

  11. assessment of the activity of glucose-6-phosphate dehydrogenase

    African Journals Online (AJOL)

    Uwaifoh

    2012-10-31

    Oct 31, 2012 ... Chemical Pathology, Irrua Specialist Teaching Hospital, Irrua, Edo ... in type 2 diabetes mellitus patients and control subjects using enzymatic ... inappropriate antioxidation process. ... without a previous diagnosis of diabetes develop a ... variants. It confers protection against malaria, which accounts for its ...

  12. Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver

    NARCIS (Netherlands)

    Griffini, P.; Vigorelli, E.; Bertone, V.; Freitas, I.; van Noorden, C. J.

    1994-01-01

    The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate

  13. The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

    Directory of Open Access Journals (Sweden)

    Mariana Igoillo-Esteve

    2007-12-01

    Full Text Available Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.Trypanosoma cruzi é altamente sensível ao estresse oxidativo causado por espécies reativas do oxigênio. Tripanotiona, o principal protetor do parasita contra o estresse oxidativo, é mantido reduzido pela tripanotiona redutase, pela presença deNADPH; a principal fonte da coenzima reduzida parece ser a via da pentose fosfato. As sete enzimas dessa via estão presentes nos quatro principais estágios do ciclo biológico do parasita; nós clonamos e expressamos as enzimas em Escherichia coli como proteínas ativas. Glucose 6-fosfato desidrogenase, que controla o fluxo da glucose da

  14. Effects of thiamine and benfotiamine on intracellular glucose metabolism and relevance in the prevention of diabetic complications.

    Science.gov (United States)

    Beltramo, Elena; Berrone, Elena; Tarallo, Sonia; Porta, Massimo

    2008-09-01

    Thiamine (vitamin B1) is an essential cofactor in most organisms and is required at several stages of anabolic and catabolic intermediary metabolism, such as intracellular glucose metabolism, and is also a modulator of neuronal and neuro-muscular transmission. Lack of thiamine or defects in its intracellular transport can cause a number of severe disorders. Thiamine acts as a coenzyme for transketolase (TK) and for the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes, enzymes which play a fundamental role for intracellular glucose metabolism. In particular, TK is able to shift excess fructose-6-phosphate and glycerhaldeyde-3-phosphate from glycolysis into the pentose-phosphate shunt, thus eliminating these potentially damaging metabolites from the cytosol. Diabetes might be considered a thiamine-deficient state, if not in absolute terms at least relative to the increased requirements deriving from accelerated and amplified glucose metabolism in non-insulin dependent tissues that, like the vessel wall, are prone to complications. A thiamine/TK activity deficiency has been described in diabetic patients, the correction of which by thiamine and/or its lipophilic derivative, benfotiamine, has been demonstrated in vitro to counteract the damaging effects of hyperglycaemia on vascular cells. Little is known, however, on the positive effects of thiamine/benfotiamine administration in diabetic patients, apart from the possible amelioration of neuropathic symptoms. Clinical trials on diabetic patients would be necessary to test this vitamin as a potential and inexpensive approach to the prevention and/or treatment of diabetic vascular complications.

  15. [Glucose-6-phosphatase from nuclear envelope in rat liver].

    Science.gov (United States)

    González-Mujica, Freddy

    2008-06-01

    Nuclear envelope (NE) and microsomal glucosa-6-phosphatase (G-6-Pase) activities were compared. Intact microsomes were unable to hydrolyze mannose-6-phosphate (M-6-P), on the other hand, intact NE hydrolyzes this substrate. Galactose-6-phosphate showed to be a good substrate for both NE and microsomal enzymes, with similar latency to that obtained with M-6-P using microsomes. In consequence, this substrate was used to measure the NE integrity. The kinetic parameters (Kii and Kis) of the intact NE G-6-Pase for the phlorizin inhibition using glucose-6-phosphate (G-6-P) and M-6-P as substrates, were very similar. The NE T1 transporter was more sensitive to amiloride than the microsomal T1. The microsomal system was more sensitive to N-ethylmalemide (NEM) than the NE and the latter was insensitive to anion transport inhibitors DIDS and SITS, which strongly affect the microsomal enzyme. The above results allowed to postulate the presence of a hexose-6-phosphate transporter in the NE which is able to carry G-6-P and M-6-P, and perhaps other hexose-6-phosphate which could be different from that present in microsomes or, if it is the same, its activity could by modified by the membrane system where it is included. The higher PPi hydrolysis activity of the intact NE G-6-Pase in comparison to the intact microsomal, suggests differences between the Pi/PPi transport (T2) of both systems. The lower sensitivity of the NE G-6-Pase to NEM suggests that the catalytic subunit of this system has some differences with the microsomal isoform.

  16. Glyceraldehyde-3-phosphate dehydrogenase from Chironomidae showed differential activity towards metals.

    Science.gov (United States)

    Chong, Isaac K W; Ho, Wing S

    2013-09-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to interact with different biomolecules and was implicated in many novel cellular activities including programmed cell death, nuclear RNA transport unrelated to the commonly known carbohydrate metabolism. We reported here the purification of GAPDH from Chironomidae larvae (Insecta, Diptera) that showed different biologic activity towards heavy metals. It was inhibited by copper, cobalt nickel, iron and lead but was activated by zinc. The GAPDH was purified by ammonium sulphate fractionation and Chelating Sepharose CL-6B chromatography followed by Blue Sepharose CL-6B chromatography. The 150-kDa tetrameric GAPDH showed optimal activity at pH 8.5 and 37°C. The multiple alignment of sequence of the Chironomidae GAPDH with other known species showed 78 - 88% identity to the conserved regions of the GADPH. Bioinformatic analysis unveils substantial N-terminal sequence similarity of GAPDH of Chironomidae larvae to mammalian GADPHs. However, the GADPH of Chironomidae larvae showed different biologic activities and cytotoxicity towards heavy metals. The GAPDH enzyme would undergo adaptive molecular changes through binding at the active site leading to higher tolerance to heavy metals.

  17. Radiometric assays for glycerol, glucose, and glycogen

    International Nuclear Information System (INIS)

    Bradley, D.C.; Kaslow, H.R.

    1989-01-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays

  18. Active site of Zn2+-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

    Directory of Open Access Journals (Sweden)

    Jin-Suk Han

    2005-01-01

    Full Text Available The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261 is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/ H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

  19. A mediator-free glucose biosensor based on glucose oxidase/chitosan/α-zirconium phosphate ternary biocomposite.

    Science.gov (United States)

    Liu, Li-Min; Wen, Jiwu; Liu, Lijun; He, Deyong; Kuang, Ren-yun; Shi, Taqing

    2014-01-15

    A novel glucose oxidase/chitosan/α-zirconium phosphate (GOD/chitosan/α-ZrP) ternary biocomposite was prepared by co-intercalating glucose oxidase (GOD) and chitosan into the interlayers of α-zirconium phosphate (α-ZrP) via a delamination-reassembly procedure. The results of X-ray diffraction, infrared spectroscopy, circular dichroism, and ultraviolet spectrum characterizations indicated not only the layered and hybrid structure of the GOD/chitosan/α-ZrP ternary biocomposite but also the recovered activity of the intercalated GOD improved by the co-intercalated chitosan. By depositing the GOD/chitosan/α-ZrP biocomposite film onto a glassy carbon electrode, the direct electrochemistry of the intercalated GOD was achieved with a fast electron transfer rate constant, k(s), of 7.48±3.52 s(-1). Moreover, this GOD/chitosan/α-ZrP biocomposite modified electrode exhibited a sensitive response to glucose in the linear range of 0.25-8.0 mM (R=0.9994, n=14), with a determination limit of 0.076 mM. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. A self-powered glucose biosensor based on pyrolloquinoline quinone glucose dehydrogenase and bilirubin oxidase operating under physiological conditions.

    Science.gov (United States)

    Kulkarni, Tanmay; Slaughter, Gymama

    2017-07-01

    A novel biosensing system capable of simultaneously sensing glucose and powering portable electronic devices such as a digital glucometer is described. The biosensing system consists of enzymatic glucose biofuel cell bioelectrodes functionalized with pyrolloquinoline quinone glucose dehydrogenase (PQQ-GDH) and bilirubin oxidase (BOD) at the bioanode and biocathode, respectively. A dual-stage power amplification circuit is integrated with the single biofuel cell to amplify the electrical power generated. In addition, a capacitor circuit was incorporated to serve as the transducer for sensing glucose. The open circuit voltage of the optimized biofuel cell reached 0.55 V, and the maximum power density achieved was 0.23 mW/ cm 2 at 0.29 V. The biofuel cell exhibited a sensitivity of 0.312 mW/mM.cm 2 with a linear dynamic range of 3 mM - 20 mM glucose. The overall self-powered glucose biosensor is capable of selectively screening against common interfering species, such as ascorbate and urate and exhibited an operational stability of over 53 days, while maintaining 90 % of its activity. These results demonstrate the system's potential to replace the current glucose monitoring devices that rely on external power supply, such as a battery.

  1. Central Nervous System Symptoms Due to Transient Methemoglobinemia in a Child With G6PD Deficiency.

    Science.gov (United States)

    Sharma, Shreya; Srinivasaraghavan, Rangan; Krishnamurthy, Sriram

    2017-01-01

    The authors herein report a 5-year-old child who presented with massive hemolysis, irritability, and cyanosis. The final diagnosis was glucose-6-phosphate dehydrogenase deficiency with associated central nervous system symptoms probably because of concomitantly acquired methemoglobinemia following oxidant drug exposure. The associated acute-onset anemia would have contributed to the development of cerebral anoxia-related seizures and encephalopathy.

  2. Labelling of the pineal gland with 99mTc-glucose-6-phosphate

    International Nuclear Information System (INIS)

    Ribeiro, M.J.; Santos, A.C.; De Lima, J.J.P.

    1998-01-01

    Lately, the pineal body has been the subject of a large variety of studies. Only recently it has been understood the role played by this endocrine gland to maintain the balance of the human body and also in animal models. Although small in dimensions, the pineal body is a very active organ, able to transmit precise temporal information. It probably participates in the synchronization of several organic functions. The present work aims to study a possible use of 99m Tc-glucose-6-P as a tracer for the pineal gland. Histoautoradiographic studies have been performed in Wistar rats. Tomoscintigraphic studies were acquired in patients and in albine rabbits (oryctolagus cuniculus hyplus). The labelling efficiency and the radiochemical purity of the labelled products have always been tested. Animal and human SPECT exams, show an activity focus projected over the area corresponding to the pineal body localization. Autoradiographic studies using [1- 14 C]-glucose-6-P did not reveal a more relevant activity at the pineal level, probably due to its hepatic conversion to 14 C-glucose. (author)

  3. Immobilisation and characterisation of glucose dehydrogenase immobilised on ReSyn: a proprietary polyethylenimine support matrix

    CSIR Research Space (South Africa)

    Twala, BV

    2010-01-01

    Full Text Available Immobilisation of enzymes is of considerable interest due to the advantages over soluble enzymes, including improved stability and recovery. Glucose Dehydrogenase (GDH) is an important biocatalytic enzyme due to is ability to recycle the biological...

  4. Vanadate influence on metabolism of sugar phosphates in fungus Phycomyces blakesleeanus.

    Directory of Open Access Journals (Sweden)

    Milan Žižić

    Full Text Available The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+ on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC, and polyphosphates, UDPG and ATP (31P NMR was also established. Increase of sugar phosphates content was not observed after addition of vanadyl (V4+, indicating that only vanadate influences its metabolism. Obtained results from in vivo experiments indicate catalytic/inhibitory vanadate action on enzymes involved in reactions of glycolysis and glycogenesis i.e., phosphoglucomutase, phosphofructokinase and glycogen phosphorylase in filamentous fungi.

  5. Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

    Directory of Open Access Journals (Sweden)

    Facincani Agda Paula

    2003-01-01

    Full Text Available The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor, Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

  6. Engineering of Class II Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

    DEFF Research Database (Denmark)

    Ortiz, Roberto; Rahman, Mahbubur; Zangrilli, Beatrice

    2017-01-01

    The front cover artwork is provided by Prof. Lo Gorton from Lund University (Sweden) and his co-workers. The image shows mutated cellobiose dehydrogenase (CDH) immobilized on a graphite electrode and how preferentially glucose is oxidized by this enzyme. Read the full text of the Article at 10.1002...

  7. Photolabeling identifies an interaction between phosphatidylcholine and glycerol-3-phosphate dehydrogenase (Gut2p) in yeast mitochondria

    DEFF Research Database (Denmark)

    Janssen, Marjolein J F W; van Voorst, Frank; Ploeger, Ginette E J

    2002-01-01

    In search of mitochondrial proteins interacting with phosphatidylcholine (PC), a photolabeling approach was applied, in which photoactivatable probes were incorporated into isolated yeast mitochondria. Only a limited number of proteins were labeled upon photoactivation, using either the PC analogue......-dependent mitochondrial glycerol-3-phosphate dehydrogenase. This was confirmed by the lack of specific labeling in mitochondria from a gut2 deletion strain. Only under conditions where the inner membrane was accessible to the probe, Gut2p was labeled by [125I]TID-PC, in parallel with increased labeling of the phosphate...

  8. THE CYTOSOLIC AND GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM TRYPANOSOMA-BRUCEI - KINETIC-PROPERTIES AND COMPARISON WITH HOMOLOGOUS ENZYMES

    NARCIS (Netherlands)

    LAMBEIR, AM; LOISEAU, AM; KUNTZ, DA; VELLIEUX, FM; MICHELS, PAM; OPPERDOES, FR

    1991-01-01

    The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like

  9. Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets.

    Science.gov (United States)

    Marimuthu, Srinivasan; Chivukula, Raghavender S V; Alfonso, Lloyd F; Moridani, Majid; Hagen, Fred K; Bhat, G Jayarama

    2011-11-01

    Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate and post-translationally modify cellular proteins in HCT-116 human colon cancer cells to understand the potential mechanisms by which it may exerts anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immuno-affinity purification, mass spectrometry and immuno-blotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeleton proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiment showed that aspirin also acetylated glucose-6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. In vitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6 phosphate dehydrogenase activity. Similar results were also observed in HT-29 human colon cancer cells. Selective inhibition of glucose-6-phosphate dehydrogenase may represent an important mechanism by which aspirin may exert its anti-cancer effects through inhibition of ribonucleotide synthesis.

  10. Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization.

    Science.gov (United States)

    Jayashree, Shanmugam; Vadivukkarasi, Ponnusamy; Anand, Kirupanithi; Kato, Yuko; Seshadri, Sundaram

    2011-08-01

    Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP-BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l(-l)), MDW 80 (301 mg l(-l)), M. komagatae (279 mg l(-l)), and MSF 34 (202 mg l(-l)), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs.

  11. The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver

    NARCIS (Netherlands)

    Frederiks, W. M.; Ouwerkerk, I. J.; Bosch, K. S.; Marx, F.; Kooij, A.; van Noorden, C. J.

    1993-01-01

    The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25 degrees C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated

  12. Research and engineering assessment of biological solubilization of phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, R.D.; McIlwain, M.E.; Losinski, S.J.; Taylor, D.D.

    1993-03-01

    This research and engineering assessment examined a microbial phosphate solubilization process as a method of recovering phosphate from phosphorus containing ore compared to the existing wet acid and electric arc methods. A total of 860 microbial isolates, collected from a range of natural environments were tested for their ability to solubilize phosphate from rock phosphate. A bacterium (Pseudomonas cepacia) was selected for extensive characterization and evaluation of the mechanism of phosphate solubilization and of process engineering parameters necessary to recover phosphate from rock phosphate. These studies found that concentration of hydrogen ion and production of organic acids arising from oxidation of the carbon source facilitated microbial solubilization of both pure chemical insoluble phosphate compounds and phosphate rock. Genetic studies found that phosphate solubilization was linked to an enzyme system (glucose dehydrogenase). Process-related studies found that a critical solids density of 1% by weight (ore to liquid) was necessary for optimal solubilization. An engineering analysis evaluated the cost and energy requirements for a 2 million ton per year sized plant, whose size was selected to be comparable to existing wet acid plants.

  13. Chemienzymatic synthesis of Uridine. Nucleotides labeled with [15N] and [13C

    DEFF Research Database (Denmark)

    Gilles, Anne-Marie; Cristea, Ioan; Palibroda, Nicolae

    1995-01-01

    +necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and a-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N,13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended...... and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy....

  14. (13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.

    Science.gov (United States)

    Gebril, Hoda M; Avula, Bharathi; Wang, Yan-Hong; Khan, Ikhlas A; Jekabsons, Mika B

    2016-02-01

    Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [(13)C]lactate labeling from metabolism of [1,2-(13)C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28 ± 2% and 7.7 ± 0.2% of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52 ± 6% of glucose was metabolized by glycolysis, compared to 19 ± 2% by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29 ± 8% by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62 ± 6% of glucose was converted to pyruvate, the metabolism of which resulted in 16 ± 1% of glucose oxidized by mitochondria and 46 ± 6% exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a

  15. X-Linked G6PD Deficiency Protects Hemizygous Males but Not Heterozygous Females against Severe Malaria

    OpenAIRE

    Guindo, Aldiouma; Fairhurst, Rick M; Doumbo, Ogobara K; Wellems, Thomas E; Diallo, Dapa A

    2007-01-01

    Editors' Summary Background. “Favism” is a condition that results from a deficiency in an enzyme called glucose-6-phosphate dehydrogenase (G6PD), and this disorder is thought to be the commonest enzyme-deficiency disease worldwide. The disease is named favism after the Italian word for broad beans (fava), which cause a classic reaction when eaten by people with G6PD deficiency. The G6PD enzyme is particularly important in red blood cells, where it protects against damage that can be caused by...

  16. iTRAQ-based proteomic profile analysis of ISKNV-infected CPB cells with emphasizing on glucose metabolism, apoptosis and autophagy pathways.

    Science.gov (United States)

    Wu, Shiwei; Yu, Lujun; Fu, Xiaozhe; Yan, Xi; Lin, Qiang; Liu, Lihui; Liang, Hongru; Li, Ningqiu

    2018-05-04

    Infectious spleen and kidney necrosis virus (ISKNV) has caused significant losses in the cultured mandarin fish (Siniperca chuatsi) industry. The molecular mechanisms that underlie interaction between ISKNV and hosts are not fully understood. In this study, the proteomic profile of CPB cells at progressive time points after ISKNV infection was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 2731 proteins corresponding to 6363 novel peptides (false discovery rate analysis of several proteins as G6PDH, β-tubulin and RPL11 were done to validate iTRAQ data. Among those differentially expressed proteins, several glucose metabolism-related enzymes, including glucose-6-phosphate dehydrogenase (G6PDH), pyruvate dehydrogenase phosphatase (PDP) and fumarate hydratase (FH), were up-regulated, while pyruvate dehydrogenase kinase (PDK) and enolase (ENO) were down-regulated at 24 h poi, suggesting that ISKNV enhanced glucose metabolism in CPB cells in early-stage infection. Simultaneously, expression of apoptosis-related proteins including Caspase 8, phosphoinositide 3-kinases (PI3Ks), and regulatory-associated protein of mTOR-like isoform X3 changed upon ISKNV infection, indicating that ISKNV induced apoptosis of CPB cells. Autophagy-related proteins including LC3 and PI3Ks were up-regulated at 24 h poi, indicating that ISKNV induced autophagy of CPB cells in early-stage infection. These findings may improve the understanding of ISKNV and host interaction and help clarify its pathogenesis mechanisms. Copyright © 2018. Published by Elsevier Ltd.

  17. Mediterranean glucose-6-phosphate dehydrogenase (G6PDC563T) mutation among jordanian females with acute hemolytic crisis

    International Nuclear Information System (INIS)

    Jabbar, A.A.; Kanakiri, N.; Kamil, M.; Rimawi, H.S.A.

    2010-01-01

    To evaluate the G6PDC563T Mediterranean mutation among Jordanian females who were admitted to Princess Rahma Teaching Hospital (PRTH) with/or previous history of favism. Study Design: A descriptive study. Place and Duration of Study: Jordanian University of Science and Technology and PRTH, from October 2003 to October 2004. Methodology: After obtaining approval from the Ethics Committee of Jordanian University of Science and Technology, a total of 32 females were included in this study. Samples from 15 healthy individual females were used as a negative control. Blood samples from these patients were collected and analyzed by allele-specific polymerase chain reaction (AS-PCR) to determine the G6PDC563T mutation. Results: Twenty one out of 32 patients were found to be G6PDC563T Mediterranean mutation (65.6%) positive. Three out of 21 patients were homozygous and remaining 18 were heterozygous for G6PDC563T Mediterranean mutation. Eleven (34.4%) out of 32 patients were found to be negative for G6PDC563T mutation indicating the presence of other G6PD mutations in the study sample. Conclusion: G6PDC563T Mediterranean mutation accounted for 65.6% of the study sample with favism in the North of Jordan. There is likely to be another G6PD deficiency variant implicated in acute hemolytic crisis (favism). (author)

  18. 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...

  19. Isotope inequilibrium of glucose metabolites in intact cells and particlefree supernatants of Ehrlich ascites tumor

    International Nuclear Information System (INIS)

    Daehnfeldt, J.L.; Winge, P.

    1975-01-01

    With an enzyme degradative technique, isotope inequilibrium of glucose metabolites was demonstrated in intact cells and particle-free supernatants of Ehrlich ascites tumor using I- 14 C-glucose as tracer. Inequilibrium was found between glucose and glucose-6-phosphate, glucose and fructose-6-phosphate, glucose and 6-phosphogluconate, while glucose-6-phosphate and fructose-6-phosphate were found to be in near equilibrium within the incubation time investigated. Glucose and lactate were found to be in near equilibrium after 8 min in intact cells. Calculations based on the equilibrium levels found, showed that these inequilibria could not be explained by the effects of the pentose cycle. (U.S.)

  20. The Multiple Localized Glyceraldehyde-3-Phosphate Dehydrogenase Contributes to the Attenuation of the Francisella tularensis dsbA Deletion Mutant

    Czech Academy of Sciences Publication Activity Database

    Pavkova, I.; Kopečková, M.; Klimentová, J.; Schmidt, M.; Sheshko, V.; Sobol, Margaryta; Žáková, J.; Hozák, Pavel; Stulík, J.

    2017-01-01

    Roč. 7, zima (2017), č. článku 503. ISSN 2235-2988 Institutional support: RVO:68378050 Keywords : DsbA * SILAC * glyceraldehyde-3-phosphate dehydrogenase * Francisella tularensis * moonlighting Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.300, year: 2016

  1. Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation.

    Science.gov (United States)

    de Freitas-Silva, Larisse; Rodríguez-Ruiz, Marta; Houmani, Hayet; da Silva, Luzimar Campos; Palma, José M; Corpas, Francisco J

    2017-11-01

    Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20μM glyphosate, we analyzed H 2 O 2 , ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H 2 0 2 , nitric oxide or peroxynitrite. Copyright © 2017 Elsevier GmbH. All rights reserved.

  2. Biochemical characteristics of glucose-6-phosphate dehydrogenase variants among the Malays of Singapore with report of a new non-deficient (GdSingapore) and three deficient variants.

    Science.gov (United States)

    Saha, N; Hong, S H; Wong, H A; Jeyaseelan, K; Tay, J S

    1991-12-01

    Biochemical characteristics of one non-deficient fast G6PD variant (GdSingapore) and six different deficient variants (three new, two Mahidol, one each of Indonesian and Mediterranean) were studied among the Malays of Singapore. The GdSingapore variant had normal enzyme activity (82%) and fast electrophoretic mobilities (140% in TEB buffer, 160% in phosphate and 140% in Tris-HCl buffer systems respectively). This variant is further characterized by normal Km for G6P; utilization of analogues (Gal6P, 2dG6P; dAmNADP), heat stability and pH optimum. The other six deficient G6PD variants had normal electrophoretic mobility in TEB buffer with enzyme activities ranging from 1 to 12% of GdB+. The biochemical characteristics identity them to be 2 Mahidol, 1 Indonesian and 1 Mediterranean variants and three new deficient variants.

  3. Sequence of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Nicotiana plumbaginifolia and phylogenetic origin of the gene family.

    Science.gov (United States)

    Habenicht, A; Quesada, A; Cerff, R

    1997-10-01

    A cDNA-library has been constructed from Nicotiana plumbaginifolia seedlings, and the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN, EC 1.2.1.9) was isolated by plaque hybridization using the cDNA from pea as a heterologous probe. The cDNA comprises the entire GapN coding region. A putative polyadenylation signal is identified. Phylogenetic analysis based on the deduced amino acid sequences revealed that the GapN gene family represents a separate ancient branch within the aldehyde dehydrogenase superfamily. It can be shown that the GapN gene family and other distinct branches of the superfamily have its phylogenetic origin before the separation of primary life-forms. This further demonstrates that already very early in evolution, a broad diversification of the aldehyde dehydrogenases led to the formation of the superfamily.

  4. Complex formation between uranium(VI) and α-D-glucose 1-phosphate

    International Nuclear Information System (INIS)

    Koban, A.; Geipel, G.; Bernhard, G.

    2003-01-01

    The complex formation of uranium(VI) with α-D-glucose 1-phosphate (C 6 H 11 O 6 PO 3 2- , G1P) was determined by time-resolved laser-induced fluorescence spectroscopy (TRLFS) at pH 4 and potentiometric titration in the pH range from 3 to 10. Both measurements show the formation of a 1 : 1 complex at lower pH values. The formation constant of UO 2 (C 6 H 11 O 6 PO 3 ) was calculated from TRLFS measurements to be log β 11 = 5.72±0.12, and from potentiometric titration log β 11 = 5.40±0.25, respectively. It was found by potentiometric titration that at higher pH values the complexation changes to a 1 : 2 complex. The stability constant for this complex was calculated to be log β 12 = 8.96±0.18. (orig.)

  5. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    Science.gov (United States)

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. A novel glucose dehydrogenase from the white-rot fungus Pycnoporus cinnabarinus: production in Aspergillus niger and physicochemical characterization of the recombinant enzyme.

    Science.gov (United States)

    Piumi, François; Levasseur, Anthony; Navarro, David; Zhou, Simeng; Mathieu, Yann; Ropartz, David; Ludwig, Roland; Faulds, Craig B; Record, Eric

    2014-12-01

    Data on glucose dehydrogenases (GDHs) are scarce and availability of these enzymes for application purposes is limited. This paper describes a new GDH from the fungus Pycnoporus cinnabarinus CIRM BRFM 137 that is the first reported GDH from a white-rot fungus belonging to the Basidiomycota. The enzyme was recombinantly produced in Aspergillus niger, a well-known fungal host producing an array of homologous or heterologous enzymes for industrial applications. The full-length gene that encodes GDH from P. cinnabarinus (PcGDH) consists of 2,425 bp and codes for a deduced protein of 620 amino acids with a calculated molecular mass of 62.5 kDa. The corresponding complementary DNA was cloned and placed under the control of the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The signal peptide of the glucoamylase prepro sequence of A. niger was used to target PcGDH secretion into the culture medium, achieving a yield of 640 mg L(-1), which is tenfold higher than any other reported value. The recombinant PcGDH was purified twofold to homogeneity in a one-step procedure with a 41 % recovery using a Ni Sepharose column. The identity of the recombinant protein was further confirmed by immunodetection using western blot analysis and N-terminal sequencing. The molecular mass of the native PcGDH was 130 kDa, suggesting a homodimeric form. Optimal pH and temperature were found to be similar (5.5 and 60 °C, respectively) to those determined for the previously characterized GDH, i.e., from Glomerella cingulata. However PcGDH exhibits a lower catalytic efficiency of 67 M(-1) s(-1) toward glucose. This substrate is by far the preferred substrate, which constitutes an advantage over other sugar oxidases in the case of blood glucose monitoring. The substrate-binding domain of PcGDH turns out to be conserved as compared to other glucose-methanol-choline (GMCs) oxidoreductases. In addition, the ability of PcGDH to reduce oxidized quinones or radical

  7. Glucose metabolism and astrocyte-neuron interactions in the neonatal brain.

    Science.gov (United States)

    Brekke, Eva; Morken, Tora Sund; Sonnewald, Ursula

    2015-03-01

    Glucose is essentially the sole fuel for the adult brain and the mapping of its metabolism has been extensive in the adult but not in the neonatal brain, which is believed to rely mainly on ketone bodies for energy supply. However, glucose is absolutely indispensable for normal development and recent studies have shed light on glycolysis, the pentose phosphate pathway and metabolic interactions between astrocytes and neurons in the 7-day-old rat brain. Appropriately (13)C labeled glucose was used to distinguish between glycolysis and the pentose phosphate pathway during development. Experiments using (13)C labeled acetate provided insight into the GABA-glutamate-glutamine cycle between astrocytes and neurons. It could be shown that in the neonatal brain the part of this cycle that transfers glutamine from astrocytes to neurons is operating efficiently while, in contrast, little glutamate is shuttled from neurons to astrocytes. This lack of glutamate for glutamine synthesis is compensated for by anaplerosis via increased pyruvate carboxylation relative to that in the adult brain. Furthermore, compared to adults, relatively more glucose is prioritized to the pentose phosphate pathway than glycolysis and pyruvate dehydrogenase activity. The reported developmental differences in glucose metabolism and neurotransmitter synthesis may determine the ability of the brain at various ages to resist excitotoxic insults such as hypoxia-ischemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. The Glucose-Insulin Control System

    DEFF Research Database (Denmark)

    Hallgreen, Christine Erikstrup; Korsgaard, Thomas Vagn; Hansen, RenéNormann N.

    2008-01-01

    This chapter reviews the glucose-insulin control system. First, classic control theory is described briefly and compared with biological control. The following analysis of the control system falls into two parts: a glucose-sensing part and a glucose-controlling part. The complex metabolic pathways...... are divided into smaller pieces and analyzed via several small biosimulation models that describe events in beta cells, liver, muscle and adipose tissue etc. In the glucose-sensing part, the beta cell are shown to have some characteristics of a classic PID controller, but with nonlinear properties...... control, the analysis shows that the system has many more facets than just keeping the glucose concentration within narrow limits. After glucose enters the cell and is phosphorylated to glucose-6-phosphate, the handling of glucose-6-phosphate is critical for glucose regulation. Also, this handling...

  9. Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Knudsen, Jan Dines; Johanson, Ted; Eliasson Lantz, Anna

    2015-01-01

    A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by p...

  10. Substrate specificity of glucose dehydrogenase and carbon source utilization pattern of pantoea dispersa strain P2 and its radiation induced mutants

    International Nuclear Information System (INIS)

    Lee, Young Keun; Murugesan, Senthilkumar

    2009-01-01

    Mineral phosphate solubilizing pantoea dispersa strain P2 produced 5.5 mM and 42.6 mM of gluconic acid on 24 h and 72 h incubation, respectively. Strain P2 exhibited glucose dehydrogenase (GDH) specific activity of 0.32 IU mg -1 protein. We have studied the substrate specificity of GDH as well as carbon source utilization pattern of strain P2. GDH of strain P2 did not use ribose as substrate. Utilization of lactose with specific activity of 0.65 IU mg -1 protein indicated that the enzyme belongs to GDH type B isozyme. Arabinose, galactose, ribose, sucrose and xylose did not induce the synthesis of GDH enzyme while mannose induced the synthesis of GDH with highest specific activity of 0.58 IU mg -1 protein. Through radiation mutagenesis, the substrate specificity of GDH was modified in order to utilize side range of sugars available in root exudates. Ribose, originally not a substrate for GDH of strain P2 was utilized as substrate by mutants P2-M5 with specific activity of 0.44 and 0.57 IU mg -1 protein, respectively. Specific activity of GDH on the media containing lactose and galactose was also improved to 1.2 and 0.52 IU mg -1 protein in P2-M5 and P2-M6 respectively. Based on the carbon source availability in root exudate, the mutants can be selected and utilized as efficient biofertilizer under P-deficient soil conditions

  11. Effects of lead nitrate on the activity of metabolic enzymes during early developmental stages of the African catfish, Clarias gariepinus (Burchell, 1822)

    NARCIS (Netherlands)

    Osman, A.G.M.; Mekkawy, Imam A.; Verreth, J.A.J.; Kirschbaum, Frank

    2007-01-01

    Glucose-6-phosphate dehydrogenase (G6PDH), lactate dehydrogenase (LDH) and pyruvate kinase (PK) are key metabolic enzymes. G6PDH has been used as a biomarker of pollution-induced carcinogenesis in fish. LDH has been used as marker of lesions in toxicology and clinical chemistry, and PK catalyses the

  12. Engineering of Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

    DEFF Research Database (Denmark)

    Ortiz, Roberto; Rahman, Mahbubur; Zangrilli, Beatrice

    2017-01-01

    Cellobiose dehydrogenase (CDH) is a fungal extracellular flavocytochrome capable of direct electron transfer (DET). Unlike other CDHs, the pH optimum for CDHs from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) is close to the human physiological pH in blood (7.4). These are......, therefore, interesting candidates for glucose measurements in human blood and the application in enzymatic fuel cells is, however, limited by their relatively low activity with this substrate. In this work, the substrate specificities of CtCDH and HiCDH have been altered by a single cysteine to tyrosine...... substitution in the active sites of CtCDH (position 291) and HiCDH (position 285), which resulted in improved kinetic constants with glucose while decreasing the activity with several disaccharides, including maltose. The DET properties of the generated CDH variants were tested in the absence...

  13. Lack of skeletal muscle IL-6 influences hepatic glucose metabolism in mice during prolonged exercise

    DEFF Research Database (Denmark)

    Bertholdt, Lærke; Gudiksen, Anders; Schwartz, Camilla Lindgren

    2017-01-01

    The liver is essential in maintaining and regulating glucose homeostasis during prolonged exercise. IL-6 has been shown to be secreted from skeletal muscle during exercise and has been suggested to signal to the liver. Therefore, the aim of this study was to investigate the role of skeletal muscle...... IL-6 on hepatic glucose regulation and substrate choice during prolonged exercise. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice (age, 12-14 wk) and littermate lox/lox (Control) mice were either rested (Rest) or completed a single bout of exercise for 10, 60, or 120 min, and the liver....... Furthermore, IL-6 MKO mice had higher hepatic pyruvate dehydrogenase (PDH)Ser232 and PDHSer300 phosphorylation than control mice at rest. In conclusion, hepatic gluconeogenic capacity in mice is increased during prolonged exercise independent of muscle IL-6. Furthermore, Skeletal muscle IL-6 influences...

  14. Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes

    NARCIS (Netherlands)

    Gustafson, LA; Neeft, M; Reijngoud, DJ; Kuipers, F; Sauerwein, HP; Romijn, JA; Herling, AW; Burger, HJ; Meijer, AJ

    2001-01-01

    We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also

  15. Sequence analysis and molecular characterization of Clonorchis sinensis hexokinase, an unusual trimeric 50-kDa glucose-6-phosphate-sensitive allosteric enzyme.

    Directory of Open Access Journals (Sweden)

    Tingjin Chen

    Full Text Available Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis, is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK, the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small

  16. Sequence Analysis and Molecular Characterization of Clonorchis sinensis Hexokinase, an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

    Science.gov (United States)

    Chen, Tingjin; Ning, Dan; Sun, Hengchang; Li, Ran; Shang, Mei; Li, Xuerong; Wang, Xiaoyun; Chen, Wenjun; Liang, Chi; Li, Wenfang; Mao, Qiang; Li, Ye; Deng, Chuanhuan; Wang, Lexun; Wu, Zhongdao; Huang, Yan; Xu, Jin; Yu, Xinbing

    2014-01-01

    Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors

  17. Reproductive strategies and genetic variability in tropical freshwater fish

    Directory of Open Access Journals (Sweden)

    Maria Dolores Peres Lassala

    2007-01-01

    Full Text Available We estimated the genetic variability of nine fish species from the Brazilian upper Paraná River floodplain (Astyanax altiparanae, Hoplias malabaricus, Leporinus lacustris, Loricariichthys platymetopon, Parauchenipterus galeatus, Pimelodus maculatus, Rhaphiodon vulpinus, Roeboides paranensis and Serrasalmus marginatus based on data for 36 putative allozyme loci obtained using corn starch gel electrophoresis of 13 enzymatic systems: aspartate aminotransferase (EC 2.6.1.1, acid phosphatase (EC 3.1.3.2, esterase (EC 3.1.1.1, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8, glucose-6-phosphate dehydrogenase (EC 1.1.1.49, glucose-6-phosphate isomerase (EC 5.3.1.9, Iditol dehydrogenase (EC 1.1.1.14, isocitrate dehydrogenase - NADP+ (EC 1.1.1.42, L-lactate dehydrogenase (EC 1.1.1.27, malate dehydrogenase (EC 1.1.1.37, malate dehydrogenase-NADP+ (EC 1.1.1.40, phosphoglucomutase (EC 5.4.2.2 and superoxide dismutase, (EC 1.15.1.1. The mean expected heterozygosity varied from zero to 0.147. When data from the literature for 75 species of tropical fish were added to the nine species of this study, the heterozygosity values differed significantly among the groups of different reproductive strategies. The highest mean heterozygosity was for the non-migratory without parental care, followed by the long-distance migratory, and the lowest mean was for the non-migratory with parental care or internal fecundation.

  18. Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae.

    Science.gov (United States)

    Satake, Ryoko; Ichiyanagi, Atsushi; Ichikawa, Keiichi; Hirokawa, Kozo; Araki, Yasuko; Yoshimura, Taro; Gomi, Keiko

    2015-11-01

    Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Underestimation of glucose turnover measured with [6-3H]- and [6,6-2H]- but not [6-14C]glucose during hyperinsulinemia in humans

    International Nuclear Information System (INIS)

    McMahon, M.M.; Schwenk, W.F.; Haymond, M.W.; Rizza, R.A.

    1989-01-01

    Recent studies indicate that hydrogen-labeled glucose tracers underestimate glucose turnover in humans under conditions of high flux. The cause of this underestimation is unknown. To determine whether the error is time-, pool-, model-, or insulin-dependent, glucose turnover was measured simultaneously with [6-3H]-, [6,6-2H2]-, and [6-14C]glucose during a 7-h infusion of either insulin (1 mU.kg-1.min-1) or saline. During the insulin infusion, steady-state glucose turnover measured with both [6-3H]glucose (8.0 +/- 0.5 mg.kg-1.min-1) and [6,6-2H2]glucose (7.6 +/- 0.5 mg.kg-1.min-1) was lower (P less than .01) than either the glucose infusion rate required to maintain euglycemia (9.8 +/- 0.7 mg.kg-1.min-1) or glucose turnover determined with [6-14C]glucose and corrected for Cori cycle activity (9.8 +/- 0.7 mg.kg-1.min-1). Consequently negative glucose production rates (P less than .01) were obtained with either [6-3H]- or [6,6-2H2]- but not [6-14C]glucose. The difference between turnover estimated with [6-3H]glucose and actual glucose disposal (or 14C glucose flux) did not decrease with time and was not dependent on duration of isotope infusion. During saline infusion, estimates of glucose turnover were similar regardless of the glucose tracer used. High-performance liquid chromatography of the radioactive glucose tracer and plasma revealed the presence of a tritiated nonglucose contaminant. Although the contaminant represented only 1.5% of the radioactivity in the [6-3H]glucose infusate, its clearance was 10-fold less (P less than .001) than that of [6-3H]glucose. This resulted in accumulation in plasma, with the contaminant accounting for 16.6 +/- 2.09 and 10.8 +/- 0.9% of what customarily is assumed to be plasma glucose radioactivity during the insulin or saline infusion, respectively (P less than .01)

  20. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.L.; Rajagopalan, K.V. (Duke Univ. Medical Center, Durham, NC (USA)); London, R.E. (National Institute of Environmental Health Science, Research Triangle Park, NC (USA))

    1989-09-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and {sup 31}P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.

  1. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    International Nuclear Information System (INIS)

    Johnson, J.L.; Rajagopalan, K.V.; London, R.E.

    1989-01-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and 31 P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well

  2. Expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of glyceraldehyde-3-phosphate dehydrogenase from Campylobacter jejuni

    International Nuclear Information System (INIS)

    Tourigny, David S.; Elliott, Paul R.; Edgell, Louise J.; Hudson, Gregg M.; Moody, Peter C. E.

    2010-01-01

    The cloning, expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of C. jejuni glyceraldehyde-3-phosphate dehydrogenase is reported. The genome of the enteric pathogen Campylobacter jejuni encodes a single glyceraldehyde-3-phosphate dehydrogenase that can utilize either NADP + or NAD + as coenzymes for the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of both the wild type and an active-site mutant of the enzyme are presented. Preliminary X-ray analysis revealed that in both cases the crystals diffracted to beyond 1.9 Å resolution. The space group is shown to be I4 1 22, with unit-cell parameters a = 90.75, b = 90.75, c = 225.48 Å, α = 90.46, β = 90.46, γ = 222.79°; each asymmetric unit contains only one subunit of the tetrameric enzyme

  3. Characterization of different FAD-dependent glucose dehydrogenases for possible use in glucose-based biosensors and biofuel cells.

    Science.gov (United States)

    Zafar, Muhammad Nadeem; Beden, Najat; Leech, Dónal; Sygmund, Christoph; Ludwig, Roland; Gorton, Lo

    2012-02-01

    In this study, different flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) were characterized electrochemically after "wiring" them with an osmium redox polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)(10)Cl](+) on graphite electrodes. One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH). The performance of the Os-polymer "wired" GDHs on graphite electrodes was tested with glucose as the substrate. Optimal operational conditions and analytical characteristics like sensitivity, linear ranges and current density of the different FADGDHs were determined. The performance of all three types of FADGDHs was studied at physiological conditions (pH 7.4). The current densities measured at a 20 mM glucose concentration were 494 ± 17, 370 ± 24, and 389 ± 19 μA cm(-2) for GcGDH, rGcGDH, and AspGDH, respectively. The sensitivities towards glucose were 2.16, 1.90, and 1.42 μA mM(-1) for GcGDH, rGcGDH, and AspGDH, respectively. Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which was indeed found. GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars.

  4. Glucose and phosphate modulation of intracellular 45Ca incorporated into pancreatic islets during culture in the absence and presence of serum

    International Nuclear Information System (INIS)

    Bergsten, P.

    1985-01-01

    The effects of glucose and phosphate on the intracellular 45 Ca content were measured in β cell-rich pancreatic islets cultured in media containing or lacking serum. Irrespective of the glucose and serum concentrations there were no or very small increments of 45 Ca contents when phosphate was raised from 0.8 to 5.8 mM during culture for 1 day. However, after 7 days of culture in serum-free medium there was a massive accumulation of 45 Ca in the islets in response to the higher phosphate concentration. Glucose markedly reduced, and serum eliminated, the extensive accumulation probably due to increased cell viability. In the cells cultured in the presence of serum, raising the glucose concentration from 1.0 to 5.5 mM resulted in an increased incorporation of 45 Ca. This effect was particularly pronounced after culture for 7 days in 5.8 mM phosphate. A further increase of glucose to 20 mM reduced the 45 Ca content. The results are consistent with the concept that glucose both stimulates 45 Ca uptake into different β-cell pools and degranulates the cell with associated loss of intracellular calcium from the granular calcium pool. (author)

  5. Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

    Science.gov (United States)

    Gerszon, Joanna; Serafin, Eligiusz; Buczkowski, Adam; Michlewska, Sylwia; Bielnicki, Jakub Antoni; Rodacka, Aleksandra

    2018-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

  6. Incorporation of phosphate into glycogen by glycogen synthase.

    Science.gov (United States)

    Contreras, Christopher J; Segvich, Dyann M; Mahalingan, Krishna; Chikwana, Vimbai M; Kirley, Terence L; Hurley, Thomas D; DePaoli-Roach, Anna A; Roach, Peter J

    2016-05-01

    The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [β-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Substrate specificity of glucose dehydrogenase and carbon source utilization pattern of pantoea dispersa strain P2 and its radiation induced mutants

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Keun; Murugesan, Senthilkumar [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-06-15

    Mineral phosphate solubilizing pantoea dispersa strain P2 produced 5.5 mM and 42.6 mM of gluconic acid on 24 h and 72 h incubation, respectively. Strain P2 exhibited glucose dehydrogenase (GDH) specific activity of 0.32 IU mg{sup -1} protein. We have studied the substrate specificity of GDH as well as carbon source utilization pattern of strain P2. GDH of strain P2 did not use ribose as substrate. Utilization of lactose with specific activity of 0.65 IU mg{sup -1} protein indicated that the enzyme belongs to GDH type B isozyme. Arabinose, galactose, ribose, sucrose and xylose did not induce the synthesis of GDH enzyme while mannose induced the synthesis of GDH with highest specific activity of 0.58 IU mg{sup -1} protein. Through radiation mutagenesis, the substrate specificity of GDH was modified in order to utilize side range of sugars available in root exudates. Ribose, originally not a substrate for GDH of strain P2 was utilized as substrate by mutants P2-M5 with specific activity of 0.44 and 0.57 IU mg{sup -1} protein, respectively. Specific activity of GDH on the media containing lactose and galactose was also improved to 1.2 and 0.52 IU mg{sup -1} protein in P2-M5 and P2-M6 respectively. Based on the carbon source availability in root exudate, the mutants can be selected and utilized as efficient biofertilizer under P-deficient soil conditions.

  8. Erythrocyte glucose-6-phosphate dehydrogenase deficiency in male newborn babies and its relationship with neonatal jaundice Deficiência de glicose-6-fosfato desidrogenase eritrocitária em recém-nascidos do sexo masculino e sua relação com a icterícia neonatal

    Directory of Open Access Journals (Sweden)

    Marli Auxiliadora C. Iglessias

    2010-01-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency, the commonest red cell enzymopathy in humans, has an X-linked inheritance. The major clinical manifestations are drug induced hemolytic anemia, neonatal jaundice and chronic nonspherocytic hemolytic anemia. The incidence of neonatal hyperbilirubinemia is much greater in G6PD-deficient neonates than babies without this deficiency. The aim of this study was to ascertain the presence of neonatal jaundice in erythrocyte G6PD-deficient male newborns. Samples of umbilical cord blood from a total of 204 male newborns of the Januário Cicco School Maternity located in Natal, Rio Grande do Norte, Brazil were analyzed. The G6PD deficiency was identified by the methemoglobin reduction test (Brewer's test. The deficiency was confirmed by quantitative spectrophotometric assay for enzyme activity and cellulose acetate electrophoresis was used to identify the G6PD variant. Eight newborns were found to be G6PD deficient with four of them exhibiting jaundice during the first 48 hours after birth with bilirubin levels higher than 10 mg/dL. All deficient individuals presented the G6PD A- variant at electrophoresis. Our findings confirmed the association between G6PD deficiency and neonatal jaundice. Hence, early diagnosis of the deficiency at birth is essential to control the appearance of jaundice and to prevent the exposure of these newborns to known hemolytic agents.A deficiência de glicose-6-fosfato desidrogenase (G6PD é a anormalidade enzimática hereditária mais frequente. É transmitida como caráter recessivo ligado ao cromossomo X e as principais manifestações clínicas são hemólise induzida por fármacos, icterícia neonatal e anemia hemolítica não esferocítica. O objetivo do estudo foi determinar a presença de icterícia neonatal em recém-nascidos do sexo masculino deficientes de glicose-6-fosfato desidrogenase. Foram analisadas 204 amostras de sangue umbilical de recém-nascidos do sexo

  9. Redox Balance in Lactobacillus reuteri DSM20016: Roles of Iron-Dependent Alcohol Dehydrogenases in Glucose/ Glycerol Metabolism.

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    Lu Chen

    Full Text Available Lactobacillus reuteri, a heterofermentative bacterium, metabolizes glycerol via a Pdu (propanediol-utilization pathway involving dehydration to 3-hydroxypropionaldehyde (3-HPA followed by reduction to 1,3-propandiol (1,3-PDO with concomitant generation of an oxidized cofactor, NAD+ that is utilized to maintain cofactor balance required for glucose metabolism and even for oxidation of 3-HPA by a Pdu oxidative branch to 3-hydroxypropionic acid (3-HP. The Pdu pathway is operative inside Pdu microcompartment that encapsulates different enzymes and cofactors involved in metabolizing glycerol or 1,2-propanediol, and protects the cells from the toxic effect of the aldehyde intermediate. Since L. reuteri excretes high amounts of 3-HPA outside the microcompartment, the organism is likely to have alternative alcohol dehydrogenase(s in the cytoplasm for transformation of the aldehyde. In this study, diversity of alcohol dehydrogenases in Lactobacillus species was investigated with a focus on L. reuteri. Nine ADH enzymes were found in L. reuteri DSM20016, out of which 3 (PduQ, ADH6 and ADH7 belong to the group of iron-dependent enzymes that are known to transform aldehydes/ketones to alcohols. L. reuteri mutants were generated in which the three ADHs were deleted individually. The lagging growth phenotype of these deletion mutants revealed that limited NAD+/NADH recycling could be restricting their growth in the absence of ADHs. Notably, it was demonstrated that PduQ is more active in generating NAD+ during glycerol metabolism within the microcompartment by resting cells, while ADH7 functions to balance NAD+/NADH by converting 3-HPA to 1,3-PDO outside the microcompartment in the growing cells. Moreover, evaluation of ADH6 deletion mutant showed strong decrease in ethanol level, supporting the role of this bifuctional alcohol/aldehyde dehydrogenase in ethanol production. To the best of our knowledge, this is the first report revealing both internal and

  10. Effect of guava (Psidium guajava Linn.) leaf soluble solids on glucose metabolism in type 2 diabetic rats.

    Science.gov (United States)

    Shen, Szu-Chuan; Cheng, Fang-Chi; Wu, Ning-Jung

    2008-11-01

    This study investigated the effect of aqueous and ethanol soluble solid extracts of guava (Psidium guajava Linn.) leaves on hypoglycemia and glucose metabolism in type 2 diabetic rats. Low-dose streptozotocin (STZ) and nicotinamide were injected into Sprague-Dawley (SD) rats to induce type 2 diabetes. Acute and long-term feeding tests were carried out, and an oral glucose tolerance test (OGTT) to follow the changes in plasma glucose and insulin levels was performed to evaluate the antihyperglycemic effect of guava leaf extracts in diabetic rats.The results of acute and long-term feeding tests showed a significant reduction in the blood sugar level in diabetic rats fed with either the aqueous or ethanol extract of guava leaves (p guava leaf extracts increased the plasma insulin level and glucose utilization in diabetic rats. The results also indicated that the activities of hepatic hexokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase in diabetic rats fed with aqueous extracts were higher than in the normal diabetic group (p guava leaf extract and the health function of guava leaves against type 2 diabetes.

  11. Characterization of different FAD-dependent glucose dehydrogenases for possible use in glucose-based biosensors and biofuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Zafar, Muhammad Nadeem; Beden, Najat; Gorton, Lo [Lund University, Department of Biochemistry and Structural Biology, Lund (Sweden); Leech, Donal [National University of Ireland Galway, School of Chemistry, Galway (Ireland); Sygmund, Christoph; Ludwig, Roland [BOKU-University of Natural Resources and Life Sciences Vienna, Food Biotechnology Laboratory, Department of Food Sciences and Technology, Wien (Austria)

    2012-02-15

    In this study, different flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) were characterized electrochemically after ''wiring'' them with an osmium redox polymer [Os(4,4'-dimethyl-2,2'-bipyridine){sub 2}(PVI){sub 10}Cl]{sup +} on graphite electrodes. One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH). The performance of the Os-polymer ''wired'' GDHs on graphite electrodes was tested with glucose as the substrate. Optimal operational conditions and analytical characteristics like sensitivity, linear ranges and current density of the different FADGDHs were determined. The performance of all three types of FADGDHs was studied at physiological conditions (pH 7.4). The current densities measured at a 20 mM glucose concentration were 494 {+-} 17, 370 {+-} 24, and 389 {+-} 19 {mu}A cm{sup -2} for GcGDH, rGcGDH, and AspGDH, respectively. The sensitivities towards glucose were 2.16, 1.90, and 1.42 {mu}A mM{sup -1} for GcGDH, rGcGDH, and AspGDH, respectively. Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which was indeed found. GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars. (orig.)

  12. Cloning, expression, purification, crystallization and preliminary crystallographic studies of UgdG, an UDP-glucose dehydrogenase from Sphingomonas elodea ATCC 31461

    International Nuclear Information System (INIS)

    Rocha, Joana; Granja, Ana Teresa; Sá-Correia, Isabel; Fialho, Arsénio; Frazão, Carlos

    2009-01-01

    Crystals of S. elodea ATCC 31461 UDP-glucose dehydrogenase (EC 1.1.1.22) were obtained in space groups P622 and P4 3 2 1 2 and diffracted to 2.4 and 3.4 Å resolution, respectively. Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC 1.1.1.22) is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40 Å resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein

  13. Lithium iron phosphate with high-rate capability synthesized through hydrothermal reaction in glucose solution

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Guangchuan; Wang, Li; Ou, Xiuqin; Zhao, Xia; Xu, Shengzhao [Institute of Power Source and Ecomaterials Science, Box 1055, Hebei University of Technology, 300130 Tianjin (China)

    2008-10-01

    Carbon-coated lithium iron phosphate (LiFePO{sub 4}/C) was hydrothermally synthesized from commercial LiOH, FeSO{sub 4} and H{sub 3}PO{sub 4} as raw materials and glucose as carbon precursor in aqueous solution at 180 C for 6 h followed by being fired at 750 C for 6 h. The samples were characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM) and constant current charge-discharge cycling test. The results show that the synthesized powders are in situ coated with carbon precursor produced from glucose. At ambient temperature (25{+-}2 C), the specific discharge capacities are 154 mAh g{sup -1} at 0.2C and 136 mAh g{sup -1} at 5 C rate, and the cycling capacity retention rate reaches 98% over 90 cycles. The excellent electrochemical performance can be correlated with the in situ formation of carbon precursor/carbon, thus leading to the even distribution of carbon and the enhancement of conductibility of individual grains. (author)

  14. A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

    Energy Technology Data Exchange (ETDEWEB)

    Kohan, Alison B.; Talukdar, Indrani; Walsh, Callee M. [Department of Biochemistry, West Virginia University, Morgantown, WV (United States); Salati, Lisa M., E-mail: lsalati@hsc.wvu.edu [Department of Biochemistry, West Virginia University, Morgantown, WV (United States)

    2009-10-09

    Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as {beta}-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser{sup 307} phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.

  15. Radioprotective effects of ocimum flavonoids on leukocyte oxidants and antioxidants in oral cancer

    International Nuclear Information System (INIS)

    Reshma, K.; Rao, A.V.; Dinesh, M.; Vasudevan, D.M.

    2008-01-01

    Oxidants (NADPH oxidase and myeloperoxidase) and antioxidants (GSH, GSH peroxidase, SOD and glucose 6 phosphate dehydrogenase, that provides NADPH for antioxidants) were assayed in the neutrophils from oral cancer patients, in three stages viz, baseline samples, 15 days after radiation and 30 days following radiation. These samples were obtained from 2 groups of patients. Group A that received radiation alone and Group B that received radiation and ocimum flavonoids, a radioprotector. The results showed a significant fall in the SOD levels in the second follow up of group B. Glucose 6 phosphate dehydrogenase showed significant increase only in the first follow up of patients who received Ocimum flavonoids. Except for these findings all other parameters remained statistically nonsignificant. (author)

  16. Radioprotective effects of ocimum flavonoids on leukocyte oxidants and antioxidants in oral cancer

    Energy Technology Data Exchange (ETDEWEB)

    Reshma, K [Department of Biochemistry, Kasturba Medical College, Mangalore (India); Rao, A V [Fr. Muller' s Medical College, Mangalore (India); Dinesh, M [Department of Oncology, Amritha Institute of Medical Sciences, Cochin (India); Vasudevan, D M [Dept. of Biochemistry, Amritha Institute of Medical Sciences, Cochin (India)

    2008-04-15

    Oxidants (NADPH oxidase and myeloperoxidase) and antioxidants (GSH, GSH peroxidase, SOD and glucose 6 phosphate dehydrogenase, that provides NADPH for antioxidants) were assayed in the neutrophils from oral cancer patients, in three stages viz, baseline samples, 15 days after radiation and 30 days following radiation. These samples were obtained from 2 groups of patients. Group A that received radiation alone and Group B that received radiation and ocimum flavonoids, a radioprotector. The results showed a significant fall in the SOD levels in the second follow up of group B. Glucose 6 phosphate dehydrogenase showed significant increase only in the first follow up of patients who received Ocimum flavonoids. Except for these findings all other parameters remained statistically nonsignificant. (author)

  17. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish; (UAB)

    2009-06-08

    The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate

  18. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    Directory of Open Access Journals (Sweden)

    Chattopadhyay Debasish

    2009-02-01

    Full Text Available Abstract Background The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. Results We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2Å resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in

  19. Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

    Directory of Open Access Journals (Sweden)

    Joanna Gerszon

    Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149. Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9. Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149 which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

  20. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

    International Nuclear Information System (INIS)

    Yeh, Joanne I.; Chinte, Unmesh; Du, Shoucheng

    2008-01-01

    Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 (angstrom) resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes.

  1. Activation of Wnt Signaling in Cortical Neurons Enhances Glucose Utilization through Glycolysis.

    Science.gov (United States)

    Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L Felipe; Inestrosa, Nibaldo C

    2016-12-09

    The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer's disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the capacity of the brain to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with the Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

    Science.gov (United States)

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

    2016-04-01

    Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

  3. Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

    Science.gov (United States)

    Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

    2008-11-01

    Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

  4. Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice.

    Directory of Open Access Journals (Sweden)

    Jan Korte

    2016-12-01

    Full Text Available Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors.

  5. Engineering a synthetic anaerobic respiration for reduction of xylose to xylitol using NADH output of glucose catabolism by Escherichia coli AI21.

    Science.gov (United States)

    Iverson, Andrew; Garza, Erin; Manow, Ryan; Wang, Jinhua; Gao, Yuanyuan; Grayburn, Scott; Zhou, Shengde

    2016-04-16

    Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp 6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over

  6. Trehalose-6-phosphate synthesis controls yeast gluconeogenesis downstream and independent of SNF1.

    Science.gov (United States)

    Deroover, Sofie; Ghillebert, Ruben; Broeckx, Tom; Winderickx, Joris; Rolland, Filip

    2016-06-01

    Trehalose-6-P (T6P), an intermediate of trehalose biosynthesis, was identified as an important regulator of yeast sugar metabolism and signaling. tps1Δ mutants, deficient in T6P synthesis (TPS), are unable to grow on rapidly fermentable medium with uncontrolled influx in glycolysis, depletion of ATP and accumulation of sugar phosphates. However, the exact molecular mechanisms involved are not fully understood. We show that SNF1 deletion restores the tps1Δ growth defect on glucose, suggesting that lack of TPS hampers inactivation of SNF1 or SNF1-regulated processes. In addition to alternative, non-fermentable carbon metabolism, SNF1 controls two major processes: respiration and gluconeogenesis. The tps1Δ defect appears to be specifically associated with deficient inhibition of gluconeogenesis, indicating more downstream effects. Consistently, Snf1 dephosphorylation and inactivation on glucose medium are not affected, as confirmed with an in vivo Snf1 activity reporter. Detailed analysis shows that gluconeogenic Pck1 and Fbp1 expression, protein levels and activity are not repressed upon glucose addition to tps1Δ cells, suggesting a link between the metabolic defect and persistent gluconeogenesis. While SNF1 is essential for induction of gluconeogenesis, T6P/TPS is required for inactivation of gluconeogenesis in the presence of glucose, downstream and independent of SNF1 activity and the Cat8 and Sip4 transcription factors. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. A high-performance liquid chromatography-based radiometric assay for sucrose-phosphate synthase and other UDP-glucose requiring enzymes

    International Nuclear Information System (INIS)

    Salvucci, M.E.; Crafts-Brandner, S.J.

    1991-01-01

    A method for product analysis that eliminates a problematic step in the radiometric sucrose-phosphate synthase assay is described. The method uses chromatography on a boronate-derivatized high-performance liquid chromatography column to separate the labeled product, [14C]sucrose phosphate, from unreacted uridine 5'-diphosphate-[14C]glucose (UDP-Glc). Direct separation of these compounds eliminates the need for treatment of the reaction mixtures with alkaline phosphatase, thereby avoiding the problem of high background caused by contaminating phosphodiesterase activity in alkaline phosphatase preparations. The method presented in this paper can be applied to many UDP-Glc requiring enzymes; here the authors show its use for determining the activities of sucrose-phosphate synthase, sucrose synthase, and uridine diphosphate-glucose pyrophosphorylase in plant extracts

  8. Reduction of quinones and phenoxy radicals by extracellular glucose dehydrogenase from Glomerella cingulata suggests a role in plant pathogenicity.

    Science.gov (United States)

    Sygmund, Christoph; Klausberger, Miriam; Felice, Alfons K; Ludwig, Roland

    2011-11-01

    The plant-pathogenic fungus Glomerella cingulata (anamorph Colletotrichum gloeosporoides) secretes high levels of an FAD-dependent glucose dehydrogenase (GDH) when grown on tomato juice-supplemented media. To elucidate its molecular and catalytic properties, GDH was produced in submerged culture. The highest volumetric activity was obtained in shaking flasks after 6 days of cultivation (3400 U l⁻¹, 4.2 % of total extracellular protein). GDH is a monomeric protein with an isoelectric point of 5.6. The molecular masses of the glycoforms ranged from 95 to 135 kDa, but after deglycosylation, a single 68 kDa band was obtained. The absorption spectrum is typical for an FAD-containing enzyme with maxima at 370 and 458 nm and the cofactor is non-covalently bound. The preferred substrates are glucose and xylose. Suitable electron acceptors are quinones, phenoxy radicals, 2,6-dichloroindophenol, ferricyanide and ferrocenium hexafluorophosphate. In contrast, oxygen turnover is very low. The GDH-encoding gene was cloned and phylogenetic analysis of the translated protein reveals its affiliation to the GMC family of oxidoreductases. The proposed function of this quinone and phenoxy radical reducing enzyme is to neutralize the action of plant laccase, phenoloxidase or peroxidase activities, which are increased in infected plants to evade fungal attack.

  9. Glucose-6-phosphate dehydrogenase deficiency and the risk of malaria: A meta-analysis and trial sequential analysis

    Science.gov (United States)

    Sun, Fengmei; Zhang, Juan; Pu, Yuepu

    2017-10-01

    This study is designed to perform a meta-analysis and trial sequential analysis (TSA) to investigate whether people with G6PD deficiency suffered less malarial infection. We searched from PubMed, Science Direct, Springer Link, CNKI, and Wan Fang databases for case-control study, cohort study or cross section study until April 2017. TSA was used to determine the state of evidence and calculate the required sample size. Eight case-control studies and five cross-sectional studies (30,683participants) were included in this meta-analysis. Compared with normal control group, we found significant protection from severe malaria (OR 0.644, 95% CI [0.493-0.842]; P=0.001) among people with decreasing G6PD activity. People with variations of G6PD gene at nucleotide 202(G6PD A-) were also found to be associated with resistance on severe malaria pooled (OR 0.851, 95% CI [0.779-0.930]; P =0.0001). Sex-stratified test suggested that protection of severe malaria is conferred to both G6PD A-males and heterozygous females (with a single copy of the variant). In conclusion, our study found a significant protection from severe malaria among G6PD deficient people compared to the

  10. BAG3 elevation inhibits cell proliferation via direct interaction with G6PD in hepatocellular carcinomas.

    Science.gov (United States)

    Kong, De-Hui; Li, Si; Du, Zhen-Xian; Liu, Chuan; Liu, Bao-Qin; Li, Chao; Zong, Zhi-Hong; Wang, Hua-Qin

    2016-01-05

    Bcl-2 associated athanogene 3 (BAG3) contains multiple protein-binding motifs to mediate potential interactions with chaperons and/or other proteins, which is possibly ascribed to the multifaceted functions assigned to BAG3. The current study demonstrated that BAG3 directly interacted with glucose 6 phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). BAG3 suppressed the PPP flux, de novo DNA synthesis and cell growth in hepatocellular carcinomas (HCCs). The growth defect of HCCs with forced BAG3 expression can be rescued by enforced G6PD expression. However, BAG3 elevation did not cause a reduction in cellular NADPH concentrations, another main product of G6PD. In addition, supplement of nucleosides alone was sufficient to recover the growth defect mediated by BAG3 elevation. Collectively, the current study established a tumor suppressor-like function of BAG3 via direct interaction with G6PD in HCCs at the cellular level.

  11. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast

    DEFF Research Database (Denmark)

    Schifferdecker, Anna Judith; Siurkus, Juozas; Andersen, Mikael Rørdam

    2016-01-01

    Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we...... developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter...... TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions...

  12. Primaquine

    Science.gov (United States)

    ... deficiency (a genetic condition), glucose-6-phosphate dehydrogenase (G6PD) deficiency (a genetic condition), or if you or someone ... eye drops, creams, patches, and inhalers) are not child-resistant and young children can open them easily. ...

  13. Dabrafenib

    Science.gov (United States)

    ... have ever had diabetes; glucose-6-phosphate dehydrogenase (G6PD) deficiency (a genetic condition); bleeding problems; eye problems; heart, ... pregnant, or if you plan to father a child. You or your partner should not become pregnant ...

  14. Kawasaki disease with G6PD deficiency--report of one case and literature review.

    Science.gov (United States)

    Chen, Chia-Hao; Lin, Li-Yan; Yang, Kuender D; Hsieh, Kai-Sheng; Kuo, Ho-Chang

    2014-06-01

    Kawasaki disease (KD) is a systemic vasculitis primarily affecting children who are younger than 5 years. The most serious complications are coronary artery aneurysms and sequelae of vasculitis with the subsequent development of coronary artery aneurysm. According to the literature, intravenous immunoglobulin (IVIG) plus high-dose aspirin (acetylsalicylic acid) were standard treatment for KD, whereas low-dose aspirin (3-5 mg/kg/day) was used for thrombocytosis in KD via antiplatelet effect. However, aspirin has been reported to have hemolytic potential in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. We report a child with G6PD-deficiency who has KD, and review the literature. Copyright © 2012. Published by Elsevier B.V.

  15. Validation of 123I-6-deoxy-6-iodo-D-glucose (6-DIC) as tracer for the in-vivo glucose transport

    International Nuclear Information System (INIS)

    Perret, P.; Ghezzi, C.; Mathieu, J.P.; Morin, C.; Vidal, M.; Comet, M.; Fagret, D.

    1997-01-01

    The evaluation of the glucose transport is very important clinically because alterations of this transport were described in numerous pathologies, in neurology, oncology and endocrinology. A new analog of the 123 I-labelled has been synthesized: 123 I-6-deoxy-6-iodo-D-glucose (6-DIG). Its in-vitro biological behaviour is similar to that of 3-O-methyl-D-glucose (3-OMG), the reference tracer of glucose transport. The aim of the study was to determine if it is possible to make evident by 6-DIG a variations of in-vivo glucose transport. The studies were effected on a model of homozygote mice (db/db), genetically diabetic (NIDDM), presenting a severe insulin-resistance, characterized by deficient glucose transport in response to insulin. The studies of 6-DIG biodistribution (5 nmol/mouse) with (1.5 UI/Kg) or without exogenous insulin, were conducted in diabetic mice (db/db) and in non-diabetic (db/+) control mice. The results show that the capture of 6-DIG, as well as that of glucose, increases (by 30%) in response to insulin in most of insulin-sensitive tissues in control mice. In the insulin-resistant and hyperglycemic db/db mouse, the capture of 6-DIG is not modified, no matter whether the exogenous insulin is present. In conclusion, the 6-DIG is able to make evident a lack of glucose transport in heart, diaphragm and skeletal muscle in diabetic mouse and a physiological variation of this transport in response to insulin, in the control mouse. This result should be stressed because for the first time it is possible to evidence in-vivo variations into glucose transport with a iodated molecule

  16. Light-regulation of enzyme activity in anacystis nidulans (Richt.).

    Science.gov (United States)

    Duggan, J X; Anderson, L E

    1975-01-01

    The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

  17. Molecular Characterization of Glucose-6-Phosphate ...

    African Journals Online (AJOL)

    HP

    The total prevalence of G6PD deficiency among the subjects was 4.59 % (4/87), all of whom were. Malay males. One of the deficient subjects had G6PD Viangchan, while the other three were G6PD. Mahidol ..... J Paediatr Child Health .1991 ...

  18. Saccharomyces cerevisiae KNU5377 stress response during high-temperature ethanol fermentation.

    Science.gov (United States)

    Kim, Il-Sup; Kim, Young-Saeng; Kim, Hyun; Jin, Ingnyol; Yoon, Ho-Sung

    2013-03-01

    Fuel ethanol production is far more costly to produce than fossil fuels. There are a number of approaches to cost-effective fuel ethanol production from biomass. We characterized stress response of thermotolerant Saccharomyces cerevisiae KNU5377 during glucose-based batch fermentation at high temperature (40°C). S. cerevisiae KNU5377 (KNU5377) transcription factors (Hsf1, Msn2/4, and Yap1), metabolic enzymes (hexokinase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, and alcohol dehydrogenase), antioxidant enzymes (thioredoxin 3, thioredoxin reductase, and porin), and molecular chaperones and its cofactors (Hsp104, Hsp82, Hsp60, Hsp42, Hsp30, Hsp26, Cpr1, Sti1, and Zpr1) are upregulated during fermentation, in comparison to S. cerevisiae S288C (S288C). Expression of glyceraldehyde-3-phosphate dehydrogenase increased significantly in KNU5377 cells. In addition, cellular hydroperoxide and protein oxidation, particularly lipid peroxidation of triosephosphate isomerase, was lower in KNU5377 than in S288C. Thus, KNU5377 activates various cell rescue proteins through transcription activators, improving tolerance and increasing alcohol yield by rapidly responding to fermentation stress through redox homeostasis and proteostasis.

  19. Source/ sink interactions underpin crop yield: the case for trehalose 6-phosphate/ SnRK1 in improvement of wheat

    Directory of Open Access Journals (Sweden)

    Matthew ePaul

    2014-08-01

    Full Text Available Considerable interest has been evoked by the analysis of the regulatory pathway in carbohydrate metabolism and cell growth involving trehalose (TRE. TRE is at small concentrations in mesophytes such as Triticum aestivum. Studies of TRE metabolism, and genetic modification of it, have shown a very wide and important role of the pathway in regulation of many processes in development, growth and photosynthesis. It has now been established that trehalose 6-phosphate (T6P, is formed from glucose-6-phosphate and UDP-glucose, derived from sucrose, by the action of trehalose phosphate synthase (TPS and broken down by trehalose-6-phosphate phosphatase (TPP providing for subtle regulation. The concentration of T6P increases with sucrose concentration. Many of the effects of T6P on metabolism and growth occur via the interaction of T6P with the SnRK1 protein kinase system. A large concentration of sucrose increases T6P and thereby inhibits SnRK1, so stimulating growth of cells and their metabolism. The T6P/SnRK1 mechanism offers an important new view of how the distribution of assimilates to organs, such as developing cereal grains, is achieved. Changing T6P concentrations by genetically modifying TPS and TPP has altered photosynthesis, sugar metabolism, growth and development which affect responses to, and recovery from, environmental factors. This review briefly summarizes the factors determining, and limiting, yield of wheat, particularly mass/grain which is highly conserved. The interactions between the source and sink relations are addressed together with how T6P/SnRK1 might function to determine grain number, size, and yield. The possibility of how these might be increased by modifying trehalose metabolism is considered. Cereal yields globally are not increasing and careful targeting of T6P may offer a way of optimizing grain growth and thus increasing yield in wheat.

  20. Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI) by high-throughput screening of existing drugs.

    Science.gov (United States)

    Eltahan, Rana; Guo, Fengguang; Zhang, Haili; Xiang, Lixin; Zhu, Guan

    2018-04-01

    Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (K m  = 0.309 mM, V max  = 31.72 nmol/μg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC 50  = 8.33 μM; K i  = 36.33 μM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC 50  = 165 μM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC 50 on HCT-8 cells = 700 μM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Interstrain polymorphisms of isoenzyme profiles and mitochondrial DNA fingerprints among seven strains assigned to Acanthamoeba polyphaga.

    Science.gov (United States)

    Kong, H H; Park, J H; Chung, D I

    1995-12-01

    Interstrain polymorphisms of isoenzyme profiles and mitochondrial (Mt) DNA fingerprints were observed among seven strains of Acanthamoeba isolated from different sources and morphologically assigned to A. polyphaga. Mt DNA fingerprints by eight restriction endonucleases (Bgl II, Sca I, Cla I, EcoR I, Xba I, Kpn I, Sal I, and Sst I) revealed considerable interstrain polymorphisms. Isoenzyme profiles revealed considerable interstrain polymorphisms for acid phosphatase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase while those for glucose phosphate isomerase, leucine aminopeptidase, and malate dehydrogenase showed similarity. Despite of the interstrain polymorphisms, the isoenzyme profiles and Mt DNA fingerprints of the strain Ap were found to be identical with those of the strain Jones. Mt DNA fingerprinting was found to be highly applicable for the strain identification, characterization, and differentiation.

  2. A CuNi/C Nanosheet Array Based on a Metal-Organic Framework Derivate as a Supersensitive Non-Enzymatic Glucose Sensor

    Science.gov (United States)

    Zhang, Li; Ye, Chen; Li, Xu; Ding, Yaru; Liang, Hongbo; Zhao, Guangyu; Wang, Yan

    2018-06-01

    Bimetal catalysts are good alternatives for non-enzymatic glucose sensors owing to their low cost, high activity, good conductivity, and ease of fabrication. In the present study, a self-supported CuNi/C electrode prepared by electrodepositing Cu nanoparticles on a Ni-based metal-organic framework (MOF) derivate was used as a non-enzymatic glucose sensor. The porous construction and carbon scaffold inherited from the Ni-MOF guarantee good kinetics of the electrode process in electrochemical glucose detection. Furthermore, Cu nanoparticles disturb the array structure of MOF derived films and evidently enhance their electrochemical performances in glucose detection. Electrochemical measurements indicate that the CuNi/C electrode possesses a high sensitivity of 17.12 mA mM-1 cm-2, a low detection limit of 66.67 nM, and a wider linearity range from 0.20 to 2.72 mM. Additionally, the electrode exhibits good reusability, reproducibility, and stability, thereby catering to the practical use of glucose sensors. Similar values of glucose concentrations in human blood serum samples are detected with our electrode and with the method involving glucose-6-phosphate dehydrogenase; the results further demonstrate the practical feasibility of our electrode.

  3. Sulfamethoxazole/Trimethoprim (Bactrim or Septra) and Pregnancy

    Science.gov (United States)

    ... condition known as glucose-6- phosphate dehydrogenase deficiency (G6PD deficiency). However, it is not always necessary to stop ... may affect a man’s ability to father a child. There are no studies looking at risk for ...

  4. Ubiquitin-Specific Protease 2 Regulates Hepatic Gluconeogenesis and Diurnal Glucose Metabolism Through 11β-Hydroxysteroid Dehydrogenase 1

    Science.gov (United States)

    Molusky, Matthew M.; Li, Siming; Ma, Di; Yu, Lei; Lin, Jiandie D.

    2012-01-01

    Hepatic gluconeogenesis is important for maintaining steady blood glucose levels during starvation and through light/dark cycles. The regulatory network that transduces hormonal and circadian signals serves to integrate these physiological cues and adjust glucose synthesis and secretion by the liver. In this study, we identified ubiquitin-specific protease 2 (USP2) as an inducible regulator of hepatic gluconeogenesis that responds to nutritional status and clock. Adenoviral-mediated expression of USP2 in the liver promotes hepatic glucose production and exacerbates glucose intolerance in diet-induced obese mice. In contrast, in vivo RNA interference (RNAi) knockdown of this factor improves systemic glycemic control. USP2 is a target gene of peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α), a coactivator that integrates clock and energy metabolism, and is required for maintaining diurnal glucose homeostasis during restricted feeding. At the mechanistic level, USP2 regulates hepatic glucose metabolism through its induction of 11β-hydroxysteroid dehydrogenase 1 (HSD1) and glucocorticoid signaling in the liver. Pharmacological inhibition and liver-specific RNAi knockdown of HSD1 significantly impair the stimulation of hepatic gluconeogenesis by USP2. Together, these studies delineate a novel pathway that links hormonal and circadian signals to gluconeogenesis and glucose homeostasis. PMID:22447855

  5. The rare sugar D-allose acts as a triggering molecule of rice defence via ROS generation.

    Science.gov (United States)

    Kano, Akihito; Fukumoto, Takeshi; Ohtani, Kouhei; Yoshihara, Akihide; Ohara, Toshiaki; Tajima, Shigeyuki; Izumori, Ken; Tanaka, Keiji; Ohkouchi, Takeo; Ishida, Yutaka; Nishizawa, Yoko; Ichimura, Kazuya; Tada, Yasuomi; Gomi, Kenji; Akimitsu, Kazuya

    2013-11-01

    Only D-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to D-allose. D-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-D-allose, a structural derivative of D-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding D-allose kinase to increase D-allose 6-phosphate synthesis were more sensitive to D-allose, but E. coli AlsI encoding D-allose 6-phosphate isomerase expression to decrease D-allose 6-phosphate reduced sensitivity. A D-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, D-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of D-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of D-allose to D-allose 6-phosphate, and treatment with D-allose might prove to be useful for reducing disease development in rice.

  6. Sustained high plasma mannose less sensitive to fluctuating blood glucose in glycogen storage disease type Ia children

    NARCIS (Netherlands)

    Nagasaka, Hironori; Yorifuji, Tohru; Bandsma, Robert H. J.; Takatani, Tomozumi; Asano, Hisaki; Mochizuki, Hiroshi; Takuwa, Mayuko; Tsukahara, Hirokazu; Inui, Ayano; Tsunoda, Tomoyuki; Komatsu, Haruki; Hiejima, Eitaro; Fujisawa, Tomoo; Hirano, Ken-ichi; Miida, Takashi; Ohtake, Akira; Taguchi, Tadao; Miwa, Ichitomo

    Plasma mannose is suggested to be largely generated from liver glycogen-oriented glucose-6-phosphate. This study examined plasma mannose in glycogen storage disease type Ia (GSD Ia) lacking conversion of glucose-6-phosphate to glucose in the liver. We initially examined fasting-and postprandial 2

  7. Distinguishing two types of gray mullet, Mugil cephalus L. (Mugiliformes: Mugilidae), by using glucose-6-phosphate isomerase (GPI) allozymes with special reference to enzyme activities.

    Science.gov (United States)

    Huang, C S; Weng, C F; Lee, S C

    2001-06-01

    The resident and migratory types of gray mullet, Mugil cephalus, on the coast of Taiwan can not be separated morphologically. Allozyme analysis was applied to estimate genetic variation between the two types of gray mullet and to test whether they belong to different populations. After starch gel electrophoresis, different allelic frequency spectra of glucose-6-phosphate isomerase-A (GPI-A) between stocks was observed. The resident stock contained Gpi-A(135) and Gpi-A(100), whereas the migratory type contained Gpi-A(100) only. In addition, GPI activities of locus A showed two distinct profiles between the two alleles. The results broadly revealed that Gpi-A allelic frequency was not regulated by temperature changes even after 6 months of thermal acclimation. This suggests that natural selection may play a role in shaping the allelic frequency change during the migratory journey. These findings suggest that the Gpi-A allelic difference can be used for population discrimination.

  8. Dexamethasone increases glucose cycling, but not glucose production, in healthy subjects

    International Nuclear Information System (INIS)

    Wajngot, A.; Khan, A.; Giacca, A.; Vranic, M.; Efendic, S.

    1990-01-01

    We established that measurement of glucose fluxes through glucose-6-phosphatase (G-6-Pase; hepatic total glucose output, HTGO), glucose cycling (GC), and glucose production (HGP), reveals early diabetogenic changes in liver metabolism. To elucidate the mechanism of the diabetogenic effect of glucocorticoids, we treated eight healthy subjects with oral dexamethasone (DEX; 15 mg over 48 h) and measured HTGO with [2-3H]glucose and HGP with [6-3H]glucose postabsorptively and during a 2-h glucose infusion (11.1 mumol.kg-1.min-1). [2-3H]- minus [6-3H]glucose equals GC. DEX significantly increased plasma glucose, insulin, C peptide, and HTGO, while HGP was unchanged. In controls and DEX, glucose infusion suppressed HTGO (82 vs. 78%) and HGP (87 vs. 91%). DEX increased GC postabsorptively (three-fold) P less than 0.005 and during glucose infusion (P less than 0.05) but decreased metabolic clearance and glucose uptake (Rd), which eventually normalized, however. Because DEX increased HTGO (G-6-Pase) and not HGP (glycogenolysis + gluconeogenesis), we assume that DEX increases HTGO and GC in humans by activating G-6-Pase directly, rather than by expanding the glucose 6-phosphate pool. Hyperglycemia caused by peripheral effects of DEX can also contribute to an increase in GC by activating glucokinase. Therefore, measurement of glucose fluxes through G-6-Pase and GC revealed significant early effects of DEX on hepatic glucose metabolism, which are not yet reflected in HGP

  9. Prevalence and risk factors of anaemia in paediatric patients in ...

    African Journals Online (AJOL)

    South African Journal of Child Health ... deficiency 42.3% (p=0.001), glucose-6 phosphate dehydrogenase (G6PD) deficiency 24.8% (p=0.02), ... Malaria and iron deficiency remain common among ill children <5 years old who are anaemic.

  10. Differences in associations between markers of antioxidative defense and asthma are sex specific

    DEFF Research Database (Denmark)

    Malling, Tine Halsen; Sigsgaard, Torben; Andersen, Helle R

    2010-01-01

    on a screening questionnaire, random sampling, or both. Serum selenium concentrations and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase [GPX], glutathione reductase [GR], and glucose-6-phosphate dehydrogenase [G6PD]) in erythrocytes were measured. Asthma was defined as either...

  11. Molecular Characterization of Glucose-6-Phosphate ...

    African Journals Online (AJOL)

    G6PD) deficiency among staff and students of a university community in Malaysia as well as to identify molecular genetics by determination of G6PD mutations. Methods: Cross-sectional and experimental studies were carried out on the staff ...

  12. Increased response to insulin of glucose metabolism in the 6-day unloaded rat soleus muscle

    Science.gov (United States)

    Henriksen, Erik J.; Tischler, Marc E.; Johnson, David G.

    1986-01-01

    Hind leg muscles of female rats were unloaded by tail cast suspension for 6 days. In the fresh-frozen unloaded soleus, the significantly greater concentration of glycogen correlated with a lower activity ratio of glycogen phosphorylase (p less than 0.02). The activity ratio of glycogen synthase also was lower (p less than 0.001), possibly due to the higher concentration of glycogen. In isolated unloaded soleus, insulin (0.1 milliunit/ml) increased the oxidation of D(U-C-14) glucose, release of lactate and pyruvate, incorporation of D-(U-C-14) glucose into glycogen, and the concentration of glucose 6-phosphate more (p less than 0.05) than in the weight-bearing soleus. At physiological doses of insulin, the percent of maximal uptake of 2-deoxy-D-(1,2-H-3) glucose/muscle also was greater in the unloaded soleus. Unloading of the soleus increased, by 50 percent the concentration of insuling receptors, due to no decrease in total receptor number during muscle atrophy. This increase may account for the greater response of glucose metabolism to insulin in this muscle. The extensor digitorum longus, which generally shows little response to unloading, displayed no differential response of glucose metabolism to insulin.

  13. The metabolism of carbohydrates and lipid peroxidation in lead-exposed workers.

    Science.gov (United States)

    Kasperczyk, Aleksandra; Dobrakowski, Michal; Ostałowska, Alina; Zalejska-Fiolka, Jolanta; Birkner, Ewa

    2015-12-01

    The present study was undertaken to estimate the effect of occupational exposure to lead on the blood concentration of glucose and several enzymes involved in glycolysis, the citric acid cycle, and the pentose phosphate pathway. To estimate the degree of lipid peroxidation, the concentrations of conjugated dienes were determined. The examined group included 145 healthy male employees of lead-zinc works. Taking into account the mean blood lead levels, the examined group was divided into two subgroups. The control group was composed of 36 healthy male administrative workers. The markers of lead exposure were significantly elevated in both subgroups when compared with the controls. There were no significant changes in fasting glucose concentration and fructose-1,6-bisphosphate aldolase activity in the study population. The concentration of conjugated dienes was significantly higher in both subgroups, whereas the activity of malate dehydrogenase was significantly higher only in the group with higher exposure. The activities of lactate dehydrogenase and sorbitol dehydrogenase were significantly decreased in the examined subgroups. The activity of glucose-6-phosphate dehydrogenase decreased significantly in the group with higher exposure and could be the cause of the elevated concentrations of conjugated dienes. It is possible to conclude that lead interferes with carbohydrate metabolism, but compensatory mechanisms seem to be efficient, as glucose homeostasis in lead-exposed workers was not disturbed. © The Author(s) 2013.

  14. Are free glucose and glucose-6-phosphate in milk indicators of specific physiological states in the cow?

    DEFF Research Database (Denmark)

    Larsen, Torben; Moyes, Kasey M

    2015-01-01

    that they are not hydrolysis product from lactose post secretion, but rather reflecting the energy status of the mammary epithelial cells pre-secretion. Wide variation in range of these metabolites, that is, from 90 to 630 μM and 5 to 324 μM, for glucose and G6P, respectively, was observed. During the first 21 weeks in milk....... In addition, lactose, protein, fat, citrate and β-hydroxybutyrate were determined and comparisons between these variables were made. Data were analysed using GLM model for the effect of parity, breed, time from last milking and stage of lactation on variations in parameters in milk. Pearson’s correlations...

  15. Evidence for carbon flux shortage and strong carbon/nitrogen interactions in pea nodules at early stages of water stress.

    Science.gov (United States)

    Gálvez, Loli; González, Esther M; Arrese-Igor, Cesar

    2005-09-01

    Symbiotic N2 fixation in legume nodules declines under a wide range of environmental stresses. A high correlation between N2 fixation decline and sucrose synthase (SS; EC 2.4.1.13) activity down-regulation has been reported, although it has still to be elucidated whether a causal relationship between SS activity down-regulation and N2 fixation decline can be established. In order to study the likely C/N interactions within nodules and the effects on N2 fixation, pea plants (Pisum sativum L. cv. Sugar snap) were subjected to progressive water stress by withholding irrigation. Under these conditions, nodule SS activity declined concomitantly with apparent nitrogenase activity. The levels of UDP-glucose, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate decreased in water-stressed nodules compared with unstressed nodules. Drought also had a marked effect on nodule concentrations of malate, succinate, and alpha-ketoglutarate. Moreover, a general decline in nodule adenylate content was detected. NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42) was the only enzyme whose activity increased as a result of water deficit, compensating for a possible C/N imbalance and/or supplying NADPH in circumstances that the pentose phosphate pathway was impaired, as suggested by the decline in glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) activity. The overall results show the occurrence of strong C/N interactions in nodules subjected to water stress and support a likely limitation of carbon flux that might be involved in the decline of N2 fixation under drought.

  16. Performance of strip-based glucose meters and cassette-based blood gas analyzer for monitoring glucose levels in a surgical intensive care setting.

    Science.gov (United States)

    Claerhout, Helena; De Prins, Martine; Mesotten, Dieter; Van den Berghe, Greet; Mathieu, Chantal; Van Eldere, Johan; Vanstapel, Florent

    2016-01-01

    We verified the analytical performance of strip-based handheld glucose meters (GM) for prescription use, in a comparative split-sample protocol using blood gas samples from a surgical intensive care unit (ICU). Freestyle Precision Pro (Abbott), StatStrip Connectivity Meter (Nova), ACCU-CHEK Inform II (Roche) were evaluated for recovery/linearity, imprecision/repeatability. The GMs and the ABL90 (Radiometer) blood gas analyzer (BGA) were tested for relative accuracy vs. the comparator hexokinase glucose-6-phosphate-dehydrogenase (HK/G6PDH) assay on a Cobas c702 analyzer (Roche). Recovery of spiked glucose was linear up to 19.3 mmol/L (347 mg/dL) with a slope of 0.91-0.94 for all GMs. Repeatability estimated by pooling duplicate measurements on samples below (n=9), in (n=51) or above (n=80) the 4.2-5.9 mM (74-106 mg/dL) range were for Freestyle Precision Pro: 4.2%, 4.0%, 3.6%; StatStrip Connectivity Meter: 4.0%, 4.3%, 4.5%; and ACCU-CHEK Inform II: 1.4%, 2.5%, 3.5%. GMs were in agreement with the comparator method. The BGA outperformed the GMs, with a MARD of 3.9% compared to 6.5%, 5.8% and 4.4% for the FreeStyle, StatStrip and ACCU-CHEK, respectively. Zero % of the BGA results deviated more than the FDA 10% criterion as compared to 9.4%, 3.7% and 2.2% for the FreeStyle, StatStrip and ACCU-CHEK, respectively. For all GMs, icodextrin did not interfere. Variation in the putative influence factors hematocrit and O2 tension could not explain observed differences with the comparator method. GMs quantified blood glucose in whole blood at about the 10% total error criterion, proposed by the FDA for prescription use.

  17. Electrochemical biosensor based on glucose oxidase encapsulated within enzymatically synthesized poly(1,10-phenanthroline-5,6-dione).

    Science.gov (United States)

    Ciftci, Hakan; Oztekin, Yasemin; Tamer, Ugur; Ramanaviciene, Almira; Ramanavicius, Arunas

    2014-11-01

    This study is focused on the investigation of electrocatalytic effect of glucose oxidase (GOx) immobilized on the graphite rod (GR) electrode. The enzyme modified electrode was prepared by encapsulation of immobilized GOx within enzymatically formed poly(1,10-phenanthroline-5,6-dione) (pPD) film. The electrochemical responses of such enzymatic electrode (pPD/GOx/GR) vs. different glucose concentrations were examined chronoamperometrically in acetate-phosphate buffer solution (A-PBS), pH 6.0, under aerobic or anaerobic conditions. Amperometric signals of the pPD/GOx/GR electrode exhibited well-defined hyperbolic dependence upon glucose concentration. Amperometric signals at 100mM of glucose were 41.17 and 32.27 μA under aerobic and anaerobic conditions, respectively. Amperometric signals of the pPD/GOx/GR electrode decreased by 6% within seven days. The pPD/GOx/GR electrode showed excellent selectivity in the presence of dopamine and uric acid. Furthermore it had a good reproducibility and repeatability with standard deviation of 9.4% and 8.0%, respectively. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Glucose 6-phosphate compartmentation and the control of glycogen synthesis

    NARCIS (Netherlands)

    Meijer, Alfred

    2002-01-01

    Using adenovirus-mediated gene transfer into FTO-2B cells, a rat hepatoma cell line, we have overexpressed hexokinase I, (HK I), glucokinase (GK), liver glycogen synthase (LGS), muscle glycogen synthase (MGS), and combinations of each of the two glucose phosphorylating enzymes with each one of the

  19. Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producer Ralstonia eutropha H16 by incubation with high substrate levels.

    Science.gov (United States)

    Franz, A; Rehner, R; Kienle, A; Grammel, H

    2012-01-01

    The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering. Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system. A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far. The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics. © 2011 The Authors. Letters in Applied Microbiology ©2011 The Society for Applied Microbiology.

  20. Diammonium phosphate stimulates transcription of L-lactate dehydrogenase leading to increased L-lactate production in the thermotolerant Bacillus coagulans strain.

    Science.gov (United States)

    Sun, Lifan; Li, Yanfeng; Wang, Limin; Wang, Yanping; Yu, Bo

    2016-08-01

    Exploration of cost-effective fermentation substrates for efficient lactate production is an important economic objective. Although some organic nitrogen sources are also cheaper, inorganic nitrogen salts for lactate fermentation have additional advantages in facilitating downstream procedures and significantly improving the commercial competitiveness of lactate production. In this study, we first established an application of diammonium phosphate to replace yeast extract with a reduced 90 % nitrogen cost for a thermotolerant Bacillus coagulans strain. In vivo enzymatic and transcriptional analyses demonstrated that diammonium phosphate stimulates the gene expression of L-lactate dehydrogenase, thus providing higher specific enzyme activity in vivo and increasing L-lactic acid production. This new information provides a foundation for establishing a cost-effective process for polymer-grade L-lactic acid production in an industrial setting.

  1. [Discovery of the target genes inhibited by formic acid in Candida shehatae].

    Science.gov (United States)

    Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

    2014-01-04

    At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

  2. Glucose metabolism and recycling by hepatocytes of OB/OB and ob/ob mice

    International Nuclear Information System (INIS)

    Lahtela, J.T.; Wals, P.A.; Katz, J.

    1990-01-01

    Hepatocytes were prepared from livers of ob/ob (obese diabetic) mice and their lean (OB/OB) siblings that had been fasted for 24 h. The hepatocytes were incubated with [U-14C, 2-3H]-, [U-14C, 3-3H]-, and [U-14C, 6-3H]glucose at concentrations from 20 to 120 mM. 14C was recovered mainly in CO2, glycogen, and lactate. Tritium was recovered in water and glycogen. The yield in labeled products from [2-3H]glucose ranged from two to three times that from [U-14C]glucose. The yields from [3-3H]- and [6-3H]glucose were similar, and 1.3-1.7 times that from [U-14C]glucose. At 40 mM, total utilization of glucose by obese mice was about twice that for lean mice, but there was little difference at 120 mM. The rate of recycling between glucose and glucose 6-phosphate was calculated. An equation to calculate the rate of recycling of glucose from the 2-3H/U-14C ratio in glycogen is derived in the APPENDIX. Our results show that (1) the utilization of glucose by hepatocytes from obese diabetic mice exceeds that of their lean controls, (2) the rate of glucose phosphorylation in both groups greatly exceeds glucose uptake and the rate of glycogen synthesis, (3) glucose phosphorylation represents a difference between a high glucokinase rate and hydrolysis of glucose 6-phosphate, and (4) recycling of glucose carbon between glucose 6-phosphate and pyruvate occurs within mouse hepatocytes

  3. 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.

  4. Direct analysis of [6,6-(2)H2]glucose and [U-(13)C6]glucose dry blood spot enrichments by LC-MS/MS.

    Science.gov (United States)

    Coelho, Margarida; Mendes, Vera M; Lima, Inês S; Martins, Fátima O; Fernandes, Ana B; Macedo, M Paula; Jones, John G; Manadas, Bruno

    2016-06-01

    A liquid chromatography tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM) in a triple-quadrupole scan mode was developed and comprehensively validated for the determination of [6,6-(2)H2]glucose and [U-(13)C6]glucose enrichments from dried blood spots (DBS) without prior derivatization. The method is demonstrated with dried blood spots obtained from rats administered with a primed-constant infusion of [U-(13)C6]glucose and an oral glucose load enriched with [6,6-(2)H2]glucose. The sensitivity is sufficient for analysis of the equivalent to blood and the overall method was accurate and precise for the determination of DBS isotopic enrichments. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Hydrogen peroxide produced by glucose oxidase affects the performance of laccase cathodes in glucose/oxygen fuel cells: FAD-dependent glucose dehydrogenase as a replacement.

    Science.gov (United States)

    Milton, Ross D; Giroud, Fabien; Thumser, Alfred E; Minteer, Shelley D; Slade, Robert C T

    2013-11-28

    Hydrogen peroxide production by glucose oxidase (GOx) and its negative effect on laccase performance have been studied. Simultaneously, FAD-dependent glucose dehydrogenase (FAD-GDH), an O2-insensitive enzyme, has been evaluated as a substitute. Experiments focused on determining the effect of the side reaction of GOx between its natural electron acceptor O2 (consumed) and hydrogen peroxide (produced) in the electrolyte. Firstly, oxygen consumption was investigated by both GOx and FAD-GDH in the presence of substrate. Relatively high electrocatalytic currents were obtained with both enzymes. O2 consumption was observed with immobilized GOx only, whilst O2 concentration remained stable for the FAD-GDH. Dissolved oxygen depletion effects on laccase electrode performances were investigated with both an oxidizing and a reducing electrode immersed in a single compartment. In the presence of glucose, dramatic decreases in cathodic currents were recorded when laccase electrodes were combined with a GOx-based electrode only. Furthermore, it appeared that the major loss of performance of the cathode was due to the increase of H2O2 concentration in the bulk solution induced laccase inhibition. 24 h stability experiments suggest that the use of O2-insensitive FAD-GDH as to obviate in situ peroxide production by GOx is effective. Open-circuit potentials of 0.66 ± 0.03 V and power densities of 122.2 ± 5.8 μW cm(-2) were observed for FAD-GDH/laccase biofuel cells.

  6. Astrocytic mitochondrial membrane hyperpolarization following extended oxygen and glucose deprivation.

    Directory of Open Access Journals (Sweden)

    Andrej Korenić

    Full Text Available Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψ(m in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD, OGD and combinations of both conditions varying in duration and sequence. Changes in Δψ(m, visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψ(m during reperfusion, whereas GD caused a robust Δψ(m negativation. In case no Δψ(m negativation was observed after OGD, subsequent chemical oxygen deprivation (OD induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψ(m hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen and their hyperpolarizing effect on Δψ(m during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury.

  7. Investigation on the Metabolic Regulation of pgi gene knockout Escherichia coli by Enzyme Activities and Intracellular Metabolite Concentrations

    Directory of Open Access Journals (Sweden)

    Nor ‘Aini, A. R.

    2006-01-01

    Full Text Available An integrated analysis of the cell growth characteristics, enzyme activities, intracellular metabolite concentrations was made to investigate the metabolic regulation of pgi gene knockout Escherichia coli based on batch culture and continuous culture which was performed at the dilution rate of 0.2h-1. The enzymatic study identified that pathways of pentose phosphate, ED pathway and glyoxylate shunt were all active in pgi mutant. The glycolysis enzymes i.e glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphatase, pyruvate kinase, triose phosphate isomerase were down regulated implying that the inactivation of pgi gene reduced the carbon flux through glycolytic pathway. Meanwhile, the pentose phosphate pathway was active as a major route for intermediary carbohydrate metabolism instead of glycolysis. The pentose phosphate pathway generates most of the major reducing co-factor NADPH as shown by the increased of NADPH/NADP+ ratio in the mutant when compared with the parent strain. The fermentative enzymes such as acetate kinase and lactate dehydrogenase were down regulated in the mutant. Knockout of pgi gene results in the significant increase in the intracellular concentration of glucose-6-phosphate and decrease in the concentration of oxaloacetate. The slow growth rate of the mutant was assumed to be affected by the accumulation of glucose-6-phosphate and imbalance of NADPH reoxidation.

  8. AglM and VNG1048G, Two Haloarchaeal UDP-Glucose Dehydrogenases, Show Different Salt-Related Behaviors

    OpenAIRE

    Kandiba, Lina; Eichler, Jerry

    2016-01-01

    Haloferax volcanii AglM and Halobacterium salinarum VNG1048G are UDP-glucose dehydrogenases involved in N-glycosylation in each species. Despite sharing >60% sequence identity and the ability of VNG1048G to functionally replace AglM in vivo, these proteins behaved differently as salinity changed. Whereas AglM was active in 2–4 M NaCl, VNG1048G lost much of its activity when salinity dropped below 3 M NaCl. To understand the molecular basis of this phenomenon, each protein was examined by s...

  9. Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.

    Science.gov (United States)

    Holtgrefe, Simone; Gohlke, Jochen; Starmann, Julia; Druce, Samantha; Klocke, Susanne; Altmann, Bianca; Wojtera, Joanna; Lindermayr, Christian; Scheibe, Renate

    2008-06-01

    Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

  10. CHANGES IN SERUM ENZYMES LEVELS ASSOCIATED WITH LIVER FUNCTIONS IN STRESSED MARWARI GOAT

    Directory of Open Access Journals (Sweden)

    Kataria N.

    2011-03-01

    Full Text Available Serum enzyme levels were determined in goats of Marwari breed belonging to farmers’ stock of arid tract of Rajasthan state, India. The animals were grouped into healthy and stressed comprising of gastrointestinal parasiticised, pneumonia affected, and drought affected. The serum enzymes determined were sorbitol dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, ornithine carbamoyl transferase, gamma-glutamayl transferase, 5’nucleotidase, glucose-6-phosphatase, arginase, and aldolase. In stressed group the mean values of all the enzymes increased significantly (p≤0.05 as compared to respective healthy mean value. All the enzymes showed highest values in the gastrointestinal parasiticised animals and least values in the animals having pneumonia. In gastrointestinal parasiticised animals maximum change was observed in G-6-Pase activity and minimum change was observed in malate dehydrogenase mean value. It was concluded that Increased activity of all the serum enzymes was due to modulation of liver functions directly or indirectly.

  11. A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

    International Nuclear Information System (INIS)

    Macedo, Edangelo M.S. de; Silva, Maria G.V.; Wiggers, Helton J.; Montanari, Carlos A.; Braz-Filho, Raimundo; Andricopulo, Adriano D.

    2009-01-01

    A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi- 10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (K i ) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 +-2.47 μmol L -1 . The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease. (author)

  12. Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

    Science.gov (United States)

    Gordon, G L; Doelle, H W

    1976-08-16

    The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

  13. Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis

    DEFF Research Database (Denmark)

    Carobbio, Stefania; Frigerio, Francesca; Rubi, Blanca

    2009-01-01

    Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been...... tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin...... secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored...

  14. Effect of trichloroethylene (TCE) toxicity on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in kidney and other rat tissues.

    Science.gov (United States)

    Khan, Sheeba; Priyamvada, Shubha; Khan, Sara A; Khan, Wasim; Farooq, Neelam; Khan, Farah; Yusufi, A N K

    2009-07-01

    Trichloroethylene (TCE), an industrial solvent, is a major environmental contaminant. Histopathological examinations revealed that TCE caused liver and kidney toxicity and carcinogenicity. However, biochemical mechanism and tissue response to toxic insult are not completely elucidated. We hypothesized that TCE induces oxidative stress to various rat tissues and alters their metabolic functions. Male Wistar rats were given TCE (1000 mg/kg/day) in corn oil orally for 25 d. Blood and tissues were collected and analyzed for various biochemical and enzymatic parameters. TCE administration increased blood urea nitrogen, serum creatinine, cholesterol and alkaline phosphatase but decreased serum glucose, inorganic phosphate and phospholipids indicating kidney and liver toxicity. Activity of hexokinase, lactate dehydrogenase increased in the intestine and liver whereas decreased in renal tissues. Malate dehydrogenase and glucose-6-phosphatase and fructose-1, 6-bisphosphatase decreased in all tissues whereas increased in medulla. Glucose-6-phosphate dehydrogenase increased but NADP-malic enzyme decreased in all tissues except in medulla. The activity of BBM enzymes decreased but renal Na/Pi transport increased. Superoxide dismutase and catalase activities variably declined whereas lipid peroxidation significantly enhanced in all tissues. The present results indicate that TCE caused severe damage to kidney, intestine, liver and brain; altered carbohydrate metabolism and suppressed antioxidant defense system.

  15. Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells.

    Science.gov (United States)

    Lee, Su Min; Koh, Ho-Jin; Park, Dong-Chan; Song, Byoung J; Huh, Tae-Lin; Park, Jeen-Woo

    2002-06-01

    NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.

  16. Salinity stress effects on [14C-1]- and [14C-6]-glucose metabolism of a salt-tolerant and salt-susceptible variety of wheat

    International Nuclear Information System (INIS)

    Krishnaraj, S.; Thorpe, T.A.

    1996-01-01

    The effect of salt (sodium sulfate) on carbohydrate metabolism was studied in a salt-tolerant (Kharchia-65) variety and a salt-susceptible (Fielder) variety of wheat (Triticum aestivum L.) by comparing their responses under control and stress conditions. Leaf segments of Kharchia-65 showed increased activity through both the pentose phosphate pathway (PPP) and the glycolytic pathway of glucose oxidation, with the former being comparatively more active in response to salt. In Fielder, there was an increase in PPP activity at the expense of glycolytic pathway activity. Label from glucose was found in the lipid, neutral sugar, amino acid, organic acid, and phosphate ester fractions in all treatments. On the basis of the label distribution patterns, it appears that Fielder leaves incubated with [ 14 C-6]-glucose were not able to utilize glucose efficiently under saline conditions. This finding was further supported by decreased label incorporation into all the fractions, especially the amino acid and organic acid fractions. Adenosine phosphate and reduced pyridine nucleotide concentrations were consistent with these observations. We conclude therefore that the salt-tolerant variety had an enhanced metabolic activity compared with the salt-susceptible variety, which contributed to its ability to overcome the adverse effects of salt. (author)

  17. Increasing synthetic serum substitute (SSS) concentrations in P1 glucose/phosphate-free medium improves implantation rate: a comparative study.

    Science.gov (United States)

    Ben-Yosef, D; Yovel, I; Schwartz, T; Azem, F; Lessing, J B; Amit, A

    2001-11-01

    To assess the comparative efficacy of IVF medium (MediCult, with 5.2 mM glucose) and a glucose/phosphate-free medium, P1 (Irvine Scientific), and to investigate the influence of increasing the serum supplementation (synthetic serum substitute; SSS; Irvine Scientific) to P1 on embryo development and implantation. Patients were randomly assigned to IVF medium (Group 1, cycles n = 172) or P1 supplemented with 10% SSS (Group 2, cycles n = 229) according to the medium scheduled for use on the day of oocyte retrieval. Another 555 IVF consequent cycles (Group 3) were performed using increased SSS concentrations (20%) in P1 medium. In this large series of IVF cycles, we herein demonstrate that significantly higher pregnancy and implantation rates were found when embryos were cultured in glucose/phosphate-free medium P1 supplemented with 20% SSS compared to supplementation with the lower SSS concentration and with IVF medium.

  18. Adhesion activity of glyceraldehyde-3-phosphate dehydrogenase in a Chinese Streptococcus suis type 2 strain.

    Science.gov (United States)

    Wang, Kaicheng; Lu, Chengping

    2007-01-01

    A total of 36 streptococcal strains, including seven S. equi ssp.zooepidemicus, two S. suis type 1 (SS1), 24 SS2, two SS9, and one SS7, were tested for glyceraldehyde-3-phosphate dehydrogenase gene (gapdh). Except from non-virulent SS2 strain T1 5, all strains harboured gapdh. The gapdh of Chinese Sichuan SS2 isolate ZY05719 and Jiangsu SS2 isolate HA9801 were sequenced and then compared with published sequences in the GenBank. The comparison revealed a 99.9 % and 99.8 % similarity of ZY05719 and HA9801, respectively, with the published sequence. Adherence assay data demonstrated a significant ((p<0.05)) reduction in adhesion of SS2 in HEp-2 cells pre-incubated with purified GAPDH compared to non pre-incubated controls, suggesting the GAPDH mediates SS2 bacterial adhesion to host cells.

  19. Heat stress induced changes in metabolic regulators of donkeys from arid tracts in India

    Directory of Open Access Journals (Sweden)

    Kataria N.

    2012-05-01

    Full Text Available To find out heat stress induced changes in metabolic regulators of donkeys from arid tracts in India, blood samples were collected to harvest the serum during moderate and extreme hot ambiences. The metabolic enzymes determined were sorbitol dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, ornithine carbamoyl transferase, gammaglutamayl transferase, 5’nucleotidase, glucose-6-phosphatase, arginase, and aldolase. The mean values of all the serum enzymes increased significantly (p≤0.05 during hot ambience as compared to respective values during moderate ambience. It was concluded that increased activity of all the enzymes in the serum was due to modulation of metabolic reactions to combat the effect of hot ambience on the animals. Activation of gluconeogenesis along with hexose monophosphate shunt and urea cycle probably helped the animals to combat the heat stress.

  20. Metabolic Control Analysis aimed at the ribose synthesis pathways of tumor cells: a new strategy for antitumor drug development

    NARCIS (Netherlands)

    Boren, Joan; Montoya, Antonio Ramos; de Atauri, Pedro; Comin-Anduix, Begoña; Cortes, Antonio; Centelles, Josep J.; Frederiks, Wilma M.; van Noorden, Cornelis J. F.; Cascante, Marta

    2002-01-01

    Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with lower concentrations of drug, if an enzyme with a high control coefficient on tumor growth is being inhibited. Here we measure glucose-6-phosphate dehydrogenase (G6PDH) control coefficient on in vivo

  1. Lactobacillus reuteri glyceraldehyde-3-phosphate dehydrogenase functions in adhesion to intestinal epithelial cells.

    Science.gov (United States)

    Zhang, Wen-Ming; Wang, Hai-Feng; Gao, Kan; Wang, Cong; Liu, Li; Liu, Jian-Xin

    2015-05-01

    This study was aimed to identify key surface proteins mediating the adhesion of lactobacilli to intestinal epithelial cells. By using Caco-2 and IPEC-J2 cells labeled with sulfo-NHS-biotin in the western blotting, a protein band of an approximately 37 kDa was detected on the surface layer of Lactobacillus reuteri strains ZJ616, ZJ617, ZJ621, and ZJ623 and Lactobacillus rhamnosus GG. Mass spectrometry analysis using the adhesion-related protein from L. reuteri ZJ617 showed that it was 100% homologous to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. reuteri JCM 1112 (GenBank: YP_001841377). The ability of L. reuteri ZJ617 to adhere to epithelial cells decreased significantly by treatment with LiCl or by blocking with an anti-GAPDH antibody, in comparison with the untreated strain (p reuteri ZJ617. The results indicated that the GAPDH protein of L. reuteri ZJ617 acts as an adhesion component that plays an important role in binding to the intestinal epithelial cells.

  2. Trehalose 6-phosphate signal is closely related to sorbitol in apple (Malus domestica Borkh. cv. Gala

    Directory of Open Access Journals (Sweden)

    Wen Zhang

    2017-02-01

    Full Text Available Trehalose-6-phosphate (Tre6P is a precursor of trehalose, which is widespread in nature and greatly influences plant growth and development. Tre6P acts as a signal of carbon availability in many plants, but little is known about the function of Tre6P in rosaceous plants, which have specific sorbitol biosynthesis and transportation pathways. In the present study, Tre6P levels and Sorbitol:Tre6P ratios were analyzed in apple (Malus domestica, Borkh. cv. Gala. Tre6P levels were positively correlated with sorbitol content but negatively correlated with sucrose, glucose, and fructose content in developing fruit. However, under sorbitol-limited conditions, Tre6P levels were positively correlated with both sorbitol and sucrose. In the presence of different exogenous sugar supply, Tre6P levels increased corresponding with sorbitol, but this was not the case with sucrose. In addition, Tre6P content and sorbitol:Tre6P ratios were more highly correlated with ADP-glucose levels under sorbitol-limited conditions and fruit development stages, respectively. These results suggest that Tre6P is more closely related to sorbitol than other soluble sugars and has an important role in influencing carbon metabolism in apple.

  3. Trehalose 6-phosphate signal is closely related to sorbitol in apple (Malus domestica Borkh. cv. Gala)

    Science.gov (United States)

    Zhang, Wen; Lunn, John E.; Feil, Regina; Wang, Yufei; Zhao, Jingjing; Tao, Hongxia; Zhao, Zhengyang

    2017-01-01

    ABSTRACT Trehalose-6-phosphate (Tre6P) is a precursor of trehalose, which is widespread in nature and greatly influences plant growth and development. Tre6P acts as a signal of carbon availability in many plants, but little is known about the function of Tre6P in rosaceous plants, which have specific sorbitol biosynthesis and transportation pathways. In the present study, Tre6P levels and Sorbitol:Tre6P ratios were analyzed in apple (Malus domestica, Borkh. cv. Gala). Tre6P levels were positively correlated with sorbitol content but negatively correlated with sucrose, glucose, and fructose content in developing fruit. However, under sorbitol-limited conditions, Tre6P levels were positively correlated with both sorbitol and sucrose. In the presence of different exogenous sugar supply, Tre6P levels increased corresponding with sorbitol, but this was not the case with sucrose. In addition, Tre6P content and sorbitol:Tre6P ratios were more highly correlated with ADP-glucose levels under sorbitol-limited conditions and fruit development stages, respectively. These results suggest that Tre6P is more closely related to sorbitol than other soluble sugars and has an important role in influencing carbon metabolism in apple. PMID:28069587

  4. Effect of CoO nanoparticles on the carbohydrate metabolism of the brain of

    Directory of Open Access Journals (Sweden)

    Shamshad M. Shaikh

    2016-10-01

    Full Text Available The effect of CoO nanoparticles (NPs on the brain of mice administered through gastrointestinal tract for a period of 30 days was studied. AAS analysis revealed that NPs administered orally were retained by cerebellum, cerebral cortex, medulla oblongata and olfactory bulb. This retention of nanoparticles by the brain promoted a significant increase in glucose, pyruvate, lactate and glycogen levels along with the concomitant increase in hexokinase, glucose 6 phosphatase, and lactate dehydrogense activities. However, a decrease in glucose 6 phosphate dehydrogenase activity was observed in the brain regions indicating a deterioration of the pentose phosphate pathway. Thus, the present study suggests that the CoO NPs affect the carbohydrate metabolism of the brain.

  5. Metabolic engineering of an ATP-neutral Embden-Meyerhof-Parnas pathway in Corynebacterium glutamicum: growth restoration by an adaptive point mutation in NADH dehydrogenase.

    Science.gov (United States)

    Komati Reddy, Gajendar; Lindner, Steffen N; Wendisch, Volker F

    2015-03-01

    Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

  6. Underestimation of glucose turnover corrected with high-performance liquid chromatography purification of [6-3H]glucose

    International Nuclear Information System (INIS)

    Schwenk, W.F.; Butler, P.C.; Haymond, M.W.; Rizza, R.A.

    1990-01-01

    We have recently reported that during infusion of commercially available [6-3H]glucose, a radioactive nonglucose contaminant may accumulate in plasma causing errors in the measurement of glucose turnover. To determine whether purification of this tracer by HPLC (high-performance liquid chromatography) before infusion would eliminate the contaminant in plasma and remove the underestimation of glucose turnover reported during hyperinsulinemia, four normal subjects each underwent two 5-h euglycemic clamps during infusion of insulin (1 mU.kg-1.min-1). Glucose turnover was measured with either commercially available [6-3H]glucose or with HPLC-purified [6-3H]glucose. HPLC analysis of samples from the clamps done with commercially available [6-3H]glucose showed that 9.7% of the infused tracer and 26% of the plasma glucose 3H radioactivity were contaminants. In contrast, no contaminant was observed in the plasma during infusion of HPLC-purified [6-3H]glucose. During the last hour of the clamp, mean glucose turnover using commercially available [6-3H]glucose was less (P less than 0.01) than the mean glucose infusion rate (7.6 +/- 0.3 vs. 10.5 +/- 0.3 mg.kg-1.min-1) yielding apparent negative (P less than 0.001) hepatic glucose release. In contrast, when HPLC-purified [6-3H]glucose was employed, glucose turnover equaled the glucose infusion rate (10.4 +/- 0.9 vs. 10.2 +/- 0.9 mg.kg-1.min-1) and hepatic glucose release was no longer negative. We conclude that removal of a tritiated nonglucose contaminant in [6-3H]glucose by HPLC yields correct estimations of glucose turnover at steady state

  7. 31P NMR saturation-transfer measurements in Saccharomyces cerevisiae: characterization of phosphate exchange reactions by iodoacetate and antimycin A inhibition

    International Nuclear Information System (INIS)

    Campbell-Burk, S.L.; Jones, K.A.; Shulman, R.G.

    1987-01-01

    31 P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. The results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A. Intracellular phosphate (P/sub i/) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state ATP/sub λ/ saturation and were used in combination with ST data to determine P/sub i/ consumption rates. 13 C NMR and O 2 electrode measurements were also conducted to monitor changes in rates of glucose consumption and O 2 consumption, respectively, under the various metabolic conditions examined. The results suggest that GAPDH/PGK-catalyzed P/sub i/-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobic and aerobic glucose-fed yeast. Kinetics through GAPDH/PGK were found to depend on metabolic conditions. The coupled system appears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol. Additionally, mitochondrial ATPase activity appears to be responsible for the transfer observed in iodoacetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally

  8. Browse Title Index

    African Journals Online (AJOL)

    Items 1 - 50 of 60 ... Vol 1, No 2 (2012), Assessment of the activity of glucose-6-phosphate dehydrogenase in ... Vol 5, No 3 (2016), Effect of aqueous extract of alligator pepper ... antigen sero-positivity and Hepatitis C virus among voluntary blood ...

  9. Heterologous expression of pyrroloquinoline quinone (pqq) gene cluster confers mineral phosphate solubilization ability to Herbaspirillum seropedicae Z67.

    Science.gov (United States)

    Wagh, Jitendra; Shah, Sonal; Bhandari, Praveena; Archana, G; Kumar, G Naresh

    2014-06-01

    Gluconic acid secretion mediated by the direct oxidation of glucose by pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) is responsible for mineral phosphate solubilization in Gram-negative bacteria. Herbaspirillum seropedicae Z67 (ATCC 35892) genome encodes GDH apoprotein but lacks genes for the biosynthesis of its cofactor PQQ. In this study, pqqE of Erwinia herbicola (in plasmid pJNK1) and pqq gene clusters of Pseudomonas fluorescens B16 (pOK53) and Acinetobacter calcoaceticus (pSS2) were over-expressed in H. seropedicae Z67. Transformants Hs (pSS2) and Hs (pOK53) secreted micromolar levels of PQQ and attained high GDH activity leading to secretion of 33.46 mM gluconic acid when grown on 50 mM glucose while Hs (pJNK1) was ineffective. Hs (pJNK1) failed to solubilize rock phosphate, while Hs (pSS2) and Hs (pOK53) liberated 125.47 μM and 168.07 μM P, respectively, in minimal medium containing 50 mM glucose under aerobic conditions. Moreover, under N-free minimal medium, Hs (pSS2) and Hs (pOK53) not only released significant P but also showed enhanced growth, biofilm formation, and exopolysaccharide (EPS) secretion. However, indole acetic acid (IAA) production was suppressed. Thus, the addition of the pqq gene cluster, but not pqqE alone, is sufficient for engineering phosphate solubilization in H. seropedicae Z67 without compromising growth under nitrogen-fixing conditions.

  10. Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.

    2009-05-14

    ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

  11. Bimolecular Rate Constants for FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus and Organic Electron Acceptors.

    Science.gov (United States)

    Tsuruoka, Nozomu; Sadakane, Takuya; Hayashi, Rika; Tsujimura, Seiya

    2017-03-10

    The flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus species require suitable redox mediators to transfer electrons from the enzyme to the electrode surface for the application of bioelectrical devices. Although several mediators for FAD-GDH are already in use, they are still far from optimum in view of potential, kinetics, sustainability, and cost-effectiveness. Herein, we investigated the efficiency of various phenothiazines and quinones in the electrochemical oxidation of FAD-GDH from Aspergillus terreus . At pH 7.0, the logarithm of the bimolecular oxidation rate constants appeared to depend on the redox potentials of all the mediators tested. Notably, the rate constant of each molecule for FAD-GDH was approximately 2.5 orders of magnitude higher than that for glucose oxidase from Aspergillus sp. The results suggest that the electron transfer kinetics is mainly determined by the formal potential of the mediator, the driving force of electron transfer, and the electron transfer distance between the redox active site of the mediator and the FAD, affected by the steric or chemical interactions. Higher k ₂ values were found for ortho-quinones than for para-quinones in the reactions with FAD-GDH and glucose oxidase, which was likely due to less steric hindrance in the active site in the case of the ortho-quinones.

  12. Bimolecular Rate Constants for FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus and Organic Electron Acceptors

    Directory of Open Access Journals (Sweden)

    Nozomu Tsuruoka

    2017-03-01

    Full Text Available The flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH from Aspergillus species require suitable redox mediators to transfer electrons from the enzyme to the electrode surface for the application of bioelectrical devices. Although several mediators for FAD-GDH are already in use, they are still far from optimum in view of potential, kinetics, sustainability, and cost-effectiveness. Herein, we investigated the efficiency of various phenothiazines and quinones in the electrochemical oxidation of FAD-GDH from Aspergillus terreus. At pH 7.0, the logarithm of the bimolecular oxidation rate constants appeared to depend on the redox potentials of all the mediators tested. Notably, the rate constant of each molecule for FAD-GDH was approximately 2.5 orders of magnitude higher than that for glucose oxidase from Aspergillus sp. The results suggest that the electron transfer kinetics is mainly determined by the formal potential of the mediator, the driving force of electron transfer, and the electron transfer distance between the redox active site of the mediator and the FAD, affected by the steric or chemical interactions. Higher k2 values were found for ortho-quinones than for para-quinones in the reactions with FAD-GDH and glucose oxidase, which was likely due to less steric hindrance in the active site in the case of the ortho-quinones.

  13. HL-1 mouse cardiomyocyte injury and death after simulated ischemia and reperfusion: roles of pH, Ca2+-independent phospholipase A2, and Na+/H+ exchange

    DEFF Research Database (Denmark)

    Andersen, Ann-Dorit; Poulsen, Kristian Arild; Lambert, Ian H

    2009-01-01

    activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA(2)-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet...

  14. Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials.

    Science.gov (United States)

    Zafar, Muhammad Nadeem; Wang, Xiaoju; Sygmund, Christoph; Ludwig, Roland; Leech, Dónal; Gorton, Lo

    2012-01-03

    A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration. © 2011 American Chemical Society

  15. A review of metabolism of labeled glucoses for use in measuring glucose recycling

    International Nuclear Information System (INIS)

    Russell, R.W.; Young, J.W.

    1990-01-01

    The fate of tritium from each carbon of D-glucose and the metabolism of L-glucose and 2-deoxy-D-glucose are known. Differences in metabolism of labeled glucoses can be used to quantify physical and chemical recycling of glucose. Only physical recycling is measured by [1- 3 H]-L-glucose, whereas [U- 14 C]-D-glucose measures total recycling. The difference between [1- 3 H]-L-glucose and [U- 14 C]-D-glucose, therefore, is chemical recycling. Recycling from extracellular binding sites and hepatic glucose 6-phosphate can be measured by difference between [1,2- 3 H]-2-deoxy-D-glucose and [1- 3 H]-L-glucose, and the difference in irreversible loss of the two will measure extrahepatic uptake of D-glucose. Recycling via Cori-alanine cycle plus CO 2 is the difference in irreversible loss measured by using [6- 3 H]-glucose and [U- 14 C]-D-glucose. Recycling via the hexose monophosphate pathway can be determined by difference in irreversible loss between [1- 3 H]-D-glucose and [6- 3 H]-D-glucose. Recycling via CO 2 and glycerol must be measured directly with [U- 14 C]glucose, bicarbonate, and glycerol. Recycling via hepatic glycogen can be estimated by subtracting all other measured chemical recycling from total chemical recycling. This review describes means to quantify glucose recycling in vivo, enabling studies of mechanisms for conservation and utilization of glucose. 54 references

  16. Toxicity of Nitrification Inhibitors on Dehydrogenase Activity in Soils

    OpenAIRE

    Ferisman Tindaon; Gero Benckiser; Johannes C. G. Ottow

    2011-01-01

    The objective of this research was to determine the effects of nitrification inhibitors (NIs) such as 3,4-dimethylpyrazolephosphate=DMPP, 4-Chlor-methylpyrazole phosphate=ClMPP and dicyandiamide,DCD) which might be expected to inhibit microbial activity, on dehydrogenase activity (DRA),in three different soils in laboratory conditions. Dehydrogenase activity were assessed via reduction of 2-p-Iodophenyl-3-p-nitrophenyl-5-phenyltetrazoliumchloride (INT). The toxicity and dose response curve of...

  17. An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

    International Nuclear Information System (INIS)

    Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B.

    1989-01-01

    UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ([beta-35S]UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with [beta-35S]. UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue

  18. Adaptation to a high protein, carbohydrate-free diet induces a marked reduction of fatty acid synthesis and lipogenic enzymes in rat adipose tissue that is rapidly reverted by a balanced diet.

    Science.gov (United States)

    Brito, S M R C; Moura, M A F; Kawashita, N H; Festuccia, W T L; Garófalo, M A R; Kettelhut, I C; Migliorini, R H

    2005-06-01

    We have previously shown that in vivo lipogenesis is markedly reduced in liver, carcass, and in 4 different depots of adipose tissue of rats adapted to a high protein, carbohydrate-free (HP) diet. In the present work, we investigate the activity of enzymes involved in lipogenesis in the epididymal adipose tissue (EPI) of rats adapted to an HP diet before and 12 h after a balanced diet was introduced. Rats fed an HP diet for 15 days showed a 60% reduction of EPI fatty acid synthesis in vivo that was accompanied by 45%-55% decreases in the activities of pyruvate dehydrogenase complex, ATP-citrate lyase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and malic enzyme. Reversion to a balanced diet for 12 h resulted in a normalization of in vivo EPI lipogenesis, and in a restoration of acetyl-CoA carboxylase activity to levels that did not differ significantly from control values. The activities of ATP-citrate lyase and pyruvate dehydrogenase complex increased to about 75%-86% of control values, but the activities of glucose-6-phosphate dehydrogenase and malic enzyme remained unchanged 12 h after diet reversion. The data indicate that in rats, the adjustment of adipose tissue lipogenic activity is an important component of the metabolic adaptation to different nutritional conditions.

  19. Genetic variability in Oligosarcus paranensis (Teleostei: Characiformes from the São Francisco river, Ivaí river basin – Paraná State, Brazil - doi: 10.4025/actascibiolsci.v35i3.14179

    Directory of Open Access Journals (Sweden)

    Michele Rocha dos Santos

    2013-08-01

    Full Text Available The genetic variability of Oligosarcus paranensis was estimated from a population collected in São Francisco river, Prudentópolis county in Paraná State (Brazil using the electrophoresis in starch gel technique. Eleven enzymatic systems were analyzed: Aspartate aminotransaminase (AAT; E. C. 2.6.1, Alcohol dehydrogenase (ADH; E. C. 1.1.1.1, Esterase (EST; E. C. 3.1.1.1, Glucose-6-phosphate isomerase (GPI; E. C. 5.3.1.9, Glycerol-3-Phosphate dehydrogenase (G3PDH; E. C. 1.1.1, Isocitrate dehydrogenase (IDH; E. C. 1.1.1.42, L-lactate dehydrogenase (LDH; E. C. 1.1.1.27, Malate dehydrogenase (MDH; E. C. 1.1.1.37 , Malate dehydrogenase NADP (ME; E. C. 1.1.1.40, Phosphoglucomutase (PGM; E. C. 5.4.2.2 and Sorbitol dehydrogenase (SORB; E.C. 1.1.1.14. Twenty loci were identified through 15% corn starch gel electrophoresis of which nine (45% were polymorphic. The average expected heterozygosity was estimated as 0.1229 ± 0.1728, and the observed was 0.0586 ± 0.1069, indicating high genetic variability. The average value of FIS = 0.5145 indicates homozygote excess.  

  20. Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Is Pyruvylated during 3-Bromopyruvate Mediated Cancer Cell Death

    Science.gov (United States)

    Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H.; Kunjithapatham, Rani; Buijs, Manon; Vossen, Josephina A.; Tchernyshyov, Irina; Cole, Robert N.; Syed, Labiq H.; Rao, Pramod P.; Ota, Shinichi; Vali, Mustafa

    2013-01-01

    Background The pyruvic acid analog 3-bromopyruvate (3BrPA) is an alkylating agent known to induce cancer cell death by blocking glycolysis. The anti-glycolytic effect of 3BrPA is considered to be the inactivation of glycolytic enzymes. Yet, there is a lack of experimental documentation on the direct interaction of 3BrPA with any of the suggested targets during its anticancer effect. Methods and Results In the current study, using radiolabeled (14C) 3BrPA in multiple cancer cell lines, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the primary intracellular target of 3BrPA, based on two-dimensional (2D) gel electrophoretic autoradiography, mass spectrometry and immunoprecipitation. Furthermore, in vitro enzyme kinetic studies established that 3BrPA has marked affinity to GAPDH. Finally, Annexin V staining and active caspase-3 immunoblotting demonstrated that apoptosis was induced by 3BrPA. Conclusion GAPDH pyruvylation by 3BrPA affects its enzymatic function and is the primary intracellular target in 3BrPA mediated cancer cell death. PMID:20044597

  1. Glucose phosphorylation is not rate limiting for accumulation of glycogen from glucose in perfused livers from fasted rats

    International Nuclear Information System (INIS)

    Youn, J.H.; Ader, M.; Bergman, R.N.

    1989-01-01

    Incorporation of Glc and Fru into glycogen was measured in perfused livers from 24-h fasted rats using [6-3H]Glc and [U-14C]Fru. For the initial 20 min, livers were perfused with low Glc (2 mM) to deplete hepatic glycogen and were perfused for the following 30 min with various combinations of Glc and Fru. With constant Fru (2 mM), increasing perfusate Glc increased the relative contribution of Glc carbons to glycogen (7.2 +/- 0.4, 34.9 +/- 2.8, and 59.1 +/- 2.7% at 2, 10, and 20 mM Glc, respectively; n = 5 for each). During perfusion with substrate levels seen during refeeding (10 mM Glc, 1.8 mumol/g/min gluconeogenic flux from 2 mM Fru), Fru provided 54.7 +/- 2.7% of the carbons for glycogen, while Glc provided only 34.9 +/- 2.8%, consistent with in vivo estimations. However, the estimated rate of Glc phosphorylation was at least 1.10 +/- 0.11 mumol/g/min, which exceeded by at least 4-fold the glycogen accumulation rate (0.28 +/- 0.04 mumol of glucose/g/min). The total rate of glucose 6-phosphate supply via Glc phosphorylation and gluconeogenesis (2.9 mumol/g/min) exceeded reported in vivo rates of glycogen accumulation during refeeding. Thus, in perfused livers of 24-h fasted rats there is an apparent redundancy in glucose 6-phosphate supply. These results suggest that the rate-limiting step for hepatic glycogen accumulation during refeeding is located between glucose 6-phosphate and glycogen, rather than at the step of Glc phosphorylation or in the gluconeogenic pathway

  2. 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

  3. Recovery of active pathogenesis-related enzymes from the apoplast ...

    African Journals Online (AJOL)

    Overall protease activity intensity was higher in the symplast. Maximum symplast contamination of the apoplast was 2% as estimated by glucose 6-phosphate dehydrogenase activity, a biochemical marker for symplast. Accumulation of pathogenesis-related enzymatic activities in the apoplast of M. acuminata leaf tissue was ...

  4. Effect of aspirin and prostaglandins on the carbohydrate metabolism in albino rats.: glucose oxidation through different pathways and glycolytic enzymes

    International Nuclear Information System (INIS)

    Balasubramanian, A.; Ramakrishnan, S.

    1980-01-01

    The effect of chronic and acute doses of aspirin and prostaglandins F2α and E2 individually on the oxidation of glucose through Embden Meyerhof-TCA cycle and pentose phosphate pathways and some key glycolytic enzymes of liver were studied in male albino rats. Studies were extended to find the combined effect of PGF2α and E2 with an acute dose of aspirin. There was increased utilisation of both 1- 14 C glucose and 6- 14 C glucose on aspirin treatment. However, the metabolism through the EM-TCA pathway was more pronounced as shown by a reduced ratio of 14 CO 2 from 1- 14 C and 6- 14 C glucose. Two hepatic key glycolytic enzymes viz. hexokinase and pyruvate kinase were increased due to aspirin treatment. Withdrawal of aspirin corrected the above impaired carbohydrate metabolism in liver. Prostaglandin F2α also caused a reduction in the utilisation of 1- 14 C glucose, while PGE2 recorded an increase in the utilisation of both 1- 14 C and 6- 14 C glucose when compared to controls, indicating that different members of prostaglandins could affect metabolisms and differently. Administration of the PGs and aspirin together showed an increase in the utilisation of 6- 14 C glucose. (auth.)

  5. Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

    International Nuclear Information System (INIS)

    Krogstad, D.J.; Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

    1981-01-01

    Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10 5 and greater than or equal to 3 X 10 6 USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10 5 and greater than or equal to10 4 USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described

  6. Increased muscle glucose uptake after exercise

    DEFF Research Database (Denmark)

    Richter, Erik; Ploug, Thorkil; Galbo, Henrik

    1985-01-01

    responsiveness of glucose uptake was noted only in controls. Analysis of intracellular glucose-6-phosphate, glucose, glycogen synthesis, and glucose transport suggested that the exercise effect on responsiveness might be due to enhancement of glucose disposal. After electrical stimulation of diabetic...... of glucose. At maximal insulin concentrations, the enhancing effect of exercise on glucose uptake may involve enhancement of glucose disposal, an effect that is probably less in muscle from diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)......It has recently been shown that insulin sensitivity of skeletal muscle glucose uptake and glycogen synthesis is increased after a single exercise session. The present study was designed to determine whether insulin is necessary during exercise for development of these changes found after exercise...

  7. The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains

    DEFF Research Database (Denmark)

    Jeppsson, M.; Johansson, B.; Jensen, Peter Ruhdal

    2003-01-01

    production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wildtype level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose...

  8. p73 regulates basal and starvation-induced liver metabolism in vivo

    OpenAIRE

    He, Zhaoyue; Agostini, Massimiliano; Liu, He; Melino, Gerry; Simon, Hans-Uwe

    2015-01-01

    As a member of the p53 gene family, p73 regulates cell cycle arrest, apoptosis, neurogenesis, immunity and inflammation. Recently, p73 has been shown to transcriptionally regulate selective metabolic enzymes, such as cytochrome c oxidase subunit IV isoform 1, glucose 6-phosphate dehydrogenase and glutaminase-2, resulting in significant effects on metabolism, including hepatocellular lipid metabolism, glutathione homeostasis and the pentose phosphate pathway. In order to further investigate th...

  9. Metabolic response to MMS-mediated DNA damage in Saccharomyces cerevisiae is dependent on the glucose concentration in the medium.

    Science.gov (United States)

    Kitanovic, Ana; Walther, Thomas; Loret, Marie Odile; Holzwarth, Jinda; Kitanovic, Igor; Bonowski, Felix; Van Bui, Ngoc; Francois, Jean Marie; Wölfl, Stefan

    2009-06-01

    Maintenance and adaptation of energy metabolism could play an important role in the cellular ability to respond to DNA damage. A large number of studies suggest that the sensitivity of cells to oxidants and oxidative stress depends on the activity of cellular metabolism and is dependent on the glucose concentration. In fact, yeast cells that utilize fermentative carbon sources and hence rely mainly on glycolysis for energy appear to be more sensitive to oxidative stress. Here we show that treatment of the yeast Saccharomyces cerevisiae growing on a glucose-rich medium with the DNA alkylating agent methyl methanesulphonate (MMS) triggers a rapid inhibition of respiration and enhances reactive oxygen species (ROS) production, which is accompanied by a strong suppression of glycolysis. Further, diminished activity of pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase upon MMS treatment leads to a diversion of glucose carbon to glycerol, trehalose and glycogen accumulation and an increased flux through the pentose-phosphate pathway. Such conditions finally result in a significant decline in the ATP level and energy charge. These effects are dependent on the glucose concentration in the medium. Our results clearly demonstrate that calorie restriction reduces MMS toxicity through increased respiration and reduced ROS accumulation, enhancing the survival and recovery of cells.

  10. Acute interleukin-6 administration does not impair muscle glucose uptake or whole-body glucose disposal in healthy humans

    DEFF Research Database (Denmark)

    Steensberg, Adam; Fischer, Christian P; Sacchetti, Massimo

    2003-01-01

    adrenaline (epinephrine). IL-6 infusion, irrespective of dose, did not result in any changes to endogenous glucose production, whole-body glucose disposal or leg- glucose uptake. These data demonstrate that acute IL-6 administration does not impair whole-body glucose disposal, net leg-glucose uptake......The cytokine interleukin (IL)-6 has recently been linked with type 2 diabetes mellitus and has been suggested to affect glucose metabolism. To determine whether acute IL-6 administration affects whole-body glucose kinetics or muscle glucose uptake, 18 healthy young men were assigned to one of three...... the cessation of infusion (recovery) to determine endogenous glucose production and whole-body glucose disposal. Infusion with HiIL-6 and LoIL-6 resulted in a marked (P

  11. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Gowda, Giri; Sagurthi, Someswar Rao [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

    2008-02-01

    The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.

  12. Precision of glucose measurements in control sera by isotope dilution/mass spectrometry: proposed definitive method compared with a reference method

    International Nuclear Information System (INIS)

    Pelletier, O.; Arratoon, C.

    1987-01-01

    This improved isotope-dilution gas chromatographic/mass spectrometric (GC/MS) method, in which [ 13 C]glucose is the internal standard, meets the requirements of a Definitive Method. In a first study with five reconstituted lyophilized sera, a nested analysis of variance of GC/MS values indicated considerable among-vial variation. The CV for 32 measurements per serum ranged from 0.5 to 0.9%. However, concentration and uncertainty values (mmol/L per gram of serum) assigned to one serum by the NBS Definitive Method (7.56 +/- 0.28) were practically identical to those obtained with the proposed method (7.57 +/- 0.20). In the second study, we used twice more [ 13 C]glucose diluent to assay four serum pools and two lyophilized sera. The CV ranged from 0.26 to 0.5% for the serum pools and from 0.28 to 0.59% for the lyophilized sera. In comparison, results by the hexokinase/glucose-6-phosphate dehydrogenase reference method agreed within acceptable limits with those by the Definitive Method but tended to be slightly higher (up to 3%) for lyophilized serum samples or slightly lower (up to 2.5%) for serum pools

  13. Effect of dietary fiber from banana (Musa paradisiaca) on metabolism of carbohydrates in rats fed cholesterol free diet.

    Science.gov (United States)

    Usha, V; Vijayammal, P L; Kurup, P A

    1989-05-01

    Effect of feeding isolated dietary fiber from M. paradisiaca on the metabolism of carbohydrates in the liver has been studied. Fiber fed rats showed significantly lower levels of fasting blood glucose and higher concentration of liver glycogen. Activity of glycogen phosphorylase, glucose-1-phosphate, uridyl transferase and glycogen synthase was significantly higher while phosphoglucomutase activity showed lower activity. Activity of some glycolytic enzymes, viz. hexokinase and pyruvic kinase was lower. Glucose-6-phosphatase showed higher activity while fructose 1-6 diphosphatase activity was not affected. Glucose-6-phosphate dehydrogenase on the other hand showed higher activity. The changes in these enzyme activities have been attributed due to the effect of higher concentration of bile acids produced in the liver as a result of feeding fiber. Evidence for this has been obtained by studying the in vitro effect of cholic acid and chenodeoxy cholic acid.

  14. Browse Title Index

    African Journals Online (AJOL)

    Items 51 - 100 of 150 ... HU Nwanjo, G Oze. Vol 14, No 2 (2005), Incidence of Glucose-6-Phosphate Dehydrogenase (G-6-PD) Deficiency in Apparently Healthy Individuals in Some Parts of North Central Nigeria, Abstract. A Abubakar, A Musa, B Lliyasu, J Adetunji, O Olaniru, T Akande, A T Mokogwu. Vol 14, No 1 (2005) ...

  15. Histochemical demonstration of creatine kinase activity using polyvinyl alcohol and auxiliary enzymes

    NARCIS (Netherlands)

    Frederiks, W. M.; Marx, F.; van Noorden, C. J.

    1987-01-01

    Creatine kinase activity (EC 2.7.3.2.) has been demonstrated in myocardium and skeletal muscle from rats by a method based on the incubation of cryostat sections with a polyvinyl alcohol-containing medium and the use of auxiliary enzymes. Hexokinase and glucose-6-phosphate dehydrogenase were spread

  16. Radiosynthesis of 6-[C-11]-D-glucose

    International Nuclear Information System (INIS)

    Grierson, J.R.; Biskupiak, J.E.; Link, J.M.; Krohn, K.A.

    1993-01-01

    Availability of 6-[C-11]-D-glucose will permit positron emission tomography (PET) investigations of glucose utilization derived from the pentose shunt which supports biosynthesis in tissues. The first radiosynthesis of 6-[C-11]-D-glucose is described. As much as 1 mCi of 6-[C-11]-D-glucose, sufficient for animal studies, is obtained from [C-11]CO 2 after 100 min with a 16% radiochemical yield (EOB). The radiosynthesis has many attractive features. The method uses [C-11]CH 3 I and combines a Wittig reaction and a stereoselective OsO 4 catalyzed alkene hydroxylation. The OsO 4 hydroxylation of the [C-11]-labeled alkene (9) is accomplished in less than 10 min with high stereoselectivity (94:6) in favor of the 6-[C-11]-D-gluco-isomer. HPLC purification (C-18) of the protected labeled sugar removes the undesired 6-[C-11]-L-ido-sugar at an early stage and avoids the use of an expensive low-capacity ion-exchange HPLC column. OsO 4 , a highly toxic reagent, is removed in the process by adsorption and inactivation on polymer-bound triphenylphosphine. (Author)

  17. The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite's 6-phosphogluconate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Famin O.

    2003-03-01

    Full Text Available Ferriprotoporphyrin IX (FPIX is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleoside monophosphate kinase and pyrimidine 5'- nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-α, phoshoglycerate kinase, glyceraldehyde 3- phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6- phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.

  18. Thermal decomposition of specifically phosphorylated D-glucoses and their role in the control of the Maillard reaction.

    Science.gov (United States)

    Yaylayan, Varoujan A; Machiels, David; Istasse, Louis

    2003-05-21

    One of the main shortcomings of the information available on the Maillard reaction is the lack of knowledge to control the different pathways, especially when it is desired to direct the reaction away from the formation of carcinogenic and other toxic substances to more aroma and color generation. The use of specifically phosphorylated sugars may impart some elements of control over the aroma profile generated by the Maillard reaction. Thermal decomposition of 1- and 6-phosphorylated glucoses was studied in the presence and absence of ammonia and selected amino acids through pyrolysis/gas chromatography/mass spectrometry using nonpolar PLOT and medium polar DB-1 columns. The analysis of the data has indicated that glucose-1-phosphate relative to glucose undergoes more extensive phosphate-catalyzed ring opening followed by formation of sugar-derived reactive intermediates as was indicated by a 9-fold increase in the amount of trimethylpyrazine and a 5-fold increase in the amount of 2,3-dimethylpyrazine, when pyrolyzed in the presence of glycine. In addition, glucose-1-phosphate alone generated a 6-fold excess of acetol as compared to glucose. On the other hand, glucose-6-phosphate enhanced retro-aldol reactions initiated from a C-6 hydroxyl group and increased the subsequent formation of furfural and 4-cyclopentene-1,3-dione. Furthermore, it also stabilized 1- and 3-deoxyglucosone intermediates and enhanced the formation of six carbon atom-containing Maillard products derived directly from them through elimination reactions such as 1,6-dimethyl-2,4-dihydroxy-3-(2H)-furanone (acetylformoin), 2-acetylpyrrole, 5-methylfurfural, 5-hydroxymethylfurfural, and 4-hydroxy-2,5-dimethyl-3-(2H)-furanone (Furaneol), due to the enhanced leaving group ability of the phosphate moiety at the C-6 carbon. However, Maillard products generated through the nucleophilic action of the C-6 hydroxyl group such as 2-acetylfuran and 2,3-dihydro-3,5-dihydroxy-4H-pyran-4-one were retarded, due

  19. Enhanced glucose cycling and suppressed de novo synthesis of glucose-6-phosphate result in a net unchanged hepatic glucose output in ob/ob mice

    NARCIS (Netherlands)

    Bandsma, RHJ; Grefhorst, A; van Dijk, TH; van der Sluijs, FH; Hammer, A; Reijngoud, DJ; Kuipers, F

    2004-01-01

    Aims/hypothesis. Leptin-deficient ob/ob mice are hyperinsulinaemic and hyperglycaemic; however, the cause of hyperglycaemia remains largely unknown. Methods. Glucose metabolism in vivo in 9-h fasted ob/ob mice and lean littermates was studied by infusing [U-C-13]-glucose, [2-C-13]-glycerol,

  20. Naturally occurring genetic variation affecting the expression of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.

    Science.gov (United States)

    Laurie-Ahlberg, C C; Bewley, G C

    1983-10-01

    Genetic variation among second and third chromosomes from natural populations of Drosophila melanogaster affects the activity level of sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8; GPDH) at both the larval and the adult stages. The genetic effects, represented by differences among chromosome substitution lines with coisogenic backgrounds, are very repeatable over time and are generally substantially larger than environmental and measurement error effects. Neither the GPDH allozyme, the geographic origin, nor the karyotype of the chromosome contributes significantly to GPDH activity variation. The strong relationship between GPDH activity level and GPDH-specific CRM level, as well as our failure to find any thermostability variation among the lines, indicates that most, if not all, of the activity variation is due to variation in the steady-state quantity of enzyme rather than in its catalytic properties. The lack of a strong relationship between adult and larval activity levels suggests the importance of stage- or isozyme-specific effects.

  1. Pathways of hepatic glycogen formation in humans following ingestion of a glucose load in the fed state

    International Nuclear Information System (INIS)

    Magnusson, I.; Chandramouli, V.; Schumann, W.C.; Kumaran, K.; Wahren, J.; Landau, B.R.

    1989-01-01

    The relative contributions of the direct and the indirect pathways to hepatic glycogen formation following a glucose load given to humans four hours after a substantial breakfast have been examined. Glucose loads labeled with [6-( 14 )C]glucose were given to six healthy volunteers along with diflunisal (1 g) or acetaminophen (1.5 g), drugs excreted in urine as glucuronides. Distribution of 14 C in the glucose unit of the glucuronide was taken as a measure of the extent to which glucose was deposited directly in liver glycogen (ie, glucose----glucose-6-phosphate----glycogen) rather than indirectly (ie, glucose----C3-compound----glucose-6-phosphate----glycogen). The maximum contribution to glycogen formation by the direct pathway was estimated to be 77% +/- 4%, which is somewhat higher than previous estimates in humans fasted overnight (65% +/- 1%, P less than 0.05). Thus, the indirect pathway of liver glycogen formation following a glucose load is operative in both the overnight fasted and the fed state, although its contribution may be somewhat less in the fed state

  2. Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides

    Science.gov (United States)

    McDonald, Tanya S; Tan, Kah Ni; Hodson, Mark P; Borges, Karin

    2014-01-01

    Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain. PMID:24169853

  3. Porous, Dexamethasone-loaded polyurethane coatings extend performance window of implantable glucose sensors in vivo.

    Science.gov (United States)

    Vallejo-Heligon, Suzana G; Brown, Nga L; Reichert, William M; Klitzman, Bruce

    2016-01-01

    Continuous glucose sensors offer the promise of tight glycemic control for insulin dependent diabetics; however, utilization of such systems has been hindered by issues of tissue compatibility. Here we report on the in vivo performance of implanted glucose sensors coated with Dexamethasone-loaded (Dex-loaded) porous coatings employed to mediate the tissue-sensor interface. Two animal studies were conducted to (1) characterize the tissue modifying effects of the porous Dex-loaded coatings deployed on sensor surrogate implants and (2) investigate the effects of the same coatings on the in vivo performance of Medtronic MiniMed SOF-SENSOR™ glucose sensors. The tissue response to implants was evaluated by quantifying macrophage infiltration, blood vessel formation, and collagen density around implants. Sensor function was assessed by measuring changes in sensor sensitivity and time lag, calculating the Mean Absolute Relative Difference (MARD) for each sensor treatment, and performing functional glucose challenge test at relevant time points. Implants treated with porous Dex-loaded coatings diminished inflammation and enhanced vascularization of the tissue surrounding the implants. Functional sensors with Dex-loaded porous coatings showed enhanced sensor sensitivity over a 21-day period when compared to controls. Enhanced sensor sensitivity was accompanied with an increase in sensor signal lag and MARD score. These results indicate that Dex-loaded porous coatings were able to elicit an attenuated tissue response, and that such tissue microenvironment could be conducive towards extending the performance window of glucose sensors in vivo. In the present article, a coating to extend the functionality of implantable glucose sensors in vivo was developed. Our study showed that the delivery of an anti-inflammatory agent with the presentation of micro-sized topographical cues from coatings may lead to improved long-term glucose sensor function in vivo. We believe that

  4. Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tsung-Pao; Pan, Yun-Ru; Fu, Chien-Yu; Chang, Hwan-You, E-mail: hychang@life.nthu.edu.tw

    2010-10-15

    UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellular spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.

  5. Significant improvement of thermal stability of glucose 1-dehydrogenase by introducing disulfide bonds at the tetramer interface.

    Science.gov (United States)

    Ding, Haitao; Gao, Fen; Liu, Danfeng; Li, Zeli; Xu, Xiaohong; Wu, Min; Zhao, Yuhua

    2013-12-10

    Rational design was applied to glucose 1-dehydrogenase (LsGDH) from Lysinibacillus sphaericus G10 to improve its thermal stability by introduction of disulfide bridges between subunits. One out of the eleven mutants, designated as DS255, displayed significantly enhanced thermal stability with considerable soluble expression and high specific activity. It was extremely stable at pH ranging from 4.5 to 10.5, as it retained nearly 100% activity after incubating at different buffers for 1h. Mutant DS255 also exhibited high thermostability, having a half-life of 9900min at 50°C, which was 1868-fold as that of its wild type. Moreover, both of the increased free energy of denaturation and decreased entropy of denaturation of DS255 suggested that the enzyme structure was stabilized by the engineered disulfide bonds. On account of its robust stability, mutant DS255 would be a competitive candidate in practical applications of chiral chemicals synthesis, biofuel cells and glucose biosensors. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. A reduction in age-enhanced gluconeogenesis extends lifespan.

    Science.gov (United States)

    Hachinohe, Mayumi; Yamane, Midori; Akazawa, Daiki; Ohsawa, Kazuhiro; Ohno, Mayumi; Terashita, Yuzu; Masumoto, Hiroshi

    2013-01-01

    The regulation of energy metabolism, such as calorie restriction (CR), is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan.

  7. A reduction in age-enhanced gluconeogenesis extends lifespan.

    Directory of Open Access Journals (Sweden)

    Mayumi Hachinohe

    Full Text Available The regulation of energy metabolism, such as calorie restriction (CR, is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan.

  8. Twofold reduction of phosphofructokinase activity in Lactococcus lactis results in strong decreases in growth rate and in glycolytic flux

    DEFF Research Database (Denmark)

    Andersen, Heidi Winterberg; Solem, Christian; Hammer, Karin

    2001-01-01

    reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions, One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one...... kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further...... of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth fate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6- phosphate...

  9. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris.

    Science.gov (United States)

    Sygmund, Christoph; Staudigl, Petra; Klausberger, Miriam; Pinotsis, Nikos; Djinović-Carugo, Kristina; Gorton, Lo; Haltrich, Dietmar; Ludwig, Roland

    2011-12-12

    FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp. Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L⁻¹ of GDH activity (57 mg L⁻¹). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

  10. Whole body glucose kinetics in type I diabetes studied with [6,6-2H] and [U-13C]-glucose and the artificial B-cell

    International Nuclear Information System (INIS)

    Darmaun, D.; Cirillo, D.; Koziet, J.; Chauvet, D.; Young, V.R.; Robert, J.J.

    1988-01-01

    Dynamic aspects of whole body glucose metabolism were assessed in ten young adult insulin-dependent (type I) diabetic men. Using a primed, continuous intravenous infusion of [6,6- 2 H]glucose and [U- 13 C]glucose, endogenous production, tissue uptake, carbon recycling, and oxidation of glucose were measured in the postabsorptive state. These studies were undertaken after blood glucose had been maintained overnight at 5.9 +/- 0.4 mmol/L (n = 10), and on another night at 10.5 +/- 0.4 mmol/L (n = 4) or 15.2 +/- 0.6 mmol/L (n = 6). In the normoglycemic state, endogenous glucose production averaged 2.15 +/- 0.13 mg x kg-1 x min-1. This value, as well as the rate of glucose carbon recycling (0.16 +/- 0.04 mg x kg-1 x min-1) and glucose oxidation (1.52 +/- 0.16 mg x kg-1 x min-1) are comparable to those found in nondiabetic controls. In the hyperglycemic states at 10 or 15 mmol/L, endogenous glucose production was increased by 11% (P less than .01) and 60% (P less than .01) compared to the normoglycemic states, respectively. Glucose carbon recycling contributed only a small percentage to this variation in glucose production (15% at the 15 mmol/L glucose level). This suggests that if gluconeogenesis participates in the increased glucose output, it is not dependent on a greater systemic supply of three-carbon precursors. The increased rate of glucose production in the hyperglycemic state was quantitatively offset by a rise in urinary glucose excretion. Glucose tissue uptake, as well as glucose oxidation, did not vary between normoglycemic and hyperglycemic states

  11. Sucrose dependent mineral phosphate solubilization in Enterobacter asburiae PSI3 by heterologous overexpression of periplasmic invertases.

    Science.gov (United States)

    Kumar, Chanchal; Wagh, Jitendra; Archana, G; Naresh Kumar, G

    2016-12-01

    Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.

  12. Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

    Science.gov (United States)

    Lee, Won-Heong; Chin, Young-Wook; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

    2011-08-01

    Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

  13. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    Energy Technology Data Exchange (ETDEWEB)

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the

  14. Subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of Trypanosoma rangeli.

    Science.gov (United States)

    Rondón-Mercado, Rocío; Acosta, Héctor; Cáceres, Ana J; Quiñones, Wilfredo; Concepción, Juan Luis

    2017-09-01

    Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Central and South America. Although this parasite is not pathogenic for human, it is being studied because it shares with Trypanosoma cruzi, the etiological agent of Chagas' disease, biological characteristics, geographic distribution, vectors and vertebrate hosts. Several metabolic studies have been performed with T. cruzi epimastigotes, however little is known about the metabolism of T. rangeli. In this work we present the subcellular distribution of the T. rangeli enzymes responsible for the conversion of glucose to pyruvate, as determined by epifluorescense immunomicroscopy and subcellular fractionation involving either selective membrane permeabilization with digitonin or differential and isopycnic centrifugation. We found that in T. rangeli epimastigotes the first six enzymes of the glycolytic pathway, involved in the conversion of glucose to 1,3-bisphosphoglycerate are located within glycosomes, while the last four steps occur in the cytosol. In contrast with T. cruzi, where three isoenzymes (one cytosolic and two glycosomal) of phosphoglycerate kinase are expressed simultaneously, only one enzyme with this activity is detected in T. rangeli epimastigotes, in the cytosol. Consistent with this latter result, we found enzymes involved in auxiliary pathways to glycolysis needed to maintain adenine nucleotide and redox balances within glycosomes such as phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, pyruvate phosphate dikinase and glycerol-3-phosphate dehydrogenase. Glucokinase, galactokinase and the first enzyme of the pentose-phosphate pathway, glucose-6-phosphate dehydrogenase, were also located inside glycosomes. Furthermore, we demonstrate that T. rangeli epimastigotes growing in LIT medium only consume glucose and do not excrete ammonium; moreover, they are unable to survive in partially-depleted glucose medium. The

  15. Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for rasburicase therapy in the context of G6PD deficiency genotype.

    Science.gov (United States)

    Relling, M V; McDonagh, E M; Chang, T; Caudle, K E; McLeod, H L; Haidar, C E; Klein, T; Luzzatto, L

    2014-08-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with development of acute hemolytic anemia (AHA) induced by a number of drugs. We provide guidance as to which G6PD genotypes are associated with G6PD deficiency in males and females. Rasburicase is contraindicated in G6PD-deficient patients due to the risk of AHA and possibly methemoglobinemia. Unless preemptive genotyping has established a positive diagnosis of G6PD deficiency, quantitative enzyme assay remains the mainstay of screening prior to rasburicase use. The purpose of this article is to help interpret the results of clinical G6PD genotype tests so that they can guide the use of rasburicase. Detailed guidelines on other aspects of the use of rasburicase, including analyses of cost-effectiveness, are beyond the scope of this document. Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines are published and updated periodically on https://www.pharmgkb.org/page/cpic to reflect new developments in the field.

  16. Identification of Electronic and Structural Descriptors of Adenosine Analogues Related to Inhibition of Leishmanial Glyceraldehyde-3-Phosphate Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Norka B. H. Lozano

    2013-04-01

    Full Text Available Quantitative structure–activity relationship (QSAR studies were performed in order to identify molecular features responsible for the antileishmanial activity of 61 adenosine analogues acting as inhibitors of the enzyme glyceraldehyde 3-phosphate dehydrogenase of Leishmania mexicana (LmGAPDH. Density functional theory (DFT was employed to calculate quantum-chemical descriptors, while several structural descriptors were generated with Dragon 5.4. Variable selection was undertaken with the ordered predictor selection (OPS algorithm, which provided a set with the most relevant descriptors to perform PLS, PCR and MLR regressions. Reliable and predictive models were obtained, as attested by their high correlation coefficients, as well as the agreement between predicted and experimental values for an external test set. Additional validation procedures were carried out, demonstrating that robust models were developed, providing helpful tools for the optimization of the antileishmanial activity of adenosine compounds.

  17. Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve

    International Nuclear Information System (INIS)

    Suzuki, Ryuichiro; Kim, Byung-Jun; Shibata, Tsuyoshi; Iwamoto, Yuki; Katayama, Takane; Ashida, Hisashi; Wakagi, Takayoshi; Shoun, Hirofumi; Fushinobu, Shinya; Yamamoto, Kenji

    2010-01-01

    Xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from B. breve was overexpressed and crystallized. The crystals belonged to the tetragonal space group I422 and diffracted to beyond 1.7 Å resolution. The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl 2 , 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å, and diffracted to beyond 1.7 Å resolution

  18. Evolutionary history of glucose-6-phosphatase encoding genes in vertebrate lineages: towards a better understanding of the functions of multiple duplicates.

    Science.gov (United States)

    Marandel, Lucie; Panserat, Stéphane; Plagnes-Juan, Elisabeth; Arbenoits, Eva; Soengas, José Luis; Bobe, Julien

    2017-05-02

    Glucose-6-phosphate (G6pc) is a key enzyme involved in the regulation of the glucose homeostasis. The present study aims at revisiting and clarifying the evolutionary history of g6pc genes in vertebrates. g6pc duplications happened by successive rounds of whole genome duplication that occurred during vertebrate evolution. g6pc duplicated before or around Osteichthyes/Chondrichthyes radiation, giving rise to g6pca and g6pcb as a consequence of the second vertebrate whole genome duplication. g6pca was lost after this duplication in Sarcopterygii whereas both g6pca and g6pcb then duplicated as a consequence of the teleost-specific whole genome duplication. One g6pca duplicate was lost after this duplication in teleosts. Similarly one g6pcb2 duplicate was lost at least in the ancestor of percomorpha. The analysis of the evolution of spatial expression patterns of g6pc genes in vertebrates showed that all g6pc were mainly expressed in intestine and liver whereas teleost-specific g6pcb2 genes were mainly and surprisingly expressed in brain and heart. g6pcb2b, one gene previously hypothesised to be involved in the glucose intolerant phenotype in trout, was unexpectedly up-regulated (as it was in liver) by carbohydrates in trout telencephalon without showing significant changes in other brain regions. This up-regulation is in striking contrast with expected glucosensing mechanisms suggesting that its positive response to glucose relates to specific unknown processes in this brain area. Our results suggested that the fixation and the divergence of g6pc duplicated genes during vertebrates' evolution may lead to adaptive novelty and probably to the emergence of novel phenotypes related to glucose homeostasis.

  19. Kinetics of formation of acrylamide and Schiff base intermediates from asparagine and glucose

    DEFF Research Database (Denmark)

    Hedegaard, Rikke Susanne Vingborg; Frandsen, Henrik; Skibsted, Leif H.

    2008-01-01

    From the concentration of glucose and asparagine as reactants and of acrylamide as product each determined by LC-MS during reaction in an acetonitrile/water (68:32) model system at pH 7.6 (0.04 M phosphate buffer) and from the relative concentration of the Schiff base intermediate, the decarboxyl......From the concentration of glucose and asparagine as reactants and of acrylamide as product each determined by LC-MS during reaction in an acetonitrile/water (68:32) model system at pH 7.6 (0.04 M phosphate buffer) and from the relative concentration of the Schiff base intermediate...

  20. Antidiabetic Effects of Momordica charantia (Karela in Male long Evans Rat

    Directory of Open Access Journals (Sweden)

    Nurul Karim

    2012-07-01

    Full Text Available The hypoglycemic effect of Momordica charantia (Karela has been reported from many laboratories. To our knowledge the underlying biochemical mechanism of action of this important clinical effect has not been reported. During the course of investigation of this aspect of the herbal fruit, it was reported from our laboratory that ethanolic extract of Momordica charantia suppressed gluconeogenesis in normal and streptozotocin (STZ induced diabetic rats by depressing the hepatic gluconeogenic enzymes fructose-1,6-bisphosphatase and glucose-6-phosphatase. The herbal extract had also enhanced the activity of glucose-6-phosphate dehydrogenase, the rate limiting enzyme of hexose monophosphate shunt (a pathway for the oxidation of glucose.

  1. Tritiated 2-deoxy-D-glucose as a probe for cell membrane permeability studies

    International Nuclear Information System (INIS)

    Walum, E.; Peterson, A.

    1982-01-01

    Tritiated 2-deoxy-D-glucose was taken up and phosphorylated by cultured cells of neuronal (NIE 115), glial (138 MG), muscle (L 6) and liver (BRL 123) origin. Upon perfusion the cells slowly released 2-deoxy-D-glucose 6-phosphate. The following values for rate constants, half-lives, and activation energies for the efflux were obtained: NIE 115: 0.0048 min -1 , 143 min, and 72 kJ mol -1 ; 138 MG: 0.0013 min -1 , 547 min, and 85 kJ mol -1 ; L 6: 0.0022 min -1 , 311 min, and 60 kJ mol -1 ; and BRL 123: 0.0013 min -1 , 528 min and 63 kJ mol -1 . When the cultures were perfused with buffer containing Triton X-100 a time- and concentration-dependent increase in the rate of efflux of 2-deoxy-D-glucose 6-phosphate was obtained. It is suggested that 2-deoxy-D-[ 3 H]glucose can be used as a probe in studies of general cell membrane permeability changes

  2. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Sygmund Christoph

    2011-12-01

    Full Text Available Abstract Background FAD dependent glucose dehydrogenase (GDH currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum spp. Results Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L-1 of GDH activity (57 mg L-1. Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. Conclusions The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

  3. Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

    Science.gov (United States)

    Itakura, Masanori; Nakajima, Hidemitsu; Semi, Yuko; Higashida, Shusaku; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

    2015-11-13

    The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Association of glucose-6-phosphate dehydrogenase deficiency and X-linked chronic granulomatous disease in a child with anemia and recurrent infections

    OpenAIRE

    Agudelo-Florez, P.; Costa-Carvalho, Beatriz Tavares [UNIFESP; Lopez, J. A.; Redher, J.; Newburger, P. E.; Alla-Saad, S. T.; Condino-Neto, A.

    2004-01-01

    Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 +/- 2.1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus...

  5. 17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

    International Nuclear Information System (INIS)

    Mendoza-Hernandez, G.; Lopez-Solache, I.; Rendon, J.L.; Diaz-Sanchez, V.; Diaz-Zagoya, J.C.

    1988-01-01

    The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively

  6. Jinggangmycin-suppressed reproduction in the small brown planthopper (SBPH), Laodelphax striatellus (Fallen), is mediated by glucose dehydrogenase (GDH).

    Science.gov (United States)

    Ding, Jun; Wu, You; You, Lin-Lin; Xu, Bin; Ge, Lin-Quan; Yang, Guo-Qing; Wu, Jin-Cai

    2017-06-01

    The small brown planthopper (SBPH), Laodelphax striatellus (Fallen), is a serious pest insect of rice, wheat, and maize in China. SBPH not only sucks plant sap but also transmits plant disease viruses, causing serious damage. These viruses include rice striped virus disease (RSV disease), black streaked dwarf, and maize rough disease virus. SBPH outbreaks are related to the overuse of pesticides in China. Some pesticides, such as triazophos, stimulate the reproduction of SBPH, but an antibiotic fungicide jinggangmycin (JGM) suppresses its reproduction. However, mechanisms of decreased reproduction of SBPH induced by JGM remain unclear. The present findings show that JGM suppressed reproduction of SBPH (↓approximately 35.7%) and resulted in the down-regulated expression of glucose dehydrogenase (GDH). GDH-silenced control females (control+dsGDH) show that the number of eggs laid was reduced by 48.6% compared to control females. Biochemical tests show that the total lipid and fatty acid contents in JGM-treated and control+dsGDH females decreased significantly. Thus, we propose that the suppression of reproduction in SBPH induced by JGM is mediated by GDH via metabolic pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Metabolic shift in lung alveolar cell mitochondria following acrolein exposure.

    Science.gov (United States)

    Agarwal, Amit R; Yin, Fei; Cadenas, Enrique

    2013-11-15

    Acrolein, an α,β unsaturated electrophile, is an environmental pollutant released in ambient air from diesel exhausts and cooking oils. This study examines the role of acrolein in altering mitochondrial function and metabolism in lung-specific cells. RLE-6TN, H441, and primary alveolar type II (pAT2) cells were exposed to acrolein for 4 h, and its effect on mitochondrial oxygen consumption rates was studied by XF Extracellular Flux analysis. Low-dose acrolein exposure decreased mitochondrial respiration in a dose-dependent manner because of alteration in the metabolism of glucose in all the three cell types. Acrolein inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, leading to decreased substrate availability for mitochondrial respiration in RLE-6TN, H441, and pAT2 cells; the reduced GAPDH activity was compensated in pAT2 cells by an increase in the activity of glucose-6-phosphate dehydrogenase, the regulatory control of the pentose phosphate pathway. The decrease in pyruvate from glucose metabolism resulted in utilization of alternative sources to support mitochondrial energy production: palmitate-BSA complex increased mitochondrial respiration in RLE-6TN and pAT2 cells. The presence of palmitate in alveolar cells for surfactant biosynthesis may prove to be the alternative fuel source for mitochondrial respiration. Accordingly, a decrease in phosphatidylcholine levels and an increase in phospholipase A2 activity were found in the alveolar cells after acrolein exposure. These findings have implications for understanding the decrease in surfactant levels frequently observed in pathophysiological situations with altered lung function following exposure to environmental toxicants.

  8. Remote Sensing and Geographic Information Systems as Decision Support Tools for Malaria Control in the Republic of Korea

    Science.gov (United States)

    2001-05-30

    remote sensing and GIS techniques to monitor vectors and vector-borne disease in Mexico, Belize and, more recently, Peru (Roberts et al. 1999...parasite is severe hemolytic anemia in persons who are deficient in glucose-6- phosphate dehydrogenase (G-6-PD). Primaquine is the only drug currently...the denaturation of hemoglobin, resulting in hemoglobinuria, kidney damage and anemia . Erythrocytes are protected from oxidation by the hexose

  9. Structure of the Bacillus anthracis dTDP- L -rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA)

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Jackson; Lee, Jesi; Halavaty, Andrei S.; Minasov, George; Anderson, Wayne F.; Kuhn, Misty L. (NWU); (SFSU)

    2017-10-30

    L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), fromBacillus anthraciswas determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs. However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.

  10. The Mycobacterium tuberculosis Complex has a Pathway for the Biosynthesis of 4-Formamido-4,6-Dideoxy-d-Glucose.

    Science.gov (United States)

    Brown, Haley A; Vinogradov, Evgeny; Gilbert, Michel; Holden, Hazel M

    2018-05-15

    Recent studies have demonstrated that the O-antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N-formylated sugars (3-formamido-3,6-dideoxy-d-glucose or 4-formamido-4,6-dideoxy-d-glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6-dehydratase, a pyridoxal 5'-phosphate or PLP-dependent aminotransferase, and an N-formyltransferase. To date, there have been no published reports of N-formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N-formyltransferase. Given that M. tuberculosis produces l-rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6-dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N-formylated sugar in M. tuberculosis, namely a PLP-dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP-4-formamido-4,6-dideoxy-d-glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.

  11. Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

    Science.gov (United States)

    Lee, Jung-Kul; Pan, Cheol-Ho

    2013-01-01

    D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

  12. Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

    Directory of Open Access Journals (Sweden)

    Woo-Suk Jung

    Full Text Available D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26, which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD, catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi. Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

  13. Glycolysis and the pentose phosphate pathway after human traumatic brain injury: microdialysis studies using 1,2-13C2 glucose

    Science.gov (United States)

    Jalloh, Ibrahim; Carpenter, Keri L H; Grice, Peter; Howe, Duncan J; Mason, Andrew; Gallagher, Clare N; Helmy, Adel; Murphy, Michael P; Menon, David K; Carpenter, T Adrian; Pickard, John D; Hutchinson, Peter J

    2015-01-01

    Increased ‘anaerobic' glucose metabolism is observed after traumatic brain injury (TBI) attributed to increased glycolysis. An alternative route is the pentose phosphate pathway (PPP), which generates putatively protective and reparative molecules. To compare pathways we employed microdialysis to perfuse 1,2-13C2 glucose into the brains of 15 TBI patients and macroscopically normal brain in six patients undergoing surgery for benign tumors, and to simultaneously collect products for nuclear magnetic resonance (NMR) analysis. 13C enrichment for glycolytic 2,3-13C2 lactate was the median 5.4% (interquartile range (IQR) 4.6–7.5%) in TBI brain and 4.2% (2.4–4.4%) in ‘normal' brain (Pbrain and 6.7% (6.3–8.9%) in ‘normal' brain. An inverse relationship was seen for PPP-glycolytic lactate ratio versus PbtO2 (r=−0.5, P=0.04) in TBI brain. Thus, glycolytic lactate production was significantly greater in TBI than ‘normal' brain. Several TBI patients exhibited PPP–lactate elevation above the ‘normal' range. There was proportionally greater PPP-derived lactate production with decreasing PbtO2. The study raises questions about the roles of the PPP and glycolysis after TBI, and whether they can be manipulated to achieve a better outcome. This study is the first direct comparison of glycolysis and PPP in human brain. PMID:25335801

  14. Feedback Inhibition of Starch Degradation in Arabidopsis Leaves Mediated by Trehalose 6-Phosphate1[W][OPEN

    Science.gov (United States)

    Martins, Marina Camara Mattos; Hejazi, Mahdi; Fettke, Joerg; Steup, Martin; Feil, Regina; Krause, Ursula; Arrivault, Stéphanie; Vosloh, Daniel; Figueroa, Carlos María; Ivakov, Alexander; Yadav, Umesh Prasad; Piques, Maria; Metzner, Daniela; Stitt, Mark; Lunn, John Edward

    2013-01-01

    Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by β-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µm in the cytosol, 0.2 to 0.5 µm in the chloroplasts, and 0.05 µm in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night. PMID:24043444

  15. Electronically type-sorted carbon nanotube-based electrochemical biosensors with glucose oxidase and dehydrogenase.

    Science.gov (United States)

    Muguruma, Hitoshi; Hoshino, Tatsuya; Nowaki, Kohei

    2015-01-14

    An electrochemical enzyme biosensor with electronically type-sorted (metallic and semiconducting) single-walled carbon nanotubes (SWNTs) for use in aqueous media is presented. This research investigates how the electronic types of SWNTs influence the amperometric response of enzyme biosensors. To conduct a clear evaluation, a simple layer-by-layer process based on a plasma-polymerized nano thin film (PPF) was adopted because a PPF is an inactive matrix that can form a well-defined nanostructure composed of SWNTs and enzyme. For a biosensor with the glucose oxidase (GOx) enzyme in the presence of oxygen, the response of a metallic SWNT-GOx electrode was 2 times larger than that of a semiconducting SWNT-GOx electrode. In contrast, in the absence of oxygen, the response of the semiconducting SWNT-GOx electrode was retained, whereas that of the metallic SWNT-GOx electrode was significantly reduced. This indicates that direct electron transfer occurred with the semiconducting SWNT-GOx electrode, whereas the metallic SWNT-GOx electrode was dominated by a hydrogen peroxide pathway caused by an enzymatic reaction. For a biosensor with the glucose dehydrogenase (GDH; oxygen-independent catalysis) enzyme, the response of the semiconducting SWNT-GDH electrode was 4 times larger than that of the metallic SWNT-GDH electrode. Electrochemical impedance spectroscopy was used to show that the semiconducting SWNT network has less resistance for electron transfer than the metallic SWNT network. Therefore, it was concluded that semiconducting SWNTs are more suitable than metallic SWNTs for electrochemical enzyme biosensors in terms of direct electron transfer as a detection mechanism. This study makes a valuable contribution toward the development of electrochemical biosensors that employ sorted SWNTs and various enzymes.

  16. Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney.

    Science.gov (United States)

    Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V; Park, Kwon Moo

    2009-03-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/(GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK(1) cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R.

  17. Pleurocidin Peptide Enhances Grouper Anti-Vibrio harveyi Immunity Elicited by Poly(lactide-co-glycolide)-Encapsulated Recombinant Glyceraldehyde-3-phosphate Dehydrogenase.

    Science.gov (United States)

    Chuang, Shu-Chun; Huang, Wan-Ling; Kau, Sau-Wei; Yang, Yun-Pei; Yang, Chung-Da

    2014-05-14

    Outer membrane proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are considered immunodominant antigens for eliciting protective immunity against Vibrio harveyi, the main etiological agent of vibriosis in fish. Cationic antimicrobial peptides (AMPs), such as pleurocidin (PLE), play important roles in activating and recruiting immune cells, thereby contributing to subsequent innate and adaptive immune responses. In the present study, we aimed to use PLE peptide as a potent adjuvant to improve the immunogenicity of V. harveyi recombinant GAPDH (rGAPDH). In order to prepare a controlled-release vaccine, PLE peptide and rGAPDH protein were simultaneously encapsulated into polymeric microparticles made from the biodegradable poly(lactide-co-glycolide) (PLG) polymer. The resulting PLG-encapsulated PLE plus rGAPDH (PLG-PLE/rGAPDH) microparticles, 3.21-6.27 μm in diameter, showed 72%-83% entrapment efficiency and durably released both PLE and rGAPDH for a long 30-day period. Following peritoneal immunization in grouper (Epinephelus coioides), PLG-PLE/rGAPDH microparticles resulted in significantly higher (p PLE/rGAPDH microparticles conferred a high survival rate (85%), which was significantly higher (p PLE peptide exhibits an efficacious adjuvant effect to elicit not only improved immunity, but also enhanced protection against V. harveyi in grouper induced by rGAPDH protein encapsulated in PLG microparticles.

  18. Enhanced performance of a glucose/O(2) biofuel cell assembled with laccase-covalently immobilized three-dimensional macroporous gold film-based biocathode and bacterial surface displayed glucose dehydrogenase-based bioanode.

    Science.gov (United States)

    Hou, Chuantao; Yang, Dapeng; Liang, Bo; Liu, Aihua

    2014-06-17

    The power output and stability of enzyme-based biofuel cells (BFCs) is greatly dependent on the properties of both the biocathode and bioanode, which may be adapted for portable power production. In this paper, a novel highly uniform three-dimensional (3D) macroporous gold (MP-Au) film was prepared by heating the gold "supraspheres", which were synthesized by a bottom-up protein templating approach, and followed by modification of laccase on the MP-Au film by covalent immobilization. The as-prepared laccase/MP-Au biocathode exihibited an onset potential of 0.62 V versus saturated calomel electrode (SCE, or 0.86 V vs NHE, normal hydrogen electrode) toward O2 reduction and a high catalytic current of 0.61 mAcm(-2). On the other hand, mutated glucose dehydrogenase (GDH) surface displayed bacteria (GDH-bacteria) were used to improve the stability of the glucose oxidation at the bioanode. The as-assembled membraneless glucose/O2 fuel cell showed a high power output of 55.8 ± 2.0 μW cm(-2) and open circuit potential of 0.80 V, contributing to the improved electrocatalysis toward O2 reduction at the laccase/MP-Au biocathode. Moreover, the BFC retained 84% of its maximal power density even after continuous operation for 55 h because of the high stability of the bacterial surface displayed GDH mutant toward glucose oxidation. Our findings may be promising for the development of more efficient glucose BFC for portable battery or self-powered device applications.

  19. Breaking the Dogma of Aldolase Specificity: Simple Aliphatic Ketones and Aldehydes are Nucleophiles for Fructose-6-phosphate Aldolase.

    Science.gov (United States)

    Roldán, Raquel; Sanchez-Moreno, Israel; Scheidt, Thomas; Hélaine, Virgil; Lemaire, Marielle; Parella, Teodor; Clapés, Pere; Fessner, Wolf-Dieter; Guérard-Hélaine, Christine

    2017-04-11

    d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Expression of 11beta-hydroxysteroid dehydrogenase 1 and 2 in subcutaneous adipose tissue of lean and obese women with and without polycystic ovary syndrome

    DEFF Research Database (Denmark)

    Svendsen, P F; Madsbad, S; Nilas, L

    2009-01-01

    OBJECTIVE: To investigate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 and 2 and hexose-6-phosphate dehydrogenase (H6PDH) mRNA in subcutaneous abdominal tissue from lean and obese women with and without polycystic ovary syndrome (PCOS), and to investigate...... assessment insulin resistance index. Body composition was evaluated by dual X-ray absorptiometry. Adipose mRNA expression of leptin and adiponectin were determined by real-time PCR. RESULTS: Polycystic ovary syndrome (P... distribution (PPolycystic ovary syndrome and obesity are independently associated with increased expression of 11beta-HSD1. This may lead to increased conversion of cortisone to cortisol...

  1. Synthesis and crystal structure of 4-fluorobenzylammonium dihydrogen phosphate, [FC6H4CH2NH3]H2PO4

    Directory of Open Access Journals (Sweden)

    Ali Rayes

    2016-12-01

    Full Text Available The asymmetric unit of the title salt, [p-FC6H4CH2NH3]+·H2PO4−, contains one 4-fluorobenzylammonium cation and one dihydrogen phosphate anion. In the crystal, the H2PO4− anions are linked by O—H...O hydrogen bonds to build corrugated layers extending parallel to the ab plane. The FC6H4CH2NH3+ cations lie between these anionic layers to maximize the electrostatic interactions and are linked to the H2PO4− anions through N—H...O hydrogen bonds, forming a three-dimensional supramolecular network. Two hydrogen atoms belonging to the dihydrogen phosphate anion are statistically occupied due to disorder along the OH...HO direction.

  2. [Carbohydrates metabolism disturbances when simulating prenatal alcohol intoxication].

    Science.gov (United States)

    Kurch, N M; Vysokogorskiĭ, V E

    2013-01-01

    The influence of prenatal alcohol intoxication on carbohydrate metabolism markers has been investigated at different terms of postnatal offspring development (15, 30 and 60 days). Plasma glucose decreased as compared with the same in control group was detected. In the liver homogenates an increase of phosphorylase activity and a decrease of glucose-6-phosphatase, aldolase and glucose-6-phosphate dehydrogenase activities were found. These changes were accompanied by the incease in the lactate/pyruvate index attributed to increased lactate content in the liver tissue. The obtained data indicate essential disturbances of carbohydrate metabolism markers in prenatal alcoholized offspring, which include stable hypoglycemia, suppression of glycolytic and pentosephosphate pathways of glucose metabolism and lactate accumulation in the liver.

  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. Characterization of the L-lactate dehydrogenase from Aggregatibacter actinomycetemcomitans.

    Directory of Open Access Journals (Sweden)

    Stacie A Brown

    Full Text Available Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a K(m of approximately 150 microM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans.

  5. Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

    Science.gov (United States)

    Chin, Young-Wook; Park, Jin-Byung; Park, Yong-Cheol; Kim, Kyoung Heon; Seo, Jin-Ho

    2013-06-01

    Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

  6. Analysis of phosphate esters in plant material. Extraction and purification.

    Science.gov (United States)

    Isherwood, F A; Barrett, F C

    1967-09-01

    1. A critical study was made of the quantitative extraction of nucleotide and sugar phosphates from plant tissue by either boiling aqueous ethanol or cold trichloroacetic acid. The effect of the extraction technique on the inactivation of the enzymes in the plant tissue and the possibility of adsorption of the phosphate esters on the cell wall were especially considered. 2. In the recommended method the plant tissue was frozen in liquid nitrogen, ground to a powder and then blended with cold aqueous trichloroacetic acid containing 8-hydroxyquinoline to prevent adsorption. 3. The extract contained large amounts of trichloroacetic acid, cations, chloride, sugars, amino acids, hydroxy organic acids, phytic acid, orthophosphoric acid and high-molecular-weight material including some phosphorus-containing compounds. All of these were removed as they were liable to interfere with the chromatographic or enzymic assay of the individual nucleotide or sugar phosphates. 4. The procedure was as follows: the last traces of trichloroacetic acid were extracted with ether after the solution had been passed through a column of Dowex AG 50 in the hydrogen form to remove all cations. High-molecular-weight compounds were removed by ultrafiltration and low-molecular-weight solutes by a two-stage chromatography on cellulose columns with organic solvents. In the first stage, sugars, amino acids, chloride and phytic acid were separated by using a basic solvent (propan-1-ol-water-aqueous ammonia) and, in the second stage, the organic acids and orthophosphoric acid were separated by using an acidic solvent (di-isopropyl ether-formic acid-2-methylpropan-2-ol-water). The final solution of nucleotide and sugar phosphates was substantially free from other solutes and was suitable for the detection of individual phosphate esters by either chromatography or enzymic assay. 5. The recovery of d-glucose 6-phosphate or adenosine 5'-triphosphate added to a trichloroacetic acid extract simulating that

  7. Expression of 11beta-hydroxysteroid dehydrogenase 1 and 2 in subcutaneous adipose tissue of lean and obese women with and without polycystic ovary syndrome.

    Science.gov (United States)

    Svendsen, P F; Madsbad, S; Nilas, L; Paulsen, S K; Pedersen, S B

    2009-11-01

    To investigate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 and 2 and hexose-6-phosphate dehydrogenase (H6PDH) mRNA in subcutaneous abdominal tissue from lean and obese women with and without polycystic ovary syndrome (PCOS), and to investigate the association between these enzymes and different measures of insulin sensitivity. Cross-sectional study. A total of 60 women, 36 women with PCOS, 17 lean (lean PCOS, LP) and 19 obese (obese PCOS, OP) and 24 age- and weight-matched control women, 8 lean (lean controls, LC) and 16 obese (obese controls, OC). Subcutaneous adipose tissue was collected from the abdomen. Peripheral insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp and determined as glucose disposal rate and insulin sensitivity index. Whole-body insulin sensitivity was calculated using homeostasis model assessment insulin resistance index. Body composition was evaluated by dual X-ray absorptiometry. Adipose mRNA expression of leptin and adiponectin were determined by real-time PCR. Polycystic ovary syndrome (PPCOS or obesity on11beta-HSD2 or H6PDH mRNA expression. Decreased peripheral insulin sensitivity (P<0.001) and increased upper body fat distribution (P<0.01) were associated with increased expression of 11beta-HSD1, but neither 11beta-HSD2 nor H6PDH. Polycystic ovary syndrome and obesity are independently associated with increased expression of 11beta-HSD1. This may lead to increased conversion of cortisone to cortisol in the peripheral adipose tissue and subsequently increased glucocorticoid activity. Decreased peripheral insulin sensitivity and central obesity was associated with increased expression of 11beta-HSD1.

  8. Relations of enzymes inAspergillus repens grown under sodium chloride stress.

    Science.gov (United States)

    Kelavkar, U P; Chhatpar, H S

    1993-09-01

    Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2M sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2M NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na(+) (as NaCl) when added up to 100MM in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.

  9. Prokaryotic Expression and Serodiagnostic Potential of Glyceraldehyde-3-Phosphate Dehydrogenase and Thioredoxin Peroxidase from Baylisascaris schroederi

    Directory of Open Access Journals (Sweden)

    Yu Li

    2017-10-01

    Full Text Available Baylisascaris schroederi, a roundworm parasite of giant pandas, badly affects the health of its hosts. Diagnosis of this disease currently depends mainly on sedimentation floatation and Polymerase Chain Reaction (PCR methods to detect the eggs. However, neither of these methods is suitable for diagnosis of early-stage panda baylisascariasis and no information on early diagnosis of this disease is available so far. Therefore, to develop an effective serologic diagnostic method, this study produced recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH and thioredoxin peroxidase (Tpx proteins from B. schroederi using a prokaryotic expression system. We determined the immunological characteristics of these proteins and their location in the parasite. Indirect enzyme-linked immunosorbent assays (ELISAs were established to detect B. schroederi infection in giant pandas based on GAPDH and Tpx respectively. The open reading frame of the GAPDH gene (1083 bp encoded a 39 kDa protein, while the predicted molecular weight of Tpx (588 bp was 21.6 kDa. Western-blotting analysis revealed that both recombinant proteins could be recognized with positive serum of pandas infected with B. schroederi. Immunohistochemical staining showed that the endogenous GAPDH of B. schroederi was widely distributed in the worm while Tpx was mainly localized in the muscle, eggs, gut wall, uterus wall and hypodermis. Serological tests showed that the GAPDH-based indirect ELISA had a sensitivity of 95.83% and specificity of 100%, while the test using Tpx as the antigen had sensitivity of 75% and specificity of 91.7%. Thus, B. schroederi Tpx is unsuitable as a diagnostic antigen for baylisascariasis, but B. schroederi GAPDH is a good candidate diagnostic antigen for B. schroederi in pandas.

  10. Adaptation of red cell enzymes and intermediates in metabolic disorders.

    Science.gov (United States)

    Goebel, K M; Goebel, F D; Neitzert, A; Hausmann, L; Schneider, J

    1975-01-01

    The metabolic activity of the red cell glycolytic pathway hexose monophosphate shunt (HMP) with dependent glutathione system was studied in patients with hyperthyroidism (n = 10), hyperlipoproteinemia (n = 16), hypoglycemia (n = 25) and hyperglycemia (n = 23). In uncontrolled diabetics and patients with hyperthyroidism the mean value of glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G-6-PD), glutathione reductase (GR) was increased, whereas these enzyme activities were reduced in patients with hypoglycemia. Apart from a few values of hexokinase (HK) which were lower than normal the results in hyperlipoproteinemia patients remained essentially unchanged, including the intermediates such as 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP) and reduced glutathione (GSH). While increased rates of 2,3-DPG and ATP in hypoglycemia patients were obtained, these substrates were markedly reduced in diabetics.

  11. Direct electrochemistry and electrocatalysis of glucose oxidase on three-dimensional interpenetrating, porous graphene modified electrode

    International Nuclear Information System (INIS)

    Cui, Min; Xu, Bing; Hu, Chuangang; Shao, Hui Bo; Qu, Liangti

    2013-01-01

    Direct electrochemistry of glucose oxidase (GOD) on three-dimensional (3D) interpenetrating porous graphene electrodes has been reported, which have been fabricated by one-step electrochemical reduction of graphene oxide (GO) from its aqueous suspension. The electrochemically reduced GO (ERGO) modified electrodes exhibited excellent electron transfer properties for GOD and enhanced the enzyme activity and stability by the assistance of chitosan. The immobilized GOD shows a fast electron transfer with the rate constant (k s ) of 6.05 s −1 . It is worth mentioning that in the air-saturated phosphate buffer solution without any mediator, the resultant modified electrodes exhibited low detection limit of 1.7 μM with wide linear range of 0.02–3.2 mM and high sensitivity and high selectivity for measuring glucose. It would also be extended to various enzymes and bioactive molecules to develop the biosensor or other bio-electrochemical devices

  12. Sensitive, specific radioisotope assay for L-glutamine-D-fructose-6- phosphat aminotransferase

    International Nuclear Information System (INIS)

    Callahan, M.; Tourian, A.; Hung, W.Y.

    1981-01-01

    A sensitive and specific radioassay for L-glutamine-D-fructose-6-phosphate aminotransferase (EC 5.3.1.19) activity is presented. Picomoles of product are measurable, and the assay can be applied to systems having limited quantities of available protein, particularly in extracts of either cell or organ cultures. The assay is at least 10,000 times more sensitive under K 1 concentrations of fructose 6-phosphate than the modified Elson-Morgan colorimetric assay and 20 times more sensitive under saturating conditions of fructose 6-phosphate. As little as 0.5 μg of cell-extract protein will yield measurable product. In contrast, 280 μg of crude-extract protein from colon is required with the modified Elson-Morgan colorimetric assay

  13. Rapid screening for glucose-6-phosphate dehydrogenase deficiency and haemoglobin polymorphisms in Africa by a simple high-throughput SSOP-ELISA method

    DEFF Research Database (Denmark)

    Enevold, Anders; Vestergaard, Lasse S; Lusingu, John

    2005-01-01

    was available. METHODS: A simple and rapid technique was developed to detect the most prominent single nucleotide polymorphisms (SNPs) in the HbB and G6PD genes. The method is able to detect the different haemoglobin polymorphisms A, S, C and E, as well as G6PD polymorphisms B, A and A- based on PCR......-amplification followed by a hybridization step using sequence-specific oligonucleotide probes (SSOPs) specific for the SNP variants and quantified by ELISA. RESULTS: The SSOP-ELISA method was found to be specific, and compared well to the commonly used PCR-RFLP technique. Identical results were obtained in 98......% (haemoglobin) and 95% (G6PD) of the tested 90 field samples from a high-transmission area in Tanzania, which were used to validate the new technique. CONCLUSION: The simplicity and accuracy of the new methodology makes it suitable for application in settings where resources are limited. It would serve...

  14. Small-angle X-ray scattering studies on the X-ray induced aggregation of ribonnuclease, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and serum albumin. A comparison with malate synthase

    International Nuclear Information System (INIS)

    Zipper, P.; Gatterer, H.G.; Schutz, J.; Durchschlag, H.

    1980-01-01

    The X-ray induced aggregation of ribonuclease, lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and serum albumin in aqueous solution was monitored in situ by means of small-angle X-ray scattering. Measurements carried out with ribonuclease, LDH and serum albumin in the absence of dithiothreitol (DTT) and with GAPDH in the presence of 0.2mM DTT established the following series for the rates of aggregation of the proteins under these conditions: ribonuclease >LDH> >GAPDH> serum albumin. Within six hours from the beginning of irradiation (i.e. about the time required for the exposure of one complete scattering curve under the conditions of our experiments) the following increases of R tilde resulted: ribonuclease 9%, LDH 7%, GAPDH 4%, serum albumin <1%. Changes of R tilde exceeding 1% are, of course, too high to be tolerated in conventional scattering experiments. Measurements carried out with LDH and GAPDH in the presence of 2mM DTT established a strong protective effect of DTT against the X-ray induced aggregation of these enzymes. The initial increase of R tilde upon irradiation of LDH and GAPDH in the presence of 2mM DTT was found to be even lower than the increase of R tilde observed when serum albumin was irradiated in the absence of DTT. However, the observed decrease of anti x of LDH and GAPDH at the early stages of irradiation suggested the occurrence of fragmentation of the enzymes as another consequence of radiation damage. This finding is discussed in context with the results from previous scattering experiments and electrophoretic studies on malate synthase. (author)

  15. Electrochemical quartz crystal impedance study on immobilization of glucose oxidase in a polymer grown from dopamine oxidation at an Au electrode for glucose sensing

    International Nuclear Information System (INIS)

    Li Mingrui; Deng Chunyan; Xie Qingji; Yang Yang; Yao Shouzhuo

    2006-01-01

    Glucose oxidase (GOD) was codeposited into a polymer grown from oxidation of dopamine (DA) at an Au electrode in a neutral phosphate aqueous solution for the first time. The electrochemical quartz crystal impedance analysis (EQCIA) method was used to monitor the GOD-immobilization process. Effects of concentrations of phosphate buffer, DA and GOD were investigated, and the optimal concentrations were found to be 20.0mM phosphate buffer (pH 7.0), 30.0mM DA and 5.00mgml -1 GOD. A glucose biosensor was thus constructed, and effects of various experimental parameters on the sensor performance, including applied potential, solution pH and electroactive interferents, were examined. At an optimal potential of 0.6V versus the KCl-saturated calomel electrode (SCE), the current response of the biosensor in the selected phosphate buffer (pH 7.0) was linear with the concentration of glucose from 0.05 to 9mM, with a lower detection limit of 3μM (S/N=3), short response time (within 15s) and good anti-interferent ability. The Michaelis constant (K m app ) was estimated to be 9.6mM. The biosensor exhibited good storage stability, i.e. 96% of its initial response was retained after 7-day storage in the selected phosphate buffer at 4deg. C, and even after another 3 weeks the biosensor retained 86% of its initial response. In addition, the enzymatic specific activity and enzymatic relative activity of the GOD immobilized in the polymer from dopamine oxidation (PFDO) were estimated from the EQCIA method to be 1.43kUg -1 and 3.7%, respectively, which were larger than the relevant values obtained experimentally using poly(o-aminophenol) and poly(N-methylpyrrole) matrices, suggesting that the PFDO is a better matrix to immobilize GOD

  16. Effect of an aqueous extract of Scoparia dulcis on blood glucose, plasma insulin and some polyol pathway enzymes in experimental rat diabetes.

    Science.gov (United States)

    Latha, M; Pari, L

    2004-04-01

    The effects of an aqueous extract of the plant Scoparia dulcis (200 mg/kg) on the polyol pathway and lipid peroxidation were examined in the liver of streptozotocin adult diabetic male albino Wistar rats. The diabetic control rats (N = 6) presented a significant increase in blood glucose, sorbitol dehydrogenase, glycosylated hemoglobin and lipid peroxidation markers such as thiobarbituric acid reactive substances (TBARS) and hydroperoxides, and a significant decrease in plasma insulin and antioxidant enzymes such as glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione (GSH) compared to normal rats (N = 6). Scoparia dulcis plant extract (SPEt, 200 mg kg-1 day-1) and glibenclamide (600 microg kg-1 day-1), a reference drug, were administered by gavage for 6 weeks to diabetic rats (N = 6 for each group) and significantly reduced blood glucose, sorbitol dehydrogenase, glycosylated hemoglobin, TBARS, and hydroperoxides, and significantly increased plasma insulin, GPx, GST and GSH activities in liver. The effect of the SPEt was compared with that of glibenclamide. The effect of the extract may have been due to the decreased influx of glucose into the polyol pathway leading to increased activities of antioxidant enzymes and plasma insulin and decreased activity of sorbitol dehydrogenase. These results indicate that the SPEt was effective in attenuating hyperglycemia in rats and their susceptibility to oxygen free radicals.

  17. Effect of an aqueous extract of Scoparia dulcis on blood glucose, plasma insulin and some polyol pathway enzymes in experimental rat diabetes

    Directory of Open Access Journals (Sweden)

    M. Latha

    2004-04-01

    Full Text Available The effects of an aqueous extract of the plant Scoparia dulcis (200 mg/kg on the polyol pathway and lipid peroxidation were examined in the liver of streptozotocin adult diabetic male albino Wistar rats. The diabetic control rats (N = 6 presented a significant increase in blood glucose, sorbitol dehydrogenase, glycosylated hemoglobin and lipid peroxidation markers such as thiobarbituric acid reactive substances (TBARS and hydroperoxides, and a significant decrease in plasma insulin and antioxidant enzymes such as glutathione peroxidase (GPx, glutathione-S-transferase (GST and reduced glutathione (GSH compared to normal rats (N = 6. Scoparia dulcis plant extract (SPEt, 200 mg kg-1 day-1 and glibenclamide (600 µg kg-1 day-1, a reference drug, were administered by gavage for 6 weeks to diabetic rats (N = 6 for each group and significantly reduced blood glucose, sorbitol dehydrogenase, glycosylated hemoglobin, TBARS, and hydroperoxides, and significantly increased plasma insulin, GPx, GST and GSH activities in liver. The effect of the SPEt was compared with that of glibenclamide. The effect of the extract may have be