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Sample records for 6-phosphate uncovering enzyme

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

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    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.

  2. Multiple independent fusions of glucose-6-phosphate dehydrogenase with enzymes in the pentose phosphate pathway.

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    Nicholas A Stover

    Full Text Available Fusions of the first two enzymes in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD and 6-phosphogluconolactonase (6PGL, have been previously described in two distant clades, chordates and species of the malarial parasite Plasmodium. We have analyzed genome and expressed sequence data from a variety of organisms to identify the origins of these gene fusion events. Based on the orientation of the domains and range of species in which homologs can be found, the fusions appear to have occurred independently, near the base of the metazoan and apicomplexan lineages. Only one of the two metazoan paralogs of G6PD is fused, showing that the fusion occurred after a duplication event, which we have traced back to an ancestor of choanoflagellates and metazoans. The Plasmodium genes are known to contain a functionally important insertion that is not seen in the other apicomplexan fusions, highlighting this as a unique characteristic of this group. Surprisingly, our search revealed two additional fusion events, one that combined 6PGL and G6PD in an ancestor of the protozoan parasites Trichomonas and Giardia, and another fusing G6PD with phosphogluconate dehydrogenase (6PGD in a species of diatoms. This study extends the range of species known to contain fusions in the pentose phosphate pathway to many new medically and economically important organisms.

  3. Identification of glucoselysine-6-phosphate deglycase, an enzyme involved in the metabolism of the fructation product glucoselysine.

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    Wiame, Elsa; Lamosa, Pedro; Santos, Helena; Van Schaftingen, Emile

    2005-01-01

    The metabolism of the glycation product fructose-epsilon-lysine in Escherichia coli involves its ATP-dependent phosphorylation by a specific kinase (FrlD), followed by the conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine by fructoselysine-6-phosphate deglycase (FrlB), which is distantly related to the isomerase domain of glucosamine-6-phosphate synthase. As shown in the present work, several bacterial operons comprise: (1) a homologue of fructoselysine-6-phosphate ...

  4. Determination of the inhibitory effect of green tea extract on glucose-6-phosphate dehydrogenase based on multilayer capillary enzyme microreactor.

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    Camara, Mohamed Amara; Tian, Miaomiao; Liu, Xiaoxia; Liu, Xin; Wang, Yujia; Yang, Jiqing; Yang, Li

    2016-08-01

    Natural herbal medicines are an important source of enzyme inhibitors for the discovery of new drugs. A number of natural extracts such as green tea have been used in prevention and treatment of diseases due to their low-cost, low toxicity and good performance. The present study reports an online assay of the activity and inhibition of the green tea extract of the Glucose 6-phosphate dehydrogenase (G6PDH) enzyme using multilayer capillary electrophoresis based immobilized enzyme microreactors (CE-IMERs). The multilayer CE-IMERs were produced with layer-by-layer electrostatic assembly, which can easily enhance the enzyme loading capacity of the microreactor. The activity of the G6PDH enzyme was determined and the enzyme inhibition by the inhibitors from green tea extract was investigated using online assay of the multilayer CE-IMERs. The Michaelis constant (Km ) of the enzyme, the IC50 and Ki values of the inhibitors were achieved and found to agree with those obtained using offline assays. The results show a competitive inhibition of green tea extract on the G6PDH enzyme. The present study provides an efficient and easy-to-operate approach for determining G6PDH enzyme reaction and the inhibition of green tea extract, which may be beneficial in research and the development of natural herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Amelioration by glucose-6-phosphate and NADP of potato glycoalkaloid inhibition in cell, enzyme and liposome assays.

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    Roddick, J G; Leonard, A L

    1999-05-01

    Lysis of human erythrocytes by 20 microM chaconine was reduced by 0.5 mM glucose-6-phosphate (G6P) and NADP. Both compounds caused approximately 50% inhibition of haemolysis at 1 mM. Glucose, glucose-1-phosphate, rhamnose, galactose and galactose-6-phosphate were ineffective; NAD was effective, although not to the extent of NADP. Of the tested sugars, only G6P reduced solanine-induced haemolysis. G6P also reduced the synergistic haemolytic action of solanine and chaconine in combination. G6P and NADP at or above 5 mM antagonised chaconine-induced betanin loss from excised red beet root discs; NADP was more effective than G6P. Disruption of PC/cholesterol liposomes by chaconine and inhibition of acetylcholinesterase by chaconine or solanine, were unaffected by up to 10 mM NADP or 50 mM G6P.

  6. Glucose-6-phosphate dehydrogenase

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    ... medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a protein that helps red ...

  7. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

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    Kuzu, Muslum; Aslan, Abdulselam; Ahmed, Ishtiaq; Comakli, Veysel; Demirdag, Ramazan; Uzun, Naim

    2016-04-01

    Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K(M) and V(max) values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K(i) value was calculated by using Cheng-Prusoff equation.

  8. Control of Enzyme IIscr and Sucrose-6-Phosphate Hydrolase Activities in Streptococcus mutans by Transcriptional Repressor ScrR Binding to the cis-Active Determinants of the scr Regulon

    OpenAIRE

    Wang, Bing; Kuramitsu, Howard K.

    2003-01-01

    In Streptococcus mutans, enzyme IIscr and sucrose-6-phosphate hydrolase are two important enzymes in the transport and metabolism of dietary sucrose. The scr regulon of S. mutans is composed of three genes, scrA and scrB, which code for enzyme IIscr and sucrose-6-phosphate hydrolase, respectively, and scrR, which codes for a GalR-LacI-type transcription regulator. It was previously shown that expression of both scrA and scrB is similarly induced by sucrose. Mutation in the scrR gene resulted ...

  9. Somatic-cell selection is a major determinant of the blood-cell phenotype in heterozygotes for glucose-6-phosphate dehydrogenase mutations causing severe enzyme deficiency

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    Filosa, S.; Giacometti, N.; Wangwei, C.; Martini, G. [Istituto Internazionale di Genetica e Biofisica, Naples (Italy)] [and others

    1996-10-01

    X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G{r_arrow}A) and two with G6PD Bari (1187C{r_arrow}T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis. 65 refs., 6 figs., 3 tabs.

  10. 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...

  11. Glucose-6-phosphate dehydrogenase deficiency

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    ... medlineplus.gov/ency/article/000528.htm Glucose-6-phosphate dehydrogenase deficiency To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a condition in which ...

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

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    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

  13. 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

  14. {open_quotes}The effects of diabetes on the activity of the enzyme glutamine: fructose-6-phosphate amindotransferase{close_quotes}

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    Nelson, S.P.

    1994-12-31

    Hexsoamine synthetic pathway (HexNSP) controls the supply of essential substrates for glycoprotein synthesis. In vitro studies suggest that increased flux of glucose via the hexsoamine synthetic pathway may play a role in glucose induced insulin resistance of glucose transport. Glutamine: fructose-6-phosphate amindotransferase (GFAT) controls flux into the hexsoamine synthetic pathway; the major products are UDPN-acetylhexosamines (UDP.HexNac=UDP.GlcNAc= UDP.GalNac). I examined whether diabetes ({approximately} 7 days post intravenous streptozotocin, and genetically linked) affects the activity of glutamine: fructose-6-phosphate in rat and mouse skeletal muscle in vivo. Nucleotide linked HexNAc were analyzed by high pressure liquid chromatography(HPLC) in deproteinized hind limb muscle extracts.

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

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    ... enzyme is involved in the normal processing of carbohydrates. It also protects red blood cells from the ... of glucose-6-phosphate dehydrogenase or alter its structure, this enzyme can no longer play its protective ...

  16. [Frequency of color blindness and glucose-6-phosphate dehydrogenase enzyme deficiency in non-industrialized populations in the state of Nuevo León, México].

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    Ceda-Flores, R M; Arriaga-Ríos, G; Muñoz-Campos, J; Bautista-Peña, V A; Angeles Rojas-Alvarado, M; González-Quiróga, G; Leal-Garza, C H; Garza-Chapa, R

    1990-01-01

    In order to know if there would be genetic structural differences among non industrial and industrial populations, two genetic markers were studied: color-blindness (CPC) and glucose-6-phosphate dehydrogenase deficiency (G6PD), in students, males and females that were resident in five non industrial populations in the State of Nuevo Leon. The results were compared with the information for industrial zone from the Monterrey Metropolitan area (AMM). It was found that the frequencies of CPC and G6PD in non industrial populations (2.57 and 0.00 per cent), were lower than the ones in the industrial AMM (4.0 and 0.66 per cent), probably as a result that in the first populations, with minor urbanization, the main factors that influence are: natural selection, interacial mixed or genetic drift and the second population is the immigration, since 1940 to present time, of Mexican populations with greater influence from the Indians and Africans.

  17. Uncovering a new role for peroxidase enzymes as drivers of angiogenesis.

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    Panagopoulos, Vasilios; Zinonos, Irene; Leach, Damien A; Hay, Shelley J; Liapis, Vasilios; Zysk, Aneta; Ingman, Wendy V; DeNichilo, Mark O; Evdokiou, Andreas

    2015-11-01

    Peroxidases are heme-containing enzymes released by activated immune cells at sites of inflammation. To-date their functional role in human health has mainly been limited to providing a mechanism for oxidative defence against invading bacteria and other pathogenic microorganisms. Our laboratory has recently identified a new functional role for peroxidase enzymes in stimulating fibroblast migration and collagen biosynthesis, offering a new insight into the causative association between inflammation and the pro-fibrogenic events that mediate tissue repair and regeneration. Peroxidases are found at elevated levels within and near blood vessels however, their direct involvement in angiogenesis has never been reported. Here we report for the first time that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are readily internalised by human umbilical vein endothelial cells (HUVEC) where they promote cellular proliferation, migration, invasion, and stimulate angiogenesis both in vitro and in vivo. These pro-angiogenic effects were attenuated using the specific peroxidase inhibitor 4-ABAH, indicating the enzyme's catalytic activity is essential in mediating this response. Mechanistically, we provide evidence that MPO and EPO regulate endothelial FAK, Akt, p38 MAPK, ERK1/2 phosphorylation and stabilisation of HIF-2α, culminating in transcriptional regulation of key angiogenesis pathways. These findings uncover for the first time an important and previously unsuspected role for peroxidases as drivers of angiogenesis, and suggest that peroxidase inhibitors may have therapeutic potential for the treatment of angiogenesis related diseases driven by inflammation.

  18. Characterization of a Mannose-6-Phosphate Isomerase from Bacillus amyloliquefaciens and Its Application in Fructose-6-Phosphate Production.

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    Sujan Sigdel

    Full Text Available The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8 was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P. The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6-phosphate demonstrating the potential for using BaM6PI in industrial applications.

  19. Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species.

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    Grill, J P; Crociani, J; Ballongue, J

    1995-07-01

    Fructose 6 phosphate phosphoketolases (F6PPKs) were purified from Bifidobacterium longum BB536, B. dentium ATCC 27534, B. globosum ATCC 25864, and Bifidobacterium animalis ATCC 25527. Concerning ions (Cu++, Zn++, Ca++, Mg++, Fe++, Co++, Mn++) and common enzyme inhibitors (fructose, ammonium sulfate, iodoacetate, and parachloromercuribenzoic acid), no difference appeared between the enzymes. Cu++, parachloromercuribenzoic acid (pCMB), and mercuric acetate induced high enzymatic inhibition. The study of pCMB demonstrated a noncompetitive inhibition. Additional results showed that the sulfhydryl group was not involved in catalytic reaction. Photooxidation experiments and determination of ionizable group pKas (5.16-7.17) suggested the presence of one or more histidines necessary for the catalytic reaction and explained the inhibition observed with pCMB. In light of the noncompetitive inhibition, this group was not directly involved in substrate binding. Determination of Km demonstrated that the affinities for fructose 6 phosphate in the case of animal and human origin strains were close. In addition, the same enzymatic efficiency (Kcat/Km) was obtained for each strain. The F6PPK activity was regulated by sodium pyrophosphate, ATP, and especially by ADP.

  20. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

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    In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation dr...

  1. Priapism and glucose-6-phosphate dehydrogenase deficiency: An underestimated correlation?

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    Aldo Franco De Rose

    2016-10-01

    Full Text Available Priapism is a rare clinical condition characterized by a persistent erection unrelated to sexual excitement. Often the etiology is idiopathic. Three cases of priapism in glucose-6-phosphate dehydrogenase (G6PD deficiency patients have been described in literature. We present the case of a 39-year-old man with glucose- 6-phosphate dehydrogenase deficiency, who reached out to our department for the arising of a non-ischemic priapism without arteriolacunar fistula. We suggest that the glucose-6-phosphate dehydrogenase deficiency could be an underestimated risk factor for priapism.

  2. [Attempt at characterization of 2 erythrocyte variants of glucose-6-phosphate dehydrogenase in a patient with a partial enzymatic deficit].

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    Bansard-Desmidt, N

    1975-09-01

    The electrophoresis shows, in red blood cells of a North African man affected by a glucose-6-phosphate dehydrogenase deficiency, the presence of two enzymes differing by their electrophoretic mobilities: one of them presents in the same mobility as variant Gd (+) B, the other being faster. After partial purification of the enzymes by ionic exchange chromatography on cellex D BIO-RAD, the preparation obtained shows some kinetic abnormalities: an increased value of 2-deoxy-glucose-6-phosphate utilisation and a non linear plot of 1/v versus 1/s, inadequate for Km determination. Assuming that our preparation contains two enzymes differing by their affinities for glucose-6-phosphate, were carried out a study of their Michaelis constants for glucose-6-phosphate by a method based on the densitometric determination of colored spots corresponding to these two variants after electrophoretic separation on cellogel strips. One of these variants is similar to Gd (+) B, the other being characterised by increased values of: electrophoretic mobility (+ 110%), Km for glucose-6-hosphate (194 +/- 38 muM, normal range being 55 to 70 muM), utilisation coefficient of 2-deoxy-glucose-6-phosphate.

  3. Characterization of three putative xylulose 5-phosphate/fructose 6-phosphate phosphoketolases in the cyanobacterium Anabaena sp. PCC 7120.

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    Moriyama, Takashi; Tajima, Naoyuki; Sekine, Kohsuke; Sato, Naoki

    2015-01-01

    Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) is a key enzyme in the central carbohydrate metabolism in heterofermentative bacteria, in which enzymatic property of Xfps is well characterized. This is not the case in other microbes. The cyanobacterium Anabaena sp. PCC 7120 possesses three putative genes encoding Xfp, all1483, all2567, and alr1850. We purified three putative Xfps as recombinant proteins. The results of gel filtration indicated that these proteins form homomultimer complex. All1483 and All2567 showed phosphoketolase activity, whereas Alr1850 did not show the activity. Kinetic analyses demonstrated that substrates, fructose 6-phosphate and inorganic phosphate, are cooperatively bound to enzymes positively and negatively, respectively.

  4. Effects of some drugs on human erythrocyte glucose 6-phosphate dehydrogenase: an in vitro study.

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    Akkemik, Ebru; Budak, Harun; Ciftci, Mehmet

    2010-12-01

    Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of human have been investigated. For this purpose, at the beginning, erythrocyte glucose 6-phosphate dehydrogenase was purified 2256 times in a yield of 44.22% by using ammonium sulphate precipitation and 2', 5'-ADP Sepharose 4B affinity gel. Temperature of +4°C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. Ketotifen, dacarbazine, thiocolchicoside, meloxicam, methotrexate, furosemide, olanzapine, methylprednizolone acetate, paricalcitol, ritodrine hydrochloride, and gadobenate-dimeglumine were used as drugs. All the drugs indicated the inhibitory effects on the enzyme. Ki constants for glucose 6-phosphate dehydrogenase were found by means of Lineweaver-Burk graphs. While methylprednizolone acetate showed competitive inhibition, the others displayed non-competitive inhibition. In addition, IC(50) values of the drugs were determined by plotting Activity% vs [I].

  5. Glucose-6-phosphate dehydrogenase mutations and haplotypes in Mexican Mestizos.

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    Arámbula, E; Aguilar L, J C; Vaca, G

    2000-08-01

    In a screening for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in 1985 unrelated male subjects from the general population (Groups A and B) belonging to four states of the Pacific coast, 21 G-6-PD-deficient subjects were detected. Screening for mutations at the G-6-PD gene by PCR-restriction enzyme in these 21 G-6-PD-deficient subjects as well as in 14 G-6-PD-deficient patients with hemolytic anemia belonging to several states of Mexico showed two common G-6-PD variants: G-6-PD A-(202A/376G) (19 cases) and G-6-PD A-(376G/968C) (9 cases). In 7 individuals the mutations responsible for the enzyme deficiency remain to be determined. Furthermore, four silent polymorphic sites at the G-6-PD gene (PvuII, PstI, 1311, and NlaIII) were investigated in the 28 individuals with G-6-PD A- variants and in 137 G-6-PD normal subjects. As expected, only 10 different haplotypes were observed. To date, in our project aiming to determine the molecular basis of G-6-PD deficiency in Mexico, 60 unrelated G-6-PD-deficient Mexican males-25 in previous studies and 35 in the present work-have been studied. More than 75% of these individuals are from states of the Pacific coast (Sinaloa, Nayarit, Jalisco, Michoacán, Guerrero, Oaxaca, and Chiapas). The results show that although G-6-PD deficiency is heterogeneous at the DNA level in Mexico, only three polymorphic variants have been observed: G-6-PD A-(202A/376G) (36 cases), G-6-PD A-(376G/968C) (13 cases), and G-6-PD Seattle(844C) (2 cases). G-6-PD A- variants are relatively distributed homogeneously and both variants explain 82% of the overall prevalence of G-6-PD deficiency. The variant G-6-PD A-(202A/376G) represents 73% of the G-6-PD A- alleles. Our data also show that the variant G-6-PD A-(376G/968C)-which has been observed in Mexico in the context of two different haplotypes-is more common than previously supposed. The three polymorphic variants that we observed in Mexico are on the same haplotypes as found in subjects from

  6. Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

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    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.

  7. Inactivation of Bakers' yeast glucose-6-phosphate dehydrogenase by aluminum

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Sungwoo; Joshi, J.G. (Univ. of Tennessee, Knoxville (USA))

    1989-04-18

    Preincubation of yeast glucose-6-phosphate dehydrogenase (G6PD) with Al(III) produced an inactive enzyme containing 1 mol of Al(III)/mol of enzyme subunit. None of the enzyme-bound Al(III) was dissociated by dialysis against 10 mM Tris-HCl, pH 7.0, containing 0.2 mM EDTA at 4{degree}C for 24 h. Citrate, NADP{sup +}, EDTA, or NaF protected the enzyme against the Al(III) inactivation. The Al(III)-inactivated enzyme, however, was completely reactivated only by citrate and NaF. The dissociation constant for the enzyme-aluminum complex was calculated to be 4 {times} 10{sup {minus}6} M with NaF, a known reversible chelator for aluminum. Modification of histidine and lysine residues of the enzyme with diethyl pyrocarbonate and acetylsalicylic acid, respectively, inactivated the enzyme. However, the modified enzyme still bound 1 mol of Al(III)/mol of enzyme subunit. Circular dichroism studies showed that the binding of Al(III) to the enzyme induced a decrease in {alpha}-helix and {beta}-sheet and an increase in random coil. Therefore, it is suggested that inactivation of G6PD by Al(III) is due to the conformational change induced by Al(III) binding.

  8. Gold Nanoparticles Decorated with Mannose-6-phosphate Analogues

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    Stéphanie Combemale

    2014-01-01

    Full Text Available Herein, the preparation of neoglycoconjugates bearing mannose-6-phosphate analogues is described by: (a synthesis of a cyclic sulfate precursor to access the carbohydrate head-group by nucleophilic displacement with an appropriate nucleophile; (b introduction of spacers on the mannose-6-phosphate analogues via Huisgen’s cycloaddition, the Julia reaction, or the thiol-ene reaction under ultrasound activation. With the resulting compounds in hand, gold nanoparticles could be functionalized with various carbohydrate derivatives (glycoconjugates and then tested for angiogenic activity. It was observed that the length and flexibility of the spacer separating the sugar analogue from the nanoparticle have little influence on the biological response. One particular nanoparticle system substantially inhibits blood vessel growth in contrast to activation by the corresponding monomeric glycoconjugate, thereby demonstrating the importance of multivalency in angiogenic activity.

  9. Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design.

    Science.gov (United States)

    Wojciechowski, Marek; Milewski, Sławomir; Mazerski, Jan; Borowski, Edward

    2005-01-01

    Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ming V. [Program of Cardiovascular Sciences, Houston, TX 77030 (United States); Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Chen, Weiqin [Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Harmancey, Romain N. [Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip [Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Taegtmeyer, Heinrich [Division of Cardiology, The University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Chan, Lawrence, E-mail: lchan@bcm.tmc.edu [Program of Cardiovascular Sciences, Houston, TX 77030 (United States); Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); St. Luke' s Episcopal Hospital, Houston, TX 77030 (United States)

    2010-05-07

    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.

  11. Glucose-6 phosphate dehydrogenase deficiency and psychotic illness

    Directory of Open Access Journals (Sweden)

    Vijender Singh

    2012-01-01

    Full Text Available Mr. T, a 28-year-old unmarried male, a diagnosed case of Glucose-6 Phosphate Dehydrogenase (G6PD deficiency since childhood, presented with 13 years of psychotic illness and disturbed biological functions. He showed poor response to antipsychotics and mood stabilizers and had three prior admissions to Psychiatry. There was a family history of psychotic illness. The General Physical Examination and Systemic Examination were unremarkable. Mental Status Examination revealed increased psychomotor activity, pressure of speech, euphoric affect, prolixity, delusion of persecution, delusion of grandiosity, delusion of control, thought withdrawal and thought insertion, and second and third person auditory hallucinations, with impaired judgment and insight. A diagnosis of schizophrenia paranoid type, with a differential diagnosis of schizoaffective disorder manic subtype, was made. This case is being reported for its rarity and atypicality of clinical presentation, as well as a course of psychotic illness in the G6PD Deficiency state,with its implications on management.

  12. Conjugated bilirubin in neonates with glucose-6-phosphate dehydrogenase deficiency.

    Science.gov (United States)

    Kaplan, M; Rubaltelli, F F; Hammerman, C; Vilei, M T; Leiter, C; Abramov, A; Muraca, M

    1996-05-01

    We used a system capable of measuring conjugated bilirubin and its monoconjugated and diconjugated fractions in serum to assess bilirubin conjugation in 29 glucose-6-phosphate dehydrogenase (G6PD)-deficient, term, male newborn infants and 35 control subjects; all had serum bilirubin levels > or = 256 mumol/L (15 mg/dI). The median value for diconjugated bilirubin was lower in the G6PD-deficient neonates than in control subjects (0.06 (range 0.00 to 1.84) vs 0.21 (range 0.00 to 1.02) mumol/L, p = 0.006). Diglucuronide was undetectable in 11 (38.9%) of the G6PD-deficient infants versus 3 (8.6%) of the control subjects (p = 0.015). These findings imply a partial defect of bilirubin conjugation not previously demonstrated in G6PD-deficient newborn infants.

  13. A joint analysis of transcriptomic and metabolomic data uncovers enhanced enzyme-metabolite coupling in breast cancer

    Science.gov (United States)

    Auslander, Noam; Yizhak, Keren; Weinstock, Adam; Budhu, Anuradha; Tang, Wei; Wang, Xin Wei; Ambs, Stefan; Ruppin, Eytan

    2016-07-01

    Disrupted regulation of cellular processes is considered one of the hallmarks of cancer. We analyze metabolomic and transcriptomic profiles jointly collected from breast cancer and hepatocellular carcinoma patients to explore the associations between the expression of metabolic enzymes and the levels of the metabolites participating in the reactions they catalyze. Surprisingly, both breast cancer and hepatocellular tumors exhibit an increase in their gene-metabolites associations compared to noncancerous adjacent tissues. Following, we build predictors of metabolite levels from the expression of the enzyme genes catalyzing them. Applying these predictors to a large cohort of breast cancer samples we find that depleted levels of key cancer-related metabolites including glucose, glycine, serine and acetate are significantly associated with improved patient survival. Thus, we show that the levels of a wide range of metabolites in breast cancer can be successfully predicted from the transcriptome, going beyond the limited set of those measured.

  14. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo

    2015-06-01

    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

  15. 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

    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......, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6...... on the growth rates, increasing 59 % in the Δpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed...

  16. INCIDENCE OF ERYTHROCYTE GLUCOSE-6-PHOSPHATE- DEHYDROGENASE DEFICIENCY

    Directory of Open Access Journals (Sweden)

    Sh. Rahbar

    1974-06-01

    The fluorescent spot technique was used for screening and qualitative determination of G-6-PD in erythrocytes. This technique was compared with other methods of G-6-PD enzyme assay and proved to be very reliable. Qualitative enzyme estimation was carried out with spectrophotometer methods. A total of 738 specimens tested and some degree of enzyme deficiencies were detected. In 20 specimens there was a complete enzyme deficiency and in 5 cases the enzyme activity was between 15 to 50 percent of normal subject. The data suggests, the blood bank should be warned of transfusion of enzyme deficient bloods to the patients with fauvism.

  17. Glucose 6-phosphate regulates hepatic glycogenolysis through inactivation of phosphorylase.

    Science.gov (United States)

    Aiston, Susan; Andersen, Birgitte; Agius, Loranne

    2003-06-01

    High glucose concentration suppresses hepatic glycogenolysis by allosteric inhibition and dephosphorylation (inactivation) of phosphorylase-a. The latter effect is attributed to a direct effect of glucose on the conformation of phosphorylase-a. Although glucose-6-phosphate (G6P), like glucose, stimulates dephosphorylation of phosphorylase-a by phosphorylase phosphatase, its physiological role in regulating glycogenolysis in intact hepatocytes has not been tested. We show in this study that metabolic conditions associated with an increase in G6P, including glucokinase overexpression and incubation with octanoate or dihydroxyacetone, cause inactivation of phosphorylase. The latter conditions also inhibit glycogenolysis. The activity of phosphorylase-a correlated inversely with the G6P concentration within the physiological range. The inhibition of glycogenolysis and inactivation of phosphorylase-a caused by 10 mmol/l glucose can be at least in part counteracted by inhibition of glucokinase with 5-thioglucose, which lowers G6P. In conclusion, metabolic conditions that alter the hepatic G6P content affect glycogen metabolism not only through regulation of glycogen synthase but also through regulation of the activation state of phosphorylase. Dysregulation of G6P in diabetes by changes in activity of glucokinase or glucose 6-phosphatase may be a contributing factor to impaired suppression of glycogenolysis by hyperglycemia.

  18. Nitrogen Assimilation, Abiotic Stress and Glucose 6-Phosphate Dehydrogenase: The Full Circle of Reductants.

    Science.gov (United States)

    Esposito, Sergio

    2016-05-11

    Glucose 6 phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is well-known as the main regulatory enzyme of the oxidative pentose phosphate pathway (OPPP) in living organisms. Namely, in Planta, different G6PDH isoforms may occur, generally localized in cytosol and plastids/chloroplasts. These enzymes are differently regulated by distinct mechanisms, still far from being defined in detail. In the last decades, a pivotal function for plant G6PDHs during the assimilation of nitrogen, providing reductants for enzymes involved in nitrate reduction and ammonium assimilation, has been described. More recently, several studies have suggested a main role of G6PDH to counteract different stress conditions, among these salinity and drought, with the involvement of an ABA depending signal. In the last few years, this recognized vision has been greatly widened, due to studies clearly showing the non-conventional subcellular localization of the different G6PDHs, and the peculiar regulation of the different isoforms. The whole body of these considerations suggests a central question: how do the plant cells distribute the reductants coming from G6PDH and balance their equilibrium? This review explores the present knowledge about these mechanisms, in order to propose a scheme of distribution of reductants produced by G6PDH during nitrogen assimilation and stress.

  19. Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

    Science.gov (United States)

    Farelli, Jeremiah D; Galvin, Brendan D; Li, Zhiru; Liu, Chunliang; Aono, Miyuki; Garland, Megan; Hallett, Olivia E; Causey, Thomas B; Ali-Reynolds, Alana; Saltzberg, Daniel J; Carlow, Clotilde K S; Dunaway-Mariano, Debra; Allen, Karen N

    2014-07-01

    Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

  20. Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

    Directory of Open Access Journals (Sweden)

    Jeremiah D Farelli

    2014-07-01

    Full Text Available Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

  1. 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.

  2. Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

    Science.gov (United States)

    Mouilleron, Stéphane; Badet-Denisot, Marie-Ange; Pecqueur, Ludovic; Madiona, Karine; Assrir, Nadine; Badet, Bernard; Golinelli-Pimpaneau, Béatrice

    2012-10-01

    The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.

  3. Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

    Science.gov (United States)

    Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

    2016-04-01

    Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

  4. Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase

    Science.gov (United States)

    Chevreux, Guillaume; Atmanene, Cédric; Lopez, Philippe; Ouazzani, Jamal; Van Dorsselaer, Alain; Badet, Bernard; Badet-Denisot, Marie-Ange; Sanglier-Cianférani, Sarah

    2011-03-01

    Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using 14N versus 15N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.

  5. Structural Diversity Within the Mononuclear and Binuclear Active Sites of N-Acetyl-D-Glucosamine-6-Phosphate Deacetylase

    Energy Technology Data Exchange (ETDEWEB)

    Hall,R.; Brown, S.; Fedorov, A.; Fedorov, E.; Xu, C.; Babbitt, P.; Almo, S.; Raushel, F.

    2007-01-01

    NagA catalyzes the hydrolysis of N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-D-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the {alpha}-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.

  6. NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis.

    Science.gov (United States)

    Abbas, Shahzada Nadeem; Mok, Kenneth Hun; Rashid, Naeem; Xie, Yongjing; Ruether, Manuel; O'Brien, John; Akhtar, Muhammad

    2016-06-01

    The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.

  7. Binding Mode and Selectivity of Steroids towards Glucose-6-phosphate Dehydrogenase from the Pathogen Trypanosoma cruzi.

    Science.gov (United States)

    Ortiz, Cecilia; Moraca, Francesca; Medeiros, Andrea; Botta, Maurizio; Hamilton, Niall; Comini, Marcelo A

    2016-03-17

    Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3β-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme.

  8. Engineering sorbitol-6-phosphate Dehydrogenase encoding gene in the lactose operon of Lactobacillus casei.

    OpenAIRE

    Nissen, Lorenzo; Yebra, María Jesús; Sgobarti, Barbara; Pérez Martínez, Gaspar

    2005-01-01

    Sorbitol is a sugar polyol claimed to have health-promoting properties. D-sorbitol-6-phosphate dehydrogeanse (StolPDh) is required for sorbose and sorbitol metabolism in Lactobacillus casei. StolPDh catalyzes the oxidation of Sorbitol-6-phosphate and also the reverse reaction, or reather, the reduction of fructose-6-phosphate with NAD+ regeneration. In order to test this function in vivo, a new food grade recombinant strain of L. casei was constructed by the integration of a StolPDh-encodi...

  9. A new paper-based analytical device for detection of Glucose-6-phosphate dehydrogenase deficiency.

    Science.gov (United States)

    Kaewarsa, Phuritat; Laiwattanapaisal, Wanida; Palasuwan, Attakorn; Palasuwan, Duangdao

    2017-03-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic haemolytic disorder. Most persons with G6PD deficiency are asymptomatic, but exposure to oxidant drugs, such as the anti-malarial drug primaquine, may induce haemolysis, which is commonly found in Asian countries. A reliable test is necessary for diagnosing the deficiency to prevent an acute haemolytic crisis. This study proposes a novel quantitative method to detect G6PD deficiency using paper-based analytical devices (G6PDD-PAD). Wax printing was utilized for fabricating circular reaction zone patterns in paper. The colorimetric assay is based on the formation of formazan via a reduction of tetra-nitro blue tetrazolium (TNBT) by the G6PD enzyme on G6PDD-PAD. Detection was achieved by capturing the colour using a desktop scanner and the colour intensity was analysed with Adobe Photoshop C56. The results showed that the G6PD activity analysed by G6PDD-PAD was highly correlated with the standard biochemical assay (SBA) (r(2)=0.87, pPAD and the SBA (mean bias 1.4 IU/gHb). The detection limit was 0 IU/gHb of G6PD activity. This study demonstrates the feasibility of using G6PDD-PAD. This simple, low-cost test ($0.1/test) should be useful for diagnosing G6PD deficiency in resource-limited settings.

  10. Glucose-6-phosphate dehydrogenase deficiency in northern Mexico and description of a novel mutation

    Indian Academy of Sciences (India)

    N. García-Magallanes; F. Luque-Ortega; E. M. Aguilar-Medina; R. Ramos-Payán; C. Galaviz-Hernández; J. G. Romero-Quintana; L. Del Pozo-Yauner; H. Rangel-Villalobos; E. Arámbula-Meraz

    2014-08-01

    Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme pathology in humans; it is X-linked inherited and causes neonatal hyperbilirubinaemia, chronic nonspherocytic haemolytic anaemia and drug-induced acute haemolytic anaemia. G6PD deficiency has scarcely been studied in the northern region of Mexico, which is important because of the genetic heterogeneity described in Mexican population. Therefore, samples from the northern Mexico were biochemically screened for G6PD deficiency, and PCR-RFLPs, and DNA sequencing used to identify mutations in positive samples. The frequency of G6PD deficiency in the population was 0.95% ($n = 1993$); the mutations in 86% of these samples were G6PD A-202A/376G, G6PD A-376G/968C and G6PD Santamaria376G/542T. Contrary to previous reports, we demonstrated that G6PD deficiency distribution is relatively homogenous throughout the country $(P = 0.48336)$, and the unique exception with high frequency of G6PD deficiency does not involve a coastal population (Chihuahua: 2.4%). Analysis of eight polymorphic sites showed only 10 haplotypes. In one individual we identified a new G6PD mutation named Mexico DF193A>G (rs199474830), which probably results in a damaging functional effect, according to PolyPhen analysis. Proteomic impact of the mutation is also described.

  11. Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency in south-east Sicily.

    Science.gov (United States)

    Cittadella, R; Civitelli, D; Manna, I; Azzia, N; Di Cataldo, A; Schilirò, G; Brancati, C

    1997-05-01

    In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in south-east Sicily, we have analysed the G6PD gene in 25 unrelated males with abnormal G6PD activity and/or electrophoretic mobility, by using the analysis of the appropriate PCR-amplified fragment of DNA and subsequent digestion by appropriate restriction-enzymes, looking for the presence of certain known G6PD mutations. We amplified the entire G6PD coding sequence into eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis and sequencing of those individual fragments that were found to be abnormal by SSCP. Through these methods we found a total of twelve G6PD Mediterranean variants with the association of a silent mutation 1311 (also known as polymorphic site Bcl I), one G6PD Mediterranean without this association, four G6PD A-Val 68 and two G6PD Santamaria and five G6PD Chatham. In a subject with normal activity a mutation was found in exon 5, designated as G6PD Sao Borja. This is the first report on the molecular analysis of G6PD mutations in Sicily and we have obtained evidence for four distinct classes of variants.

  12. 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

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

    Directory of Open Access Journals (Sweden)

    Saúl Gómez-Manzo

    2016-12-01

    Full Text Available 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.

  14. Isolation and characterization of the trehalose-6-phosphate synthase gene from Locusta migratoria manflensis

    Institute of Scientific and Technical Information of China (English)

    Shu-Yan Cui; Yu-Xian Xia

    2009-01-01

    Trehalose plays an important role in protecting organisms from various stresses.Trehalose-6-phosphate synthase (TPS) is the key enzyme in trehalose synthesis, but in in-sects only a few TPS genes have been identified and their function has not been well characterized. To better understand the function of TPS in insects, a complete TPS com-plementary DNA (eDNA) clone was obtained from the fat body of the locust Locusta migratoria manilensis (GenBank accession number: EU 131894). The full-length cDNA is 2 806 bp, including an open reading frame of 2 442 bp, which encodes an 813 amino acids protein with a calculated molecular weight of 91 976 Daltons and an isoelectric point of 6.14. The deduced amino acid sequence is highly similar to other published insect TPS and its C-terminal also has a region homologous to trehalose phosphate phsophatase (TPP).Semi-quantitative analysis indicated that the TPS transcript was expressed not only in fat body, but also in gut, hemolymph and leg muscle. These data may facilitate studies of TPS function in insects and improve our understanding of trehalose metabolism.

  15. Glucose-6-Phosphate Dehydrogenase deficiency presented with convulsion: a rare case

    Directory of Open Access Journals (Sweden)

    Alparslan Merdin

    2014-03-01

    Full Text Available Red blood cells carry oxygen in the body and Glucose-6-Phosphate Dehydrogenase protects these cells from oxidative chemicals. If there is a lack of Glucose-6-Phosphate Dehydrogenase, red blood cells can go acute hemolysis. Convulsion is a rare presentation for acute hemolysis due to Glucose-6-Phosphate Dehydrogenase deficiency. Herein, we report a case report of a Glucose-6-Phosphate Dehydrogenase deficiency diagnosed patient after presentation with convulsion. A 70 year-old woman patient had been hospitalized because of convulsion and fatigue. She has not had similar symptoms before. She had ingested fava beans in the last two days. Her hypophyseal and brain magnetic resonance imaging were normal. Blood transfusion was performed and the patient recovered.

  16. Glucose-6-phosphate dehydrogenase deficiency in Tunisia: molecular data and phenotype-genotype association.

    Science.gov (United States)

    Laouini, N; Bibi, A; Ammar, H; Kazdaghli, K; Ouali, F; Othmani, R; Amdouni, S; Haloui, S; Sahli, C A; Jouini, L; Hadj Fredj, S; Siala, H; Ben Romdhane, N; Toumi, N E; Fattoum, S; Messsaoud, T

    2013-02-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. In this study, we aimed to perform a molecular investigation of G6PD deficiency in Tunisia and to associate clinical manifestations and the degree of deficiency with the genotype. A total of 161 Tunisian subjects of both sexes were screened by spectrophotometric assay for enzyme activity. Out of these, 54 unrelated subjects were selected for screening of the most frequent mutations in Tunisia by PCR/RFLP, followed by size-based separation of double-stranded fragments under non-denaturing conditions on a denaturing high performance liquid chromatography system. Of the 56 altered chromosomes examined, 75 % had the GdA(-) mutation, 14.28 % showed the GdB(-) mutation and no mutations were identified in 10.72 % of cases. Hemizygous males with GdA(-) mutation were mostly of class III, while those with GdB(-) mutation were mainly of class II. The principal clinical manifestation encountered was favism. Acute hemolytic crises induced by drugs or infections and neonatal jaundice were also noted. Less severe clinical features such as low back pain were present in heterozygous females and in one homozygous female. Asymptomatic individuals were in majority heterozygote females and strangely one hemizygous male. The spectrum of mutations seems to be homogeneous and similar to that of Mediterranean countries; nevertheless 10.72 % of cases remain with undetermined mutation thus suggesting a potential heterogeneity of the deficiency at the molecular level. On the other hand, we note a better association of the molecular defects with the severity of the deficiency than with clinical manifestations.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Glucose-6-Phosphate Dehydrogenase Regulation in Anoxia Tolerance of the Freshwater Crayfish Orconectes virilis

    Directory of Open Access Journals (Sweden)

    Benjamin Lant

    2011-01-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PDH, the enzyme which catalyzes the rate determining step of the pentose phosphate pathway (PPP, controls the production of nucleotide precursor molecules (R5P and powerful reducing molecules (NADPH that support multiple biosynthetic functions, including antioxidant defense. G6PDH from hepatopancreas of the freshwater crayfish (Orconectes virilis showed distinct kinetic changes in response to 20 h anoxic exposure. Km values for both substrates decreased significantly in anoxic crayfish; Km NADP+ dropped from 0.015±0.008 mM to 0.012±0.008 mM, and Km G6P decreased from 0.13±0.02 mM to 0.08±0.007 mM. Two lines of evidence indicate that the mechanism involved is reversible phosphorylation. In vitro incubations that stimulated protein kinase or protein phosphatase action mimicked the effects on anoxia on Km values, whereas DEAE-Sephadex chromatography showed the presence of two enzyme forms (low- and high-phosphate whose proportions changed during anoxia. Incubation studies implicated protein kinase A and G in mediating the anoxia-responsive changes in G6PDH kinetic properties. In addition, the amount of G6PDH protein (measured by immunoblotting increased by ∼60% in anoxic hepatopancreas. Anoxia-induced phosphorylation of G6PDH could contribute to modifying carbon flow through the PPP under anoxic conditions, potentially maintaining NADPH supply for antioxidant defense during prolonged anoxia-induced hypometabolism.

  19. Cloning and Characterization of a Salt Tolerance-Associated Gene Encoding Trehalose-6-Phosphate Synthase in Sweetpotato

    Institute of Scientific and Technical Information of China (English)

    JIANG Tao; ZHAI Hong; WANG Fei-bing; ZHOU Hua-nan; SI Zeng-zhi; HE Shao-zhen; LIU Qing-chang

    2014-01-01

    Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes:trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). In the present study, a TPS gene, named IbTPS, was ifrst isolated from sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lushu 3 by rapid ampliifcation of cDNA ends (RACE). The open reading frame (ORF) contained 2 580 nucleotides encoding 859 amino acids with a molecular weight of 97.433 kDa and an isoelectric point (pI) of 5.7. The deduced amino acid sequence showed high identities with TPS of other plants. Real-time quantitative PCR analysis revealed that the expression level of IbTPS gene was signiifcantly higher in stems of Lushu 3 than in its leaves and roots. Subcellular localization analysis in onion epidermal cells indicated that IbTPS gene was located in the nucleus. Transgenic tobacco (cv. Wisconsin 38) plants over-expressing IbTPS gene exhibited signiifcantly higher salt tolerance compared with the control plant. Trehalose and proline content was found to be signiifcantly more accumulated in transgenic tobacco plants than in the wild-type and several stress tolerance related genes were up-regulated. These results suggest that IbTPS gene may enhance salt tolerance of plants by increasing the amount of treahalose and proline and regulating the expression of stress tolerance related genes.

  20. 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

  1. Evaluation of the blue formazan spot test for screening glucose 6 phosphate dehydrogenase deficiency.

    Science.gov (United States)

    Pujades, A; Lewis, M; Salvati, A M; Miwa, S; Fujii, H; Zarza, R; Alvarez, R; Rull, E; Corrons, J L

    1999-06-01

    Several screening tests for glucose 6 phosphate dehydrogenase (G6PD) deficiency have been reported thus far, and a standardized method of testing was proposed by the International Council for Standardization in Hematology (ICSH). The screening test used in any particular laboratory depends upon a number of factors such as cost, time required, temperature, humidity, and availability of reagents. In this study, a direct comparison between three different G6PD screening methods has been undertaken. In 71 cases (50 hematologically normal volunteers, 9 hemizygous G6PD-deficient males, and 12 heterozygous deficient females), the blue formazan spot test (BFST) was compared with the conventional methemoglobin reduction test (HiRT) and the ICSH-recommended fluorescent spot test (FST-ICSH). In all cases, the results obtained with the three screening tests were correlated with the enzyme activity assayed spectrophotometrically. In hemizygous G6PD-deficient males, all cases were equally detected with the three methods: BFST (4.7-6.64, controls: 11.1-13.4), BMRT (score +3 in all 9 cases), and FST (no fluorescence in 9 cases). In heterozygous G6PD-deficient females, two methods detected 7 out of 12 cases (BFST: 8.71-11.75, controls: 11.1-13.4; and BMRT: score +3 in 7 cases), whereas the FST-ICSH missed all 12 cases that presented a variable degree of fluorescence. Although the sensitivity for G6PD-deficient carrier detection is the same for the BMRT and the BFST, the latter has the advantage of being semiquantitative and not merely qualitative. Unfortunately, none of the three screening tests compared here allowed the detection of the 100% heterozygote carrier state of G6PD deficiency.

  2. Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Camel Liver

    Directory of Open Access Journals (Sweden)

    Mahmoud A. Ibrahim

    2014-01-01

    Full Text Available Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The Km value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with Ki value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver.

  3. Glutathione metabolism and glucose 6-phosphate dehydrogenase activity in experimental liver injury.

    Directory of Open Access Journals (Sweden)

    Watanabe,Akiharu

    1983-12-01

    Full Text Available Increased activities of liver glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49 and 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.44 in the pentose phosphate cycle were accompanied with a depletion of reduced glutathione (GSH following an intragastric administration of carbon tetrachloride (CCl4 to rats. Oxidized glutathione (GSSG also decreased remarkably, keeping the GSSG: GSH ratio constant. No significant alteration of glutathione reductase (EC 1.6.4.2., glutathione peroxidase (EC 1.11.1.9 and malic enzyme (EC 1.1.1.40 activities in the supernatant and gamma-glutamyl transpeptidase (gamma-GTP, EC 2.3.2.2 activity in the homogenate of the injured liver were observed. Furthermore, no marked difference in the GSH-synthesizing activity was found between control and CCl4-intoxicated liver. An intraperitoneal injection of GSH produced a significant increase in liver GSH content in control rats but not in CCl4-treated rats; G6PD activity was not affected. Intraperitoneal injections of diethylmaleate resulted in continuously diminished levels of liver GSH without any alteration of liver G6PD activity. In vitro disappearance of GSH added to the liver homogenate from CCl4-treated rats occurred enzymatically and could not be prevented by the addition of a NADPH-generating system. The results suggest that increased G6PD activity in CCl4-injured liver does not play an important role in the maintenance of glutathione in the reduced form and that the decreased GSH content in the injured liver might be caused by enhanced GSH catabolism not due to gamma-GTP.

  4. Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

    2014-08-01

    Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.

  5. Cloning and characterization of a glucose 6-phosphate/phosphate translocator from Oryza sativa

    Institute of Scientific and Technical Information of China (English)

    姜华武; 佃蔚敏; 刘非燕; 吴平

    2003-01-01

    Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy, and glucose 6-phosphate is the preferred hexose phosphate taken up by nongreen plastids. A cDNA clone encoding glucose 6-phosphate/phosphate translocator (GPT) was isolated from a cDNA library of immature seeds of rice and named as OsGPT. The cDNA has one uninterrupted open reading frame encoding a 42 kDa polypeptide possessing transit peptide consisting of 70 amino acid residues. The OsGPT gene maps on chromosome 8 of rice and is linked to the quantitative trait locus for 1000-grain weight. The expression of OsGPT is mainly restricted to heterotrophic tissues. These results suggest that glucose 6-phosphate imported via GPT can be used for starch biosynthesis in rice nongreen plastids.

  6. 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.

  7. Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production.

    Science.gov (United States)

    Maleki, Susan; Mærk, Mali; Valla, Svein; Ertesvåg, Helga

    2015-05-15

    The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.

  8. False-Positive Newborn Screen Using the Beutler Spot Assay for Galactosemia in Glucose-6-Phosphate Dehydrogenase Deficiency.

    Science.gov (United States)

    Stuhrman, Grace; Perez Juanazo, Stefanie J; Crivelly, Kea; Smith, Jennifer; Andersson, Hans; Morava, Eva

    2017-01-12

    Classical galactosemia is detected through newborn screening by measuring galactose-1-phosphate uridylyltransferase (GALT) in the USA primarily via the Beutler spot assay. We report on an 18-month-old patient with glucose-6-phosphate dehydrogenase (G6PD) deficiency that was originally diagnosed with classical galactosemia. The patient presented with elevated liver function enzymes and bilirubinemia and was immediately treated with soy-based formula. Confirmatory tests revealed deficiency of the GALT enzyme, however, full-sequencing of GALT was normal, suggestive of a different ideology. The Beutler spot assay uses three other enzymatic steps in addition to GALT. A deficiency in either of these enzymes can result in suspected decreased GALT activity when using the Beutler assay. Congenital Disorders of Glycosylation screening for phosphoglucomutase-1 deficiency was negative. Quantitative analysis of G6PD enzyme in red blood cells showed a severe deficiency and a deletion in G6PD. Soy-formula, the standard treatment for galactosemia, has been reported to trigger hemolysis in G6PD deficient patients. G6PD and phosphoglucomutase-1 deficiencies should be considered when confirmatory tests are negative for pathogenic variants in GALT and galactose-1-phosphate level is normal.

  9. 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

  10. 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...

  11. L-Ribose isomerase and mannose-6-phosphate isomerase: properties and applications for L-ribose production.

    Science.gov (United States)

    Xu, Zheng; Sha, Yuanyuan; Liu, Chao; Li, Sha; Liang, Jinfeng; Zhou, Jiahai; Xu, Hong

    2016-11-01

    L-Ribose is a synthetic L-form monosaccharide. It is a building block of many novel nucleotide analog anti-viral drugs. Bio-production of L-ribose relies on a two-step reaction: (i) conversion of L-arabinose to L-ribulose by the catalytic action of L-arabinose isomerase (L-AI) and (ii) conversion of L-ribulose to L-ribose by the catalytic action of L-ribose isomerase (L-RI, EC 5.3.1.B3) or mannose-6-phosphate isomerase (MPI, EC 5.3.1.8, alternately named as phosphomannose isomerase). Between the two enzymes, L-RI is a rare enzyme that was discovered in 1996 by Professor Izumori's group, whereas MPI is an essential enzyme in metabolic pathways in humans and microorganisms. Recent studies have focused on their potentials for industrial production of L-ribose. This review summarizes the applications of L-RI and MPI for L-ribose production.

  12. Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length.

    Science.gov (United States)

    Brockhausen, Inka; Nair, Dileep G; Chen, Min; Yang, Xiaojing; Allingham, John S; Szarek, Walter A; Anastassiades, Tassos

    2016-04-01

    Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

  13. Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation.

    Directory of Open Access Journals (Sweden)

    Boris Hespeels

    Full Text Available The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS and seven putative trehalase (TRE gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process.

  14. N-acetylglucosamine 6-phosphate deacetylase (nagA is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus.

    Directory of Open Access Journals (Sweden)

    Vikas Yadav

    Full Text Available Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum. For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r; named as ΔnagA via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.

  15. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  16. 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

  17. 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}.

  18. Glucose-6-phosphate Reduces Calcium Accumulation in Rat Brain Endoplasmic Reticulum

    Science.gov (United States)

    2012-04-01

    low millimolar range. Most Ca2+ is sequestered within organelles , including the endoplasmic reticulum (ER), Golgi, mitochondria , and nucleus (Carafoli...G6P and thapsigargin caused generalized reduction in Ca2+ accumulation in remarkably similar patterns with no apparent gray matter regional...with glucose-6-phosphate (10 mM) or thapsigargin (1 µM), revealed very similar pattern of generalized reduction in 45Ca2+ accumulation in gray and

  19. Identification of the trehalose-6-phosphate synthase gene family in winter wheat and expression analysis under conditions of freezing stress

    Indian Academy of Sciences (India)

    D. W. Xie; X. N. Wang; L. S. Fu; J. Sun; W. Zheng; Z. F. Li

    2015-03-01

    Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in plants. Trehalose contents are potentially modulated by trehalose-6-phosphate synthase (TPS), which is a key enzyme in the trehalose biosynthetic pathway. Using available wheat expressed sequence tag sequence information from NCBI and two wheat genome databases, we identified 12 wheat TPS genes and performed a comprehensive study on their structural, evolutionary and functional properties. The estimated divergence time of wheat TPS gene pairs and wheat–rice orthologues suggested that wheat and rice have a common ancestor. The number of TPS genes in the wheat genome was estimated to be at least 12, which is close to the number found in rice, Arabidopsis and soybean. Moreover, it has been reported earlier in other plants that TPS genes respond to abiotic stress, however, our study mainly analysed the TPS gene family under freezing conditions in winter wheat, and determined that most of the TPS gene expression in winter wheat was induced by freezing conditions, which further suggested that wheat TPS genes were involved in winter wheat freeze-resistance signal transduction pathways. Taken together, the current study represents the first comprehensive study of TPS genes in winter wheat and provides a foundation for future functional studies of this important gene family in Triticeae.

  20. Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana.

    Science.gov (United States)

    Riegler, Heike; Herter, Thomas; Grishkovskaya, Irina; Lude, Anja; Ryngajllo, Malgorzata; Bolger, Marie E; Essigmann, Bernd; Usadel, Björn

    2012-04-15

    GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30-40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.

  1. 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.

  2. The tertiary origin of the allosteric activation of E. coli glucosamine-6-phosphate deaminase studied by sol-gel nanoencapsulation of its T conformer.

    Directory of Open Access Journals (Sweden)

    Sergio Zonszein

    Full Text Available The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P deaminase from Escherichia coli (EcGNPDA as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P. We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the

  3. Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1

    Directory of Open Access Journals (Sweden)

    Qiushi Chen

    2015-10-01

    Full Text Available Glutamine-fructose-6-phosphate transaminase 1 (GFPT1 is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A, and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.

  4. A novel c.197T ® A variant among Brazilian neonates with glucose-6-phosphate dehydrogenase deficiency

    Directory of Open Access Journals (Sweden)

    José Pereira de Moura Neto

    2008-01-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49 deficiency is the most common enzyme deficiency worldwide, causing a spectrum of diseases including neonatal hyperbilirubinemia and acute or chronic hemolysis. We used the methemoglobin reduction test and G6PD electrophoresis to screen 655 neonates (354 females and 301 males for common G6PD mutations in the city of Salvador in the Northeastern Brazilian state Bahia and found that 66 (10.1% were G6PD-deficient (41 females and 25 males. The 66 (10.1% G6PD-deficient neonates were assessed for the c.376 A -> G (exon 5 and c.202 G -> A (exon 4 mutations using the polymerase chain reaction and restriction enzyme fragment length polymorphism (PCR-RFLP analysis and the results validated by DNA sequencing. Of the 66 G6PD-deficient neonates investigated we found that 54 (81.8% presented the c.376 A -> G (p.Asn126Asp and c.202 G -> A (p.Val68Met mutations, two (3% had the c.376 A -> G mutation only, two (3% had the c.202 G -> A mutation only, five (7.6% exhibited a previously unrecorded 197T -> A (p.Phe66Thr substitution in exon 4 and three showed no mutations at any of these sites. Of the five neonates exhibiting the new 197T -> A (p.Phe66Thr substitution, four (6.1% also presented the c.202 G -> A and c.376 A -> G mutations and one (1.5% had the c.[197T -> A / 202 G -> A] combination. We propose to name the new variant G6PD Bahia.

  5. Signal amplification of glucosamine-6-phosphate based on ribozyme glmS.

    Science.gov (United States)

    Zhao, Yongyun; Chen, Haodong; Du, Feng; Yasmeen, Afshan; Dong, Juan; Cui, Xin; Tang, Zhuo

    2014-12-15

    Ribozyme glmS based isothermal amplification assay is developed for the colorimetric detection of glucosamine-6-phosphate (GlcN6P). Upon binding to the metabolite target GlcN6P, self-cleavage of glmS ribozyme is initiated to release RNA fragment that can trigger the cascade signal amplification to release large amount of G-quadruplex DNAzymes as reporter for colorimetric detection. Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. This assay not only offers a convenient detection of GlcN6P with high specificity and sensitivity, but also provides a platform for high-throughput screening of antibiotics based on glmS riboswitches.

  6. Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase.

    Science.gov (United States)

    Cardi, Manuela; Chibani, Kamel; Cafasso, Donata; Rouhier, Nicolas; Jacquot, Jean-Pierre; Esposito, Sergio

    2011-07-01

    Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants.

  7. [The role glucose-6-phosphate dehydrogenase in pathogenesis of anemia in leptospirosis].

    Science.gov (United States)

    Avdeeva, M G; Moĭsova, D L; Gorodin, V N; Kostomarov, A M; Zotov, S V; Cherniavskaia, O V

    2002-01-01

    The activity of glucose-6-phosphate dehydrogenase (G-6-PDG) of plasma and erythrocytes, levels of erythrocytes, hemoglobin, free hemoglobin and reticulocytes in different periods of illness were assessed in 30 men with severe icteric leptospirosis and anemia. Elevation of plasma G-6-PDG activity, levels of free hemoglobin (FH) and reticulocytes were found. Hemolysis was more pronounced at the height of the disease. One of the causes of hemolysis in severe leptospiral jaundice may be deficiency of erythrocytic G-6-PDG which is inheritable male disease. Low G-6-PDG activity of erythrocytes on the second week of the illness preceded anemia in 83.3% cases which could be a prognostic criterion of developing anemic syndrome. Combined treatment of leptospirosis with administration of tocopherol acetate resulted in a significant fall of plasma FH and G-6-PDG, of severity and duration of anemia versus the control group.

  8. Cloning and expression of trehalose-6-phosphate synthase 1 from Rhizopus oryzae.

    Science.gov (United States)

    Ozer Uyar, Ebru; Yücel, Meral; Hamamcı, Haluk

    2016-05-01

    Trehalose is a reducing disaccharide acting as a protectant against environmental stresses in many organisms. In fungi, Trehalose-6-phosphate synthase 1 (TPS1) plays a key role in the biosynthesis of trehalose. In this study, a full-length cDNA from Rhizopus oryzae encoding TPS1 (designated as RoTPS1) was isolated. The RoTPS1 cDNA is composed of 2505 nucleotides and encodes a protein of 834 amino acids with a molecular mass of 97.8 kDa. The amino acid sequence of RoTPS1 has a relatively high homology with the TPS1s in several other filamentous fungi. RoTPS1 was cloned into Saccharomyces cerevisiae and secretively expressed.

  9. Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch.

    Science.gov (United States)

    Fei, Xiang; Holmes, Thomas; Diddle, Julianna; Hintz, Lauren; Delaney, Dan; Stock, Alex; Renner, Danielle; McDevitt, Molly; Berkowitz, David B; Soukup, Juliane K

    2014-12-19

    The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereus glmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

  10. 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

  11. Glucosamine: fructose-6-phosphate amidotransferase in the white shrimp Litopenaeus vannamei: characterization and regulation under alkaline and cadmium stress.

    Science.gov (United States)

    Liu, Y; Cai, D X; Wang, L; Li, J Z; Wang, W N

    2015-10-01

    Heavy metal residues and chemical contaminators considered as relevant sources of aquatic environmental pollutants have a generally immunosuppressive effect on aquatic organisms, depressing metabolic activities and immune response. Glutamine: fructose-6-phosphate aminotransferase (GFAT, EC2.6.1.16) is the first, and rate-limiting, enzyme in the hexosamine biosynthetic pathway, and is involved in the regulation of chitin biosynthesis and glycosylation of proteins. We have isolated and characterized GFAT from the white shrimp Litopenaeus vannamei. Amino acid sequence similarity of the Lv-GFAT (L.vannamei-GFAT) was highest to GFATs isolated from insects and mammals (83 % similarity to that of Haemaphysalis longicornis). The open-reading frame of the Lv-GFAT codes for a protein of 41.6 kDa with a calculated isoelectric point of 5.03. RT-PCR assays showed that endogenous Lv-GFAT mRNA is most strongly expressed in the intestine. Further analysis of Lv-GFAT gene expression in hepatopancreas by quantitative real-time PCR demonstrated that Lv-GFAT transcript levels increased when the shrimp were exposed to alkaline pH (9.3) and cadmium stress, but the time when its mRNA expression level peaked differed under these stresses. We also first expressed the recombinant protein of GFAT from shrimps in Escherichia coli. Western blot analyses confirmed that the Lv-GFAT protein was strongly expressed in the hepatopancreas after exposure to the LC-Cd stress. These results suggest that Lv-GFAT expression is stimulated by alkaline pH and cadmium stress and that it may play important roles in resistance of shrimp to environmental stresses.

  12. Glucose-6-phosphate dehydrogenase (G6PD-deficient epithelial cells are less tolerant to infection by Staphylococcus aureus.

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    Yi-Ting Hsieh

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is a key enzyme in the pentose phosphate pathway and provides reducing energy to all cells by maintaining redox balance. The most common clinical manifestations in patients with G6PD deficiency are neonatal jaundice and acute hemolytic anemia. The effects of microbial infection in patients with G6PD deficiency primarily relate to the hemolytic anemia caused by Plasmodium or viral infections and the subsequent medication that is required. We are interested in studying the impact of bacterial infection in G6PD-deficient cells. G6PD knock down A549 lung carcinoma cells, together with the common pathogen Staphylococcus aureus, were employed in our cell infection model. Here, we demonstrate that a lower cell viability was observed among G6PD-deficient cells when compared to scramble controls upon bacterial infection using the MTT assay. A significant increase in the intracellular ROS was detected among S. aureus-infected G6PD-deficient cells by observing dichlorofluorescein (DCF intensity within cells under a fluorescence microscope and quantifying this signal using flow cytometry. The impairment of ROS removal is predicted to enhance apoptotic activity in G6PD-deficient cells, and this enhanced apoptosis was observed by annexin V/PI staining under a confocal fluorescence microscope and quantified by flow cytometry. A higher expression level of the intrinsic apoptotic initiator caspase-9, as well as the downstream effector caspase-3, was detected by Western blotting analysis of G6PD-deficient cells following bacterial infection. In conclusion, we propose that bacterial infection, perhaps the secreted S. aureus α-hemolysin in this case, promotes the accumulation of intracellular ROS in G6PD-deficient cells. This would trigger a stronger apoptotic activity through the intrinsic pathway thereby reducing cell viability when compared to wild type cells.

  13. Expression of glutamine:fructose-6-phosphate amidotransferase in human tissues: evidence for high variability and distinct regulation in diabetes.

    Science.gov (United States)

    Nerlich, A G; Sauer, U; Kolm-Litty, V; Wagner, E; Koch, M; Schleicher, E D

    1998-02-01

    Recent in vitro and in vivo studies suggested that the increased flux of glucose through the hexosamine biosynthetic pathway may contribute to glucose-induced insulin resistance and to the induction of the synthesis of growth factors. Because glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first and rate-limiting step in the formation of hexosamine products, this enzyme is the key regulator in this pathway and is therefore possibly also involved in the alterations occurring in preclinical or manifest diabetic patients. To study the expression of GFAT in human tissues, we produced and characterized a peptic antiserum specifically recognizing GFAT protein and a riboprobe for the detection of GFAT mRNA. Immunohistochemical and nonradioactive in situ hybridization analysis revealed high levels of expression of GFAT protein and mRNA in adipocytes and skeletal muscle. Furthermore, a marked GFAT expression was found in vascular smooth muscle cells with unexpectedly high variability and lower levels in other cells, e.g., peripheral nerve sheath cells or endocrine-active cells, including the pancreatic islet cell. GFAT protein expression was below detection level in endothelium, osteocytes, lymphocytes, granulocytes, and in most quiescent fibroblasts. In renal tissue, GFAT was expressed in tubular epithelial cells, while glomerular cells remained essentially unstained. Renal sections obtained from patients with diabetic nephropathy showed significant GFAT expression in some glomerular epithelial and mesangial cells, indicating that GFAT expression may be induced by manifest diabetes. Our data indicate that GFAT is expressed in most tissues involved in the development of diabetic late complications. Furthermore, the results suggest that GFAT gene expression is highly regulated.

  14. Glucose-6-phosphate dehydrogenase regulation in the hepatopancreas of the anoxia-tolerant marine mollusc, Littorina littorea.

    Science.gov (United States)

    Lama, Judeh L; Bell, Ryan A V; Storey, Kenneth B

    2013-01-01

    Glucose-6-phosphate dehydrogenase (G6PDH) gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions. Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8) showed a 38% decrease in K m G6P and enhanced inhibition by urea, whereas in pH 6 assays K m NADP and maximal activity changed significantly between the two states. The mechanism underlying anoxia-responsive changes in enzyme properties proved to be a change in the phosphorylation state of G6PDH. This was documented with immunoblotting using an anti-phosphoserine antibody, in vitro incubations that stimulated endogenous protein kinases versus protein phosphatases and significantly changed K m G6P, and phosphorylation of the enzyme with (32)P-ATP. All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG) and protein phosphatases (PP1 or PP2C). Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the transition

  15. Glucose-6-phosphate dehydrogenase regulation in the hepatopancreas of the anoxia-tolerant marine mollusc, Littorina littorea

    Directory of Open Access Journals (Sweden)

    Judeh L. Lama

    2013-02-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PDH gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions. Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8 showed a 38% decrease in Km G6P and enhanced inhibition by urea, whereas in pH 6 assays Km NADP and maximal activity changed significantly between the two states. The mechanism underlying anoxia-responsive changes in enzyme properties proved to be a change in the phosphorylation state of G6PDH. This was documented with immunoblotting using an anti-phosphoserine antibody, in vitro incubations that stimulated endogenous protein kinases versus protein phosphatases and significantly changed Km G6P, and phosphorylation of the enzyme with 32P-ATP. All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG and protein phosphatases (PP1 or PP2C. Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the

  16. A novel R198H mutation in the glucose-6-phosphate dehydrogenase gene in the tribal groups of the Nilgiris in Southern India.

    Science.gov (United States)

    Chalvam, R; Kedar, P S; Colah, R B; Ghosh, K; Mukherjee, M B

    2008-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common red cell enzymopathy among humans. In India, G6PD Mediterranean, G6PD Orissa, and G6PD Kerala-Kalyan are the three common mutations which account almost 90% of G6PD deficiency. Here we describe G6PD Coimbra, an unreported variant from India, and a novel 593 G --> A mutation in exon 6 with an amino acid change of Arg 198 His, among the tribal groups of the Nilgiris in Southern India. Further, this novel mutation was structurally characterized and it was found that the mutation is located at the end of the coenzyme domain, which may cause enzyme instability.

  17. Glucose-6-phosphate dehydrogenase Guadalajara--a case of chronic non-spherocytic haemolytic anaemia responding to splenectomy and the role of splenectomy in this disorder.

    Science.gov (United States)

    Hamilton, J W; Jones, F G C; McMullin, Mary Frances

    2004-08-01

    Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme of the pentose phosphate shunt pathway a major function of which is to prevent cellular oxidative damage. Deficiency in red blood cells is associated with a number of varied clinical manifestations. Chronic non-spherocytic haemolytic anaemia is uncommon but is usually characterized by chronic haemolysis, often with severe anaemia. In the past splenectomy in this condition has been thought to be of questionable benefit. We report a case of G6PD Guadalajara where splenectomy produced transfusion independence and have reviewed the literature. Those cases with exon 10 mutations often have a severe clinical phenotype, which responds to splenectomy. This procedure should be considered in this condition.

  18. Marked differences in drug-induced methemoglobinemia in sheep are not due to RBC glucose-6-phosphate dehydrogenase, reduced glutathione, or methemoglobin reductase activity

    Energy Technology Data Exchange (ETDEWEB)

    Martin, D.G.; Guertler, A.T.; Lagutchik, M.S.; Woodard, C.L.; Leonard, D.A.

    1993-05-13

    Benzocaine is a commonly used topical anesthetic that is structurally similar to current candidates for cyanide prophylaxis. Benzocaine induces profound methemoglobinemia in some sheep but not others. After topical benzocaine administration certain sheep respond to form MHb (elevated MHb 16-50% after a 56-280 mg dose, a 2-10 second spray with benzocine), while other phenotypically similar sheep fail to significantly form MHb (less than a 2% increase from baseline). Deficiencies in Glucose-6-phosphate dehydrogenase (G-6-PD), reduced glutathione (GSH), and MHb reductase increase the susceptibility to methemoglobinemia in man and animals. Sheep are used as a model for G-6-PD deficiency in man, and differences in this enzyme level could cause the variable response seen in these sheep. Similarly, differences in GSH and MHb reductase could be responsible for the observed differences in MHb formation.

  19. Glucose-6-phosphate dehydrogenase (G6PD. Response of the human erythrocyte and another cells to the decrease in their activity.

    Directory of Open Access Journals (Sweden)

    Javier Fernando Bonilla

    2009-11-01

    Full Text Available Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway and the main intracellular source of reduced nicotidamineadenine nucleotidephosphate (NADPH, involved in diverse physiological processes such as antioxidant defense, (for instance in the erythrocyte endothelial growth modulation, erithropoyesis, vascularization and phagocitosis. G6PDH deficiency is the most common X-chromosome-linked enzymopathy in human beings. Although it is present in any type cell, its absolute deficiency is incompatible with life. According to WHO, 400 million people are affected by G6PD deficiency in the world but in Colombia, the severe form prevalence is about 3% to 7%. There are no data related to slight and moderate alterations, that also have clinical effects. This paper reviews some G6PD biomolecular aspects, its classification according to activity and electrophoretic mobility, as well as some main clinical aspects related to its activity alteration.

  20. Discovery and characterization of an F420-dependent glucose-6-phosphate dehydrogenase (Rh-FGD1) from Rhodococcus jostii RHA1.

    Science.gov (United States)

    Nguyen, Quoc-Thai; Trinco, Gianluca; Binda, Claudia; Mattevi, Andrea; Fraaije, Marco W

    2017-04-01

    Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation.

  1. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†

    Science.gov (United States)

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-01-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

  2. Uncovering the Math Curriculum

    Science.gov (United States)

    Burns, Marilyn

    2014-01-01

    Teachers often express to Marulyn Burns their worry about the need to "cover the curriculum." In response, she draws on one of her favorite quotes: "You don't want to cover a subject; you want to uncover it." This quote is from "The Having of Wonderful Ideas and Other Essays on Teaching and Learning" by Eleanor…

  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

    2014-08-01

    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.

  4. Splenic artery pseudoaneurysm due to seatbelt injury in a glucose-6-phosphate dehydrogenase-deficient adult.

    Science.gov (United States)

    Lau, Yu Zhen; Lau, Yuk Fai; Lai, Kang Yiu; Lau, Chu Pak

    2013-11-01

    A 23-year-old man presented with abdominal pain after suffering blunt trauma caused by a seatbelt injury. His low platelet count of 137 × 10(9)/L was initially attributed to trauma and his underlying hypersplenism due to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Despite conservative management, his platelet count remained persistently reduced even after his haemoglobin and clotting abnormalities were stabilised. After a week, follow-up imaging revealed an incidental finding of a pseudoaneurysm (measuring 9 mm × 8 mm × 10 mm) adjacent to a splenic laceration. The pseudoaneurysm was successfully closed via transcatheter glue embolisation; 20% of the spleen was also embolised. A week later, the platelet count normalised, and the patient was subsequently discharged. This case highlights the pitfalls in the detection of a delayed occurrence of splenic artery pseudoaneurysm after blunt injury via routine delayed phase computed tomography. While splenomegaly in G6PD may be a predisposing factor for injury, a low platelet count should arouse suspicion of internal haemorrhage rather than hypersplenism.

  5. Glucosamine and glucosamine-6-phosphate derivatives: catalytic cofactor analogues for the glmS ribozyme.

    Science.gov (United States)

    Posakony, Jeffrey J; Ferré-D'Amaré, Adrian R

    2013-05-17

    Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactors of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by SN2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl3-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogues, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz), and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogues. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection.

  6. Incidence and mutation analysis of glucose-6-phosphate dehydrogenase deficiency in eastern indonesian populations

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    Tantular,Indah S.

    2010-12-01

    Full Text Available We conducted a field survey of glucose-6-phosphate dehydrogenese (G6PD deficiency in the eastern Indonesian islands, and analyzed G6PD variants molecularly. The incidence of G6PD deficiency in 5 ethnic groups (Manggarai, Bajawa, Nage-Keo, Larantuka, and Palue on the Flores and Palue Islands was lower than that of another native group, Sikka, or a nonnative group, Riung. Molecular analysis of G6PD variants indicated that 19 cases in Sikka had a frequency distribution of G6PD variants similar to those in our previous studies, while 8 cases in Riung had a different frequency distribution of G6PD variants. On the other hand, from field surveys in another 8 ethnic groups (Timorese, Sumbanese, Savunese, Kendari, Buton, Muna, Minahasa, and Sangirese on the islands of West Timor, Sumba, Sulawesi, Muna and Bangka, a total of 49 deficient cases were detected. Thirty-nine of these 49 cases had G6PD Vanua Lava (383T>C of Melanesian origin. In our previous studies, many cases of G6PD Vanua Lava were found on other eastern Indonesian islands. Taken together, these findings may indicate that G6PD Vanua Lava is the most common variant in eastern Indonesian populations, except for Sikka.

  7. Incidence and mutation analysis of glucose-6-phosphate dehydrogenase deficiency in eastern Indonesian populations.

    Science.gov (United States)

    Tantular, Indah S; Matsuoka, Hiroyuki; Kasahara, Yuichi; Pusarawati, Suhintam; Kanbe, Toshio; Tuda, Josef S B; Kido, Yasutoshi; Dachlan, Yoes P; Kawamoto, Fumihiko

    2010-12-01

    We conducted a field survey of glucose-6-phosphate dehydrogenese (G6PD) deficiency in the eastern Indonesian islands, and analyzed G6PD variants molecularly. The incidence of G6PD deficiency in 5 ethnic groups (Manggarai, Bajawa, Nage-Keo, Larantuka, and Palue) on the Flores and Palue Islands was lower than that of another native group, Sikka, or a nonnative group, Riung. Molecular analysis of G6PD variants indicated that 19 cases in Sikka had a frequency distribution of G6PD variants similar to those in our previous studies, while 8 cases in Riung had a different frequency distribution of G6PD variants. On the other hand, from field surveys in another 8 ethnic groups (Timorese, Sumbanese, Savunese, Kendari, Buton, Muna, Minahasa, and Sangirese) on the islands of West Timor, Sumba, Sulawesi, Muna and Bangka, a total of 49 deficient cases were detected. Thirty-nine of these 49 cases had G6PD Vanua Lava (383T>C) of Melanesian origin. In our previous studies, many cases of G6PD Vanua Lava were found on other eastern Indonesian islands. Taken together, these findings may indicate that G6PD Vanua Lava is the most common variant in eastern Indonesian populations, except for Sikka.

  8. Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity.

    Science.gov (United States)

    Park, Jiyoung; Rho, Ho Kyung; Kim, Kang Ho; Choe, Sung Sik; Lee, Yun Sok; Kim, Jae Bum

    2005-06-01

    Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and diet-induced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulin-dependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.

  9. Telomerase prevents accelerated senescence in glucose-6-phosphate dehydrogenase (G6PD-deficient human fibroblasts

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    Wu Yi-Hsuan

    2009-02-01

    Full Text Available Abstract Fibroblasts derived from glucose-6-phosphate dehydrogenase (G6PD-deficient patients display retarded growth and accelerated cellular senescence that is attributable to increased accumulation of oxidative DNA damage and increased sensitivity to oxidant-induced senescence, but not to accelerated telomere attrition. Here, we show that ectopic expression of hTERT stimulates telomerase activity and prevents accelerated senescence in G6PD-deficient cells. Stable clones derived from hTERT-expressing normal and G6PD-deficient fibroblasts have normal karyotypes, and display no sign of senescence beyond 145 and 105 passages, respectively. Activation of telomerase, however, does not prevent telomere attrition in earlier-passage cells, but does stabilize telomere lengths at later passages. In addition, we provide evidence that ectopic expression of hTERT attenuates the increased sensitivity of G6PD-deficient fibroblasts to oxidant-induced senescence. These results suggest that ectopic expression of hTERT, in addition to acting in telomere length maintenance by activating telomerase, also functions in regulating senescence induction.

  10. Glucose-6-Phosphate Dehydrogenase Deficiency among Male Blood Donors in Sana’a City, Yemen

    Science.gov (United States)

    Al-Nood, Hafiz A.; Bazara, Fakiha A.; Al-Absi, Rashad; Habori, Molham AL

    2012-01-01

    Objectives To determine the prevalence of Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency among Yemeni people from different regions of the country living in the capital city, Sana’a, giving an indication of its overall prevalence in Yemen. Methods A cross-sectional study was conducted among Yemeni male blood donors attending the Department of Blood Bank at the National Centre of the Public Health Laboratories in the capital city, Sana’a, Yemen. Fluorescent spot method was used for screening, spectrophotometeric estimation of G-6-PD activity and separation by electrophoresis was done to determine the G-6-PD phenotype. Results Of the total 508 male blood donors recruited into the study, 36 were G-6-PD deficient, giving a likely G-6-PD deficiency prevalence of 7.1%. None of these deficient donors had history of anemia or jaundice. Thirty-five of these deficient cases (97.2%) showed severe G-6-PD deficiency class II (<10% of normal activity), and their phenotyping presumptively revealed a G-6-PD-Mediterranean variant. Conclusion The results showed a significant presence of G-6-PD deficiency with predominance of a severe G-6-PD deficiency type in these blood donors in Sana’a City, which could represent an important health problem through occurrence of hemolytic anemia under oxidative stress. A larger sample size is needed to determine the overall prevalence of G-6-PD deficiency, and should be extended to include DNA analysis to identify its variants in Yemen. PMID:22359725

  11. 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.

  12. Contribution of haemolysis to jaundice in Sephardic Jewish glucose-6-phosphate dehydrogenase deficient neonates.

    Science.gov (United States)

    Kaplan, M; Vreman, H J; Hammerman, C; Leiter, C; Abramov, A; Stevenson, D K

    1996-06-01

    We determined the contribution of haemolysis to the development of hyperbilirubinaemia in glucose-6-phosphate dehydrogenase (G-6-PD) deficient neonates and G-6-PD normal controls. Blood carboxyhaemoglobin (COHb), sampled on the third day of life, was measured by gas chromatography, corrected for inhaled carbon monoxide (COHbC), and expressed as a percentage of total haemoglobin concentration (Hb). Serum bilirubin was tested as clinically necessary. 37 non-jaundiced (peak serum total bilirubin (PSTB) or = 257 mumol/l) G-6-PD-deficient neonates were compared to 31 non-jaundiced and 24 jaundiced controls with comparable PSTB values, respectively. COHbC values for the entire G-6-PD deficient group were higher than in the controls (0.75 +/- 0.17% v 0.62 +/- 0.19%, P 0.05) but did in the controls (r = 0.58, P < 0.001). COHbC values were increased to a similar extent in the G-6-PD-deficient, non-jaundiced (0.72 +/- 0.16%), the G-6-PD-deficient, jaundiced (0.80 +/- 0.19%) and the control, jaundiced (0.75 +/- 0.18%) subgroups, compared to the control, non-jaundiced subgroup (0.53 +/- 0.13%) (P < 0.05). Although present in G-6-PD deficient neonates, increased haemolysis was not directly related to the PSTB.

  13. Tumor-targeted intracellular delivery of anticancer drugs through the mannose-6-phosphate/insulin-like growth factor II receptor

    NARCIS (Netherlands)

    Prakash, Jai; Beljaars, Leonie; Harapanahalli, Akshay K.; Zeinstra-Smith, Mieke; de Jager-Krikken, Alie; Hessing, Martin; Steen, Herman; Poelstra, Klaas

    2010-01-01

    Tumor-targeting of anticancer drugs is an interesting approach for the treatment of cancer since chemotherapies possess several adverse effects. In the present study, we propose a novel strategy to deliver anticancer drugs to the tumor cells through the mannose-6-phosphate/insulin-like growth factor

  14. 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...

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Diversity in expression of glucose-6-phosphate dehydrogenase deficiency in females.

    Science.gov (United States)

    Abdulrazzaq, Y M; Micallef, R; Qureshi, M; Dawodu, A; Ahmed, I; Khidr, A; Bastaki, S M; Al-Khayat, A; Bayoumi, R A

    1999-01-01

    The aims of this study were to determine the prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the United Arab Emirates (UAE), to describe the different mutations in the population, to determine its prevalence, and to study inheritance patterns in families of G6PD-deficient individuals. All infants born at Tawam Hospital, Al-Ain, UAE from January 1994 to September 1996 were screened at birth for their G6PD status. In addition, those attending well-baby clinics during the period were also screened for the disorder. Families of 40 known G6PD-deficient individuals, selected randomly from the records of three hospitals in the country, were assessed for G6PD deficiency. Where appropriate, this was followed by definition of G6PD mutations. Of 8198 infants, 746 (9.1%), comprising 15% of males and 5% of females tested, were found to be G6PD deficient. A total of 27 families were further assessed: of these, all but one family had the nt563 Mediterranean mutation. In one family, two individuals had the nt202 African mutation. The high manifestation of G6PD deficiency in women may be due to the preferential expression of the G6PD-deficient gene and X-inactivation of the normal gene, and/or to the presence of an 'enhancer' gene that makes the expression of the G6PD deficiency more likely. The high level of consanguinity which, theoretically, should result in a high proportion of homozygotes and consequently a higher proportion of females with the deficiency, was not found to be a significant factor.

  18. Evaluation of Glucose-6-Phosphate Dehydrogenase Deficiency without Hemolysis in Icteric Newborns

    Directory of Open Access Journals (Sweden)

    Farzaneh Eghbalian

    2007-04-01

    Full Text Available Objective: Glucose-6- phosphate dehydrogenase (G6PD deficiency is an inherited deficiency that may be the cause of neonatal jaundice. Our aim was to study the prevalence of G6PD deficiency without hemolysis in relation to neonatal jaundice. Material & Methods: This prospective descriptive study has been conducted on 272 icteric newborns admitted to the Ekbatan Hospital from October 2002 to September 2004. The dataset included: age, sex, total and direct bilirubin, hemoglobin, reticulocyte count, blood group and Rh of mother and newborn, direct Coombs, G6PD level and the type of treatment. All data was analyzed by using statistical method. Findings: From 272 neonates, 12 neonates (4.4% were found to have G6PD deficiency. The male to female ratio was 5 to 1 (10 male and 2 female neonates. From 12 neonates with G6PD deficiency, hemolysis was seen in 5 neonates (41.7% and the rate of G6PD deficiency without hemolysis was 2.6%. There was no difference in the mean bilirubin level, hemoglobin level and also reticulocyte count between patients with G6PD deficiency and those without G6PD deficiency (p>0.05. Out of 12 patients with G6PD deficiency, 2 patients (16.7% had blood exchange transfusion. Rh and ABO incompatibility were not seen in any of the12 patients with G6PD deficiency. Conclusion: In this study the prevalence of G6PD deficiency in icteric newborns was considerably high and most of them were non hemolytic, so we recommend G6PD test as a screening program for every newborn at the time of delivery.

  19. Transcriptome analysis of potato leaves expressing the trehalose-6-phosphate synthase 1 gene of yeast.

    Science.gov (United States)

    Kondrák, Mihály; Marincs, Ferenc; Kalapos, Balázs; Juhász, Zsófia; Bánfalvi, Zsófia

    2011-01-01

    Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast exhibit improved drought tolerance, but grow slower and have a lower carbon fixation rate and stomatal density than the wild-type. To understand the molecular basis of this phenomenon, we have compared the transcriptomes of wild-type and TPS1-transgenic plants using the POCI microarray containing 42,034 potato unigene probes. We show that 74 and 25 genes were up-, and down-regulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild-type. The differentially regulated genes were assigned into 16 functional groups. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes are involved in transcriptional and post-transcriptional regulation. Expression of genes encoding a 14-3-3 regulatory protein, and four transcription factors were down-regulated in the TPS1-transgenic leaves. To verify the microarray results, we used RNA gel blot analysis to examine the expression of eight genes and found that the RNA gel blot and microarray data correlated in each case. Using the putative Arabidopsis orthologs of the assigned potato sequences we have identified putative transcription binding sites in the promoter region of the differentially regulated genes, and putative protein-protein interactions involving some of the up- and down-regulated genes. We have also demonstrated that starch content is lower, while malate, inositol and maltose contents are higher in the TPS1-transgenic than in the wild-type leaves. Our results suggest that a complex regulatory network, involving transcription factors and other regulatory proteins, underpins the phenotypic alterations we have observed previously in potato when expressing the TPS1 gene of yeast.

  20. Transcriptome analysis of potato leaves expressing the trehalose-6-phosphate synthase 1 gene of yeast.

    Directory of Open Access Journals (Sweden)

    Mihály Kondrák

    Full Text Available Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase (TPS1 gene of yeast exhibit improved drought tolerance, but grow slower and have a lower carbon fixation rate and stomatal density than the wild-type. To understand the molecular basis of this phenomenon, we have compared the transcriptomes of wild-type and TPS1-transgenic plants using the POCI microarray containing 42,034 potato unigene probes. We show that 74 and 25 genes were up-, and down-regulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild-type. The differentially regulated genes were assigned into 16 functional groups. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes are involved in transcriptional and post-transcriptional regulation. Expression of genes encoding a 14-3-3 regulatory protein, and four transcription factors were down-regulated in the TPS1-transgenic leaves. To verify the microarray results, we used RNA gel blot analysis to examine the expression of eight genes and found that the RNA gel blot and microarray data correlated in each case. Using the putative Arabidopsis orthologs of the assigned potato sequences we have identified putative transcription binding sites in the promoter region of the differentially regulated genes, and putative protein-protein interactions involving some of the up- and down-regulated genes. We have also demonstrated that starch content is lower, while malate, inositol and maltose contents are higher in the TPS1-transgenic than in the wild-type leaves. Our results suggest that a complex regulatory network, involving transcription factors and other regulatory proteins, underpins the phenotypic alterations we have observed previously in potato when expressing the TPS1 gene of yeast.

  1. Hereditary sideroblastic anemia and glucose-6-phosphate dehydrogenase deficiency in a Negro family.

    Science.gov (United States)

    Prasad, A S; Tranchida, L; Konno, E T; Berman, L; Albert, S; Sing, C F; Brewer, G J

    1968-06-01

    Detailed clinical and genetic studies have been performed in a Negro family, which segregated for sex-linked sideroblastic anemia and glucose-6-phosphate dehydrogenase (G-6-DP) deficiency. This is the first such pedigree reported. Males affected with sideroblastic anemia had growth retardation, hypochromic microcytic anemia, elevated serum iron, decreased unsaturated iron-binding capacity, increased (59)Fe clearance, low (59)Fe incorporation into erythrocytes, normal erythrocyte survival ((51)Cr), normal hemoglobin electrophoretic pattern, erythroblastic hyperplasia of marrow with increased iron, and marked increase in marrow sideroblasts, particularly ringed sideroblasts. Perinuclear deposition of ferric aggregates was demonstrated to be intramitochondrial by electron microscopy. Female carriers of the sideroblastic gene were normal but exhibited a dimorphic population of erythrocytes including normocytic and microcytic cells. The bone marrow studies in the female (mother) showed ringed marrow sideroblasts. Studies of G-6-PD involved the methemoglobin elution test for G-6-PD activity of individual erythrocytes, quantitative G-6-PD assay, and electrophoresis. In the pedigree, linkage information was obtained from a doubly heterozygous woman, four of her sons, and five of her daughters. Three sons were doubly affected, and one was normal. One daughter appeared to be a recombinant. The genes appeared to be linked in the coupling phase in the mother. The maximum likelihood estimate of the recombination value was 0.14. By means of Price-Jones curves, the microcytic red cells in peripheral blood were quantitated in female carriers. The sideroblast count in the bone marrow in the mother corresponded closely to the percentage of microcytic cells in peripheral blood. This is the second example in which the cellular expression of a sex-linked trait has been documented in the human red cells, the first one being G-6-PD deficiency. The coexistence of the two genes in doubly

  2. Glutamine:fructose-6-phosphate amidotransferase activity in cultured human skeletal muscle cells: relationship to glucose disposal rate in control and non-insulin-dependent diabetes mellitus subjects and regulation by glucose and insulin.

    OpenAIRE

    1996-01-01

    We examined the activity of the rate-limiting enzyme for hexosamine biosynthesis, glutamine:fructose-6-phosphate amidotransferase (GFA) in human skeletal muscle cultures (HSMC), from 17 nondiabetic control and 13 subjects with non-insulin-dependent diabetes. GFA activity was assayed from HSMC treated with low (5 mM) or high (20 mM) glucose and low (22 pM) or high (30 microM) concentrations of insulin. In control subjects GFA activity decreased with increasing glucose disposal rate (r = -0.68,...

  3. Avoiding Buffer Interference in ITC Experiments: A Case Study from the Analysis of Entropy-Driven Reactions of Glucose-6-Phosphate Dehydrogenase.

    Science.gov (United States)

    Bianconi, M Lucia

    2016-01-01

    Isothermal titration calorimetry (ITC) is a label-free technique that allows the direct determination of the heat absorbed or released in a reaction. Frequently used to determining binding parameters in biomolecular interactions, it is very useful to address enzyme-catalyzed reactions as both kinetic and thermodynamic parameters can be obtained. Since calorimetry measures the total heat effects of a reaction, it is important to consider the contribution of the heat of protonation/deprotonation that is possibly taking place. Here, we show a case study of the reaction catalyzed by the glucose-6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides. This enzyme is able to use either NAD(+) or NADP(+) as a cofactor. The reactions were done in five buffers of different enthalpy of protonation. Depending on the buffer used, the observed calorimetric enthalpy (ΔH(cal)) of the reaction varied from -22.93 kJ/mol (Tris) to 19.37 kJ/mol (phosphate) for the NADP(+)-linked reaction, and -11.67 kJ/mol (Tris) to 7.32 kcal/mol or 30.63 kJ/mol (phosphate) for the NAD(+) reaction. We will use this system as an example of how to extract proton-independent reaction enthalpies from kinetic data to ensure that the reported accurately represent the intrinsic heat of reaction.

  4. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Wei, Shi-Cheng, E-mail: kqsc-wei@bjmu.edu.cn [Peking University School of Stomatology, Beijing 100081 (China); Liang, Yu-He, E-mail: kqsc-wei@bjmu.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China)

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  5. An enzymatic fluorimetric assay for glucose-6-phosphate: application in an in vitro Warburg-like effect

    OpenAIRE

    Zhu, Aiping; Romero, Roberto; Petty, Howard R.

    2009-01-01

    Recently, there has been a resurgence of interest in the regulatory role of cell metabolism in tumor biology and immunology. To assess changes in metabolite levels in cell populations and tissues, especially from small clinical samples, highly sensitive assays are required. Based upon glucose 6-phosphate’s reaction and the diaphorase-resazurin amplifying system, we have developed a fluorescence methodology to measure glucose 6-phosphate concentrations in cell extracts. In this approach, gluco...

  6. Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening.

    Science.gov (United States)

    Ma, Yi; Ghoshdastider, Umesh; Wang, Jufang; Ye, Wei; Dötsch, Volker; Filipek, Slawomir; Bernhard, Frank; Wang, Xiaoning

    2012-12-01

    Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.

  7. Regulation of hexokinase and glucose-6-phosphate dehydrogenase genes expression at norm and pathology

    Directory of Open Access Journals (Sweden)

    Marunych R. Yu.

    2013-03-01

    Full Text Available The increasing of glycolysis in tumors under aerobic conditions is known as Warburg phenomenon; the activity of the pentose phosphate pathway increases also significantly. The pentose phosphate pathway and glycolysis, especially their first steps, and the regulatory enzyme 6-phosphofrukto-2-kinase/fructose-2,6-bisphosphatase are influenced by cell signaling systems such as the system of circadian clock, the system of hypoxia-inducible factor and unfolded protein response system, that allow malignant cells to adapt to stress factors such as hypoxia, ischemia and influence of low molecular agents. The review enlightens the impact of signaling systems on the key enzymes of glycolysis and the pentose phosphate pathway gene expression in normal cells and in malignant cells, and their importance for survival of malignant cells under stress conditions.

  8. Cyclic 3',5'-adenosine monophosphate and N-acetylglucosamine-6-phosphate as regulatory signals in catabolite repression of the lac operon in Escherichia coli.

    Science.gov (United States)

    Goldenbaum, P E; Broman, R L; Dobrogosz, W J

    1970-09-01

    When an Escherichia coli mutant lacking the enzyme N-acetyl-glucosamine-6-phosphate (AcGN6P) deacetylase is grown in a succinate-mineral salts medium and exposed to an exogenous source of N-acetylglucosamine, approximately 20 to 30 pmoles of AcGN6P per mug of cell dry weight will accumulate in these cells. This accumulation occurs within 2 to 4 min after the addition of N-acetylglucosamine and is coincident with the production of a severe permanent catabolite repression of beta-galactosidase synthesis. This repression does not occur if adenosine 3',5'-cyclic phosphate (cyclic AMP) is added to the cells before AcGN6P accumulates. An immediate derepression occurs when cyclic AMP is added to cells that have already accumulated a large AcGN6P pool. These findings are consistent with the view that low-molecular-weight carbohydrate metabolites and cyclic AMP play key roles in the catabolite repression phenomenon, and that metabolites such as AcGN6P may participate in the represion mechanism by influencing either the formation or degradation of cyclic AMP in E. coli.

  9. 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.

  10. Enhanced production of epsilon-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene.

    Science.gov (United States)

    Lee, Won-Heong; Park, Jin-Byung; Park, Kyungmoon; Kim, Myoung-Dong; Seo, Jin-Ho

    2007-08-01

    Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.

  11. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1)

    Science.gov (United States)

    Zhao, Minzhi; Li, Haiyun; Ma, Yan; Gong, He; Yang, Shu; Fang, Qiaojun; Hu, Zhiyuan

    2017-01-01

    Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression (P<0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes.

  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. Metabolomic profile of glycolysis and the pentose phosphate pathway identifies the central role of glucose-6-phosphate dehydrogenase in clear cell-renal cell carcinoma.

    Science.gov (United States)

    Lucarelli, Giuseppe; Galleggiante, Vanessa; Rutigliano, Monica; Sanguedolce, Francesca; Cagiano, Simona; Bufo, Pantaleo; Lastilla, Gaetano; Maiorano, Eugenio; Ribatti, Domenico; Giglio, Andrea; Serino, Grazia; Vavallo, Antonio; Bettocchi, Carlo; Selvaggi, Francesco Paolo; Battaglia, Michele; Ditonno, Pasquale

    2015-05-30

    The analysis of cancer metabolome has shown that proliferating tumor cells require a large quantities of different nutrients in order to support their high rate of proliferation. In this study we analyzed the metabolic profile of glycolysis and the pentose phosphate pathway (PPP) in human clear cell-renal cell carcinoma (ccRCC) and evaluate the role of these pathways in sustaining cell proliferation, maintenance of NADPH levels, and production of reactive oxygen species (ROS). Metabolomic analysis showed a clear signature of increased glucose uptake and utilization in ccRCC tumor samples. Elevated levels of glucose-6-phosphate dehydrogenase (G6PDH) in association with higher levels of PPP-derived metabolites, suggested a prominent role of this pathway in RCC-associated metabolic alterations. G6PDH inhibition, caused a significant decrease in cancer cell survival, a decrease in NADPH levels, and an increased production of ROS, suggesting that the PPP plays an important role in the regulation of ccRCC redox homeostasis. Patients with high levels of glycolytic enzymes had reduced progression-free and cancer-specific survivals as compared to subjects with low levels. Our data suggest that oncogenic signaling pathways may promote ccRCC through rerouting the sugar metabolism. Blocking the flux through this pathway may serve as a novel therapeutic target.

  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

    A total of 3200 milk samples from Holstein and Jersey cows were analysed for free glucose and glucose-6-phosphate (G6P) by an enzymatic-fluorometric method that requires no pre-treatment. The cows were primiparous as well as multiparous, and samples were taken throughout the entire lactation period......, free glucose increased whereas G6P decreased. Concentration of free glucose in milk is greater for primiparous than multiparous cows and greater for Holstein than Jersey cows. Concentration of G6P was not affected by parity or breed. The use of free glucose and G6P as indicators of physiological...

  15. Comparative analysis of glucose-6-phosphate dehydrogenase levels in pre-term and term babies delivered at University of Ilorin Teaching Hospital, Nigeria

    Directory of Open Access Journals (Sweden)

    Temitope Olorunsola Obasa

    2012-03-01

    Full Text Available Glucose-6-phosphate (G6P is an enzyme in the hexose monophosphate shunt required for the production of reducing equivalents needed to mop up free radicals. thereby keeping hemoglobin in its free state. Deficiency of the enzyme can cause severe neonatal jaundice. The aim of this study was to compare G6PD levels in pre-term and term babies, and evaluate the extent to which G6PD deficiency determines the severity of jaundice in various gestational age groups. Samples of cord blood collected from consecutively delivered babies in the University of Ilorin Teaching Hospital, Nigeria, were assayed for G6PD levels, and the babies were observed for jaundice during the first week of life. Those who developed jaundice had serial serum bilirubin measured. Nine hundred and thirty-three babies had G6PD assayed, with 348 being G6PD deficient, giving a hospital based prevalence of 37.3%. Of the 644 who were followed up, 143 (22.2% were pre-term and 501(77.8% were term babies. Babies with gestational age (GA 27-29 weeks had the highest G6PD levels. However, there was no significant variation among the different gestational age groups (F=0.64, P=0.64. Jaundice occurred more in pre-term compared to term babies with a relative risk of 2.41 (χ2=60.95, P=0.00001. Occurrence of jaundice in pre-term babies was irrespective of G6PD status (χ2=0.2, P=0.66, RR=1.09, CI=0.83

  16. Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

    Science.gov (United States)

    Fikrika, H.; Ambarsari, L.; Sumaryada, T.

    2016-01-01

    Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

  17. [Regularities of organ-specific expression of enzyme systems in cattle].

    Science.gov (United States)

    Tatarenko, O F; Glazko, V I

    1992-01-01

    The organ specificity of creatine kinase, esterase, isocitrate dehydrogenase lactate dehydrogenase, nucleoside phosphorylase, adenylate kinase, hexokinase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase of black-white cattle has been studied. Esterases, creatine kinase, adenylate kinase, hexokinase and glucose-6-phosphate dehydrogenase have a very wide spectrum of the organ variabilities. Liver and heart have the largest specificity of enzymes activity. Some peculiarities of isozyme spectrum are found in ovaries and spleen.

  18. Advances in the Molecular Biological Research of Human Glucose-6-phosphate Dehydrogenase%人类葡萄糖-6-磷酸脱氢酶的分子生物学研究进展

    Institute of Scientific and Technical Information of China (English)

    刘晗; 蒋玮莹

    2009-01-01

    葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PD)缺乏症作为一种全球范围内最常见的酶缺乏症之一,受到研究者们的广泛关注.G6PD催化磷酸戊糖途径的第一步,由此酶催化生成的NADPH+H+对于对抗氧化性损伤是极其重要的.本文将从G6PD的结构与功能,SNP的研究与单体型的建立,抗疟疾选择优势与新的G6PD基因突变检测方法这几方面的研究进展综述如下.%Glucose-6-phosphate dehydrogenase(G6PD)deficiency is one of the most common enzymopathies attracting many researchers.G6PD catalyses the first committed step in the pentose phosphate pathway,and the generation of NADPH by this enzyme is essential for protection against oxidative stress.The progress in research of the structures and functions of G6PD gene'S,SNP and haplotype,new detective techniques of new mutation and recent positive selection of anti-malaria are reviewed.

  19. Glucose-6-phosphate-dehydrogenase deficiency and its correlation with other risk factors in jaundiced newborns in Southern Brazil

    Institute of Scientific and Technical Information of China (English)

    Clarissa Gutirrez Carvalho; Simone Martins Castro; Ana Paula Santin; Carina Zaleski; Felipe Gutirrez Carvalho; Roberto Giugliani

    2011-01-01

    Objective:To evaluate the correlation between glucose-6-phosphate-dehydrogenase (G6PD) deficiency and neonatal jaundice.Methods: Prospective, observational case-control study was conducted on490 newborns admitted to Hospital de Clínicas de Porto Alegre for phototherapy, who all experienced35 or more weeks of gestation, from March to December2007. Enzymatic screening ofG6PD activity was performed, followed byPCR.Results:There was prevalence of4.6% and a boy-girl ratio of3:1 in jaundiced newborns. No jaundiced neonate withABO incompatibility presented G6PD deficiency, and no Mediterranean mutation was found. A higher proportion of deficiency was observed in Afro-descendants. There was no association withUGT1A1 variants. Conclusions:G6PD deficiency is not related to severe hyperbilirubinemia and considering the high miscegenation in this area of Brazil, other gene interactions should be investigated.

  20. Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance.

    Science.gov (United States)

    Romero, C; Bellés, J M; Vayá, J L; Serrano, R; Culiáñez-Macià, F A

    1997-03-01

    The yeast trehalose-6-phosphate synthase gene (TPS1) was engineered under the control of the cauliflower mosaic virus regulatory sequences (CaMV35S) for expression in plants. Using Agrobacterium-mediated transfer, the gene was incorporated into the genomic DNA and constitutively expressed in Nicotiana tabacum L. plants. Trehalose was determined in the transformants, by anion-exchange chromatography coupled to pulsed amperometric detection. The non-reducing disaccharide accumulated up to 0.17 mg per g fresh weight in leaf extracts of transgenic plants. Trehaloseaccumulating plants exhibited multiple phenotypic alterations, including stunted growth, lancet-shaped leaves, reduced sucrose content and improved drought tolerance. These pleiotropic effects, and the fact that water loss from detached leaves was not significantly affected by trehalose accumulation, suggest that synthesis of this sugar, rather than leading to an osmoprotectant effect, had altered sugar metabolism and regulatory pathways affecting plant development and stress tolerance.

  1. Can affinity interactions influence the partitioning of glucose-6-phosphate dehydrogenase in two-phase aqueous micellar systems?

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    André M. Lopes

    2008-01-01

    Full Text Available In this work, we provide an investigation of the role and strength of affinity interactions on the partitioning of the glucose-6-phosphate dehydrogenase in aqueous two-phase micellar systems. These systems are constituted of micellar surfactant solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to biomolecules. We studied G6PD partitioning in systems composed of the nonionic surfactants, separately, in the presence and absence of affinity ligands. We observed that G6PD partitions to the micelle-poor phase, owing to the strength of excluded-volume interactions in these systems that drive the protein to the micelle-poor phase, where there is more free volume available.

  2. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1

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    Zhao MZ

    2017-03-01

    Full Text Available Minzhi Zhao,* Haiyun Li,* Yan Ma, He Gong, Shu Yang, Qiaojun Fang, Zhiyuan Hu Chinese Academy of Sciences Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: Abraxane (Abr, a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX. To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1, showed significant differential expression (P<0.05 in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes. Keywords: quantitative proteomics, nano-drug, drug efficacy, lung cancer, molecular mechanisms, abraxane

  3. First evaluation of glucose-6-phosphate dehydrogenase (G6PD deficiency in vivax malaria endemic regions in the Republic of Korea.

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    Youn-Kyoung Goo

    Full Text Available BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK. METHODS: Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK. RESULTS: Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants. CONCLUSIONS: No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.

  4. Survey of the Prevalence of Glucose-6-Phosphate Dehydrogenase (G6PD Deficiency in Admitted Men for Premarriage Tests in Zahedan-Iran Reference Laboratory

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    Nakhaee Ali Reza

    2009-09-01

    Full Text Available Background: GLucose-6-phosphate dehydrogenase (G6PD deficiency is the most common known enzymopathy in human. G6PD deficiency is usually asymptomatic, however, deficient individuals are at increased risk of developing acute hemolytic anemia and hyperbilirubinemia following intake of oxidative agents and fava. The objective of present study was to detect prevalence of G6PD deficiency in admitted males for premarriage tests in Zahedan Reference Laboratory. Also, we compared blood indices of normal and G6PD deficient individuals.Materials and Methods: This descriptive study was carried out on 1340 admitted males in Zahedan Reference Laboratory from February 2008 to March 2009. G6PD activity was determined in EDTA containing blood samples by qualitative fluorescence spot test, then G6PD deficiency was confirmed by quantitative spectrophotometric method. Total leukocyte count and RBC indices of G6PD deficient samples and the same number of normal samples were compared. The differences between two groups were compared using Sigmaplot software and t-Student test. A P-value less than 0.05 was considered statistically significant.Results: G6PD deficiency was found in 84 individuals of total 1340 by fluorescence spot test and confirmed in 79 by quantitative method. Therefore, prevalence of G6PD deficiency in Zahedan was estimated to be 5.9%. Comparison of deficient and normal individuals did not show significant difference in WBC count, RBC count, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin (MCH and RDW-SD. However, mean corpuscular volume (MCV was significantly high and mean corpuscular hemoglobin concentration (MCHC and RDW-CV were significantly low in G6PD deficient individuals compared to those with normal enzyme level.Discussion: Present study revealed that the prevalence of G6PD deficiency in Zahedan is 5.9%. Severity of G6PD deficiency in quantitative assay indicated that class I and II are probably dominant variants in

  5. Purification and Structural and Kinetic Characterization of the Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase from the Crassulacean Acid Metabolism Plant, Pineapple.

    Science.gov (United States)

    Tripodi, KEJ.; Podesta, F. E.

    1997-03-01

    Pyrphosphate-dependent phosphofructokinase (PFP) was purified to electrophoretic homogeneity from illuminated pineapple (Ananas comosus) leaves. The purified enzyme consists of a single subunit of 61.5 kD that is immunologically related to the potato tuber PFP [beta] subunit. The native form of PFP likely consists of a homodimer of 97.2 kD, as determined by gel filtration. PFP's glycolytic activity was strongly dependent on pH, displaying a maximum at pH 7.7 to 7.9. Gluconeogenic activity was relatively constant between pH 6.7 and 8.7. Activation by Fru-2,6-bisphosphate (Fru-2,6-P2) was dependent on assay pH. In the glycolytic direction, it activated about 10-fold at pH 6.7, but only 2-fold at pH 7.7. The gluconeogenic reaction was only weakly affected by Fru-2,6-P2. The true substrates for the PFP forward and reverse reactions were Fru-6-phosphate and Mg-pyrophosphate, and Fru-1,6-P2, orthophosphate, and Mg2+, respectively. The results suggest that pineapple PFP displays regulatory properties consistent with a pH-based regulation of its glycolytic activity, in which a decrease in cytosolic pH caused by nocturnal acidification during Crassulacean acid metabolism, which could curtail its activity, is compensated by a parallel increase in its sensitivity to Fru-2,6-P2. It is also evident that the [beta] subunit alone is sufficient to confer PFP with a high catalytic rate and the regulatory properties associated with activation by Fru-2,6-P2.

  6. 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

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    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

  7. A survey for isoenzymes of glucosephosphate isomerase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogenase in C3-, C 4-and crassulacean-acid-metabolism plants, and green algae.

    Science.gov (United States)

    Herbert, M; Burkhard, C; Schnarrenberger, C

    1979-01-01

    Two isoenzymes each of glucosephosphate isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were separated by (NH4)2SO4 gradient solubilization and DEAE-cellulose ion-exchange chromatography from green leaves of the C3-plants spinach (Spinacia oleracea L.), tobacco (Nicotiana tabacum L.) and wheat (Triticum aestivum L.), of the Crassulacean-acid-metabolism plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen, and from the green algae Chlorella vulgaris and Chlamydomonas reinhardii. After isolation of cell organelles from spinach leaves by isopyenic centrifugation in sucrose gradients one of two isoenzymes of each of the four enzymes was found to be associated with whole chloroplasts while the other was restricted to the soluble cell fraction, implying the same intracellular distribution of these isoenzymes also in the other species.Among C4-plants, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in only one form in corn (Zea mays L.), sugar cane (Saccharum officinarum L.) and Coix lacrymajobi L., but as two isoenzymes in Atriplex spongiosa L. and Portulaca oleracea L. In corn, the two dehydrogenases were mainly associated with isolated mesophyll protoplasts while in Atriplex spongiosa they were of similar specific activity in both mesophyll protoplasts and bundle-sheath strands. In all five C4-plants three isoenzymes of glucosephosphate isomerase and phosphoglucomutase were found. In corn two were localized in the bundle-sheath strands and the third one in the mesophyll protoplasts. The amount of activity of the enzymes was similar in each of the two cell fractions. Apparently, C4 plants have isoenzymes not only in two cell compartments, but also in physiologically closely linked cell types such as mesophyll and bundle-sheath cells.

  8. STUDY OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE (G6PD DEFICIENCY IN JAUNDICED NEONATES OF A TERTIARY CARE CENTRE OF NORTH-EAST INDIA

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    Aukifa Khamim

    2016-05-01

    Full Text Available roteins from oxidative damage. Glucose-6-Phosphate Dehydrogenase (G6PD deficiency is the commonest red cell enzyme abnormality associated with haemolysis leading to Neonatal Jaundice (NNJ. It is a genetically inherited X-linked abnormality. AIMS To find out incidence of G6PD deficiency amongst jaundiced patients and relation between G6PD deficiency and sex, peak level of Total Serum Bilirubin (TSB, significant hyperbilirubinemia, duration of phototherapy and need for exchange transfusion. SETTINGS AND DESIGN Hospital based retrospective study. METHODS AND MATERIALS This retrospective study was carried out among 1224 jaundiced neonates needing phototherapy admitted in the Neonatology Unit of Dept. of Paediatrics (March 2015 to October 2015, Assam Medical College and Hospital (AMCH, Dibrugarh, Assam. STATISTICAL ANALYSIS USED Data were entered in SPSS (Software package for statistical analysis, version 16 and analysed using Chi-Square test and Mann Whitney U test. RESULTS A total of 2574 neonates were admitted during the 8 months period, of which 1224 had NNJ (47.5%. Of these 77 (5.07% babies were G6PD deficient. Male (n=53 to female (n=24 ratio was 2:1. The commonest age at presentation was 2nd to 4th days in both G6PD deficient and G6PD normal neonates. Mean peak-TSB level in G6PD deficient cases (20.03±5.30 mg/dL was significantly higher than G6PD normal cases (16.67±3.93 mg/dL; 45% of G6PD deficient neonates developed significant hyperbilirubinemia (Indirect bilirubin more than 20 mg% and required Double Volume Exchange Transfusion (DVET. Mean duration of phototherapy in G6PD deficient NNJ babies is 2.5±1.2 days, which is significantly higher (p<0.05 when compared to G6PD normal NNJ babies where it is 2±1.1 days. In babies with significant hyperbilirubinemia, it is seen that there is signif icant difference (p<0.001 between G6PD deficient and G6PD normal babies. There was significant difference in requirement of DVET between G6PD deficient

  9. Disruption of the Candida albicans TPS1 Gene Encoding Trehalose-6-Phosphate Synthase Impairs Formation of Hyphae and Decreases Infectivity†

    Science.gov (United States)

    Zaragoza, Oscar; Blazquez, Miguel A.; Gancedo, Carlos

    1998-01-01

    The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30°C was indistinguishable from that of the wild type. However, at 42°C it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37°C, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42°C, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 106 CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation. PMID:9683476

  10. Glucose-6-phosphate isomerase is an endogenous inhibitor to myofibril-bound serine proteinase of crucian carp (Carassius auratus).

    Science.gov (United States)

    Sun, Le-Chang; Zhou, Li-Gen; Du, Cui-Hong; Cai, Qiu-Feng; Hara, Kenji; Su, Wen-Jin; Cao, Min-Jie

    2009-06-24

    Glucose-6-phosphate isomerase (GPI) was purified to homogeneity from the skeletal muscle of crucian carp ( Carassius auratus ) by ammonium sulfate fractionation, column chromatographies of Q-Sepharose, SP-Sepharose, and Superdex 200 with a yield of 8.0%, and purification folds of 468. The molecular mass of GPI was 120 kDa as estimated by gel filtration, while on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two subunits (55 and 65 kDa) were identified, suggesting that it is a heterodimer. Interestingly, GPI revealed specific inhibitory activity toward a myofibril-bound serine proteinase (MBSP) from crucian carp, while no inhibitory activity was identified toward other serine proteinases, such as white croaker MBSP and crucian carp trypsin. Kinetic analysis showed that GPI is a competitive inhibitor toward MBSP, and the K(i) was 0.32 microM. Our present results indicated that the multifunctional protein GPI is an endogenous inhibitor to MBSP and may play a significant role in the regulation of muscular protein metabolism in vivo.

  11. Identification of point mutations in Glucose-6-Phosphate Dehydrogenase gene in Timor Island people : A preliminary report

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    Widanto Hardjowasito

    2001-12-01

    Full Text Available Glucose 6 phosphate dehydrogenase (G6PD deficiency is common in malaria endemic region, however no molecular study has been performed on G6PD deficiency in Timor Island, Indonesia a malarial hyperendemic area which Proto Malay is the majority of the people in that island. To observe the frequency and molecular type of mutations in G6PD deficient Proto Malay people, 118 native people were screened using formazan ring test. Mutation in the G6PD gene were determined by MPTP (Multiple PCR using Multiple Tandem Forward Primers and a common Reserve Pimer method and confirmed by automatic sequencer. This study shows that three males have lower G6PD activity. Using MPTP method, a point mutation could be indicated in the two cases. Sequencing of the amplified products in 2 G6PD patients disclosed mutations of T383C in exon 5 and C 592 T in exon 6 in respective case. Our result documents point mutations in exon 5 and exon 6 in the G6PD gene of two Proto Malay people in Timor. These mutations are common in Asia region. (Med J Indones 2001; 10: 210-3Keywords: mutations, G6PD, Proto Malay.

  12. Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (Hordeum vulgare cv. Nure) roots.

    Science.gov (United States)

    Cardi, Manuela; Chibani, Kamel; Castiglia, Daniela; Cafasso, Donata; Pizzo, Elio; Rouhier, Nicolas; Jacquot, Jean-Pierre; Esposito, Sergio

    2013-12-01

    In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms. The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coli, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction. The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies.

  13. Alleviation of PEGylated Puerarin on Erythrocyte Hemolysis Induced by Puerarin in Glucose-6-phosphate Dehydrogenase-deficient Rats

    Institute of Scientific and Technical Information of China (English)

    LIU; Xin-yi; LI; Jian-rong; WANG; Nai-jie; ZHANG; Guang-ping; DU; Feng; YE; Zu-guang; XIANG; Da-xiong

    2013-01-01

    Objective To explore and analyze the reducing hemolytic effects of PEGylated puerarin (PEG-PUE) on erythrocytes induced by PUE in glucose-6-phosphate dehydrogenase (G6PD)-deficient rats. Methods The rat model with G6PD-deficiency was established via sc injecting 1% acetylphenyl-hydrazine. Then the G6PD-deficient erythrocyte suspension obtained from this rat model was used to evaluate the hemolytic effects of PUE and the reducing hemolytic effects of PEG-PUE via hemolytic activity and erythrocyte osmotic fragility assay. Results It was found that PUE could cause a serious hemolysis to the erythrocyte suspension with the increase of drug concentration and the prolongation of drug incubation time, the hemolytic rate of PUE was up to 40%, while the addition of PEG-PUE to the erythrocyte suspension revealed no significant hemolysis. Additionally, the result of erythrocyte osmotic fragility indicated that PEG-PUE exerted a slight effect on the erythrocyte membranes, and the NaCl concentration that induced 50% hemolysis (32 mmol/L) was about one-third PUE. Conclusion These results demonstrate that PEG-PUE could play a significant role in reducing the side effect of hemolysis induced by PUE. The low hemolytic activity of PEG-PUE makes it a favorable candidate for in vivo tests and PEG-PUE could also provide the useful insight for the further formulation development as an innovative drug.

  14. Alleviation of PEGylated Puerarin on Erythrocyte Hemolysis Induced by Puerarin in Glucose-6-phosphate Dehydrogenase-deficient Rats

    Institute of Scientific and Technical Information of China (English)

    LIU Xin-yi; LI Jian-rong; WANG Nai-jie; ZHANG Guang-ping; DU Feng; YE Zu-guang; XIANG Da-xiong

    2013-01-01

    Objective To explore and analyze the reducing hemolytic effects of PEGylated puerarin (PEG-PUE) on erythrocytes induced by PUE in glucose-6-phosphate dehydrogenase (G6PD)-deficient rats.Methods The rat model with G6PD-deficiency was established via sc injecting 1% acetylphenyl-hydrazine.Then the G6PD-deficient erythrocyte suspension obtained from this rat model was used to evaluate the hemolytic effects of PUE and the reducing hemolytic effects of PEG-PUE via hemolytic activity and erythrocyte osmotic fragility assay.Results It was found that PUE could cause a serious hemolysis to the erythrocyte suspension with the increase of drug concentration and the prolongation of drug incubation time,the hemolytic rate of PUE was up to 40%,while the addition of PEG-PUE to the erythrocyte suspension revealed no significant hemolysis.Additionally,the result of erythrocyte osmotic fragility indicated that PEG-PUE exerted a slight effect on the erythrocyte membranes,and the NaCl concentration that induced 50% hemolysis (32 mmol/L) was about one-third PUE.Conclusion These results demonstrate that PEG-PUE could play a significant role in reducing the side effect of hemolysis induced by PUE.The low hemolytic activity of PEG-PUE makes it a favorable candidate for in vivo tests and PEG-PUE could also provide the useful insight for the further formulation development as an innovative drug.

  15. 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

  16. The Class II trehalose 6-phosphate synthase gene PvTPS9 modulates trehalose metabolism in Phaseolus vulgaris nodules.

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    Aarón Barraza

    2016-11-01

    Full Text Available Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS of common bean (Phaseolus vulgaris, was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1% of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

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

    Directory of Open Access Journals (Sweden)

    Saúl Gómez-Manzo

    2016-05-01

    Full Text Available 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.

  18. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with asymptomatic malaria in a rural community in Burkina Faso

    Institute of Scientific and Technical Information of China (English)

    Abdoul Karim Ouattara; Cyrille Bisseye; Birama Diarra; Tegwind Rebeca Compaore; Florencia Djigma; Virginio Pietra; Remy Moret; Jacques Simpore

    2014-01-01

    Objective: To investigate 4 combinations of mutations responsible for glucose-6-phosphate dehydrogenase (G6PD) deficiency in a rural community of Burkina Faso, a malaria endemic country. Methods: Two hundred individuals in a rural community were genotyped for the mutations A376G, G202A, A542T, G680T and T968C using TaqMan single nucleotide polymorphism assays and polymerase chain reaction followed by restriction fragment length polymorphism. Results: The prevalence of the G6PD deficiency was 9.5% in the study population. It was significantly higher in men compared to women (14.3%vs 6.0%, P=0.049). The 202A/376G G6PD A-was the only deficient variant detected. Plasmodium falciparum asymptomatic parasitaemia was significantly higher among the G6PD-non-deficient persons compared to the G6PD-deficient (P Conclusions:This study showed that the G6PD A-variant associated with protection against asymptomatic malaria in Burkina Faso is probably the most common deficient variant.

  19. How Do Sugars Regulate Plant Growth and Development? New Insight into the Role of Trehalose-6-Phosphate

    Institute of Scientific and Technical Information of China (English)

    Liam E.O'Hara; Matthew J.Paul; Astrid Wingler

    2013-01-01

    Plant growth and development are tightly controlled in response to environmental conditions that influence the availability of photosynthetic carbon in the form of sucrose.Trehalose-6-phosphate (T6P),the precursor of trehalose in the biosynthetic pathway,is an important signaling metabolite that is involved in the regulation of plant growth and development in response to carbon availability.In addition to the plant's own pathway for trehalose synthesis,formation of T6P or trehalose by pathogens can result in the reprogramming of plant metabolism and development.Developmental processes that are regulated by T6P range from embryo development to leaf senescence.Some of these processes are regulated in interaction with phytohormones,such as auxin.A key interacting factor of T6P signaling in response to the environment is the protein kinase sucrose non-fermenting related kinase-1 (SnRK1),whose catalytic activity is inhibited by T6P.SnRK1 is most likely involved in the adjustment of metabolism and growth in response to starvation.The transcription factor bZIP11 has recently been identified as a new player in the T6P/SnRK1 regulatory pathway.By inhibiting SnRK1,T6P promotes biosynthetic reactions.This regulation has important consequences for crop production,for example,in the developing wheat grain and during the growth of potato tubers.

  20. 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

    BACKGROUND: Mutations in the haemoglobin beta-globin (HbB) and glucose-6-phosphate dehydrogenase (G6PD) genes cause widespread human genetic disorders such as sickle cell diseases and G6PD deficiency. In sub-Saharan Africa, a few predominant polymorphic variants of each gene account for a majority...

  1. Mannose-6-phosphate/insulin-like growth Factor-II receptors may represent a target for the selective delivery of mycophenolic acid to fibrogenic cells

    NARCIS (Netherlands)

    Greupink, Albert; Bakker, Hester; van Goor, H.; de Borst, M.H.; Beljaars, L.; Poelstra, Klaas

    2006-01-01

    Purpose. The insulin-like growth factor axis plays an important role in fibrogenesis. However, little is known about mannose-6-phosphate/Insulin-like growth factor-II receptor (M6P/IGF-IIR) expression during fibrosis. When expressed preferentially on fibrogenic cells, this receptor may be used to se

  2. Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation.

    Science.gov (United States)

    Frederiks, Wilma M; van Marle, Jan; van Oven, Carel; Comin-Anduix, Begonya; Cascante, Marta

    2006-01-01

    Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP, and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 +/- 4% and 43 +/- 4% higher, respectively, than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle-dependent activity.

  3. Genome-wide identification and evolution analysis of Trehalose-6-phosphate synthase gene family in Nelumbo nucifera

    Directory of Open Access Journals (Sweden)

    Qijiang Jin

    2016-09-01

    Full Text Available Trehalose-6-phosphate synthase (TPS plays a key role in plant carbohydrate metabolism and the perception of carbohydrate availability. In the present work, the publicly available Nelumbo nucifera (lotus genome sequence database was analyzed which led to identification of nine lotus TPS genes (NnTPS. It was found that at least two introns are included in the coding sequences of NnTPS genes. When the motif compositions were analyzed we found that NnTPS generally shared the similar motifs, implying that they have similar functions. The dN/dS ratios were always less than 1 for different domains and regions outside domains, suggesting purifying selection on the lotus TPS gene family. The regions outside TPS domain evolved relatively faster than NnTPS domains. A phylogenetic tree was constructed using all predicted coding sequences of lotus TPS genes, together with those from Arabidopsis, poplar, soybean and rice. The result indicated that those TPS genes could be clearly divided into two main subfamilies (I-II, where each subfamily could be further divided into 2 (I and 5 (II subgroups. Analyses of divergence and adaptive evolution show that purifying selection may have been the main force driving evolution of plant TPS genes. Some of the critical sites that contributed to divergence may have been under positive selection. Transcriptome data analysis revealed that most NnTPS genes were predominantly expressed in sink tissues. Expression pattern of NnTPS genes under copper and submergence stress indicated that NNU_014679 and NNU_022788 might play important roles in lotus energy metabolism and participate in stress response. Our results can facilitate further functional studies of TPS genes in lotus.

  4. Prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Ouest and Sud-Est departments of Haiti.

    Science.gov (United States)

    von Fricken, Michael E; Weppelmann, Thomas A; Eaton, Will T; Alam, Meer T; Carter, Tamar E; Schick, Laura; Masse, Roseline; Romain, Jean R; Okech, Bernard A

    2014-07-01

    Malaria remains a significant public health issue in Haiti, with chloroquine (CQ) used almost exclusively for the treatment of uncomplicated infections. Recently, single dose primaquine (PQ) was added to the Haitian national malaria treatment policy, despite a lack of information on the prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency within the population. G6PD deficient individuals who take PQ are at risk of developing drug induced hemolysis (DIH). In this first study to examine G6PD deficiency rates in Haiti, 22.8% (range 14.9%-24.7%) of participants were found to be G6PD deficient (class I, II, or III) with 2.0% (16/800) of participants having severe deficiency (class I and II). Differences in deficiency were observed by gender, with males having a much higher prevalence of severe deficiency (4.3% vs. 0.4%) compared to females. Male participants were 1.6 times more likely to be classified as deficient and 10.6 times more likely to be classified as severely deficient compared to females, as expected. Finally, 10.6% (85/800) of the participants were considered to be at risk for DIH. Males also had much higher rates than females (19.3% vs. 4.6%) with 4.9 times greater likelihood (p value 0.000) of having an activity level that could lead to DIH. These findings provide useful information to policymakers and clinicians who are responsible for the implementation of PQ to control and manage malaria in Haiti.

  5. Silencing the mannose 6-phosphate/IGF-II receptor differentially affects tumorigenic properties of normal breast epithelial cells.

    Science.gov (United States)

    Caixeiro, Nicole J; Martin, Janet L; Scott, Carolyn D

    2013-12-01

    Although loss of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR) in breast cancer is believed to play a role in tumorigenesis, it has not been demonstrated that M6P/IGF-IIR loss is sufficient to confer a malignant phenotype in an untransformed cell. We investigated the impact of M6P/IGF-IIR silencing using phenotypically normal (MCF-10A) and oncogenically transformed (MCF-10T, the c-Ha-ras transformed derivative of MCF-10A) human breast epithelial cell lines as model systems. In both cell lines, silencing of M6P/IGF-IIR increased cell proliferation and motility, with the effects being more pronounced in MCF-10A cells. Although anchorage-independent growth was increased by M6P/IGF-IIR silencing in MCF-10T cells, MCF-10A cells did not acquire the ability to grow in soft agar. Conversely, reduced M6P/IGF-IIR expression increased the invasive potential of MCF-10A cells, but did not enhance the already high rate of invasion of MCF-10T cells. M6P/IGF-IIR silencing had no effect on basal or IGF-II-stimulated IGF-I receptor (IGF-IR) or AKT phosphorylation in either cell line, but both were abrogated by IGF-IR kinase inhibition, which also reduced the stimulatory effect of M6P/IGF-IIR silencing on proliferation under basal and IGF-II-stimulated conditions in both cell lines. However, cell motility was neither stimulated by IGF-II nor reduced by IGF-IR inhibition, suggesting that potentiation of specific tumorigenic features in response to M6P/IGF-IIR silencing involves IGF-II- dependent and -independent mechanisms. Collectively, these data suggest that M6P/IGF-IIR silencing alone is insufficient to confer a tumorigenic phenotype, but can enhance tumorigenicity in an already transformed cell.

  6. Arg-gly-asp-mannose-6-phosphate inhibits activation and proliferation of hepatic stellate cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Lian-Sheng Wang; Ying-Wei Chen; Ding-Guo Li; Han-Ming Lu

    2006-01-01

    AIM: To investigate the effect of arg-gly-asp-mannose-6phosphate (RGD-M6P) on the activation and proliferation of primary hepatic stellate cells in vitro.METHODS: Hepatic stellate cells (HSCs) were isolated from rats by in situ collagenase perfusion of liver and 18% Nycodenz gradient centrifugation and cultured on uncoated plastic plates for 24 h with DMEM containing 10% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, HSCs were cultured in 2% FBS/DMEM with transforming growth factor β1, M6P, RGD, or RGD-M6P, respectively. Cell morphology was observed under inverted microscope, smooth muscle α-actin (α-SMA)was detected by immunocytochemistry, type Ⅲprocollagen (PCⅢ) in supernatant was determined by radioimmunoassay, and the proliferation rate of HSCs was assessed by flow cytometry.RESULTS: RGD-M6P significantly inhibited the morphological transformation and the α-SMA and PC Ⅲ expressions of HSCs in vitro and also dramatically prevented the proliferation of HSCs in vitro. Such effects were remarkably different from those of RGD or M6P.CONCLUSION: The new compound, RGD-M6P, which has a dramatic effect on primary cultured HSCs in vitro, can inhibit the transformation of HSCs in culture caused by TGFβ1, suppresses the expression of PCⅢand decreases proliferation rate of HSC. RGD-M6P can be applied as a selective drug carrier targeting at HSCs,which may be a new approach to the prevention and treatment of liver fibrosis.

  7. Effects of variant UDP-glucuronosyltransferase 1A1 gene,glucose-6-phosphate dehydrogenase deficiency and thalassemia on cholelithiasis

    Institute of Scientific and Technical Information of China (English)

    Yang-Yang Huang; Ching-Shui Huang; Sien-Sing Yang; Min-Shung Lin; May-Jen Huang; Ching-Shan Huang

    2005-01-01

    AIM: To test the hypothesis that the variant UDPglucuronosyltransferase 1A1 (UGT1A1) gene, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and thalassemia influence bilirubin metabolism and play a role in the development of cholelithiasis.METHODS: A total of 372 Taiwan Chinese with cholelithiasis who had undergone cholecystectomy and 293 healthy individuals were divided into case and control groups,respectively. PCR and restriction fragment length polymorphism were used to analyze the promoter area and nucleotides 211, 686, 1 091, and 1 456 of the UGT1A1 gene for all subjects and the gene variants for thalassemia and G6PD deficiency.RESULTS: Variation frequencies for the cholelithiasis patients were 16.1%, 25.8%, 5.4%, and 4.3% for A(TA)6TAA/A(TA)7TAA (6/7), heterozygosity within the coding region, compound heterozygosity, and homozygosity of the UGT1A1 gene, respectively. Comparing the case and control groups, a statistically significant difference in frequency was demonstrated for the homozygous variation of the UGT1A1 gene (P = 0.012, χ2 test), but not for the other variations. Further, no difference was demonstrated in a between-group comparison of the incidence of G6PD deficiency and thalassemia (2.7% vs 2.4% and 5.1% vs 5.1%, respectively). The bilirubin levels for the cholelithiasis patients with the homozygous variant-UGT1A1 gene were significantly different from the control analog (18.0±6.5 and 12.7±2.9 μmol/L, respectively; P<0.001, Student's ttest).CONCLUSION: Our results show that the homozygous variation in the UGT1A1 gene is a risk factor for the development of cholelithiasis in Taiwan Chinese.

  8. 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

  9. Frequency of Thalassemia, Iron and Glucose-6Phosphate Dehydrogenase Deficiency Among Turkish Migrating Nomad Children in Southern Iran

    Directory of Open Access Journals (Sweden)

    Mehrabani D

    2009-04-01

    Full Text Available Ferropenia and consequent iron deficiency anemia (IDA, β-thalassemia, and glucose 6-phosphate dehydrogenase (G6PD deficiency are three main common hematological problems in Iran. This study was conducted to investigate the prevalence of these problems in Turkish migrating nomads in southern Iran. From June to October 2006, the blood sample of 152 Turkish migrating nomadic children including 79 (52% males and 73 (48% females were evaluated for iron indices and G6PD deficiency in southern Iran. The family history of thalassemia, favism, and signs and symptoms related to anemia of participants were determined. RBC count, different types of Hb, Hct, MCV, MCH, MCHC, RDW, SI, TIBC and SF were measured immediately after blood sampling. Twenty-seven (17.7% children had serum ferritin (SF level <12 ng/dL, while this low serum ferritin level was similar in both genders. The low hemoglobin (Hb level had a statistical correlation with the low serum ferritin level. Among all participants, the prevalence of G6PD deficiency was 7.2% which was more frequent in males compared to females (8.9% vs. 5.5%. Seven (4.6% children had Hb  3.5 g/dL; and the prevalence of β-thalassemia trait was higher in female children compared with males (5.5% vs. 3.8%. The prevalence of IDA was 17.7%. Although this figure is less than the prevalence found in other developing countries (25-35%; but it shows that Turkish ethnic nomads in southern Iran are still behind the health statues in the industrialized countries (5-8%. The relatively high prevalence of β-thalassemia trait also is a major potential risk; and careful performance of Iranian thalassaemia program is highly suggested. It seems that G6PD deficiency is a prevalent disease in migrating Turkish nomads, and again establishment of educational programs, and investigation of dietary habits of Turkish migrating nomads on how and by whom the fava beans are consumed; seems to be a good way to prevent favism.

  10. 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.

  11. The oxidative pentose phosphate pathway in the haloarchaeon Haloferax volcanii involves a novel type of glucose-6-phosphate dehydrogenase--The archaeal Zwischenferment.

    Science.gov (United States)

    Pickl, Andreas; Schönheit, Peter

    2015-04-28

    The oxidative pentose phosphate pathway (OPPP), catalyzing the oxidation of glucose-6-phosphate to ribulose-5-phosphate is ubiquitous in eukarya and bacteria but has not yet been reported in archaea. In haloarchaea a putative 6-phosphogluconate dehydrogenase (6PGDH) is annotated, whereas a gene coding for glucose-6-phosphate dehydrogenase (Glc6PDH) could not be identified. Here we report the purification and characterization of a novel type of Glc6PDH in Haloferax volcanii that is not related to bacterial and eukaryal Glc6PDHs and the encoding gene is designated as azf (archaeal zwischenferment). Further, recombinant H. volcanii 6PGDH was characterized. Deletion mutant analyses indicate that both, Glc6PDH and 6PGDH, are functionally involved in pentose phosphate formation in vivo. This is the first report on the operation of the OPPP in the domain of archaea.

  12. Modulation effect of blu-ray irradiation combined with comprehensive therapy on serum indexes of neonatal erythrocyte glucose-6-phosphate dehydrogenase deficiency-induced hyperbilirubinemia

    Institute of Scientific and Technical Information of China (English)

    Xuan Yang

    2016-01-01

    Objective:To study the modulation effect of blu-ray irradiation combined with comprehensive therapy on serum indexes of neonatal erythrocyte glucose-6-phosphate dehydrogenase deficiency-induced hyperbilirubinemia.Methods:A total of42 cases of neonates with erythrocyte glucose-6-phosphate dehydrogenase deficiency-induced hyperbilirubinemia were chosen for study and randomly divided into observation group (n=21) and control group (n=21). Observation group received blu-ray irradiation combined with comprehensive treatment and control group only received routine treatment. Then bilirubin levels, bilirubin encephalopathy condition, anemia condition and oxidative stress degree of two groups were compared. Results:12 h, 24 h and 48 h after treatment, serum TBIL, DBIL, IBIL, Hb, GSH and CAT contents of both groups showed decreasing trend and MDA contents showed increasing trend; serum TBIL, DBIL, IBIL, Hb, GSH and CAT contents of observation group were lower than those of control group and MDA contents were higher than those of control group. 6 d, 7 d and 8 d after treatment, serum S100β and NSE contents of both groups showed decreasing trend and serum S100β and NSE contents of observation group were lower than those of control group.Conclusion:Blu-ray irradiation combined with comprehensive therapy helps to reduce bilirubin levels of neonatal erythrocyte glucose-6-phosphate dehydrogenase deficiency-induced hyperbilirubinemia and protect nerve function, but it will aggravate anemia condition and oxidative stress degree, and needs attention and intervention in clinical practice.

  13. Cyclic 3′,5′-Adenosine Monophosphate and N-Acetyl-glucosamine-6-Phosphate as Regulatory Signals in Catabolite Repression of the lac Operon in Escherichia coli1

    Science.gov (United States)

    Goldenbaum, Paul E.; Broman, Rodney L.; Dobrogosz, Walter J.

    1970-01-01

    When an Escherichia coli mutant lacking the enzyme N-acetyl-glucosamine-6-phosphate (AcGN6P) deacetylase is grown in a succinate-mineral salts medium and exposed to an exogenous source of N-acetylglucosamine, approximately 20 to 30 pmoles of AcGN6P per μg of cell dry weight will accumulate in these cells. This accumulation occurs within 2 to 4 min after the addition of N-acetylglucosamine and is coincident with the production of a severe permanent catabolite repression of β-galactosidase synthesis. This repression does not occur if adenosine 3′,5′-cyclic phosphate (cyclic AMP) is added to the cells before AcGN6P accumulates. An immediate derepression occurs when cyclic AMP is added to cells that have already accumulated a large AcGN6P pool. These findings are consistent with the view that low-molecular-weight carbohydrate metabolites and cyclic AMP play key roles in the catabolite repression phenomenon, and that metabolites such as AcGN6P may participate in the represion mechanism by influencing either the formation or degradation of cyclic AMP in E. coli. PMID:4319836

  14. Trametes versicolor carboxylate reductase uncovered

    OpenAIRE

    Winkler, Margit; Winkler, Christoph K.

    2016-01-01

    Abstract The first carboxylate reductase from Trametes versicolor was identified, cloned, and expressed in Escherichia coli. The enzyme reduces aromatic acids such as benzoic acid and derivatives, cinnamic acid, and 3-phenylpropanoic acid, but also aliphatic acids such as octanoic acid are reduced. Graphical abstract

  15. 高等植物葡萄糖-6-磷酸脱氢酶的研究进展%Research progress in glucose-6-phosphate dehydrogenase in higher plants

    Institute of Scientific and Technical Information of China (English)

    于定群; 汤浩茹; 张勇; 罗娅; 刘泽静

    2012-01-01

    Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting step of the oxidative pentose phosphate pathway, existing in both cytosolic and plastidic compartments of higher plants. Its main function is to provide reducing power (NADPH) and pentose phosphates for reductive biosynthesis and maintenance of the redox state of the cell. In addition, the expression of this enzyme is related to different biotic and abiotic stresses. In this review, we analyzed the isoenzyme, regulation and biological function of G6PDH. Meanwhile, we summarized the progress work of G6PDH involved in stress resistance, gene cloning, enzyme-deficiency and cluster analysis. Problems should be solved were also discussed.%葡萄糖-6-磷酸脱氢酶是植物戊糖磷酸途径中的一个关键性调控酶.其主要生理功能是产生供生物合成需要的NADPH及一些中间产物;此外还参与各种生物和非生物胁迫的应答反应.文中主要从葡萄糖-6-磷酸脱氢酶同工酶与调节机制等方面探讨了其生物学功能,再从胁迫耐受、基因克隆、酶的缺失和替代等方面的研究进行综述,并对已发表的高等植物中的G6PDH氨基酸序列进行聚类分析,为今后该酶的研究提供参考.

  16. 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].

  17. High Level Expression of Glucose-6-phosphate Dehydrogenase Gene PsG6PDH from Populus suaveolens in E. coli

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    In order to investigate the functions of the gene PsG6PDH and the mechanisms underlying freezing tolerance of Populus suaveolens, the recombinant expression vector pET-G (pET30a-G6PDH), which contained full encoding region of PsG6PDH gene, was established. The recombinant was identified by lawn-PCR and double enzyme digestion and then transformed into expression host XA90 and induced by isopropyl-a-D-thiogalactoside (IPTG) to express 100 kD polypeptide of G6PDH fusion protein. The results showed that the expressed amount of the fusion protein culminated after 1 mmol·L-1 IPTG treatment for 4 h and that pET-G product was predominately soluble and not extra-cellular secreting.

  18. Silencing of Entamoeba histolytica Glucosamine 6-Phosphate Isomerase by RNA Interference Inhibits the Formation of Cyst-Like Structures

    Directory of Open Access Journals (Sweden)

    Hugo Aguilar-Díaz

    2013-01-01

    Full Text Available Encystment is an essential process in the biological cycle of the human parasite Entamoeba histolytica. In the present study, we evaluated the participation of E. histolytica Gln6Pi in the formation of amoeba cyst-like structures by RNA interference assay. Amoeba trophozoites transfected with two Gln6Pi siRNAs reduced the expression of the enzyme in 85%, which was confirmed by western blot using an anti-Gln6Pi antibody. The E. histolytica Gln6Pi knockdown with the mix of both siRNAs resulted in the loss of its capacity to form cyst-like structures (CLSs and develop a chitin wall under hydrogen peroxide treatment, as evidenced by absence of both resistance to detergent treatment and calcofluor staining. Thus, only 5% of treated trophozoites were converted to CLS, from which only 15% were calcofluor stained. These results represent an advance in the understanding of chitin biosynthesis in E. histolytica and provide insight into the encystment process in this parasite, which could allow for the developing of new control strategies for this parasite.

  19. 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.

  20. Enhanced freeze tolerance of baker's yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough.

    Science.gov (United States)

    Tan, Haigang; Dong, Jian; Wang, Guanglu; Xu, Haiyan; Zhang, Cuiying; Xiao, Dongguang

    2014-08-01

    Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker's yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301(TPS1) overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301(TPS1) were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301(TPS1) was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker's yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker's yeast.

  1. Interchangeable but Essential Functions of SNX1 and SNX2 in the Association of Retromer with Endosomes and the Trafficking of Mannose 6-Phosphate Receptors▿ †

    Science.gov (United States)

    Rojas, Raul; Kametaka, Satoshi; Haft, Carol R.; Bonifacino, Juan S.

    2007-01-01

    The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function. PMID:17101778

  2. Glucose-6-phosphate dehydrogenase from brewers' yeast. The effects of pH and temperature on the steady-state kinetic parameters of the two-chain protein species.

    Science.gov (United States)

    Kuby, S A; Roy, R N

    1976-05-04

    A systematic study has been made of the pH- and temperature-dependency of the steady-state kinetic parameters of the stabilized two-subunit enzyme species of glucose-6-phosphate dehydrogenase, in the absence of superimposed association-dissociation reactions. The Vmax(app) data obtained in several buffers between pH 5 and 10 and at 18-32 degrees C lead to the postulate that at least two sets of protonic equilibria may govern the catalysis (one near pH 5.7 AT 25 DEGREES C and another near pH 9.2); furthermore, two pathways for product formation (i.e., two Vmax's) appear to be required to explain the biphasic nature of the log Vmax(app) vs. pH curves, with Vmax(basic) greater than Vmax(acidic + neutral). Of the several buffers explored, either a uniform degree of interaction or a minimal degree of buffer species interaction could be assessed from the enthalpy changes associated with the derived values for ionization constants attributed to the protonic equilibria in the enzyme-substrates ternary complexes for the case of Tris-acetate-EDTA buffers, at constant ionic strength. With the selection of this buffer at 0.1 (T/2) and at 25 and 32 degrees C, a self-consistent kinetic mechanism has emerged which allows for the random binding of the two fully ionized substrates to the enzyme via two major pathways, and product formation by both E-A--B- and HE-A--B-. As before (Kuby et al. Arch. Biochem, Biophys. 165, 153-178, 1974), a quasi-equilibrium is presumed, with rate-limiting steps (k + 5 and k + 5') at the interconversion of the ternary complexes. Values for the two sets of protonic equilibria defined by this mechanism (viz., pKk, pKH2 for the first ionizations, and pKk', pKH' for the second) could then be estimated. From their numerical values (e.g., at 25 degrees C: pKK = 5.7 PKH2 = 5.2; and pKK' = 9.1, PKH' = 8.2) and from the values for delta H degrees ioniz (e.g., delta H degrees pKK APPROXIMATELY 5.1 KCAL/MOL; DELTA H degrees pKK' APPROXIMATELY 11 KCAL/MOL), A

  3. Analysis of mucolipidosis II/III GNPTAB missense mutations identifies domains of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase involved in catalytic function and lysosomal enzyme recognition.

    Science.gov (United States)

    Qian, Yi; van Meel, Eline; Flanagan-Steet, Heather; Yox, Alex; Steet, Richard; Kornfeld, Stuart

    2015-01-30

    UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits.

  4. Uncovering Topological Structures in Unstructured Data

    Science.gov (United States)

    2015-04-20

    AFRL-OSR-VA-TR-2015-0091 Uncovering Topological Structures in Unstructured Data Keith Bowman ILLINOIS INSTITUTE OF TECHNOLOGY Final Report 04/20/2015...COVERED (From - To)      01-05-2012 to 30-04-2015 4.  TITLE AND SUBTITLE Uncovering Topological Structures in Unstructured Data 5a.  CONTRACT NUMBER 5b...scanned point-cloud data . It has two stages. In the first stage, we analyzed scan data and extracted topologically critical points. We used these critical

  5. Risks of hemolysis in glucose-6-phosphate dehydrogenase deficient infants exposed to chlorproguanil-dapsone, mefloquine and sulfadoxine-pyrimethamine as part of intermittent presumptive treatment of malaria in infants

    DEFF Research Database (Denmark)

    Poirot, Eugenie; Vittinghoff, Eric; Ishengoma, Deus;

    2015-01-01

    BACKGROUND: Chlorproguanil-dapsone (CD) has been linked to hemolysis in symptomatic glucose-6-phosphate dehydrogenase deficient (G6PDd) children. Few studies have explored the effects of G6PD status on hemolysis in children treated with Intermittent Preventive Treatment in infants (IPTi) antimala......BACKGROUND: Chlorproguanil-dapsone (CD) has been linked to hemolysis in symptomatic glucose-6-phosphate dehydrogenase deficient (G6PDd) children. Few studies have explored the effects of G6PD status on hemolysis in children treated with Intermittent Preventive Treatment in infants (IPTi...

  6. 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.

  7. Partial purification of glucose-6-phosphate dehydrogenase by aqueous two-phase poly(ethyleneglycol/phosphate systems Purificação parcial de glucose-6-fosfato desidrogenase por sistemas de duas fases aquosas poli (etilenoglicol/fosfato

    Directory of Open Access Journals (Sweden)

    Marcela Zanella Ribeiro

    2007-03-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PDH is an important enzyme used in biochemical and medical studies and in several analytical methods that have industrial and commercial application. This work evaluated the extraction of G6PDH in aqueous two-phase system (ATPS of poly(ethyleneglycol (PEG/phosphate buffer, using as enzyme source a medium prepared through commercial baker's yeast disruption. Firstly, the effects of PEG molar mass on the enzyme partition and of homogenization and rest on the system equilibrium were investigated. Afterwards, several ATPS were prepared using statistical analysis (2² factorial design. The results, including kinetic and thermodynamic parameters for the G6PDH activity, showed partial purification of this enzyme in ATPS composed of 17.5% (w/w PEG400 and 15.0% (w/w phosphate. A high enzymatic recovery value (97.7%, a high partition coefficient (351, and an acceptable purification factor (2.28 times higher than in cell homogenate were attained from the top phase. So, it was possible to attain an effective enzyme pre-purification by separating some contaminants with a simple method such as liquid-liquid extraction in aqueous two-phase systems (ATPS.Glicose-6-fosfato desidrogenase (G6PDH é uma importante enzima usada em estudos bioquímicos e médicos, bem como em diversos métodos analíticos com aplicação comercial e industrial. Neste trabalho foi avaliado a extração da G6PDH em sistemas de duas fases aquosas (ATPS constituídos por poli(etilenoglicol (PEG/tampão fosfato, usando como fonte de enzima um meio preparado por rompimento de leveduras de panificação comercial. Inicialmente foram investigados os efeitos da massa molar do PEG na partição da enzima e da homogeneização e repouso no equilíbrio do sistema. Na sequência, diversos ATPS foram preparados usando análise estatística (planejamento fatorial 2². Os resultados, incluindo parâmetros cinéticos e termodinâmicos para a atividade da G6PDH

  8. A role of Rab29 in the integrity of the trans-Golgi network and retrograde trafficking of mannose-6-phosphate receptor.

    Directory of Open Access Journals (Sweden)

    Shicong Wang

    Full Text Available Rab29 (also referred as Rab7L1 is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN, and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR, and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR or calcium independent M6PR (ciM6PR, but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G. Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs.

  9. A Role of Rab29 in the Integrity of the Trans-Golgi Network and Retrograde Trafficking of Mannose-6-Phosphate Receptor

    Science.gov (United States)

    Wang, Shicong; Ma, Zexu; Xu, Xiaohui; Wang, Zhen; Sun, Lixiang; Zhou, Yunhe; Lin, Xiaosi; Hong, Wanjin; Wang, Tuanlao

    2014-01-01

    Rab29 (also referred as Rab7L1) is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN), and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR), and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR) or calcium independent M6PR (ciM6PR), but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G). Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs. PMID:24788816

  10. Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

    Institute of Scientific and Technical Information of China (English)

    Lin Yuan-zhen; Lin Shan-zhi; Zhang Wei; Zhang Qian; Zhang Zhi-yi; Guo Huan

    2005-01-01

    A 1207 hp cDNA fragment (PsG6PDH) was amplified by PT-PCR from cold-induced total Pna of the freexing-tolerant P. Suaveolens, using primers based on the highly comserved region of published plant glucose-6-phosphate dehydrogenase (G6PDH)genes. The sepuence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted aminoacid residues. Moreover, the nucleotide sequence of psG6PDH showed 83%,82%,79%,79% and 78% identity, and the derived amino acid sequence shared 44.2%,44.7%,42.0%,40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana tabacum, Triticum aestivum, Oryxa sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to cytosolic G6PDH gene. This is the first report on clonign of the G6PDH gene from woody plants.

  11. The role of reduced glutathione during the course of acute haemolysis in glucose-6-phosphate dehydrogenase deficient patients: clinical and pharmacodynamic aspects.

    Science.gov (United States)

    Corbucci, G G

    1990-01-01

    Tissue hypoperfusion leads to cellular oxidative and peroxidative damage due to biochemical disorders in the oxygen and substrate metabolism. The metabolic turnover of glutathione (GSH) represents one the main cytoprotective systems against the peroxide attack and the depletion or defect in resynthesis of this compound is accompanied by pathological consequences. In the present study the clinical effects of glutathione depletion were investigated in conditions of acute tissue hypoxia due to marked haemolysis in glucose-6-phosphate dehydrogenase deficient patients (favism syndrome). In these subjects a significant marker of the tissue oxidative damage was represented by the uric acid blood levels, presumably linked to xanthine-hypoxanthine altered metabolism. To antagonize the effects of oxyradical pathology, reduced glutathione was administered to a group of patients and the results confirmed the cytoprotective role played by the GSH supplementation. The GSH action was evident on the tissue metabolism and this supports the opinion that reduced glutathione could represent a new and interesting therapeutic approach in marked and acute hypoxic conditions.

  12. Insulin-like growth factors (IGFs) stimulate the release of alpha 1-antichymotrypsin and soluble IGF-II/mannose 6-phosphate receptor from MCF7 breast cancer cells.

    Science.gov (United States)

    Confort, C; Rochefort, H; Vignon, F

    1995-09-01

    The growth of hormone-responsive MCF7 human breast cancer cells is controlled by steroid hormones and growth factors. By metabolic labeling of cells grown in steroid- and growth factor-stripped serum conditions, we show that insulin-like growth factors (IGF-I and IGF-II) increase by approximately 5-fold the release of several proteins including cathepsin D, alpha 1-antichymotrypsin, and soluble forms of the multifunctional IGF-II/mannose 6-phosphate (M6P) receptor. Two soluble forms of IGF-II/M6P receptors were detected, one major (approximately 260 kilodaltons) and one minor (approximately 85 kilodaltons) that probably represents a proteolytic fragment of the larger soluble molecule. IGFs increased receptor release in a dose-dependent fashion with 50-60% of newly synthesized receptor released at 5-10 nM IGFs. The release of IGF-II/M6P receptors correlated with the levels of secreted cathepsin D in different human breast cancer cells or in rats stable transfectants that are constitutively expressing variable levels of human cathepsin D. IGFs had a stronger effect on IGF-II/M6P receptor release, whereas estradiol treatment preferentially enhanced the release of protease and antiprotease. We thus demonstrate that in human breast cancer cells, IGFs not only act as strong mitogens but also regulate release of alpha 1-antichymotrypsin, IGF-II/M6P-soluble receptor, and cathepsin D; three proteins that potentially regulate cell proliferation and/or invasion.

  13. Ethanol-induced yeast flocculation directed by the promoter of TPS1 encoding trehalose-6-phosphate synthase 1 for efficient ethanol production.

    Science.gov (United States)

    Li, Qian; Zhao, Xin-Qing; Chang, Alan K; Zhang, Qiu-Mei; Bai, Feng-Wu

    2012-01-01

    Yeast flocculation is an important trait in the brewing industry as well as in ethanol production, through which biomass can be recovered by cost-effective sedimentation. However, mass transfer limitation may affect yeast growth and ethanol fermentation if the flocculation occurs earlier before fermentation is completed. In this article, a novel type of cell-cell flocculation induced by trehalose-6-phosphate synthase 1 (TPS1) promoter was presented. The linear cassette HO-P(TPS1)-FLO1(SPSC01)-KanMX4-HO was constructed to transform the non-flocculating industrial yeast S. cerevisiae 4126 by chromosome integration to obtain a new flocculating yeast strain, ZLH01, whose flocculation was induced by ethanol produced during fermentation. The experimental results illustrated that flocculation of ZLH01 was triggered by 3% (v/v) ethanol and enhanced as ethanol concentration increased till complete flocculation was achieved at ethanol concentration of 8% (v/v). Real time PCR analysis confirmed that the expression of FLO1(SPSC01) was dependent on ethanol concentration. The growth and ethanol fermentation of ZLH01 were improved significantly, compared with the constitutive flocculating yeast BHL01 engineered with the same FLO gene but directed by the constitutive 3-phosphoglycerate kinase promoter PGK1, particularly under high temperature conditions. These characteristics make the engineered yeast more suitable for ethanol production from industrial substrates under high gravity and temperature conditions. In addition, this strategy offers advantage in inducing differential expression of other genes for metabolic engineering applications of S. cerevisiae.

  14. 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 < 18 years (51 males, 83 females). Low serum ferritin (< 12 ng/dL) was present in 17.9% of children (21.7% in females and 11.8% in males). Low haemoglobin (Hb) correlated significantly with a low serum ferritin. Only 1 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.

  15. Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model.

    Science.gov (United States)

    Spencer, Netanya Y; Yan, Ziying; Cong, Le; Zhang, Yulong; Engelhardt, John F; Stanton, Robert C

    2016-02-01

    Studies to determine subcellular localization and translocation of proteins are important because subcellular localization of proteins affects every aspect of cellular function. Such studies frequently utilize mutagenesis to alter amino acid sequences hypothesized to constitute subcellular localization signals. These studies often utilize fluorescent protein tags to facilitate live cell imaging. These methods are excellent for studies of monomeric proteins, but for multimeric proteins, they are unable to rule out artifacts from native protein subunits already present in the cells. That is, native monomers might direct the localization of fluorescent proteins with their localization signals obliterated. We have developed a method for ruling out such artifacts, and we use glucose 6-phosphate dehydrogenase (G6PD) as a model to demonstrate the method's utility. Because G6PD is capable of homodimerization, we employed a novel approach to remove interference from native G6PD. We produced a G6PD knockout somatic (hepatic) cell line using CRISPR-Cas9 mediated genome engineering. Transfection of G6PD knockout cells with G6PD fluorescent mutant proteins demonstrated that the major subcellular localization sequences of G6PD are within the N-terminal portion of the protein. This approach sets a new gold standard for similar studies of subcellular localization signals in all homodimerization-capable proteins.

  16. Uncovering student ideas in physical science

    CERN Document Server

    Keeley, Page

    2014-01-01

    If you and your students can't get enough of a good thing, Volume 2 of Uncovering Student Ideas in Physical Science is just what you need. The book offers 39 new formative assessment probes, this time with a focus on electric charge, electric current, and magnets and electromagnetism. It can help you do everything from demystify electromagnetic fields to explain the real reason balloons stick to the wall after you rub them on your hair.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Nine Different Glucose-6-phosphate Dehydrogenase (G6PD Variants in a Malaysian Population with Malay, Chinese, Indian and Orang Asli (Aboriginal Malaysian Backgrounds

    Directory of Open Access Journals (Sweden)

    Isa,Zaleha Mohamed

    2008-10-01

    Full Text Available The Malaysian people consist of several ethnic groups including the Malay, the Chinese, the Indian and the Orang Asli (aboriginal Malaysians. We collected blood samples from outpatients of 2 hospitals in the State of Selangor and identified 27 glucose-6-phosphate dehydrogenase (G6PD-deficient subjects among these ethnic groups. In the Malay, G6PD Viangchan (871G>A, 1311C>T, IVS11 nt93T>C and G6PD Mahidol (487G>A types, which are common in Cambodia and Myanmar, respectively, were detected. The Malay also had both subtypes of G6PD Mediterranean:the Mediterranean subtype (563C>T, 1311C>T, IVS11 nt93T>C and the Indo-Pakistan subtype (563C>T, 1311C, IVS11 nt93T. In Malaysians of Chinese background, G6PD Kaiping (1388G>A, G6PD Canton (1376G>T and G6PD Gaohe (95A>G, which are common in China, were detected. Indian Malaysians possessed G6PD Mediterranean (Indo-Pakistan subtype and G6PD Namoru (208T>C, a few cases of which had been reported in Vanuatu and many in India. Our findings indicate that G6PD Namoru occurs in India and flows to Malaysia up to Vanuatu. We also discovered 5 G6PD-deficient cases with 2 nucleotide substitutions of 1311C>T and IVS11 nt93T>C, but without amino-acid substitution in the G6PD molecule. These results indicate that the Malaysian people have incorporated many ancestors in terms of G6PD variants.

  19. Endocytosis of receptor-bound insulin-like growth factor II is enhanced by mannose-6-phosphate in IM9 cells.

    Science.gov (United States)

    Polychronakos, C; Piscina, R

    1988-12-01

    The insulin-like growth factor II (IGF-II), and glycoproteins containing mannose 6-phosphate (M6P), bind to two different sites of the same receptor molecule (Morgan et al, Nature 329:301, 1987). To study the interactions between the two ligands on their common receptor in intact cells, we examined the effect of free M6P on IGF-II binding and endocytosis in the IM9 human lymphoblastoid cell line. M6P, up to a 3 mM concentration, had no effect on the binding of IGF-II to the cell surface receptor of intact IM9 cells at 4 degrees C. By contrast, when IM9 cells were incubated with 125I-IGF-II at 37 degrees C, 1 mM M6P increased cell-associated radioactivity by twofold. The increase was resistant to acid wash at 4 degrees C, and therefore assumed to represent endocytosed IGF-II. Acid-washable radioactivity was no different, confirming that, in intact cells, M6P does not affect IGF-II surface binding. In addition, preincubation of cells with M6P at 37 degrees C for up to 3 hours did not change the abundance of receptor on the cell surface, as measured by a subsequent 4 degrees C binding assay. We conclude that M6P causes a shift of IGF-II-occupied receptors form the cell surface to intracellular locations without affecting surface binding of this ligand in IM9 cells. The effect could be produced by the binding of M6P itself, or by the displacement of endogenous phosphomannosylated ligands.

  20. A cationic-independent mannose 6-phosphate receptor inhibitor (PXS64 ameliorates kidney fibrosis by inhibiting activation of transforming growth factor-β1.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    Full Text Available The activity of transforming growth factor-β1 (TGF-β1 is regulated by its conversion from the latent to the active form. We have previously shown that the conversion is at least in part mediated by the cationic-independent mannose 6-phosphate receptor (CI-M6PR, as the CI-M6PR inhibitor, PXS-25 has anti-fibrotic properties in human kidney tubular (HK-2 cells under high glucose conditions. However, its clinical use is limited by low bioavailability. Our aim was to determine the effects of PXS64, a pro-drug of PXS25, in in vitro and in vivo models of renal fibrosis. HK-2 cells were exposed to latent TGFβ1+/- PXS64 for 48 hours. The mRNA and protein levels of pro-fibrotic and pro-inflammatory markers were determined. A 7 day unilateral ureteric obstruction (UUO model was used and the following experimental groups were studied: (i Sham operated, (ii UUO, (iii UUO + telmisartan (iv UUO + PSX64. HK-2 cells exposed to PXS64 reduced TGFβ mediated effects on collagen IV, fibronectin, macrophage chemotactic protein-1 (MCP-1 and phospho-smad2 protein expression, consistent with inhibition of the conversion of latent to active TGF-β1. PXS 64 treated UUO mice had a lower tubulointerstitial fibrosis index, collagen IV and fibronectin protein and mRNA expression when compared to untreated UUO mice. In addition, these animals had lower MCP-1 mRNA expression, reduced inflammarory cell infiltrate, as indicated by fewer CD45, F4/80 positive cells, and reduced phospho-Smad2 protein expression when compared to untreated UUO animals. Our data demonstrates that PSX64 is an effective anti-fibrotic agent by inhibiting the activation of latent TGF-β1.

  1. Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

    Science.gov (United States)

    Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

    2015-01-01

    Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

  2. Hepatitis C virus host cell interactions uncovered

    DEFF Research Database (Denmark)

    Gottwein, Judith; Bukh, Jens

    2007-01-01

      Insights into virus-host cell interactions as uncovered by Randall et al. (1) in a recent issue of PNAS further our understanding of the hepatitis C virus (HCV) life cycle, persistence, and pathogenesis and might lead to the identification of new therapeutic targets. HCV persistently infects 180...... million individuals worldwide, causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The only approved treatment, combination therapy with IFN- and ribavirin, targets cellular pathways (2); however, a sustained virologic response is achieved only in approximately half of the patients...... treated. Therefore, there is a pressing need for the identification of novel drugs against hepatitis C. Although most research focuses on the development of HCV-specific antivirals, such as protease and polymerase inhibitors (3), cellular targets could be pursued and might allow the development of broad...

  3. Uncovering the Hidden Decisions that Shape Curricula

    Science.gov (United States)

    Harlow, Danielle Boyd

    2010-10-01

    Developing explanatory models is a central practice to scientific inquiry. When students create and test explanatory models for scientific phenomenon, they develop content knowledge, knowledge of the nature of science, and creative thinking skills. Unfortunately, such instruction rarely occurs in K-12 science. This is, in part, because teachers do not have the opportunity to develop sophisticated understandings of the process of modeling, but also because teaching in this way requires teachers to make real-time instructional decisions that are responsive to students' ideas. This is challenging for teachers, especially because this decision process is often invisible. In this talk, I will highlight the importance of providing opportunities for sophisticated science thinking for our youngest learners and consider how uncovering the decisions that shape physics courses for teachers may benefit their future students.

  4. Glucose-6-phosphate dehydrogenase deficiency and the risk of malaria and other diseases in children in Kenya: a case-control and a cohort study

    Science.gov (United States)

    Uyoga, Sophie; Ndila, Carolyne M; Macharia, Alex W; Nyutu, Gideon; Shah, Shivang; Peshu, Norbert; Clarke, Geraldine M; Kwiatkowski, Dominic P; Rockett, Kirk A; Williams, Thomas N

    2015-01-01

    Summary Background The global prevalence of X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is thought to be a result of selection by malaria, but epidemiological studies have yielded confusing results. We investigated the relationships between G6PD deficiency and both malaria and non-malarial illnesses among children in Kenya. Methods We did this study in Kilifi County, Kenya, where the G6PD c.202T allele is the only significant cause of G6PD deficiency. We tested the associations between G6PD deficiency and severe and complicated Plasmodium falciparum malaria through a case-control study of 2220 case and 3940 control children. Cases were children aged younger than 14 years, who visited the high dependency ward of Kilifi County Hospital with severe malaria between March 1, 1998, and Feb 28, 2010. Controls were children aged between 3–12 months who were born within the same study area between August 2006, and September 2010. We assessed the association between G6PD deficiency and both uncomplicated malaria and other common diseases of childhood in a cohort study of 752 children aged younger than 10 years. Participants of this study were recruited from a representative sample of households within the Ngerenya and Chonyi areas of Kilifi County between Aug 1, 1998, and July 31, 2001. The primary outcome measure for the case-control study was the odds ratio for hospital admission with severe malaria (computed by logistic regression) while for the cohort study it was the incidence rate ratio for uncomplicated malaria and non-malaria illnesses (computed by Poisson regression), by G6PD deficiency category. Findings 2863 (73%) children in the control group versus 1643 (74%) in the case group had the G6PD normal genotype, 639 (16%) versus 306 (14%) were girls heterozygous for G6PD c.202T, and 438 (11%) versus 271 (12%) children were either homozygous girls or hemizygous boys. Compared with boys and girls without G6PD deficiency, we found significant

  5. Pectic enzymes

    NARCIS (Netherlands)

    Benen, J.A.E.; Voragen, A.G.J.; Visser, J.

    2003-01-01

    The pectic enzymes comprise a diverse group of enzymes. They consist of main-chain depolymerases and esterases active on methyl- and acetylesters of galacturonosyl uronic acid residues. The depolymerizing enzymes comprise hydrolases as wel as lyases

  6. Enzyme assays.

    Science.gov (United States)

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  7. 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

  8. 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

  9. Platelet enzyme abnormalities in leukemias

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    S Sharma

    2011-01-01

    Full Text Available Aim of the Study: The aim of this study was to evaluate platelet enzyme activity in cases of leukemia. Materials and Methods: Platelet enzymes glucose-6-phosphate dehydrogenase (G6PD, pyruvate kinase (PK and hexokinase (HK were studied in 47 patients of acute and chronic leukemia patients, 16 patients with acute myeloid leukemia (AML(13 relapse, three in remission, 12 patients with acute lymphocytic leukemia (ALL (five in relapse, seven in remission, 19 patients with chronic myeloid leukemia (CML. Results: The platelet G6PD activity was significantly low in cases of AML, ALL and also in CML. G6PD activity was normalized during AML remission. G6PD activity, although persistently low during ALL remission, increased significantly to near-normal during remission (P < 0.05 as compared with relapse (P < 0.01. Platelet PK activity was high during AML relapse (P < 0.05, which was normalized during remission. Platelet HK however was found to be decreased during all remission (P < 0.05. There was a significant positive correlation between G6PD and PK in cases of AML (P < 0.001 but not in ALL and CML. G6PD activity did not correlate with HK activity in any of the leukemic groups. A significant positive correlation was however seen between PK and HK activity in cases of ALL remission (P < 0.01 and CML (P < 0.05. Conclusions: Both red cell and platelet enzymes were studied in 36 leukemic patients and there was no statistically significant correlation between red cell and platelet enzymes. Platelet enzyme defect in leukemias suggests the inherent abnormality in megakaryopoiesis and would explain the functional platelet defects in leukemias.

  10. Cobalamins uncovered by modern electronic structure calculations

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta; Ryde, Ulf

    2009-01-01

    This review describes how computational methods have contributed to the held of cobalamin chemistry since the start of the new millennium. Cobalamins are cobalt-dependent cofactors that are used for alkyl transfer and radical initiation by several classes of enzymes. Since the entry of modern...

  11. Uncovering the architecture of action semantics.

    Science.gov (United States)

    Watson, Christine E; Buxbaum, Laurel J

    2014-10-01

    Despite research suggesting that stored sensorimotor information about tool use is a component of the semantic representations of tools, little is known about the action features or organizing principles that underlie this knowledge. We used methods similar to those applied in other semantic domains to examine the "architecture" of action semantic knowledge. In Experiment 1, participants sorted photographs of tools into groups according to the similarity of their associated "use" actions and rated tools on dimensions related to action. The results suggest that the magnitude of arm movement, configuration of the hand, and manner of motion during tool use play a role in determining how tools cluster in action "semantic space." In Experiment 2, we validated the architecture uncovered in Experiment 1 using an implicit semantic task for which tool use knowledge was not ostensibly relevant (blocked cyclic word-picture matching). Using stimuli from Experiment 1, we found that participants performed more poorly during blocks of trials containing tools used with similar versus unrelated actions, and the amount of semantic interference depended on the magnitude of action similarity among tools. Thus, the degree of featural overlap between tool use actions plays a role in determining the overall semantic similarity of tools.

  12. Alterations in energy/redox metabolism induced by mitochondrial and environmental toxins: a specific role for glucose-6-phosphate-dehydrogenase and the pentose phosphate pathway in paraquat toxicity.

    Science.gov (United States)

    Lei, Shulei; Zavala-Flores, Laura; Garcia-Garcia, Aracely; Nandakumar, Renu; Huang, Yuting; Madayiputhiya, Nandakumar; Stanton, Robert C; Dodds, Eric D; Powers, Robert; Franco, Rodrigo

    2014-09-19

    Parkinson's disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat "hijacks" the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in

  13. Uncovering Wolbachia diversity upon artificial host transfer.

    Directory of Open Access Journals (Sweden)

    Daniela I Schneider

    Full Text Available The common endosymbiotic Wolbachia bacteria influence arthropod hosts in multiple ways. They are mostly recognized for their manipulations of host reproduction, yet, more recent studies demonstrate that Wolbachia also impact host behavior, metabolic pathways and immunity. Besides their biological and evolutionary roles, Wolbachia are new potential biological control agents for pest and vector management. Importantly, Wolbachia-based control strategies require controlled symbiont transfer between host species and predictable outcomes of novel Wolbachia-host associations. Theoretically, this artificial horizontal transfer could inflict genetic changes within transferred Wolbachia populations. This could be facilitated through de novo mutations in the novel recipient host or changes of haplotype frequencies of polymorphic Wolbachia populations when transferred from donor to recipient hosts. Here we show that Wolbachia resident in the European cherry fruit fly, Rhagoletis cerasi, exhibit ancestral and cryptic sequence polymorphism in three symbiont genes, which are exposed upon microinjection into the new hosts Drosophila simulans and Ceratitis capitata. Our analyses of Wolbachia in microinjected D. simulans over 150 generations after microinjection uncovered infections with multiple Wolbachia strains in trans-infected lines that had previously been typed as single infections. This confirms the persistence of low-titer Wolbachia strains in microinjection experiments that had previously escaped standard detection techniques. Our study demonstrates that infections by multiple Wolbachia strains can shift in prevalence after artificial host transfer driven by either stochastic or selective processes. Trans-infection of Wolbachia can claim fitness costs in new hosts and we speculate that these costs may have driven the shifts of Wolbachia strains that we saw in our model system.

  14. Cloning and Bioinformatic Analysis of Glucose-6-phosphate 1-dehydrogenase Gene(gsdA) from Aspergillus oryzae%米曲霉6-磷酸葡萄糖脱氢酶基因 gsdA 的克隆及生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    刘薇; 吴晶晶; 陈宏文

    2012-01-01

      6-磷酸葡萄糖脱氢酶催化6-磷酸葡萄糖生成6-磷酸葡萄糖酸,并生成NADPH,是微生物胞内磷酸戊糖途径(PPP)的关键酶。本研究以食品安全菌米曲霉CICC2012为材料,克隆获得6-磷酸葡萄糖脱氢酶基因(GenBank登录号:JN123468)。序列分析表明,该酶是由222个氨基酸组成的亲水性蛋白;128~134位氨基酸序列DHYLGKE为活性区域;170~176位氨基酸序列GTEGRGG可能为辅因子结合位点。进化树分析表明,米曲霉6-磷酸葡萄糖脱氢酶同其他丝状真菌及酵母的G6PDH较相似%  Glucose-6-phosphate 1-dehydrogenase(G6PDH) is one of the key enzymes in pentose phosphate pathway(PPP). Here, the gene encoding G6PDH is cloned from Aspergillus oryzae CICC2012. This gene was sequenced and submitted to GenBank(accession number: JN123468). The sequence was analyzed bioinformatically. The results show that G6PDH from A. oryzae is a hydrophilic enzyme consisting of 222 amino acids. The sequence from 128 to 134(DHYLGKE) is the active site, and the sequences from 170 to 176(GTEGRGG) is the possible site of coenzyme binding. The phylogenetic tree shows that G6PDH of A. oryzae is similar to other filamentous fungi and the yeast one, while distinguished from the bacterial type, the plant one, and the human one.

  15. NLM Grantee's "HealthMap" Helps Uncover Measles Vaccination Gap

    Science.gov (United States)

    ... courtesy of NLM NLM Grantee's "HealthMap" Helps Uncover Measles Vaccination Gap Inadequate vaccine coverage is likely a driving force behind the ongoing Disneyland measles outbreak, according to calculations by a research team ...

  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. The effects of salt stress cause a diversion of basal metabolism in barley roots: possible different roles for glucose-6-phosphate dehydrogenase isoforms.

    Science.gov (United States)

    Cardi, Manuela; Castiglia, Daniela; Ferrara, Myriam; Guerriero, Gea; Chiurazzi, Maurizio; Esposito, Sergio

    2015-01-01

    In this study the effects of salt stress and nitrogen assimilation have been investigated in roots of hydroponically-grown barley plants exposed to 150 mM NaCl, in presence or absence of ammonium as the sole nitrogen source. Salt stress determines a diversion of root metabolism towards the synthesis of osmolytes, such as glycine betaine and proline, and increased levels of reduced glutathione. The metabolic changes triggered by salt stress result in a decrease in both activities and protein abundance of key enzymes, namely GOGAT and PEP carboxylase, and in a slight increase in HSP70. These variations would enhance the requirement for reductants supplied by the OPPP, consistently with the observed increase in total G6PDH activity. The involvement and occurrence of the different G6PDH isoforms have been investigated, and the kinetic properties of partially purified cytosolic and plastidial G6PDHs determined. Bioinformatic analyses examining co-expression profiles of G6PDHs in Arabidopsis and barley corroborate the data presented. Moreover, the gene coding for the root P2-G6PDH isoform was fully sequenced; the biochemical properties of the corresponding protein were examined experimentally. The results are discussed in the light of the possible distinct roles and regulation of the different G6PDH isoforms during salt stress in barley roots.

  18. Macroscopic model for biological fixation and its uncover-ing idea in Chinese Mongolian traditional osteopathy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Namula; LI Xue-en; WANG Mei; HU Da-lai

    2009-01-01

    Splintage external fixation in Chinese Mongolian oste-opathy is a biological macroscopic model. In this model, the ideas of self-life "unity of mind and body" and vital natural "correspondence of nature and human" combine the physi-ological and psychological self-fixation with supplementary external fixation of fracture using small splints. This model implies macroscopic ideas of uncovering fixation and healing: structural stability integrating geometrical "dy-namic" stability with mechanical "dynamic" equilibrium and the stability of state integrating statics with dynamics, and osteoblasts with osteoclasts, and psychological stability in-tegrating closed and open systems of human and nature. These ideas indicate a trend of development in modem osteopathy.

  19. Forizymes - functionalised artificial forisomes as a platform for the production and immobilisation of single enzymes and multi-enzyme complexes.

    Science.gov (United States)

    Visser, Franziska; Müller, Boje; Rose, Judith; Prüfer, Dirk; Noll, Gundula A

    2016-01-01

    The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.

  20. The effect of bilirubin on lipid peroxidation and antioxidant enzymes in cumene hydroperoxide-treated erythrocytes.

    Science.gov (United States)

    Yeşilkaya, A; Yeğin, A; Ozdem, S; Aksu, T A

    1998-01-01

    Recently, it has been suggested that bilirubin may act as a potent biological chain-breaking antioxidant. To observe the effects of free bilirubin on antioxidant reactions in cumene hydroperoxide-treated erythrocytes (15 g hemoglobin/dl), we added bilirubin at four different concentrations (0.5, 1, 5, and 10 mg/dl). We measured the thiobarbituric acid-reactive substance and reduced glutathione levels, and some antioxidant enzyme activities, namely superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase. Thiobarbituric acid-reactive substance and chemiluminescent signals decreased during the incubation. Superoxide dismutase activities also decreased but not as much as in the control group. Glucose-6-phosphate dehydrogenase activities and reduced glutathione levels increased, but catalase activities remained the same as the control group. Our results suggest that bilirubin--in the concentrations we have used--partially prevented the oxidant effects of cumene hydroperoxide.

  1. Glucose-6-Phosphate Dehydrogenase Deficiency and Haemoglobin Drop after Sulphadoxine-Pyrimethamine Use for Intermittent Preventive Treatment of Malaria during Pregnancy in Ghana - A Cohort Study.

    Directory of Open Access Journals (Sweden)

    Ruth Owusu

    Full Text Available Sulphadoxine-Pyrimethamine (SP is still the only recommended antimalarial for use in intermittent preventive treatment of malaria during pregnancy (IPTp in some malaria endemic countries including Ghana. SP has the potential to cause acute haemolysis in G6PD deficient people resulting in significant haemoglobin (Hb drop but there is limited data on post SP-IPTp Hb drop. This study determined the difference, if any in proportions of women with significant acute haemoglobin drop between G6PD normal, partial deficient and full deficient women after SP-IPTp.Prospectively, 1518 pregnant women who received SP for IPTp as part of their normal antenatal care were enrolled. Their G6PD status were determined at enrollment followed by assessments on days 3, 7,14 and 28 to document any adverse effects and changes in post-IPTp haemoglobin (Hb levels. The three groups were comparable at baseline except for their mean Hb (10.3 g/dL for G6PD normal, 10.8 g/dL for G6PD partial deficient and 10.8 g/dL for G6PD full defect women.The prevalence of G6PD full defect was 2.3% and 17.0% for G6PD partial defect. There was no difference in the proportions with fractional Hb drop ≥ 20% as compared to their baseline value post SP-IPTp among the 3 groups on days 3, 7, 14. The G6PD full defect group had the highest median fractional drop at day 7. There was a weak negative correlation between G6PD activity and fractional Hb drop. There was no statistical difference between the three groups in the proportions of those who started the study with Hb ≥ 8g/dl whose Hb level subsequently fell below 8g/dl post-SP IPTp. No study participant required transfusion or hospitalization for severe anaemia.There was no significant difference between G6PD normal and deficient women in proportions with significant acute haemoglobin drop post SP-IPTp and lower G6PD enzyme activity was not strongly associated with significant acute drug-induced haemoglobin drop post SP-IPTp but a larger

  2. Cloning and Sequence Analysis of Mannose-6-phosphate lsomerase cDNA from Metarhizium anisopliae ZJ1109%金龟子绿僵菌甘露糖6-磷酸异构酶基因cDNA序列的克隆及分析

    Institute of Scientific and Technical Information of China (English)

    李亚

    2011-01-01

    The primers were designed according to the conservative sequence of mannose-6-phosphate isomerase gene, and the complete cDNA sequence of Metarhizium anisopliae was amplified by RT-PCR and RACE PCR. The complete cDNA sequence of mpi gene was 1513 bp and the complete ORF length was 1328 bp which encoded a protein with 441 amino acid residues. Blast analysis indicated the deduced amino acid sequence of the mpi gene from M. Anisopliae showed high homology with other fungi respectively. The analysis of protein structure showed MPI protein had the characteristic of the conservative phosphate enzyme, and mainly constructed by a-helix and random coil.%通过绿僵菌属甘露糖6-磷酸异构酶基因保守核苷酸区域设计简并性引物,采用RT-PCR及RACE-PCR技术成功克隆了金龟子绿僵菌mpi基因cDNA序列.该基因cDNA序列全长为1513bp,开放阅读框长度为1328 bp,共编码441氨基酸.BLAST分析发现该基因演绎的氨基酸序列与其它真菌同源性较高,蛋白结构分析表明MPI蛋白是较保守的蛋白磷酸酶结构特征,主要由α螺旋和不规则卷曲构成.

  3. 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.

  4. Isolation and Expression Analysis of Plastidic Glucose-6-phosphate Dehydrogenase Gene from Rice (Oryza sativa L.)%水稻质体葡萄糖-6-磷酸脱氢酶基因的克隆与表达研究

    Institute of Scientific and Technical Information of China (English)

    侯夫云; 黄骥; 陆驹飞; 王州飞; 张红生

    2006-01-01

    Glucose-6-phosphate dehydrogenase is a rate-limiting enzyme of pentose phosphate pathway, existing in cytosolic and plastidic compartments of higher plants. A novel gene encoding plastidic glucose-6-phosphate dehydrogenase was isolated from rice (Oryza sativa L.) and designated OsG6PDH2 in this article. Through semiquantitative RT-PCR approach it was found that OsG6PDH2 mRNA was weakly expressed in rice leaves, stems, immature spikes or flowered spikes, and a little higher in roots.However, the expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete opening reading frame (ORF) of OsG6PDH2 was inserted into pET30a (+), and expressed in Escherichia coli strain BL21 (DE3). The enzyme activity assay of transformed bacterial cells indicated that OsG6PDH2 encoding product had a typical function of glucose-6-phosphate dehydrogenase.%戊糖磷酸途径是高等植物中重要的代谢途径,主要生理功能是产生NADPH以及供核酸代谢的磷酸戊糖.葡萄糖-6-磷酸脱氢酶(G6PDH)是戊糖磷酸途径的关键酶,广泛存在于高等植物细胞的细胞质和质体中.本研究首次从水稻(Oryzasativa L.)幼苗中分离了核编码的质体G6PDH基因OsG6PDH2,序列分析表明OsG6PDH2编码一个具有588个氨基酸残基的多肽,等电点为8.5,分子量66 kDa.OsG6PDH2的N端有1个70个氨基酸的信号肽,推测的裂解位点为Gly55和Val56,表明OsG6PDH2编码产物可能定位于质体.多序列比较的结果表明OsG6PDH2与拟南芥、烟草、马铃薯质体G6PDH的一致性分别达81%、87%、83%.进化关系说明水稻OsG6PDH2与拟南芥(AtG6PDH3)、马铃薯(StG6PDH1)处于高等植物P2型质体G6PDH分支上,暗示了OsG6PDH2可能是一个P2型的质体蛋白.Matinspector程序分析表明,OsG6PDH2在起始密码子上游含有一个bZIP转录因子识别位点、一个ABA应答元件、一个CRT/DRE元件和1个W-box元件.半定量RT-PCR分析表明,OsG6PDH2在水稻根、茎、叶和幼

  5. Scratching below the surface to uncover HRM practices in Denmark

    DEFF Research Database (Denmark)

    Jørgensen, Frances; Ulhøi, John Parm

    2010-01-01

    firms. The empirical part of this research stems from an ongoing in-depth study aimed at uncovering and describing the relationship between HRM and innovation, highlighting possible areas for misinterpretations that have contributed to the assumption that Danish firms do not prioritize HRM....

  6. Weaving Social Foundations through Dance Pedagogy: A Pedagogy of Uncovering

    Science.gov (United States)

    Barr, Sherrie; Risner, Doug

    2014-01-01

    Today's dance educators enter classrooms populated by increasingly diverse students in which teachers' pedagogical knowledge necessitates heightened understandings of race, ethnicity, social class, gender, and sexuality. Uncovering taken-for-granted assumptions, dominant stereotypes, and educational structures that reproduce social…

  7. Uncovering Students' Thinking about Thinking Using Concept Maps

    Science.gov (United States)

    Ritchhart, Ron; Turner, Terri; Hadar, Linor

    2009-01-01

    A method for uncovering students' thinking about thinking, specifically their meta-strategic knowledge, is explored within the context of an ongoing, multi-year intervention designed to promote the development of students' thinking dispositions. The development of a concept-map instrument that classroom teachers can use and an analytic framework…

  8. Virus-Induced Gene Silencing-Based Functional Analyses Revealed the Involvement of Several Putative Trehalose-6-Phosphate Synthase/Phosphatase Genes in Disease Resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in Tomato

    Science.gov (United States)

    Zhang, Huijuan; Hong, Yongbo; Huang, Lei; Liu, Shixia; Tian, Limei; Dai, Yi; Cao, Zhongye; Huang, Lihong; Li, Dayong; Song, Fengming

    2016-01-01

    Trehalose and its metabolism have been demonstrated to play important roles in control of plant growth, development, and stress responses. However, direct genetic evidence supporting the functions of trehalose and its metabolism in defense response against pathogens is lacking. In the present study, genome-wide characterization of putative trehalose-related genes identified 11 SlTPSs for trehalose-6-phosphate synthase, 8 SlTPPs for trehalose-6-phosphate phosphatase and one SlTRE1 for trehalase in tomato genome. Nine SlTPSs, 4 SlTPPs, and SlTRE1 were selected for functional analyses to explore their involvement in tomato disease resistance. Some selected SlTPSs, SlTPPs, and SlTRE1 responded with distinct expression induction patterns to Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 as well as to defense signaling hormones (e.g., salicylic acid, jasmonic acid, and a precursor of ethylene). Virus-induced gene silencing-mediated silencing of SlTPS3, SlTPS4, or SlTPS7 led to deregulation of ROS accumulation and attenuated the expression of defense-related genes upon pathogen infection and thus deteriorated the resistance against B. cinerea or Pst DC3000. By contrast, silencing of SlTPS5 or SlTPP2 led to an increased expression of the defense-related genes upon pathogen infection and conferred an increased resistance against Pst DC3000. Silencing of SlTPS3, SlTPS4, SlTPS5, SlTPS7, or SlTPP2 affected trehalose level in tomato plants with or without infection of B. cinerea or Pst DC3000. These results demonstrate that SlTPS3, SlTPS4, SlTPS5, SlTPS7, and SlTPP2 play roles in resistance against B. cinerea and Pst DC3000, implying the importance of trehalose and tis metabolism in regulation of defense response against pathogens in tomato. PMID:27540389

  9. Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells.

    Science.gov (United States)

    Fernie, Alisdair R; Roscher, Albrecht; Ratcliffe, R. George; Kruger, Nicholas J

    2002-02-01

    The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media. 31P-NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6-bisphosphate content of each line was unaffected by 2-deoxyglucose and increased during growth on phosphate-limited media. Short-term treatment of callus with 2-deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long-term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U-14C]glucose in response to incubation with 2-deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate-depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6-bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate-deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the

  10. Deficiência da glicose-6-fosfato desidrogenase com infecções de repetição: relato de caso Glucose-6-phosphate dehydrogenase deficiency with recurrent infections: case report

    Directory of Open Access Journals (Sweden)

    Abertina Rosa-Borges

    2001-08-01

    Full Text Available OBJETIVO: relatar a ocorrência de uma deficiência funcional de neutrófilos rara, com quadro clínico e laboratorial semelhante ao da doença granulomatosa crônica. MÉTODOS: relato de caso de paciente com deficiência acentuada da glicose-6-fosfato desidrogenase e infecções de repetição. Realizada pesquisa bibliográfica utilizando as bases de dados Medline e Lilacs, abrangendo o período de 1972 a 2000. RESULTADOS: paciente com nível da glicose-6-fosfato desidrogenase extremamente reduzido e quadro de infeções graves com melhora clínica após uso de cotrimoxazol contínuo. Os leucócitos do paciente apresentam defeito no metabolismo oxidativo, similar ao da doença granulomatosa crônica. CONCLUSÕES: o diagnóstico da deficiência da glicose-6-fosfato desidrogenase em neutrófilos deve ser considerado em qualquer paciente com anemia hemolítica não esferocítica congênita no qual o nível da glicose-6-fosfato desidrogenase esteja anormalmente baixo ou apresente infeções de repetição. É diagnóstico diferencial da doença granulomatosa crônica.OBJECTIVE: To report a case of rare neutrophil functional disorder with clinical and laboratory findings similar to those of chronic granulomatous disease. METHODS: Patient with extremely reduced level of glucose-6-phosphate dehydrogenase and recurrent infections that improved after continuous use of cotrimoxazole. The patient presented leukocytes with defective respiratory burst, similar to what occurs in chronic granulomatous disease. COMMENTS: The diagnosis of glucose-6-phosphate dehydrogenase deficiency in neutrophils should be considered in any patient with hemolytic anemia whose level of G6PD is extremely low or in any patient that presents recurrent infections as differential diagnosis of chronic granulomatous disease.

  11. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

    Directory of Open Access Journals (Sweden)

    S Andrea Moreno

    2015-06-01

    Full Text Available Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF of T. b. brucei: (i fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme in glycosomes, (ii enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes and a GAPDH isoenzyme in the cytosol, (iii malate dehydrogenase in cytosol and (iv glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  12. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes.

    Science.gov (United States)

    Moreno, S Andrea; Nava, Mayerly

    2015-06-01

    Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  13. A novel point mutation in a class IV glucose-6-phosphate dehydrogenase variant (G6PD São Paulo and polymorphic G6PD variants in São Paulo State, Brazil

    Directory of Open Access Journals (Sweden)

    Raimundo Antonio G. Oliveira

    2009-01-01

    Full Text Available In this study, we used red cell glucose-6-phosphate dehydrogenase (G6PD activity to screen for G6PD-deficient individuals in 373 unrelated asymptomatic adult men who were working with insecticides (organophosphorus and carbamate in dengue prevention programs in 27 cities in São Paulo State, Brazil. Twenty-one unrelated male children suspected of having erythroenzymopathy who were attended at hospitals in São Paulo city were also studied. Fifteen of the 373 adults and 12 of the 21 children were G6PD deficient. G6PD gene mutations were investigated in these G6PD-deficient individuals by using PCR-RFLP, PCR-SSCP analysis and DNA sequencing. Twelve G6PD A-202A/376G and two G6PD Seattle844C, as well as a new variant identified as G6PD São Paulo, were detected among adults, and 11 G6PD A-202A/376G and one G6PD Seattle844C were found among children. The novel mutation c.660C > G caused the replacement of isoleucine by methionine (I220M in a region near the dimer interface of the molecule. The conservative nature of this mutation (substitution of a nonpolar aliphatic amino acid for another one could explain why there was no corresponding change in the loss of G6PD activity (64.5% of normal activity in both cases.

  14. cDNA cloning of glucose-6-phosphate isomerase from crucian carp (Carassius carassius) and expression of the active region as myofibril-bound serine proteinase inhibitor in Escherichia coli.

    Science.gov (United States)

    Han, Long; Cao, Min-Jie; Shi, Chao-lan; Wei, Xiao-Nan; Li, Huan; Du, Cui-Hong

    2014-02-01

    Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) can act as a myofibril-bound serine proteinase (MBSP) inhibitor (MBSPI) in fish. In order to better understand the biological information of the GPI and its functional domain for inhibiting MBSP, the cDNA of GPI was cloned from crucian carp (Carassius carassius) with RT-PCR, nested-PCR and 3'-RACE. The result of sequencing showed that the GPI cDNA had an open reading frame of 1662bp encoding 553 amino acid residues. After constructing and comparing the three-dimensional structures of GPI and MBSP, the middle fragment of crucian carp GPI (GPI-M) was predicted as a functional domain for inhibiting MBSP. Then the crucian carp GPI-M gene was cloned and expressed in Escherichia coli. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant GPI-M (rGPI-M) with molecular mass of approximately 21kDa in the form of inclusion bodies. The rGPI-M was obtained at an electrophoresis level purity of approximately 95% after denaturation and dialysis renaturation.

  15. L-Ribose production from L-arabinose by immobilized recombinant Escherichia coli co-expressing the L-arabinose isomerase and mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans.

    Science.gov (United States)

    Kim, Kyoung-Rok; Seo, Eun-Sun; Oh, Deok-Kun

    2014-01-01

    L-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the L-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l L-ribose from 300 g/l L-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co(2+), with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h(-1) produced an average of 100 g/l L-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for L-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from L-arabinose as the substrate.

  16. Evaluation on the Effectiveness of 2-Deoxyglucose-6-phosphate phosphatase (DOGR1 Gene as a Selectable Marker for Oil Palm (Elaeis guineensis Jacq. Embryogenic Calli Transformation Mediated by Agrobacterium tumefaciens.

    Directory of Open Access Journals (Sweden)

    Abang Masli eDayang Izawati

    2015-09-01

    Full Text Available DOGR1, which encodes for 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOGR1 gene, was transformed into oil palm embryogenic calli mediated by Agrobacterium tumefaciens strain LBA4404. Transformed embryogenic calli were exposed to 400 mg l–1 2-deoxyglucose (2-DOG as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOGR1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOGR1 gene and 2-DOG for regenerating transgenic oil palm.

  17. Food Enzymes

    Science.gov (United States)

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  18. Enzyme immunoassay

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from...

  19. Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Stéphane Delmas

    Full Text Available A key challenge in the production of second generation biofuels is the conversion of lignocellulosic substrates into fermentable sugars. Enzymes, particularly those from fungi, are a central part of this process, and many have been isolated and characterised. However, relatively little is known of how fungi respond to lignocellulose and produce the enzymes necessary for dis-assembly of plant biomass. We studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA sequencing we showed that, 24 hours after exposure to straw, gene expression of known and presumptive plant cell wall-degrading enzymes represents a huge investment for the cells (about 20% of the total mRNA. Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal arsenal of enzymes for the degradation of plant biomass. Using transcription factor deletion mutants (xlnR and creA to study the response to both lignocellulosic substrates and low carbon source concentrations, we showed that a subset of genes coding for degradative enzymes is induced by starvation. Our data support a model whereby this subset of enzymes plays a scouting role under starvation conditions, testing for available complex polysaccharides and liberating inducing sugars, that triggers the subsequent induction of the majority of hydrolases. We also showed that antisense transcripts are abundant and that their expression can be regulated by growth conditions.

  20. Uncovering introductory astronomy students' conceptual modules of lunar phases

    Science.gov (United States)

    Lindell, Rebecca; Traxler, Adrienne

    2017-01-01

    Brewe, Bruun and Bearden developed Module Analysis of Multiple Choice Responses (MAMCR) methodology for using network analysis to uncover the underlying conceptual modules of student performance on multiple-choice assessments. The Lunar Phases Concept Inventory (LPCI) assesses students understanding of lunar phases across 8 separate dimensions of understanding based on the results of a detailed qualitative phenomenology of college students' understanding of lunar phases. Unlike many concept inventories, the LPCI has multiple items for each dimension of understanding and each response corresponds to either the scientifically correct answer or to an alternative idea uncovered from the qualitative investigation. In this study, we have combined MAMCR with the database of nearly 2000 LPCI pre-test results. We will report on the preliminary different conceptual modules of lunar phases and the relationship of these modules to previous qualitative research.

  1. 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

  2. Uncovering Student Ideas in Astronomy 45 Formative Assessment Probes

    CERN Document Server

    Keeley, Page

    2012-01-01

    What do your students know-or think they know-about what causes night and day, why days are shorter in winter, and how to tell a planet from a star? Find out with this book on astronomy, the latest in NSTA's popular Uncovering Student Ideas in Science series. The 45 astronomy probes provide situations that will pique your students' interest while helping you understand how your students think about key ideas related to the universe and how it operates.

  3. Alkylating enzymes.

    Science.gov (United States)

    Wessjohann, Ludger A; Keim, Jeanette; Weigel, Benjamin; Dippe, Martin

    2013-04-01

    Chemospecific and regiospecific modifications of natural products by methyl, prenyl, or C-glycosyl moieties are a challenging and cumbersome task in organic synthesis. Because of the availability of an increasing number of stable and selective transferases and cofactor regeneration processes, enzyme-assisted strategies turn out to be promising alternatives to classical synthesis. Two categories of alkylating enzymes become increasingly relevant for applications: firstly prenyltransferases and terpene synthases (including terpene cyclases), which are used in the production of terpenoids such as artemisinin, or meroterpenoids like alkylated phenolics and indoles, and secondly methyltransferases, which modify flavonoids and alkaloids to yield products with a specific methylation pattern such as 7-O-methylaromadendrin and scopolamine.

  4. Incremento de la glucosa-6-fosfato-deshidrogenasa eritrocitaria en jóvenes con síndrome de Down tras un programa de actividad física de 12 semanas A 12-week physical activity program increases glucose-6-phosphate-dehydrogenase activity in Down syndrome adolescents

    Directory of Open Access Journals (Sweden)

    Francisco J. Ordóñez

    2005-12-01

    Full Text Available Recientemente se ha publicado que las células trisómicas presentan una mayor sensibilidad al daño oxidativo, que podría justificar la frecuente asociación de síndrome de Down a aterosclerosis, envejecimiento precoz, etc. Para conocer el posible papel de la actividad física moderada en la mejora de la capacidad antioxidante se estudió el comportamiento de la enzima glucosa-6-fosfato-deshidrogenasa (G6PDH eritrocitaria en 31 adolescentes varones (16.3 ± 1.1 años tras desarrollar un programa de 12 semanas con tres sesiones (45-60 minutos y una intensidad del 60-75% frecuencia cardíaca máxima teórica. Nuestros resultados indican una mayor actividad de G6PDH en individuos con síndrome de Down cuando se compara con controles sin trisomía ajustados a su sexo, edad e índice de masa corporal. Asimismo observamos un incremento significativo de su actividad tras completar nuestro programa de 12 semanas. Podemos concluir que la actividad física moderada mejora la capacidad antioxidante en jóvenes con síndrome de Down.In recent years it has been claimed that trisomic cells are more sensitive to oxidative stress since there is an imbalance in the hydrogen peroxide metabolism. We designed the present study to assess the activity level of antioxidant enzyme glucose-6-phosphate-dehydrogenase (G6PDH of erythrocytes in 31 male adolescents with Down syndrome (mean age 16.3 ± 1.1 after performing a 12 week aerobic training program. First of all, a significant increase of 14.9% in the catalytic activity of G6PDH was observed in male adolescents with Down syndrome when compared with age, sex and body mass-matched controls without trisomy. After 12-wk program its activity increased significantly compared to baseline value in Down syndrome individuals. Our data are consistent with previous evidence of the existence of higher oxidative stress in adolescents with Down syndrome when compared to the general population. We may also conclude that G6PDH

  5. Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases.

    Science.gov (United States)

    Oh, Doo-Byoung

    2015-08-01

    Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy.

  6. Chronic nonspherocytic hemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency: report of two families with novel mutations causing G6PD Bangkok and G6PD Bangkok Noi.

    Science.gov (United States)

    Tanphaichitr, Voravarn S; Hirono, Akira; Pung-amritt, Parichat; Treesucon, Ajjima; Wanachiwanawin, Wanchai

    2011-07-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common hereditary enzymopathies worldwide. Mostly G6PD deficient cases are asymptomatic though they may have the risk of neonatal jaundice (NNJ) and acute intravascular hemolysis during oxidative stress. Chronic nonspherocytic hemolytic anemia (CNSHA) due to G6PD deficiency is rare. In Thailand, one case was reported 40 years ago and by biochemical study this G6PD was reported to be a new variant G6PD Bangkok. We, herein, report two families with CNSHA due to G6PD deficiency. In the first family, we have been following up the clinical course of the patient with G6PD Bangkok. In addition to chronic hemolysis, he had three acute hemolytic episodes requiring blood transfusions during childhood period. Multiple gallstones were detected at the age of 27. His two daughters who inherited G6PD Bangkok from him and G6PD Vanua Lava from his wife are asymptomatic. Both of them had NNJ and persistent evidences of compensated hemolysis. Molecular analysis revealed a novel missense mutation 825 G→C predicting 275 Lys→Asn causing G6PD Bangkok. In the second family, two male siblings are affected. They had NNJ and several hemolytic episodes which required blood transfusions. On follow-up they have been diagnosed with chronic hemolysis as evidenced by reticulocytosis and indirect hyperbilirubinemia. Molecular analysis revealed combined missense mutations in exons 12 and 13. The first mutation was 1376 G→T predicting 459 Arg→Leu (known as G6PD Canton) and the second one was 1502 T→G predicting 501 Phe→Cys. We designated the resulting novel G6PD variant, G6PD Bangkok Noi.

  7. Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of southwestern China

    Institute of Scientific and Technical Information of China (English)

    YANG YinFeng; ZHU YueChun; LI DanYi; LI ZhiGang; L(U) HuiRu; WU Jing; TANG Jing; TONG ShuFen

    2007-01-01

    The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31%(19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2-13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen independent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping(1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.

  8. Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of southwestern China

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31% (19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2―13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen inde-pendent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping (1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.

  9. Acquired hemoglobin variants and exposure to glucose-6-phosphate dehydrogenase deficient red blood cell units during exchange transfusion for sickle cell disease in a patient requiring antigen-matched blood.

    Science.gov (United States)

    Raciti, Patricia M; Francis, Richard O; Spitalnik, Patrice F; Schwartz, Joseph; Jhang, Jeffrey S

    2013-08-01

    Red blood cell exchange (RBCEx) is frequently used in the management of patients with sickle cell disease (SCD) and acute chest syndrome or stroke, or to maintain target hemoglobin S (HbS) levels. In these settings, RBCEx is a category I or II recommendation according to guidelines on the use of therapeutic apheresis published by the American Society for Apheresis. Matching donor red blood cells (RBCs) to recipient phenotypes (e.g., C, E, K-antigen negative) can decrease the risk of alloimmunization in patients with multi-transfused SCD. However, this may select for donors with a higher prevalence of RBC disorders for which screening is not performed. This report describes a patient with SCD treated with RBCEx using five units negative for C, E, K, Fya, Fyb (prospectively matched), four of which were from donors with hemoglobin variants and/or glucose-6-phosphate dehydrogenase (G6PD) deficiency. Pre-RBCEx HbS quantification by high performance liquid chromatography (HPLC) demonstrated 49.3% HbS and 2.8% hemoglobin C, presumably from transfusion of a hemoglobin C-containing RBC unit during a previous RBCEx. Post-RBCEx HPLC showed the appearance of hemoglobin G-Philadelphia. Two units were G6PD-deficient. The patient did well, but the consequences of transfusing RBC units that are G6PD-deficient and contain hemoglobin variants are unknown. Additional studies are needed to investigate effects on storage, in-vivo RBC recovery and survival, and physiological effects following transfusion of these units. Post-RBCEx HPLC can monitor RBCEx efficiency and detect the presence of abnormal transfused units.

  10. Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

    Science.gov (United States)

    Buchanan, R L; Lewis, D F

    1984-08-01

    Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes.

  11. An Enzyme-based 1:2 Demultiplexer Interfaced with an Electrochemical Actuator.

    Science.gov (United States)

    Fratto, Brian E; Guz, Nataliia; Fallon, Tyler T; Katz, Evgeny

    2016-08-02

    An enzyme-based 1:2 demultiplexer is designed in a flow system composed of three cells where each one is modified with a different enzyme: hexokinase, glucose dehydrogenase and glucose-6-phosphate dehydrogenase. The Input signal activating the biocatalytic cascade is represented by glucose, while the Address signal represented by ATP is responsible for directing the Input signal to one of the output channels, depending on the logic value of the Address. The biomolecular 1:2 demultiplexer is extended to include two electrochemical actuators releasing entrapped DNA molecules in the active output channel. The modular design of the system allows for easy exchange and extension of the functional elements. The present demultiplexer can be easily integrated in various biomolecular logic systems, including different logic gates based on the enzyme- or DNA-based reactions, as well as containing different chemical actuators, for example, with a biomolecular release function.

  12. Uncovering growth-suppressive MicroRNAs in lung cancer

    DEFF Research Database (Denmark)

    Liu, Xi; Sempere, Lorenzo F; Galimberti, Fabrizio

    2009-01-01

    PURPOSE: MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growth-suppressive miRNAs in lung cancer. EXPERIMENTAL DESIGN: miRNA arrays were done on normal lung tissues...... and adenocarcinomas from wild-type and proteasome degradation-resistant cyclin E transgenic mice to reveal repressed miRNAs in lung cancer. Real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays validated these findings. Lung cancer cell lines were derived from each...... transgenic line (designated as ED-1 and ED-2 cells, respectively). Each highlighted miRNA was independently transfected into these cells. Growth-suppressive mechanisms were explored. Expression of a computationally predicted miRNA target was examined. These miRNAs were studied in a paired normal...

  13. Uncovering Transcriptional Regulatory Networks by Sparse Bayesian Factor Model

    Directory of Open Access Journals (Sweden)

    Qi Yuan(Alan

    2010-01-01

    Full Text Available Abstract The problem of uncovering transcriptional regulation by transcription factors (TFs based on microarray data is considered. A novel Bayesian sparse correlated rectified factor model (BSCRFM is proposed that models the unknown TF protein level activity, the correlated regulations between TFs, and the sparse nature of TF-regulated genes. The model admits prior knowledge from existing database regarding TF-regulated target genes based on a sparse prior and through a developed Gibbs sampling algorithm, a context-specific transcriptional regulatory network specific to the experimental condition of the microarray data can be obtained. The proposed model and the Gibbs sampling algorithm were evaluated on the simulated systems, and results demonstrated the validity and effectiveness of the proposed approach. The proposed model was then applied to the breast cancer microarray data of patients with Estrogen Receptor positive ( status and Estrogen Receptor negative ( status, respectively.

  14. DOES UNCOVERED INTEREST RATE PARITY HOLD IN TURKEY?

    Directory of Open Access Journals (Sweden)

    Ozcan Karahan

    2012-01-01

    Full Text Available Most of the earlier empirical studies focusing on developed countries failed to give evidence in favor of the Uncovered Interest Rate Parity (UIP. After intensive financial liberalization processes and mostly preferred free exchange rate regimes, a new area of research starts to involve the investigation whether UIP holds for developing economies differently. Accordingly, we tested the UIP for Turkey’s monthly interest rate and exchange rate data between 2002 and 2011. We run conventional regressions in the form of Ordinary Least Squares (OLS and used a simple Generalized Autoregressive Conditional Heteroskedasticity (GARCH analysis. The empirical results of both methods do not support the validity of UIP for Turkey. Thus, together with most of the earlier empirical studies focusing on developed countries and detecting the invalidity of UIP, we can argue that the experience of Turkey and developed economies are not different.

  15. Laughing It Off: Uncovering the Everyday Work Experience of Nurses

    Directory of Open Access Journals (Sweden)

    Valerie M. Adams

    2007-03-01

    Full Text Available During research towards her doctoral dissertation, the author noticed that nurses understated the conditions in which they worked. Seeking to understand how nursing culture shapes how nurses describe their work, she developed a “toolbox” of reflexive methods. She used metaphors of nursing and emotion expressed as laughter to identify aspects of nursing culture in semistructured interviews with nurses working in Australian residential aged care facilities. She also incorporated autoethnography, as she had worked as a registered nurse while studying economics. The inclusion of her voice in the data illustrates the difference between nursing culture and another worldview. These pluralist methods made explicit some of the effects of gendered socialization, such as understatement and self-consciousness, and demonstrate how they are embedded in nursing culture. Awareness of such norms is important for understanding marketized caring labor. This combination of methods has significance for uncovering workplace culture in other forms of marketized caring.

  16. Charge uncovering effects on flute instabilities in hot electron plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Spong, D.A.

    1985-01-01

    Recent measurements and concurrent theoretical equilibrium models of the ELMO Bumpy Torus (EBT) edge plasma region (as described by E. F. Jaeger et al. in Magnetic Well Depth in EBT and Sensitivity to Hot Electron Ring Geometry, ORNL/TM-9185 (1984)) have indicated that the hot electron ring beta ..beta../sub hot/ at the C-T transition may not always be sufficient to produce the local minimum in the magnetic field thought to be necessary for MHD stability. This has led to the examination of other mechanisms that could account for the observed stability of the T-mode. In this report, an effect known as charge uncovering, which depends not on the value of ..beta../sub hot/ but rather on the ratio n/sub hot//n/sub core/, is studied.

  17. Uncover the Ideology Behind News Reports Through Transitivity Analysis

    Institute of Scientific and Technical Information of China (English)

    董亚男

    2015-01-01

    When people read the reports relating to Occupy Central from different news papers, they get completely different feelings towards the event. To find out how this phenomenon happened, this paper is going to apply transitivity analysis to the news reports. The reports are selected from China Daily, CNN and BBC respectively. To have a deep application of this method, only verbal process wil be taken into consideration. This paper wil discuss the proportion of verbal process from the two sides (Occupy Central people as one side and people against them as the other), the message delivered by the verbal process, the sequence and the transformation of verbal process. The purpose is to uncover the ideology hidden behind the seemingly objective news reports through transitivity analysis.

  18. Uncovering Transcriptional Regulatory Networks by Sparse Bayesian Factor Model

    Science.gov (United States)

    Meng, Jia; Zhang, Jianqiu(Michelle); Qi, Yuan(Alan); Chen, Yidong; Huang, Yufei

    2010-12-01

    The problem of uncovering transcriptional regulation by transcription factors (TFs) based on microarray data is considered. A novel Bayesian sparse correlated rectified factor model (BSCRFM) is proposed that models the unknown TF protein level activity, the correlated regulations between TFs, and the sparse nature of TF-regulated genes. The model admits prior knowledge from existing database regarding TF-regulated target genes based on a sparse prior and through a developed Gibbs sampling algorithm, a context-specific transcriptional regulatory network specific to the experimental condition of the microarray data can be obtained. The proposed model and the Gibbs sampling algorithm were evaluated on the simulated systems, and results demonstrated the validity and effectiveness of the proposed approach. The proposed model was then applied to the breast cancer microarray data of patients with Estrogen Receptor positive ([InlineEquation not available: see fulltext.]) status and Estrogen Receptor negative ([InlineEquation not available: see fulltext.]) status, respectively.

  19. Uncovering transcriptional regulation of metabolism by using metabolic network topology

    DEFF Research Database (Denmark)

    Patil, Kiran Raosaheb; Nielsen, Jens

    2005-01-01

    therefore developed an algorithm that is based on hypothesis-driven data analysis to uncover the transcriptional regulatory architecture of metabolic networks. By using information on the metabolic network topology from genome-scale metabolic reconstruction, we show that it is possible to reveal patterns...... in the metabolic network that follow a common transcriptional response. Thus, the algorithm enables identification of so-called reporter metabolites (metabolites around which the most significant transcriptional changes occur) and a set of connected genes with significant and coordinated response to genetic...... changes induced by complex regulatory mechanisms coordinating the activity of different metabolic pathways. It is difficult to map such global transcriptional responses by using traditional methods, because many genes in the metabolic network have relatively small changes at their transcription level. We...

  20. TIME HORIZON AND UNCOVERED INTEREST PARITY IN EMERGING ECONOMIES

    Directory of Open Access Journals (Sweden)

    Norlida Hanim Mohd Salleh

    2011-07-01

    Full Text Available The aim of this study is to re-examine the well-known empirical puzzle of uncovered interest parity (UIP for emerging market economies with different prediction time horizons. The empirical results obtained using dynamic panel and time series techniques for monthly data from January 1995 to December 2009 eventually show that the panel data estimates are more powerful than those obtained by applying individual time series estimations and the significant contribution of the exchange rate prediction horizons in determining the status of UIP. This finding reveals that at the longer time horizon, the model has better econometric specification and thus more predictive power for exchange rate movements compared to the shorter time period. The findings can also be a signalling of well-integrated currency markets and a reliable guide to international investors as well as for the orderly conduct of monetary authorities.

  1. Reverse reaction of malic enzyme for HCO3- fixation into pyruvic acid to synthesize L-malic acid with enzymatic coenzyme regeneration.

    Science.gov (United States)

    Ohno, Yoko; Nakamori, Toshihiko; Zheng, Haitao; Suye, Shin-ichiro

    2008-05-01

    Malic enzyme [L-malate: NAD(P)(+) oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)(+)). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO(3)(-) fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD(+), and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO(3)(-) and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 degrees C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD(+).

  2. 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.

  3. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  4. 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

  5. Consolidity: Mystery of inner property of systems uncovered

    Directory of Open Access Journals (Sweden)

    Hassen T. Dorrah

    2012-10-01

    Full Text Available This paper uncovers the mystery of consolidity, an inner property of systems that was amazingly hidden. Consolidity also reveals the secrecy of why strong stable and highly controllable systems are not invulnerable of falling and collapsing. Consolidity is measured by its Consolidity Index, defined as the ratio of overall changes of output parameters over combined changes of input and system parameters, all operating in fully fuzzy environment. Under this notion, systems are classified into consolidated, quasi-consolidated, neutrally consolidated, unconsolidated, quasi-unconsolidated and mixed types. The strategy for the implementation of consolidity is elaborated for both natural and man-made existing systems as well as the new developed ones. An important critique arises that the by-product consolidity of natural or built-as-usual system could lead to trapping such systems into a completely undesired unconsolidity. This suggests that the ample number of conventional techniques that do not take system consolidity into account should gradually be changed, and adjusted with improved consolidity-based techniques. Four Golden Rules are highlighted for handling system consolidity, and applied to several illustrative case studies. These case studies cover the consolidity analysis of the Drug Concentration problem, Predator-Prey Population problem, Spread of Infectious Disease problem, AIDS Epidemic problem and Arm Race model. It is demonstrated that consolidity changes are contrary (opposite in sign to changes of both stability and controllability. This is a very significant result showing that our present practice of stressing on building strong stable and highly controllable systems could have already jeopardized the consolidity behavior of an ample family of existing real life systems. It is strongly recommended that the four Golden Rules of consolidity should be enforced as future strict regulations of systems modeling, analysis, design and

  6. Uncovering transcriptional interactions via an adaptive fuzzy logic approach

    Directory of Open Access Journals (Sweden)

    Chen Chung-Ming

    2009-12-01

    Full Text Available Abstract Background To date, only a limited number of transcriptional regulatory interactions have been uncovered. In a pilot study integrating sequence data with microarray data, a position weight matrix (PWM performed poorly in inferring transcriptional interactions (TIs, which represent physical interactions between transcription factors (TF and upstream sequences of target genes. Inferring a TI means that the promoter sequence of a target is inferred to match the consensus sequence motifs of a potential TF, and their interaction type such as AT or RT is also predicted. Thus, a robust PWM (rPWM was developed to search for consensus sequence motifs. In addition to rPWM, one feature extracted from ChIP-chip data was incorporated to identify potential TIs under specific conditions. An interaction type classifier was assembled to predict activation/repression of potential TIs using microarray data. This approach, combining an adaptive (learning fuzzy inference system and an interaction type classifier to predict transcriptional regulatory networks, was named AdaFuzzy. Results AdaFuzzy was applied to predict TIs using real genomics data from Saccharomyces cerevisiae. Following one of the latest advances in predicting TIs, constrained probabilistic sparse matrix factorization (cPSMF, and using 19 transcription factors (TFs, we compared AdaFuzzy to four well-known approaches using over-representation analysis and gene set enrichment analysis. AdaFuzzy outperformed these four algorithms. Furthermore, AdaFuzzy was shown to perform comparably to 'ChIP-experimental method' in inferring TIs identified by two sets of large scale ChIP-chip data, respectively. AdaFuzzy was also able to classify all predicted TIs into one or more of the four promoter architectures. The results coincided with known promoter architectures in yeast and provided insights into transcriptional regulatory mechanisms. Conclusion AdaFuzzy successfully integrates multiple types of

  7. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  8. Urban association rules: uncovering linked trips for shopping behavior

    CERN Document Server

    Yoshimura, Yuji; Hobin, Juan N Bautista; Ratti, Carlo; Blat, Josep

    2016-01-01

    In this article, we introduce the method of urban association rules and its uses for extracting frequently appearing combinations of stores that are visited together to characterize shoppers' behaviors. The Apriori algorithm is used to extract the association rules (i.e., if -> result) from customer transaction datasets in a market-basket analysis. An application to our large-scale and anonymized bank card transaction dataset enables us to output linked trips for shopping all over the city: the method enables us to predict the other shops most likely to be visited by a customer given a particular shop that was already visited as an input. In addition, our methodology can consider all transaction activities conducted by customers for a whole city in addition to the location of stores dispersed in the city. This approach enables us to uncover not only simple linked trips such as transition movements between stores but also the edge weight for each linked trip in the specific district. Thus, the proposed methodo...

  9. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Directory of Open Access Journals (Sweden)

    Shin-Rong Lee

    2016-03-01

    Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

  10. Uncovering disassortativity in large scale-free networks

    Science.gov (United States)

    Litvak, Nelly; van der Hofstad, Remco

    2013-02-01

    Mixing patterns in large self-organizing networks, such as the Internet, the World Wide Web, and social and biological networks, are often characterized by degree-degree dependencies between neighboring nodes. In this paper, we propose a new way of measuring degree-degree dependencies. One of the problems with the commonly used assortativity coefficient is that in disassortative networks its magnitude decreases with the network size. We mathematically explain this phenomenon and validate the results on synthetic graphs and real-world network data. As an alternative, we suggest to use rank correlation measures such as Spearman's ρ. Our experiments convincingly show that Spearman's ρ produces consistent values in graphs of different sizes but similar structure, and it is able to reveal strong (positive or negative) dependencies in large graphs. In particular, we discover much stronger negative degree-degree dependencies in Web graphs than was previously thought. Rank correlations allow us to compare the assortativity of networks of different sizes, which is impossible with the assortativity coefficient due to its genuine dependence on the network size. We conclude that rank correlations provide a suitable and informative method for uncovering network mixing patterns.

  11. Losartan ameliorates dystrophic epidermolysis bullosa and uncovers new disease mechanisms.

    Science.gov (United States)

    Nyström, Alexander; Thriene, Kerstin; Mittapalli, Venugopal; Kern, Johannes S; Kiritsi, Dimitra; Dengjel, Jörn; Bruckner-Tuderman, Leena

    2015-07-20

    Genetic loss of collagen VII causes recessive dystrophic epidermolysis bullosa (RDEB)-a severe skin fragility disorder associated with lifelong blistering and disabling progressive soft tissue fibrosis. Causative therapies for this complex disorder face major hurdles, and clinical implementation remains elusive. Here, we report an alternative evidence-based approach to ameliorate fibrosis and relieve symptoms in RDEB. Based on the findings that TGF-β activity is elevated in injured RDEB skin, we targeted TGF-β activity with losartan in a preclinical setting. Long-term treatment of RDEB mice efficiently reduced TGF-β signaling in chronically injured forepaws and halted fibrosis and subsequent fusion of the digits. In addition, proteomics analysis of losartan- vs. vehicle-treated RDEB skin uncovered changes in multiple proteins related to tissue inflammation. In line with this, losartan reduced inflammation and diminished TNF-α and IL-6 expression in injured forepaws. Collectively, the data argue that RDEB fibrosis is a consequence of a cascade encompassing tissue damage, TGF-β-mediated inflammation, and matrix remodeling. Inhibition of TGF-β activity limits these unwanted outcomes and thereby substantially ameliorates long-term symptoms.

  12. Charting the Vasculome: Uncovering the Principles of Vascular Organization

    Science.gov (United States)

    Oppenheim, Jacob; Magnasco, Marcelo

    2014-03-01

    The efficient distribution of resources in any system requires a carefully designed architecture that is both space filling and efficient. While the principles of such networks are beginning to be uncovered in plants, they remain poorly elucidated in the case of higher animals. We have developed a high-throughput, easily implemented method of mapping vascular networks in mammalian tissue. By combining high resolution, rapid fluorescence blockface imaging with serial sectioning, we are able to map the vasculature of the rat liver at a resolution of 10 microns, revealing the structure above the level of the capillaries, constituting the largest vascular dataset yet assembled. We have developed algorithms for the efficient three-dimensional reconstruction from two-dimensional images, allowing skeletonization and investigation of its geometry and topology. We are able to calculate the scaling properties of these networks as well as the frequency of loops at each level. Using sophisticated topological tools, we are beginning to elucidate the principles of their organization. Ultimately, a greater understanding of vasculature is necessary for the success of efforts in synthetic and regenerative biology along with the better understanding of the growth and development of cancers.

  13. Greenhouse gas emissions from shallow uncovered coal seams

    Institute of Scientific and Technical Information of China (English)

    Saghafi Abouna

    2014-01-01

    This study discusses a method of quantifying emissions from surface coal mining that has been trialled in Australia. The method is based on direct measurement of surface emissions from uncovered coal seams in mine pits, concurrent measurement of residual gas content of blasted coal in mine pits, and measurement of pre-mining gas content of the same seam from cores retrieved from exploration boreholes drilled away from active mining. The results from one of the mines studied are presented in this paper. In this mine, the pre-mining gas content of the target seam was measured using cores from an exploration borehole away from active mining. Gas content varied from 0.7 to 0.8 m3/t and gas composition varied from 16% to 21% CH4 (84-79% CO2). In-pit measurements included seam surface emissions and residual gas content of blasted and ripped coal. Residual gas content varied from 0.09 to 0.15 m3/t, less than twofold across the mine pit. Composition of the residual gas was in general 90%CO2 and 10%CH4, with slight var-iation between samples. Coal seam surface emissions varied from 1.03 to 7.50 mL of CO2-e per minute and per square meter of the coal seam surface, a sevenfold variation across the mine pit.

  14. Uncovering patterns of technology use in consumer health informatics.

    Science.gov (United States)

    Hung, Man; Conrad, Jillian; Hon, Shirley D; Cheng, Christine; Franklin, Jeremy D; Tang, Philip

    2013-11-01

    Internet usage and accessibility has grown at a staggering rate, influencing technology use for healthcare purposes. The amount of health information technology (Health IT) available through the Internet is immeasurable and growing daily. Health IT is now seen as a fundamental aspect of patient care as it stimulates patient engagement and encourages personal health management. It is increasingly important to understand consumer health IT patterns including who is using specific technologies, how technologies are accessed, factors associated with use, and perceived benefits. To fully uncover consumer patterns it is imperative to recognize common barriers and which groups they disproportionately affect. Finally, exploring future demand and predictions will expose significant opportunities for health IT. The most frequently used health information technologies by consumers are gathering information online, mobile health (mHealth) technologies, and personal health records (PHRs). Gathering health information online is the favored pathway for healthcare consumers as it is used by more consumers and more frequently than any other technology. In regard to mHealth technologies, minority Americans, compared with White Americans utilize social media, mobile Internet, and mobile applications more frequently. Consumers believe PHRs are the most beneficial health IT. PHR usage is increasing rapidly due to PHR integration with provider health systems and health insurance plans. Key issues that have to be explicitly addressed in health IT are privacy and security concerns, health literacy, unawareness, and usability. Privacy and security concerns are rated the number one reason for the slow rate of health IT adoption.

  15. Roles of glucose-6-phosphate dehydrogenase in obesity induced by high fat diet%葡萄糖-6-磷酸脱氢酶在高脂饲料诱导肥胖中的作用

    Institute of Scientific and Technical Information of China (English)

    吴春艳; 王芳; 芦建慧; 赵艳

    2013-01-01

    Objective To explore the roles of glucose-6-phosphate dehydrogenase (G6PD) and its downstream NADPH oxidase (NOX) in obesity induced by high fat diet.Methods 40 male Wistar rats were randomly divided into a high fat diet group (30 rats)and a control group (10 rats) fed with rat chow.After six weeks,rats fed with high fat diet were screened out obesity-prone group and obesity-resistant group based on the gain of body weight.obesity-prone group and obesity-resistant group rats continued to be fed with high fat diet.Basic diet group served as normal control.All rats were killed after 13 weeks.Biochemical markers and G6PD activity were determined in adipose tissues.The expression of G6PD and the NOX subunit of P22 gene were detected using RT-PCR.Results Blood glucose levels were higher in obesity-prone group rats than those in control rats (P < 0.05).Triglyceride levels and body fat contents were significantly higher in OP rats than that in obesity-resistant group rats(P < 0.05).Insulin,insulin resistant index,body weight were significantly higher in obesity-prone group rats than that in control and obesity-resistant group rats (P < 0.05).There was no significant difference between the control and obesity-resistant group rats.The activity and expression of G6PD in the obesity-prone group rats were lower than those in the control and obesity-resistant group rats(P < 0.05).The expression of P22 subunit in the obesity-prone group rats was significantly higher than that in the control and obesity-resistant group rats (P < 0.05),but there was no significant difference between the control and OR rats.Conclusion G6PD and its downsream NAPDH oxidase may play an important role in the pathogenesis and development of obesity.%目的 探讨葡萄糖-6-磷酸脱氢酶(G6PD)及其下游NADPH氧化酶在高脂饲料诱导肥胖中的作用.方法 40只雄性Wistar大鼠随机分为高脂饲料组(30只)和基础饲料组(10只),6周后,高脂饲料组依据体

  16. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    Science.gov (United States)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  17. Uncovering Multiple Populations in Globular Clusters with Washington Photometry

    Science.gov (United States)

    Geisler, Douglas; Cummings, Jeff; Villanova, Sandro; Carraro, Giovanni

    2015-01-01

    Globular Clusters (GCs), long considered as ideal Simple Stellar Populations, are now known to harbor a wide variety of chemical inhomogeneities. Multiple populations (MP) are being found in a growing number of Galactic globular clusters (GCs) via both photometric and spectroscopic techniques. Indeed, it has been suggested that a GC is an object that possesses MP. A definitive investigation of MP in GCs will undoubtedly provide a profound improvement in our understanding of their formation and evolution.However, most studies employ either high resolution VLT spectroscopy, HST photometry or inefficient filters from the ground. A ground-based photometric system which is both efficient and effective would be especially excellent for uncovering MP. We demonstrate that the Washington system meets these goals. The Washington C filter, in addition to being specifically designed for the purpose of detecting MPs, is both much broader and redder than competing UV filters, making it far more efficient at detecting MPs and much less sensitive to reddening and extinction.Our analysis of the well-studied GC NGC 1851 shows indeed that the C filter is both very efficient and effective at detecting its previously discovered MPs in the RGB and SGB, using relatively little telescope time on only a 1-meter telescope. Remarkably, we have also detected an intrinsically broad MS best characterized by two distinct but heavily overlapping populations that cannot be explained by binaries, field stars, or photometric errors. Detailed analysis shows that the MS distribution is in very good agreement with that seen on the RGB. This is the first time MPs in a MS have been discovered from the ground, and just as strikingly, using only a 1-meter telescope. The Washington system thus proves to be a very powerful tool for investigating MPs, and holds particular promise for extragalactic objects where photons are limited.

  18. Uncovering labially impacted teeth: apically positioned flap and closed-eruption techniques.

    Science.gov (United States)

    Vermette, M E; Kokich, V G; Kennedy, D B

    1995-01-01

    The purpose of this study was to examine the esthetic and periodontal differences between two methods of uncovering labially impacted maxillary anterior teeth: the apically positioned flap and closed-eruption techniques. The sample consisted of 30 patients who were recalled a minimum of three months after orthodontic treatment of a unilateral labially impacted maxillary anterior tooth. Eighteen of the patients had undergone an apically positioned flap (APF) procedure, and the remaining twelve had undergone the closed-eruption (CE) technique. In the CE group, clinical examination showed less width of attached gingiva on the distal surface and increased probing bone level on the facial surface of the uncovered teeth relative to their contralateral controls. Uncovered teeth in the APF group showed more apical gingival margins on the mesial and facial surfaces; greater crown length on the midfacial surface; increased probing attachment level on the facial surface; increased width of attached gingiva on the facial surface; increased probing bone level on mesial, facial, and distal surfaces; and gingival scarring. Radiographic examination showed shorter roots on the uncovered teeth in both groups. Photographic examination revealed vertical relapse of the uncovered teeth in the APF group. We conclude that labially impacted maxillary anterior teeth uncovered with an apically positioned flap technique have more unesthetic sequalae than those uncovered with a closed-eruption technique.

  19. Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

    Science.gov (United States)

    Madhavarao, Chikkathur N; Agarabi, Cyrus D; Wong, Lily; Müller-Loennies, Sven; Braulke, Thomas; Khan, Mansoor; Anderson, Howard; Johnson, Gibbes R

    2014-01-01

    Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant β-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The β-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased β-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in β-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased β-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.

  20. Uncovering cognitive processes: Different techniques that can contribute to cognitive load research and instruction

    NARCIS (Netherlands)

    Van Gog, Tamara; Kester, Liesbeth; Nievelstein, Fleurie; Giesbers, Bas; Fred, Paas

    2009-01-01

    Van Gog, T., Kester, L., Nievelstein, F., Giesbers, B., & Paas, F. (2009). Uncovering cognitive processes: Different techniques that can contribute to cognitive load research and instruction. Computers in Human Behavior, 25, 325-331.

  1. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

    Science.gov (United States)

    Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

    2015-01-01

    Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

  2. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  3. Enzymes in Glycolysis and the Citric Acid Cycle in the Yeast and Mycelial Forms of Paracoccidioides brasiliensis

    Science.gov (United States)

    Kanetsuna, Fuminori; Carbonell, Luis M.

    1966-01-01

    Kanetsuna, Fuminori (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela), and Luis M. Carbonell. Enzymes in glycolysis and the citric acid cycle in the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol. 92:1315–1320. 1966.—Enzymatic activities in glycolysis, the hexose monophosphate shunt, and the citric acid cycle in cell-free extracts of the yeast and mycelial forms of Paracoccidioides brasiliensis were examined comparatively. Both forms have the enzymes of these pathways. Activities of glucose-6-phosphate dehydrogenase and malic dehydrogenase of the mycelial form were higher than those of the yeast form. Another 15 enzymatic activities of the mycelial form were lower than those of the yeast form. The activity of glyceraldehyde-3-phosphate dehydrogenase showed the most marked difference between the two forms, its activity in the mycelial form being about 20% of that in the yeast form. PMID:5924267

  4. Optimising enzyme production by bakers yeast in continuous culture: physiological knowledge useful for process design and control

    Energy Technology Data Exchange (ETDEWEB)

    Gregory, M.E. [Centre for Process Systems Engineering, Imperial Coll. of Science, Technology and Medicine, London (United Kingdom); Bulmer, M. [Advanced Centre for Biochemical Engineering, University Coll., London (United Kingdom); Bogle, I.D.L. [Advanced Centre for Biochemical Engineering, University Coll., London (United Kingdom); Titchener-Hooker, N. [Advanced Centre for Biochemical Engineering, University Coll., London (United Kingdom)

    1996-10-01

    Saccharomyces cerevisiae was grown in aerobic continuous culture on a defined minimal medium, with glucose (40 g.l{sup -1}) as the growth-limiting carbon source, to acquire knowledge useful in process design and for model-based control. Steady-state concentrations of biomass, glucose, ethanol and activities of model products alcohol dehydrogenase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase and iso-citrate dehydrogenase were determined at dilution rates (D) between 0.06 h{sup -1} and 0.323 h{sup -1} (close to {mu}{sub max}). Enzyme activities showed productivity trends related to the transition from oxidative to oxido-reductive growth. Conclusions are drawn from the data with regard to designing a new process for production of intracellular enzymes. Issues of process stability as well as productivity are discussed. (orig.). With 5 figs.

  5. Glutathione, glutathione-related enzymes, and oxidative stress in individuals with subacute occupational exposure to lead.

    Science.gov (United States)

    Dobrakowski, Michał; Pawlas, Natalia; Hudziec, Edyta; Kozłowska, Agnieszka; Mikołajczyk, Agnieszka; Birkner, Ewa; Kasperczyk, Sławomir

    2016-07-01

    The aim of the study was to investigate the influence of subacute exposure to lead on the glutathione-related antioxidant defense and oxidative stress parameters in 36 males occupationally exposed to lead for 40±3.2days. Blood lead level in the examined population increased significantly by 359% due to lead exposure. Simultaneously, erythrocyte glutathione level decreased by 16%, whereas the activity of glutathione-6-phosphate dehydrogenase in erythrocytes and leukocytes decreased by 28% and 10%, respectively. Similarly, the activity of glutathione-S-transferase in erythrocytes decreased by 45%. However, the activity of glutathione reductase in erythrocytes and leukocytes increased by 26% and 6%, respectively, whereas the total oxidant status value in leukocytes increased by 37%. Subacute exposure to lead results in glutathione pool depletion and accumulation of lipid peroxidation products; however, it does not cause DNA damage. Besides, subacute exposure to lead modifies the activity of glutathione-related enzymes.

  6. Hereditary characteristics of enzyme deficiency and dermatoglyphics in congenital color blindness.

    Science.gov (United States)

    Wu, L Z; Zeng, L H; Ma, Q Y; Xie, Y J; Chen, Y Z; Wu, D Z

    1988-01-01

    The hereditary characteristics of enzyme deficiency and dermatoglyphics in congenital color blindness (CCB) were studied. We propose that there is a linkage between the two loci on the X-chromosome determining CCB and glucose-6-phosphate dehydrogenase (G6PD), based on our study of a high incidence of G6PD deficiency in 156 male cases with CCB. The CCB gene is closely linked with that of G6PD deficiency from our pedigree investigations. The rise in the frequency of eight or more whorls, the low value of atd angle and the presenting rate of real palmar patterns of the thenar, hypothenar and I, areas presented the hereditary traits of congenital color blindness.

  7. Activity of the glutathione antioxidant system and NADPH-generating enzymes in blood serum of rats with type 2 diabetes mellitus after administration of melatonin-correcting drugs.

    Science.gov (United States)

    Agarkov, A A; Popova, T N; Verevkin, A N; Matasova, L V

    2014-06-01

    We studied the effects of epifamin and melaxen on serum content of reduced glutathione and activities of glutathione peroxidase, glutathione reductase, and NADPH-generating enzymes (glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase) in rats with type 2 diabetes mellitus. The concentration of reduced glutathione was decreased in rats with this disease (by 1.8 times), but increased after treatment with epifamin and melaxen (by 1.6 and 1.7 times, respectively). Activities of glutathione peroxidase, glutathione reductase, and NADPH-generating enzymes returned to the control level. Correction of melatonin concentration after treatment with the test drugs was probably followed by inhibition of free radical processes. The observed changes were accompanied by normalization of activity of the glutathione antioxidant system and NADPH-generating enzymes required for normal function of this system.

  8. Deepest Image of Exploded Star Uncovers Bipolar Jets

    Science.gov (United States)

    2004-08-01

    A spectacular new image of Cassiopeia A from NASA's Chandra X-ray Observatory released today has nearly 200 times more data than the "First Light" Chandra image of this object made five years ago. The new image reveals clues that the initial explosion caused by the collapse of a massive star was far more complicated than suspected. Chandra Broadband Image of Cassiopeia A Chandra Broadband Image of Cassiopeia A "Although this young supernova remnant has been intensely studied for years, this deep observation is the most detailed ever made of the remains of an exploded star," said Martin Laming of the Naval Research Laboratory in Washington, D.C. Laming is part of a team of scientists led by Una Hwang of the Goddard Space Flight Center in Greenbelt, Maryland. "It is a gold mine of data that astronomers will be panning through for years to come." The one-million-second (about 11.5-day) observation of Cassiopeia A uncovered two large, opposed jet-like structures that extend to about 10 light years from the center of the remnant. Clouds of iron that have remained nearly pure for the approximately 340 years since the explosion were also detected. "The presence of the bipolar jets suggests that jets could be more common in relatively normal supernova explosions than supposed by astronomers," said Hwang. A paper by Hwang, Laming and others on the Cassiopeia A observation will appear in an upcoming issue of The Astrophysical Journal Letters. Chandra Enhanced Silicon Image of Cassiopeia A Chandra Enhanced Silicon Image of Cassiopeia A X-ray spectra show that the jets are rich in silicon atoms and relatively poor in iron atoms. In contrast, fingers of almost pure iron gas extend in a direction nearly perpendicular to the jets. This iron was produced in the central, hottest regions of the star. The high silicon and low iron abundances in the jets indicate that massive, matter-dominated jets were not the immediate cause of the explosion, as these should have carried out large

  9. Enzyme kinetics of conjugating enzymes: PAPS sulfotransferase.

    Science.gov (United States)

    James, Margaret O

    2014-01-01

    The sulfotransferase (SULT) enzymes catalyze the formation of sulfate esters or sulfamates from substrates that contain hydroxy or amine groups, utilizing 3'-phosphoadenosyl-5'-phosphosulfate (PAPS) as the donor of the sulfonic group. The rate of product formation depends on the concentrations of PAPS and substrate as well as the sulfotransferase enzyme; thus, if PAPS is held constant while varying substrate concentration (or vice versa), the kinetic constants derived are apparent constants. When studied over a narrow range of substrate concentrations, classic Michaelis-Menten kinetics can be observed with many SULT enzymes and most substrates. Some SULT enzymes exhibit positive or negative cooperativity during conversion of substrate to product, and the kinetics fit the Hill plot. A characteristic feature of most sulfotransferase-catalyzed reactions is that, when studied over a wide range of substrate concentrations, the rate of product formation initially increases as substrate concentration increases, then decreases at high substrate concentrations, i.e., they exhibit substrate inhibition or partial substrate inhibition. This chapter gives an introduction to sulfotransferases, including a historical note, the nomenclature, a description of the function of SULTs with different types of substrates, presentation of examples of enzyme kinetics with SULTs, and a discussion of what is known about mechanisms of substrate inhibition in the sulfotransferases.

  10. Changes in the biochemical composition and enzyme activity during dormancy release of Cyclocarya paliurus seeds

    Institute of Scientific and Technical Information of China (English)

    Fang Sheng-zuo; Wang Jia-yuan

    2007-01-01

    Cyclocarya paliurus is only propagated from seeds which have pronounced dormancy. Overcoming seed dormancy is an important component of efficient and cost-effective seedling production of Cyclocarya paliurus. Changes in biochemical composition and enzyme activity were investigated during dormancy release. The activities of all the studied enzymes in the stratified seeds increased significantly, compared to those in the control samples. Of the enzymes examined, the activities of protease increased the most (413.8%), followed by peroxidase (278.7%), lipase (161.0%), glucose-6-phosphate dehydrognase (149.1%) and amylase (60.6%) after 8 months of stratification. Crude fat and protein constituted the bulk of the storage reserves in mature seeds of C. paliurus. Compared with the seeds before stratification, about 45% of the starch, 46% of the protein and 11% of the crude fat were depleted during dormancy release of C. paliurus seeds, while the soluble sugar content was enhanced by 101.5% in the germinating seeds. Correlation analysis showed, during dormancy release of C. paliurus seeds, a close positive relationship between POD and G6PDH activity as well as soluble sugar content and amylase activity, while there was a significant negative relationship between storage substances and their related enzyme activities.

  11. Changes in renal enzyme activities following the administration of gentamicin to unilateral nephrectomized rats.

    Science.gov (United States)

    Avidan, G; Aladjem, M; Israeli, B A; Bogin, E

    1986-02-28

    The effects of unilateral nephrectomy and the impact of gentamicin administration on renal tissue enzyme activities in adult Wistar rats were investigated. Gentamicin 200 mg/kg body wt. or an equivalent volume of saline to control rats was administered subcutaneously on three consecutive days, followed by unilateral nephrectomy. Rats were killed on day 3, 7 or 14 following nephrectomy. Alkaline phosphatase, predominantly a proximal tubular brush border enzyme, rose in both the experimental and control groups, however, significantly less in the gentamicin treated rats. Aspartate aminotransferase activity, an enzyme participating in renal glucogenesis, increased transiently in the control but remained unchanged in the experimental group. No difference in glucose-6-phosphate dehydrogenase activity between the two groups was observed, probably reflecting the localization of this enzyme to distal tubular segments, a site unaffected by gentamicin. Significant and similar increases in Mg2+ and Na+ K+ ATPase were observed on day 14 in both groups. The administration of the drug resulted in a marked reduction in oxygen consumption, with a higher oxidation to phosphorylation ratio (P/O). Serum creatinine concentration was significantly higher on days 3 and 7 in the experimental group reverting to control values on the 14th day. Urea concentration increased significantly on days 3 and 7, decreasing on the 14th day to values slightly, but significantly, higher than those of the controls.

  12. Diagnosis and epidemiology of red blood cell enzyme disorders

    Directory of Open Access Journals (Sweden)

    Richard Van Wijk

    2013-03-01

    Full Text Available The red blood cell possess an active metabolic machinery that provides the cell with energy to pump ions against electrochemical gradients, to maintain its shape, to keep hemoglobin iron in the reduced (ferrous form, and to maintain enzyme and hemoglobin sulfhydryl groups. The main source of metabolic energy comes from glucose. Glucose is metabolized through the glycolytic pathway and through the hexose monophosphate shunt. Glycolysis catabolizes glucose to pyruvate and lactate, which represent the end products of glucose metabolism in the erythrocyte. Adenosine diphosphate (ADP is phosphorylated to adenosine triphosphate (ATP, and nicotinamide adenine dinucleotide (NAD+ is reduced to NADH in glycolysis. 2,3- Bisphosphoglycerate, an important regulator of the oxygen affinity of hemoglobin, is generated during glycolysis by the Rapoport-Luebering shunt. The hexose monophosphate shunt oxidizes glucose-6-phosphate, reducing NADP+ to reduced nicotinamide adenine dinucleotide phosphate (NADPH. The red cell lacks the capacity for de novo purine synthesis but has a salvage pathway that permits synthesis of purine nucleotides from purine bases...

  13. Enzymes for improved biomass conversion

    Science.gov (United States)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  14. Unhairing with enzymes

    OpenAIRE

    Crispim, A.; Mota, M.

    2003-01-01

    The use of enzymes in the leather industry is increasing and their application is being widened to include operations such as de-greasing, unhairing and other wet-end operations. Enzymes can also be used to assist with recycling leather wastes as well as to avoid pollution. The present work is devoted to illustrate the potential application of enzymes in unhairing without hair destruction. Enzymatic unhairing is based upon the weakening of the epidermis basal layer to which the hair is at...

  15. Microbial amylolytic enzymes.

    Science.gov (United States)

    Vihinen, M; Mäntsälä, P

    1989-01-01

    Starch-degrading, amylolytic enzymes are widely distributed among microbes. Several activities are required to hydrolyze starch to its glucose units. These enzymes include alpha-amylase, beta-amylase, glucoamylase, alpha-glucosidase, pullulan-degrading enzymes, exoacting enzymes yielding alpha-type endproducts, and cyclodextrin glycosyltransferase. Properties of these enzymes vary and are somewhat linked to the environmental circumstances of the producing organisms. Features of the enzymes, their action patterns, physicochemical properties, occurrence, genetics, and results obtained from cloning of the genes are described. Among all the amylolytic enzymes, the genetics of alpha-amylase in Bacillus subtilis are best known. Alpha-Amylase production in B. subtilis is regulated by several genetic elements, many of which have synergistic effects. Genes encoding enzymes from all the amylolytic enzyme groups dealt with here have been cloned, and the sequences have been found to contain some highly conserved regions thought to be essential for their action and/or structure. Glucoamylase appears usually in several forms, which seem to be the results of a variety of mechanisms, including heterogeneous glycosylation, limited proteolysis, multiple modes of mRNA splicing, and the presence of several structural genes.

  16. Adenylate-forming enzymes

    Science.gov (United States)

    Schmelz, Stefan; Naismith, James H.

    2012-01-01

    Thioesters, amides and esters are common chemical building blocks in a wide array of natural products. The formation of these bonds can be catalyzed in a variety of ways. For chemists, the use of an activating group is a common strategy and adenylate enzymes are exemplars of this approach. Adenylating enzymes activate the otherwise unreactive carboxylic acid by transforming the normal hydroxyl leaving group into adenosine monophosphate. Recently there have been a number of studies of such enzymes and in this review we suggest a new classification scheme. The review highlights the diversity in enzyme fold, active site architecture and metal coordination that has evolved to catalyze this particular reaction. PMID:19836944

  17. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD).

    Science.gov (United States)

    Mauricio, Isabel L; Yeo, Matthew; Baghaei, Mehdi; Doto, Daniela; Pratlong, Francine; Zemanova, Eva; Dedet, Jean-Pierre; Lukes, Julius; Miles, Michael A

    2006-06-01

    Multilocus enzyme electrophoresis is the gold standard for identification of Leishmania species and strains. Drawbacks include: only amino acid polymorphisms affecting electrophoretic mobility are detected; distinct allozymes can have coincident mobilities; few characters are available; and parasites must be cultured in bulk. So far, thousands of Leishmania strains have been phenotyped by multilocus enzyme electrophoresis. Here, we sequence enzyme-coding genes to provide a PCR-based higher resolution equivalent of multilocus enzyme electrophoresis, particularly for Leishmania infantum. Of 15 enzymes used for multilocus enzyme electrophoresis (MON typing) we have sequenced aspartate aminotransferase, glucose-6-phosphate isomerase, nucleoside hydrolase 1, nucleoside hydrolase 2 and 6-phosphogluconate dehydrogenase. Heterozygous alleles were common, with multiple heterozygous sites within a single locus for several of the genes. Haplotypes were resolved by allele-specific PCR and allele-specific sequencing. Heterozygous haplotypes conformed to the haplotypes of putative parents. One strain appeared to be hybrid across two genetic groups of the Leishmania donovani complex. In most cases, a single amino acid polymorphism was responsible for change in enzyme mobility. Some indistinguishable phenotypes were produced by distinct genotypes. Silent genetic polymorphisms provided enhanced discrimination over multilocus enzyme electrophoresis, for example, by subdividing the zymodeme MON-1. The PCR-based genotyping that we describe could be applied directly to clinical samples or to small volume cultures and in a multilocus sequence typing format. Furthermore, it can be used to detect recombination indirectly and for population genetics studies.

  18. Magnetically responsive enzyme powders

    Energy Technology Data Exchange (ETDEWEB)

    Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2015-04-15

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  19. A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes

    NARCIS (Netherlands)

    Maruthamuthu, Mukil; Jiménez Avella, Diego; Stevens, Patricia; van Elsas, Jan Dirk

    2016-01-01

    BACKGROUND: Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomas

  20. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M;

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  1. Cotton cellulose: enzyme adsorption and enzymic hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Beltrame, P.L.; Carniti, P.; Focher, B.; Marzetti, A.; Cattaneo, M.

    1982-01-01

    The adsorption of a crude cellulase complex from Trichoderma viride on variously pretreated cotton cellulose samples was studied in the framework of the Langmuir approach at 2-8 degrees. The saturation amount of adsorbed enzyme was related to the susceptibility of the substrates to hydrolysis. In every case the adsorption process was faster by 2-3 orders of magnitude than the hydrolysis step to give end products. For ZnCl/sub 2/-treated cotton cellulose the Langmuir parameters correlated fairly well with the value of the Michaelis constant, measured for its enzymic hydrolysis, and the adsorptive complex was indistinguishable from the complex of the Michaelis-Menten model for the hydrolysis.

  2. Using Text Mining to Uncover Students' Technology-Related Problems in Live Video Streaming

    Science.gov (United States)

    Abdous, M'hammed; He, Wu

    2011-01-01

    Because of their capacity to sift through large amounts of data, text mining and data mining are enabling higher education institutions to reveal valuable patterns in students' learning behaviours without having to resort to traditional survey methods. In an effort to uncover live video streaming (LVS) students' technology related-problems and to…

  3. Uncovering the Best Skill Multimap by Constraining the Error Probabilities of the Gain-Loss Model

    Science.gov (United States)

    Anselmi, Pasquale; Robusto, Egidio; Stefanutti, Luca

    2012-01-01

    The Gain-Loss model is a probabilistic skill multimap model for assessing learning processes. In practical applications, more than one skill multimap could be plausible, while none corresponds to the true one. The article investigates whether constraining the error probabilities is a way of uncovering the best skill assignment among a number of…

  4. 77 FR 12227 - Long Term 2 Enhanced Surface Water Treatment Rule: Uncovered Finished Water Reservoirs; Public...

    Science.gov (United States)

    2012-02-29

    ... AGENCY 40 CFR Parts 141 and 142 Long Term 2 Enhanced Surface Water Treatment Rule: Uncovered Finished Water Reservoirs; Public Meeting AGENCY: Environmental Protection Agency (EPA). ACTION: Notice of public... requirement in the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 rule). At this meeting, EPA...

  5. Uncovering the Motivating Factors behind Writing in English in en EFL Context

    Science.gov (United States)

    Büyükyavuz, Oya; Çakir, Ismail

    2014-01-01

    Writing in a language, whether the target or native, is regarded as a complex activity operating on multiple cognitive levels. This study aimed to uncover the factors which motivate teacher trainees of English to write in English in an EFL context. The study also investigated the differences in the ways teacher trainees are motivated in terms of…

  6. Uncovering Expertise-Related Differences in Troubleshooting Performance: Combining Eye Movement and Concurrent Verbal Protocol Data

    NARCIS (Netherlands)

    Van Gog, Tamara; Paas, Fred; Van Merriënboer, Jeroen

    2007-01-01

    This study explored the value of eye movement data for uncovering relatively small expertise-related differences in electrical circuit-troubleshooting performance, and describes that value in relation to concurrent verbal protocols. Results show that in the ‘problem orientation’ phase, higher expert

  7. Epistemically Virtuous Risk Management : Financial Due Diligence and Uncovering the Madoff Fraud

    NARCIS (Netherlands)

    de Bruin, Boudewijn; Luetge, Christoph; Jauernig, Johanna

    2014-01-01

    The chapter analyses how Bernard Madoff’s Ponzi scheme was uncovered by Harry Markopolos, an employee of Rampart Investment Management, LLC, and the contribution of so-called epistemic virtues to Markopolos’ success. After Rampart had informed the firm about an allegedly highly successful hedge fund

  8. 76 FR 4290 - Uncovered Innerspring Units From the People's Republic of China: Final Results of First...

    Science.gov (United States)

    2011-01-25

    ... synthetic material or woven material and then glued together in a linear fashion. Uncovered innersprings are... innerspring. Pocketed and non-pocketed innerspring units are included in this definition. Non-pocketed innersprings are typically joined together with helical wire and border rods. Non-pocketed innersprings...

  9. 77 FR 21961 - Uncovered Innerspring Units From the People's Republic of China: Final Results and Final...

    Science.gov (United States)

    2012-04-12

    ... synthetic material or woven material and then glued together in a linear fashion. Uncovered innersprings are... innerspring. Pocketed and non-pocketed innerspring units are included in this definition. Non-pocketed innersprings are typically joined together with helical wire and border rods. Non-pocketed innersprings...

  10. 40 CFR 141.714 - Requirements for uncovered finished water storage facilities.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 22 2010-07-01 2010-07-01 false Requirements for uncovered finished water storage facilities. 141.714 Section 141.714 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) WATER PROGRAMS (CONTINUED) NATIONAL PRIMARY DRINKING WATER REGULATIONS...

  11. Uncovering Influence through Social Network Analysis: The Role of Schools in Education for Sustainable Development

    Science.gov (United States)

    Kolleck, Nina

    2016-01-01

    This paper examines the implementation of Education for Sustainable Development (ESD) in Germany and explores the possibilities of Social Network Analysis (SNA) for uncovering influential actors in educational policy innovation processes. From the theoretical perspective, an actor's influence is inferred from its relative position within…

  12. EXCHANGE RATES AND VOLATILITY IN CENTRAL AND EASTERN EUROPE: A TEST FOR UNCOVERED INTEREST PARITY

    Directory of Open Access Journals (Sweden)

    DUMITRESCU Dan-Gabriel

    2009-05-01

    Full Text Available At times of heightened global capital market volatility, high-yielding currencies tend to depreciate, while low-yielding currencies tend to serve as a€śsafe heavena€ť. We present the results of a test for Uncovered Interest Parity for selected European cu

  13. Uncovering One Trilingual Child's Multi-Literacies Development across Informal and Formal Learning Contexts

    Science.gov (United States)

    Kim, Mi Song

    2016-01-01

    Due to globalisation and rapid technological change, today's educators need to help students develop multi-literacy competencies to enable them to function successfully in our culturally and linguistically diverse (CLD) and increasingly connected global and digital society. A qualitative, longitudinal case study attempted to uncover the…

  14. Feminist Approaches to Triangulation: Uncovering Subjugated Knowledge and Fostering Social Change in Mixed Methods Research

    Science.gov (United States)

    Hesse-Biber, Sharlene

    2012-01-01

    This article explores the deployment of triangulation in the service of uncovering subjugated knowledge and promoting social change for women and other oppressed groups. Feminist approaches to mixed methods praxis create a tight link between the research problem and the research design. An analysis of selected case studies of feminist praxis…

  15. Genome-wide meta-analysis uncovers novel loci influencing circulating leptin levels

    DEFF Research Database (Denmark)

    Kilpeläinen, Tuomas O; Carli, Jayne F Martin; Skowronski, Alicja A

    2016-01-01

    Leptin is an adipocyte-secreted hormone, the circulating levels of which correlate closely with overall adiposity. Although rare mutations in the leptin (LEP) gene are well known to cause leptin deficiency and severe obesity, no common loci regulating circulating leptin levels have been uncovered...

  16. Detection of enzymes dehydrogenases and proteases inBrugia malayi filarial parasites.

    Science.gov (United States)

    Bhandary, Y P; Krithika, K N; Kulkarni, Sandeep; Reddy, M V R; Harinath, B C

    2006-03-01

    Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the other two stages (PFlouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the L(3) larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size.

  17. Enzyme molecules as nanomotors.

    Science.gov (United States)

    Sengupta, Samudra; Dey, Krishna K; Muddana, Hari S; Tabouillot, Tristan; Ibele, Michael E; Butler, Peter J; Sen, Ayusman

    2013-01-30

    Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme molecules increase in the presence of the substrate, hydrogen peroxide, in a concentration-dependent manner. Employing a microfluidic device to generate a substrate concentration gradient, we show that both catalase and urease enzyme molecules spread toward areas of higher substrate concentration, a form of chemotaxis at the molecular scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chemically interconnected enzymes can be drawn together.

  18. Robust enzyme design: bioinformatic tools for improved protein stability.

    Science.gov (United States)

    Suplatov, Dmitry; Voevodin, Vladimir; Švedas, Vytas

    2015-03-01

    The ability of proteins and enzymes to maintain a functionally active conformation under adverse environmental conditions is an important feature of biocatalysts, vaccines, and biopharmaceutical proteins. From an evolutionary perspective, robust stability of proteins improves their biological fitness and allows for further optimization. Viewed from an industrial perspective, enzyme stability is crucial for the practical application of enzymes under the required reaction conditions. In this review, we analyze bioinformatic-driven strategies that are used to predict structural changes that can be applied to wild type proteins in order to produce more stable variants. The most commonly employed techniques can be classified into stochastic approaches, empirical or systematic rational design strategies, and design of chimeric proteins. We conclude that bioinformatic analysis can be efficiently used to study large protein superfamilies systematically as well as to predict particular structural changes which increase enzyme stability. Evolution has created a diversity of protein properties that are encoded in genomic sequences and structural data. Bioinformatics has the power to uncover this evolutionary code and provide a reproducible selection of hotspots - key residues to be mutated in order to produce more stable and functionally diverse proteins and enzymes. Further development of systematic bioinformatic procedures is needed to organize and analyze sequences and structures of proteins within large superfamilies and to link them to function, as well as to provide knowledge-based predictions for experimental evaluation.

  19. Enzymic lactose hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J.J.; Brand, J.C.

    1980-01-01

    Acid or enzymic hydrolysis can be used to hydrolyze lactose. Advantages of both are compared and details of enzymic hydrolysis using yeast or fungal enzymes given. The new scheme outlined involves recycling lactase. Because lactose and lactase react to ultrafiltration (UF) membranes differently separation is possible. Milk or milk products are ultrafiltered to separate a concentrate from a lactose-rich permeate which is treated with lactase in a reactor until hydrolysis reaches a required level. The lactase can be removed by UF as it does not permeate the membrane, and it is recycled back to the reactor. Permeate from the second UF stage may or may not be recombined with the concentrate from the first stage to produce a low lactose product (analysis of a typical low-lactose dried whole milk is given). Batch or continuous processes are explained and a batch process without enzyme recovery is discussed. (Refs. 4).

  20. Enzymes in Analytical Chemistry.

    Science.gov (United States)

    Fishman, Myer M.

    1980-01-01

    Presents tabular information concerning recent research in the field of enzymes in analytic chemistry, with methods, substrate or reaction catalyzed, assay, comments and references listed. The table refers to 128 references. Also listed are 13 general citations. (CS)

  1. Membrane Assisted Enzyme Fractionation

    DEFF Research Database (Denmark)

    Yuan, Linfeng

    . In this thesis, separations using crossflow elecro-membrane filtration (EMF) of amino acids, bovine serum albumin (BSA) and industrial enzymes from Novozymes were performed. The main objective of this study was to investigate the technological feasibility of EMF in the application of industrial enzyme...... fractionation, such as removal of a side activity from the main enzyme activity. As a proof-of-concept, amino acids were used as model solution to test the feasibility of EMF in the application of amphoteric molecule separation. A single amino acid was used to illustrate the effect of an electric field...... on the separation performance were very small in the investigated range. The mass transport of each enzyme can be well explained by the Extended-Nernst-Planck equation. Better separation was observed at lower feed concentration, higher solution pH in the investigated range and with a polysulfone (PS) MF membrane...

  2. Overproduction of ligninolytic enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  3. RNA-modifying enzymes.

    Science.gov (United States)

    Ferré-D'Amaré, Adrian R

    2003-02-01

    A bewildering number of post-transcriptional modifications are introduced into cellular RNAs by enzymes that are often conserved among archaea, bacteria and eukaryotes. The modifications range from those with well-understood functions, such as tRNA aminoacylation, to widespread but more mysterious ones, such as pseudouridylation. Recent structure determinations have included two types of RNA nucleobase modifying enzyme: pseudouridine synthases and tRNA guanine transglycosylases.

  4. Trehalose-6-phosphate synthase and stabilization of yeast glycolysis

    DEFF Research Database (Denmark)

    Fraenkel, Dan; Nielsen, Jens

    2016-01-01

    ‘Lost in transition: Startup of glycolysis yields subpopulations of nongrowing cells…’ (‘LIT’, van Heerden et al. 2014) is a massive paper from groups in Amsterdam and Delft, which deals with broad issues in metabolism and cell heterogeneity, as addressed for the predominant metabolic pathway......, glycolysis, in the context of a long studied but incompletely understood yeast mutant which is impaired in use of glucose without evident direct defects in the pathway. The primary approach is the quite original one of predicting, for the mutant, the dynamics of metabolism upon glucose addition, based...

  5. Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

    Directory of Open Access Journals (Sweden)

    Lushchak V.I.

    1998-01-01

    Full Text Available The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

  6. Red cell enzymes.

    Science.gov (United States)

    Paniker, N V

    1975-03-01

    As compared to other cells of the body, the mammalian red cell has one of the simplest structural organizations. As a result, this cell has been extensively used in studies involving the structure, function, and integrity of cell membranes as well as cytoplasmic events. Additionally, the metabolic activities of the red blood cell are also relatively simple. During the past quarter century or so, an ocean of knowledge has been gathered on various aspects of red cell metabolism and function. The fields of enzymes, hemoglobin, membrane, and metabolic products comprise the major portion of this knowledge. These advances have made valuable contributions to biochemistry and medicine. Despite these favorable aspects of this simple, anucleated cell, it must be conceded that our knowledge about the red cell is far from complete. We are still in the dark concerning the mechanism involved in several aspects of its membrane, hemoglobin, enzymes, and a large number of other constituents. For example, a large number of enzymes with known catalytic activity but with unknown function have eluded investigators despite active pursuit. This review will be a consolidation of our present knowledge of human red cell enzymes, with particular reference to their usefulness in the diagnosis and therapy of disease. Owing to the multitude of publications by prominent investigators on each of the approximately 50 enzymes discussed in this review, it was impossible to cite a majority of them.

  7. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-01-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C → U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics. PMID:26465508

  8. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  9. Random-walk enzymes.

    Science.gov (United States)

    Mak, Chi H; Pham, Phuong; Afif, Samir A; Goodman, Myron F

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C→U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  10. Enzyme recycling in lignocellulosic biorefineries

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Pinelo, Manuel

    2017-01-01

    platform. Cellulases are the most important enzymes required in this process, but the complex nature of lignocellulose requires several other enzymes (hemicellulases and auxiliary enzymes) for efficient hydrolysis. Enzyme recycling increases the catalytic productivity of the enzymes by reusing them...... upscaled and tested in industrial settings, mainly because of many difficulties with recycling of enzymes from the complex lignocellulose hydrolyzate at industrially relevant conditions, i.e., high solids loadings. The challenges are associated with the large number of different enzymes required...... for efficient hydrolysis, enzyme stability, and the detrimental interaction between enzyme and lignin. This review provides a comprehensive overview of the various methods for enzyme recovery and recycling, for example recycling of free enzymes, readsorption to fresh material, recycling of solids, membrane...

  11. Changes in soluble carbohydrates and related enzymes induced by low temperature during early developmental stages of quinoa (Chenopodium quinoa) seedlings.

    Science.gov (United States)

    Rosa, Mariana; Hilal, Mirna; González, Juan A; Prado, Fernando E

    2004-06-01

    Low temperature represents one of the principal limitations in species distribution and crop productivity. Responses to chilling include the accumulation of simple carbohydrates and changes in enzymes involved in their metabolism. Soluble carbohydrate levels and invertase, sucrose synthase (SS), sucrose-6-phosphate synthase (SPS) and alpha-amylase activities were analysed in cotyledons and embryonic axes of quinoa seedlings grown at 5 degrees C and 25 degrees C in the dark. Significant differences in enzyme activities and carbohydrate levels were observed. Sucrose content in cotyledons was found to be similar in both treatments, while in embryonic axes there were differences. Invertase activity was the most sensitive to temperature in both organs; however, SS and SPS activities appear to be less stress-sensitive. Results suggest that 1) metabolism in germinating perispermic seeds would be different from endospermic seeds, 2) sucrose futile cycles would be operating in cotyledons, but not in embryonic axes of quinoa seedlings under our experimental conditions, 3) low temperature might induce different regulatory mechanisms on invertase, SS and SPS enzymes in both cotyledons and embryonic axes of quinoa seedlings, and 4) low temperature rather than water uptake would be mainly responsible for the changes observed in carbohydrate and related enzyme activities.

  12. Ameliorating effect of eugenol on hyperglycemia by attenuating the key enzymes of glucose metabolism in streptozotocin-induced diabetic rats.

    Science.gov (United States)

    Srinivasan, Subramani; Sathish, Gajendren; Jayanthi, Mahadevan; Muthukumaran, Jayachandran; Muruganathan, Udaiyar; Ramachandran, Vinayagam

    2014-01-01

    Epidemiological studies have demonstrated that diabetes mellitus is a serious health burden for both governments and healthcare providers. This study was hypothesized to evaluate the antihyperglycemic potential of eugenol by determine the activities of key enzymes of glucose metabolism in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced into male albino Wistar rats by intraperitoneal administration of STZ (40 mg/kg body weight (b.w.)). Eugenol was administered to diabetic rats intragastrically at 2.5, 5, and 10 mg/kg b.w. for 30 days. The dose 10 mg/kg b.w. significantly reduced the levels of blood glucose and glycosylated hemoglobin (HbA1c) and increased plasma insulin level. The altered activities of the key enzymes of carbohydrate metabolism such as hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and liver marker enzymes (AST, ALT, and ALP), creatine kinase and blood urea nitrogen in serum and blood of diabetic rats were significantly reverted to near normal levels by the administration of eugenol. Further, eugenol administration to diabetic rats improved body weight and hepatic glycogen content demonstrated the antihyperglycemic potential of eugenol in diabetic rats. The present findings suggest that eugenol can potentially ameliorate key enzymes of glucose metabolism in experimental diabetes, and it is sensible to broaden the scale of use of eugenol in a trial to alleviate the adverse effects of diabetes.

  13. D-pinitol attenuates the impaired activities of hepatic key enzymes in carbohydrate metabolism of streptozotocin-induced diabetic rats.

    Science.gov (United States)

    Sivakumar, Selvaraj; Subramanian, Sorimuthu P

    2009-09-01

    During diabetes mellitus, endogenous hepatic glucose production is increased as a result of impaired activities of the key enzymes of carbohydrate metabolism, which leads to the condition known as hyperglycemia. D-pinitol, a bioactive constituent isolated from soybeans, has been shown to reduce hyperglycemia in experimental diabetes. We therefore designed this study to investigate the effect of oral administration of D-pinitol (50 mg/kg b. w. for 30 days) on the activities of key enzymes in carbohydrate and glycogen metabolism in the liver tissues of streptozotocin-induced diabetic rats. The efficacy was compared with glyclazide, a standard hypoglycemic drug. Oral administration of D-pinitol to diabetic group of rats showed a marked decrease in the levels of blood glucose, glycosylated hemoglobin and an increase in plasma insulin and body weight. The activities of the hepatic enzymes such as hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glycogen synthase and hepatic glycogen content were significantly (p pinitol. The results suggest that alterations in the activities of key metabolic enzymes of carbohydrate metabolism could be one of the biochemical rationale by which D-pinitol attenuates the hyperglycemic effect in diabetic rats.

  14. Uncovering highly obfuscated plagiarism cases using fuzzy semantic-based similarity model

    OpenAIRE

    Salha M. Alzahrani; Naomie Salim; Vasile Palade

    2015-01-01

    Highly obfuscated plagiarism cases contain unseen and obfuscated texts, which pose difficulties when using existing plagiarism detection methods. A fuzzy semantic-based similarity model for uncovering obfuscated plagiarism is presented and compared with five state-of-the-art baselines. Semantic relatedness between words is studied based on the part-of-speech (POS) tags and WordNet-based similarity measures. Fuzzy-based rules are introduced to assess the semantic distance between source and su...

  15. Uncovering Special Nuclear Materials by Low-energy Nuclear Reaction Imaging

    OpenAIRE

    P. B. Rose; Erickson, A. S.; Mayer, M; J. Nattress; Jovanovic, I.

    2016-01-01

    Weapons-grade uranium and plutonium could be used as nuclear explosives with extreme destructive potential. The problem of their detection, especially in standard cargo containers during transit, has been described as “searching for a needle in a haystack” because of the inherently low rate of spontaneous emission of characteristic penetrating radiation and the ease of its shielding. Currently, the only practical approach for uncovering well-shielded special nuclear materials is by use of act...

  16. Entropy and Enzyme Catalysis.

    Science.gov (United States)

    Åqvist, Johan; Kazemi, Masoud; Isaksen, Geir Villy; Brandsdal, Bjørn Olav

    2017-02-21

    The role played by entropy for the enormous rate enhancement achieved by enzymes has been debated for many decades. There are, for example, several confirmed cases where the activation free energy is reduced by around 10 kcal/mol due to entropic effects, corresponding to a rate enhancement of ∼10(7) compared to the uncatalyzed reaction. However, despite substantial efforts from both the experimental and theoretical side, no real consensus has been reached regarding the origin of such large entropic contributions to enzyme catalysis. Another remarkable instance of entropic effects is found in enzymes that are adapted by evolution to work at low temperatures, near the freezing point of water. These cold-adapted enzymes invariably show a more negative entropy and a lower enthalpy of activation than their mesophilic orthologs, which counteracts the exponential damping of reaction rates at lower temperature. The structural origin of this universal phenomenon has, however, remained elusive. The basic problem with connecting macroscopic thermodynamic quantities, such as activation entropy and enthalpy derived from Arrhenius plots, to the 3D protein structure is that the underlying detailed (microscopic) energetics is essentially inaccessible to experiment. Moreover, attempts to calculate entropy contributions by computer simulations have mostly focused only on substrate entropies, which do not provide the full picture. We have recently devised a new approach for accessing thermodynamic activation parameters of both enzyme and solution reactions from computer simulations, which turns out to be very successful. This method is analogous to the experimental Arrhenius plots and directly evaluates the temperature dependence of calculated reaction free energy profiles. Hence, by extensive molecular dynamics simulations and calculations of up to thousands of independent free energy profiles, we are able to extract activation parameters with sufficient precision for making

  17. Angiotensin-converting enzyme

    DEFF Research Database (Denmark)

    Sørensen, P G; Rømer, F K; Cortes, D

    1984-01-01

    In order to evaluate bleomycin-associated lung damage in humans, lung function parameters and serum levels of the endothelial-bound angiotensin-converting enzyme (ACE) were determined by serial measurements in 11 patients who were treated for testicular cancer. None developed clinical or radiolog......In order to evaluate bleomycin-associated lung damage in humans, lung function parameters and serum levels of the endothelial-bound angiotensin-converting enzyme (ACE) were determined by serial measurements in 11 patients who were treated for testicular cancer. None developed clinical...

  18. Thermal Behavior of a Single Spent Fuel in Water Pool Storage Under Partially Uncovered Condition

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Woo Ram; Park, Hee Sung; Song, Sub Lee; Lee, Jae Young [Handong Global Univ, Pohang (Korea, Republic of)

    2015-10-15

    LOCA in SFP can be led by a partial drain-down or a boil off scenario. In order to predict the response and consequence in such case, exact model on the partially uncovered SFP has to be established. Most studies on accidents in SFP have been done by safety analysis codes such as ATHLET-CD, ASTEC, MAAP, and MELCOR. However, an experimental investigation has not been conducted so far. Schultz et al.(2014) studied experimentally the response of air cooled BWR fuel assembly which is blocked at lower side fluid path. In this study, we experimentally investigated the thermal response of a partially uncovered single nuclear fuel rod (SNFR) in the SFP. The SNFR was 1/4 scaled down in axial length. 1-dimensional numerical analysis model was developed and compared with the result of experiment. An experimental study was conducted for obtaining transient temperature profile data of a modeled single nuclear fuel rod in heating condition under partially uncovered condition. Numerical prediction model was developed also and the prediction result was compared with the experimental result.

  19. Absence of effects of dietary wheat bran on the activities of some key enzymes of carbohydrate and lipid metabolism in mouse liver and adipose tissue.

    Science.gov (United States)

    Stanley, J C; Lambadarios, J A; Newsholme, E A

    1986-03-01

    1. The effects of a 100 g/kg dietary substitution of wheat bran on the body-weight gain, food consumption and faecal dry weight of mice given a high-sucrose diet and on the activities of some key enzymes of carbohydrate and lipid metabolism in liver and adipose tissue were studied. 2. Wheat bran had no effect on body-weight gain, food consumption or faecal dry weight. 3. Wheat bran had no effect on the activities of hepatic glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) (EC 1.1.1.40), ATP-citrate (pro-3S)-lyase (EC 4.1.3.8), pyruvate kinase (EC 2.7.1.40) and fructose-1,6-bisphosphatase (EC 3.1.3.11). The activity of hepatic 6-phosphofructokinase (EC 2.7.1.11) increased but only when expressed on a body-weight basis. 4. Wheat bran had no effect on the activities of adipose tissue glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+), ATP-citrate (pro-3S)-lyase, hexokinase (EC 2.7.1.1), 6-phosphofructokinase and pyruvate kinase. 5. These results suggest that unlike guar gum and bagasse, wheat bran does not change the flux through some pathways of lipogenesis in liver and adipose tissue when mice are given high-sucrose diets.

  20. The surface science of enzymes

    DEFF Research Database (Denmark)

    Rod, Thomas Holm; Nørskov, Jens Kehlet

    2002-01-01

    One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function......? To solve these problems we must understand in some detail how enzymes interact with reactants from its surroundings. These interactions take place at the surface of the enzyme and the question of enzyme function can be viewed as the surface science of enzymes. In this article we discuss how to describe...... catalysis by enzymes, and in particular the analogies between enzyme catalyzed reactions and surface catalyzed reactions. We do this by discussing two concrete examples of reactions catalyzed both in nature (by enzymes) and in industrial reactors (by inorganic materials), and show that although analogies...

  1. Amperometric Enzyme Electrodes

    Science.gov (United States)

    1989-12-01

    form of carbon (glascy carbon, graphite, reticulated vitreous carbon, carbon paste, fiber or foil). Carbon is favored for enzyme immoblization...interference from spurious electroactive species in blood, t proprietary multilayer membranie that includes a cellulose acetate memirane and a Nucleopore

  2. Computational enzyme design

    Science.gov (United States)

    Bolon, Daniel N.

    2002-08-01

    The long-term objective of computational enzyme design is the ability to generate efficient protein catalysts for any chemical reaction. This thesis develops and experimentally validates a general computational approach for the design of enzymes with novel function. In order to include catalytic mechanism in protein design, a high-energy state (HES) rotamer (side chain representation) was constructed. In this rotamer, substrate atoms are in a HES. In addition, at least one amino acid side chain is positioned to interact favorably with substrate atoms in their HES and facilitate the reaction. Including an amino acid side chain in the HES rotamer automatically positions substrate relative to a protein scaffold and allows protein design algorithms to search for sequences capable of interacting favorably with the substrate. Because chemical similarity exists between the transition state and the high-energy state, optimizing the protein sequence to interact favorably with the HES rotamer should lead to transition state stabilization. In addition, the HES rotamer model focuses the subsequent computational active site design on a relevant phase space where an amino acid is capable of interacting in a catalytically active geometry with substrate. Using a HES rotamer model of the histidine mediated nucleophilic hydrolysis of p-nitrophenyl acetate, the catalytically inert 108 residue E. coli thioredoxin as a scaffold, and the ORBIT protein design software to compute sequences, an active site scan identified two promising active site designs. Experimentally, both candidate ?protozymes? demonstrated catalytic activity significantly above background. In addition, the rate enhancement of one of these ?protozymes? was the same order of magnitude as the first catalytic antibodies. Because polar groups are frequently buried at enzyme-substrate interfaces, improved modeling of buried polar interactions may benefit enzyme design. By studying native protein structures, rules have been

  3. The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

    Directory of Open Access Journals (Sweden)

    VERONIKA DOUBNEROVÁ

    2009-08-01

    Full Text Available The non-photosynthetic NADP-malic enzyme EC 1.1.1.40 (NADP-ME, which catalyzes the oxidative decarboxylation of L-malate and NADP+ to produce pyruvate and NADPH, respectively, and which could be involved in plant defense responses, was isolated from Nicotiana tabacum L. leaves. The mechanism of the enzyme reaction was studied by the initial rate method and was found to be an ordered sequential one. Regulation possibilities of purified cytosolic NADP-ME by cell metabolites were tested. Intermediates of the citric acid cycle (a-ketoglutarate, succinate, fumarate, metabolites of glycolysis (pyruvate, phosphoenolpyruvate, glucose-6-phosphate, compounds connected with lipogenesis (coenzyme A, acetyl-CoA, palmitoyl-CoA and some amino acids (glutamate, glutamine, aspartate did not significantly affect the NADP-ME activity from tobacco leaves. In contrast, macroergic compounds (GTP, ATP and ADP were strong inhibitors of NADP-ME; the type of inhibition and the inhibition constants were determined in the presence of the most effective cofactors (Mn2+ or Mg2+, required by NADP-ME. Predominantly non-competitive type of inhibitions of NADP-ME with respect to NADP+ and mixed type to L-malate were found.

  4. Long circulating enzyme replacement therapy rescues bone pathology in mucopolysaccharidosis VII murine model.

    Science.gov (United States)

    Rowan, Daniel J; Tomatsu, Shunji; Grubb, Jeffrey H; Haupt, Bisong; Montaño, Adriana M; Oikawa, Hirotaka; Sosa, Angela C; Chen, Anping; Sly, William S

    2012-09-01

    Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by deficiency of the lysosomal enzyme β-glucuronidase (GUS), leading to accumulation of glycosaminoglycans (GAGs). Enzyme replacement therapy (ERT) effectively clears GAG storage in the viscera. Recent studies showed that a chemically modified form of GUS (PerT-GUS), which escaped clearance by mannose 6-phosphate and mannose receptors and showed prolonged circulation, reduced CNS storage more effectively than native GUS. Clearance of storage in bone has been limited due to the avascularity of the growth plate. To evaluate the effectiveness of long-circulating PerT-GUS in reducing the skeletal pathology, we treated MPS VII mice for 12 weeks beginning at 5 weeks of age with PerT-GUS or native GUS and used micro-CT, radiographs, and quantitative histopathological analysis for assessment of bones. Micro-CT findings showed PerT-GUS treated mice had a significantly lower BMD. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with native GUS treated mice. Long term treatment with PerT-GUS from birth up to 57 weeks also significantly improved bone lesions demonstrated by micro-CT, radiographs and quantitative histopathological assay. In conclusion, long-circulating PerT-GUS provides a significant impact to rescue of bone lesions and CNS involvement.

  5. Odontogenic keratocysts: a clinical and histological study with special reference to enzyme histochemistry.

    Science.gov (United States)

    Magnusson, B C

    1978-02-01

    Of a total of 1,420 odontogenic cysts, 52 (3.3%) were diagnosed as odontogenic keratocysts. Clinical and histological findings in these 52 cysts are reported. Frozen sections of 26 of the keratocysts were incubated to show the following enzyme activities: NADH2- and NADPH2-diaphorase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, leucine aminopeptidase and ATPase. Furthermore, keratinization was studied with the rhodamine B method and lipids with the oil red O, the OTAN and the acid hematein methods. Sections from epidermis, oral mucosa, radicular cysts, residual cysts and follicular cysts served as reference material. The oxidative enzymes showed strong activity in the keratocyst epithelium which contrasted with weak activity in the reference cysts. Acid phosphatase activity was weak in all epithelia except that in keratocysts, which displayed a marked activity. In the fibrous capsule of the keratocyst a high activity of leucine aminopeptidase was recorded. This high activity contrasted with a weak activity in the reference material. The significance of the histochemical results in relation to the aggressive behavior of the keratocyst is discussed.

  6. The Moderately Efficient Enzyme: Futile Encounters and Enzyme Floppiness.

    Science.gov (United States)

    Bar-Even, Arren; Milo, Ron; Noor, Elad; Tawfik, Dan S

    2015-08-18

    The pioneering model of Henri, Michaelis, and Menten was based on the fast equilibrium assumption: the substrate binds its enzyme reversibly, and substrate dissociation is much faster than product formation. Here, we examine this assumption from a somewhat different point of view, asking what fraction of enzyme-substrate complexes are futile, i.e., result in dissociation rather than product formation. In Knowles' notion of a "perfect" enzyme, all encounters of the enzyme with its substrate result in conversion to product. Thus, the perfect enzyme's catalytic efficiency, kcat/KM, is constrained by only the diffusion on-rate, and the fraction of futile encounters (defined as φ) approaches zero. The available data on >1000 different enzymes suggest that for ≥90% of enzymes φ > 0.99 and for the "average enzyme" φ ≥ 0.9999; namely, <1 of 10(4) encounters is productive. Thus, the "fast equilibrium" assumption holds for the vast majority of enzymes. We discuss possible molecular origins for the dominance of futile encounters, including the coexistence of multiple sub-states of an enzyme's active site (enzyme floppiness) and/or its substrate. Floppiness relates to the inherent flexibility of proteins, but also to conflicting demands, or trade-offs, between rate acceleration (the rate-determining chemical step) and catalytic turnover, or between turnover rate and accuracy. The study of futile encounters and active-site floppiness may contribute to a better understanding of enzyme catalysis, enzyme evolution, and improved enzyme design.

  7. Molecular cloning and cold-induced expression of trehalose-6-phosphate synthase gene in Harmonia axyridis (Coleoptera: Coccinellidae)%异色瓢虫海藻糖合成酶基因的克隆及低温诱导表达分析

    Institute of Scientific and Technical Information of China (English)

    秦资; 王甦; 魏苹; 徐彩娣; 唐斌; 张帆

    2012-01-01

    海藻糖是昆虫的血糖,在昆虫体内主要通过海藻糖合成酶(trehalose-6-phosphate synthase,TPS)催化合成.本研究通过同源克隆和cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术,从异色瓢虫Harmonia axyridis中克隆得到了TPS基因的cDNA全长序列,命名为HaTPS(GenBank登录号:FJ501960),全长2949 bp,包含3′非翻译区为505 bp,5′非翻译区为26bp,开放阅读框长2418 bp,共编码805个氨基酸.软件分析显示该基因编码蛋白的分子量为90.58 kD,等电点为7.01,包含两个糖基化位点,无信号肽和跨膜结构.同源比对分析发现,昆虫中TPS基因高度保守,包含两个保守的结构域.同时,采用实时荧光定量PCR技术对异色瓢虫HaTPS在不同发育阶段、低温诱导条件下的表达量进行了研究.结果表明:HaTPS在预蛹期的表达量最高;在短时低温诱导条件下,HaTPS的表达量随着温度的降低而显著升高,在降温和升温处理条件下,HaTPS的表达最呈现先升高后下降的表达趋势.结果表明,TPS基因在昆虫抗逆中起到了重要的调节作用.昆虫经过低温诱导,其TPS基因的调控能力得到提升.%Trehalose, which is the blood sugar in insects, is synthesized mainly by trehalose-6-phosphate synthase (TPS) in insects. By homologous cloning and rapid-amplification of cDNA ends (RACE), the full cDNA of the TPS gene in Harmonia axyridis was cloned and named HaTPS (GenBanfc accession number; FJ501960). This gene with 5' and 3' non-coding region of 26 bp and 505 bp, respectively, contains an open reading frame of 2 418 nucleotides encoding a protein of 805 ammo acids with the predicted molecular weight of 90.58 kDa and pI of 7.01. The encoded protein contains two potential N-glycosylation sites, without signal peptide and transmembrane domain. The homology comparison showed that insect TPS was highly conserved with two conserved domains. Real-time fluorescent quantitative PCR was conducted at different

  8. Eco-physiological studies on Indian arid zone plants. III. Effect of sodium chloride and gibberellin on the activity of the enzymes of carbohydrate metabolism in leaves of Pennisetum typhoides

    Energy Technology Data Exchange (ETDEWEB)

    Huber, W.; Rustagi, P.N.; Sankhla, N.

    1974-01-01

    Seedlings of Pennisetum typhoides were grown in sodium chloride (NaCl) and gibberellic acid (GA/sub 3/) separately and in combination, and the effects on the activity of amylase, phosphorylase, aldolase, invertase, hexose-phosphateisomerase, sucrose-synthetase and sucrose-6-phosphate-synthetase were studied. Treatment of the seedlings with NaCl caused an inhibition of the activity of amylase and invertase in the leaf homogenate, but enhanced that of phosphorylase, aldolase, sucrose-synthetase and sucrose-6-phosphate-synthetase. GA/sub 3/ alone, as observed earlier, promoted the activity of invertase but indicated no significant influence on the other enzymes tested. In combination with salt, however, GA/sub 3/ tended to counteract, partially or wholly, the effect of NaCl on the activity of severe enzymes tested. The possible significance of the similarities between the action of abscisic acid (ABA) and salinity in influencing growth and metabolism of plants during stress is discussed. 34 references, 3 figures.

  9. Relationship of lipogenic enzyme activities to the rate of rat liver fatty acid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, G.; Kelley, D.; Schmidt, P.; Virk, S.; Serrato, C.

    1986-05-01

    The mechanism by which diet regulates liver lipogenesis is unclear. Here the authors report how dietary alterations effect the activities of key enzymes of fatty acid (FA) synthesis. Male Sprague-Dawley rats, 400-500 g, were fasted for 48h and then refed a fat-free, high carbohydrate (HC) diet (75% cal. from sucrose) for 0,3,9,24 and 48h, or refed a HC diet for 48h, then fed a high-fat (HF) diet (44% cal. from corn oil) for 3,9,24 and 48h. The FA synthesis rate and the activities of acetyl CoA carboxylase (AC), fatty acid synthase (FAS), ATP citrate lyase (CL), and glucose 6-phosphate dehydrogenase (G6PDH) were determined in the livers. FA synthesis was assayed with /sup 3/H/sub 2/O, enzyme activities were measured spectrophotometrically except for AC which was assayed with /sup 14/C-bicarbonate. There was no change in the activity of AC during fasting or on the HC diet. Fasting decreased the rate of FA synthesis by 25% and the activities of FAS and CL by 50%; refeeding the HC diet induced parallel changes in FA synthesis and the activities of FAS, CL, and G6PDH. After 9h on the HF diet, FA synthesis had decreased sharply, AC activity increased significantly while no changes were detected in the other activities. Subsequently FA synthesis did not change while the activities of the enzymes decreased slowly. These enzymes did not appear to regulate FA synthesis during inhibition of lipogenesis, but FAS, CL or G6PDH may be rate limiting in the induction phase. Other key factors may regulate FA synthesis during dietary alterations.

  10. Halophilic adaptation of enzymes.

    Science.gov (United States)

    Madern, D; Ebel, C; Zaccai, G

    2000-04-01

    It is now clear that the understanding of halophilic adaptation at a molecular level requires a strategy of complementary experiments, combining molecular biology, biochemistry, and cellular approaches with physical chemistry and thermodynamics. In this review, after a discussion of the definition and composition of halophilic enzymes, the effects of salt on their activity, solubility, and stability are reviewed. We then describe how thermodynamic observations, such as parameters pertaining to solvent-protein interactions or enzyme-unfolding kinetics, depend strongly on solvent composition and reveal the important role played by water and ion binding to halophilic proteins. The three high-resolution crystal structures now available for halophilic proteins are analyzed in terms of haloadaptation, and finally cellular response to salt stress is discussed briefly.

  11. The invisible Web uncovering information sources search engines can't see

    CERN Document Server

    Sherman, Chris

    2001-01-01

    Enormous expanses of the Internet are unreachable with standard web search engines. This book provides the key to finding these hidden resources by identifying how to uncover and use invisible web resources. Mapping the invisible Web, when and how to use it, assessing the validity of the information, and the future of Web searching are topics covered in detail. Only 16 percent of Net-based information can be located using a general search engine. The other 84 percent is what is referred to as the invisible Web-made up of information stored in databases. Unlike pages on the visible Web, informa

  12. Structural insight to mutation effects uncover a common allosteric site in class C GPCRs

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Boesgaard, Michael W; Munk, Christian;

    2017-01-01

    . Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics. RESULTS: We uncover one common site for both positive and negative modulators with different amino acid layouts that can......MOTIVATION: Class C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry...

  13. Mayaro virus infection alters glucose metabolism in cultured cells through activation of the enzyme 6-phosphofructo 1-kinase.

    Science.gov (United States)

    El-Bacha, Tatiana; Menezes, Maíra M T; Azevedo e Silva, Melissa C; Sola-Penna, Mauro; Da Poian, Andrea T

    2004-11-01

    Although it is well established that cellular transformation with tumor virus leads to changes on glucose metabolism, the effects of cell infection by non-transforming virus are far to be completely elucidated. In this study, we report the first evidence that cultured Vero cells infected with the alphavirus Mayaro show several alterations on glucose metabolism. Infected cells presented a two fold increase on glucose consumption, accompanied by an increment in lactate production. This increase in glycolytic flux was also demonstrated by a significant increase on the activity of 6-phosphofructo 1-kinase, one of the regulatory enzymes of glycolysis. Analysis of the kinetic parameters revealed that the regulation of 6-phosphofructo 1-kinase is altered in infected cells, presenting an increase in Vmax along with a decrease in Km for fructose-6-phosphate. Another fact contributing to an increase in enzyme activity was the decrease in ATP levels observed in infected cells. Additionally, the levels of fructose 2,6-bisphosphate, a potent activator of this enzyme, was significantly reduced in infected cells. These observations suggest that the increase in PFK activity may be a compensatory cellular response to the viral-induced metabolic alterations that could lead to an impairment of the glycolytic flux and energy production.

  14. Effects of Oxygen Limitation on Xylose Fermentation, Intracellular Metabolites, and Key Enzymes of Neurospora crassa AS3.1602

    Science.gov (United States)

    Zhang, Zhihua; Qu, Yinbo; Zhang, Xiao; Lin, Jianqiang

    The effects of oxygen limitation on xylose fermentation of Neurospora crassa AS3.1602 were studied using batch cultures. The maximum yield of ethanol was 0.34 g/g at oxygen transfer rate (OTR) of 8.4 mmol/L·h. The maximum yield of xylitol was 0.33 g/g at OTR of 5.1 mmol/L·h. Oxygen limitation greatly affected mycelia growth and xylitol and ethanol productions. The specific growth rate (μ) decreased 82% from 0.045 to 0.008 h-1 when OTR changed from 12.6 to 8.4 mmol/L·h. Intracellular metabolites of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle were determined at various OTRs. Concentrations of most intracellular metabolites decreased with the increase in oxygen limitation. Intracellular enzyme activities of xylose reductase, xylitol dehydrogenase, and xylulokinase, the first three enzymes in xylose metabolic pathway, decreased with the increase in oxygen limitation, resulting in the decreased xylose uptake rate. Under all tested conditions, transaldolase and transketolase activities always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase played a major role in NADPH regeneration, and its activity decreased remarkably with the increase in oxygen limitation.

  15. Reprogramming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes.

    Science.gov (United States)

    Wang, Junjian; Duan, Zhijian; Nugent, Zoann; Zou, June X; Borowsky, Alexander D; Zhang, Yanhong; Tepper, Clifford G; Li, Jian Jian; Fiehn, Oliver; Xu, Jianzhen; Kung, Hsing-Jien; Murphy, Leigh C; Chen, Hong-Wu

    2016-08-10

    Metabolic reprogramming such as the aerobic glycolysis or Warburg effect is well recognized as a common feature of tumorigenesis. However, molecular mechanisms underlying metabolic alterations for tumor therapeutic resistance are poorly understood. Through gene expression profiling analysis we found that histone H3K36 methyltransferase NSD2/MMSET/WHSC1 expression was highly elevated in tamoxifen-resistant breast cancer cell lines and clinical tumors. IHC analysis indicated that NSD2 protein overexpression was associated with the disease recurrence and poor survival. Ectopic expression of NSD2 wild type, but not the methylase-defective mutant, drove endocrine resistance in multiple cell models and xenograft tumors. Mechanistically, NSD2 was recruited to and methylated H3K36me2 at the promoters of key glucose metabolic enzyme genes. Its overexpression coordinately up-regulated hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD), two key enzymes of glycolysis and the pentose phosphate pathway (PPP), as well as TP53-induced glycolysis regulatory phosphatase TIGAR. Consequently, NSD2-driven tamoxifen-resistant cells and tumors displayed heightened PPP activity, elevated NADPH production, and reduced ROS level, without significantly altered glycolysis. These results illustrate a coordinated, epigenetic activation of key glucose metabolic enzymes in therapeutic resistance and nominate methyltransferase NSD2 as a potential therapeutic target for endocrine resistant breast cancer.

  16. Treating Wastewater With Immobilized Enzymes

    Science.gov (United States)

    Jolly, Clifford D.

    1991-01-01

    Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

  17. Effect of copper on liver key enzymes of anaerobic glucose metabolism from freshwater tropical fish Prochilodus lineatus.

    Science.gov (United States)

    Carvalho, Cleoni dos Santos; Fernandes, Marisa Narciso

    2008-11-01

    We investigated the effect of copper on liver key enzymes of the anaerobic glucose metabolism (hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK; lactate dehydrogenase, LDH) as well as of the pentose pathway (glycose-6-phosphate dehydrogenase, G6PDH) from the fish Prochilodus lineatus. The fish were acclimated at either 20 degrees C or 30 degrees C at pH 7.0, transferred to water at pH 4.5 or 8.0, and exposed to 96 h-CL(50) copper concentrations. Copper accumulation in liver was higher in fish acclimated at 20 degrees C and maintained in water pH 8.0. Three-way analysis of variance revealed a significant effect of temperature on all enzymes, a significant effect of pH on all enzymes except for PK, and a significant effect of copper on only PFK, and LDH in pH 4.5 at 20 degrees C and, at 30 degrees C, on PFK and PK at pH 4.5 and 8.0, HK at pH 4.5 and G6PDH at pH 8.0. There were significant interactions between treatments for many enzymes. These changes suggest that the activity of enzymes in question is modified by a change in ambient water. At least at 30 degrees C, the overall reduction in the glycolytic enzyme activities of copper-exposed fish seems to reduce energy availability via glucose metabolism, thereby contributing to enhance copper toxic effects.

  18. The Catalytic Function of Enzymes.

    Science.gov (United States)

    Splittgerber, Allan G.

    1985-01-01

    Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

  19. From detecting astrocyte connectivity to uncovering drug effects in living tissues

    CERN Document Server

    Pires, Marcelo; Vaz, Sandra; Sebastião, Ana; Lind, Pedro G

    2013-01-01

    We introduce a simple procedure of multivariate signal analysis to uncover the connectivity structure among cells composing a living tissue and describe how to apply it for extracting insight on the effect of drugs in the tissue. The procedure is based in the covariance matrix of time resolved activity signals. By determining the time-lag that maximizes covariance one derives the weight of the corresponding connection between cells. Introducing simple constraints, it is possible to conclude if pairs of cells are connected or not and in which direction. After testing the method against synthetic data we apply it to study propagation of $Ca^{2+}$ waves in astrocytes, with the aim of uncovering the cell connectivity structure. Our method shows to be particularly suited for this type of networking signal propagation where signals are pulse-like and have short time-delays, and is shown to be superior to standard methods, namely a multivariate Granger algorithm. Finally, based the statistical analysis of the connec...

  20. Animal models of Huntington's disease: implications in uncovering pathogenic mechanisms and developing therapies

    Institute of Scientific and Technical Information of China (English)

    Lin-hui WANG; Zheng-hong QIN

    2006-01-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, which is caused by an abnormal expansion of Cytosine Adenine Guanine (CAG) trinucleotide repeat in the gene making huntingtin (Htt). Despite intensive research efforts devoted to investigate molecular mechanisms of pathogenesis, effective therapy for this devastating disease is still not available at present. The development of various animal models of HD has offered alternative approaches in the study of HD molecular pathology. Many HD models, including chemical-induced models and genetic models, mimic some aspects of HD symptoms and pathology. To date, however, there is no ideal model which replicates all of the essential features of neuropathology and progressive motor and cognitive impairments of human HD. As a result, our understanding of molecular mechanisms of pathogenesis in HD is still limited. A new model is needed in order to uncover the pathogenesis and to develop novel therapies for HD. In this review we discussed usefulness and limitations of various animal and cellular models of HD in uncovering molecular mechanisms of pathogenesis and developing novel therapies for HD.

  1. Uncovering Sundanese Values by Analyzing Symbolic Meaning of Ménak Priangan Clothing (1800-1942)

    Science.gov (United States)

    Karmila, M.; Suciati; Widiaty, I.

    2016-04-01

    This study investigates symbolic meanings found in the Sunda ethnic clothing, particularly the Menak Priangan clothing. This study aims to uncover and document those symbolic meanings found in the Menak Priangan clothing as an effort to develop Sunda cultural artefacts of West Java. This study on Menak Priangan clothing applies ethnography (visual) and aesthetic methods. The visual method is utilized in order to uncover local cultural (Sunda) values found in Menak Priangan clothing visualization, including: design, model, name, and representing colours, which then directed towards local Sundanese aesthetic concepts living within the Priangan community. Furthermore, aesthetic method is used to explore role of aesthetic values in empowering visual cultural values within certain community, particularly Sunda aesthetic values. The study results show that since the 19th century, Sunda ethnic clothing was limited to Priangan Sunda only, while traditional clothing wearing by Priangan people reflects their social strata, consisting of: a. Menak Gede (Menak pangluhurna: mayor), bearing raden title, b. Menak Leutik/Santana (mayor assistant), titles: asep, mas, agus, ujang, (Nyimas for woman), c. Somah/Cacah: ordinary people/lower class. Clothing is a cultural phenomenon within certain culture reflecting such society experiences. For Menak people, clothing and its accessories have important meanings. They wear such traditional clothing and accessories as a symbol of power they have within bureaucratic structure and as a symbol of social status they bear within traditional community structure.

  2. Uncovering signal transduction networks from high-throughput data by integer linear programming.

    Science.gov (United States)

    Zhao, Xing-Ming; Wang, Rui-Sheng; Chen, Luonan; Aihara, Kazuyuki

    2008-05-01

    Signal transduction is an important process that transmits signals from the outside of a cell to the inside to mediate sophisticated biological responses. Effective computational models to unravel such a process by taking advantage of high-throughput genomic and proteomic data are needed to understand the essential mechanisms underlying the signaling pathways. In this article, we propose a novel method for uncovering signal transduction networks (STNs) by integrating protein interaction with gene expression data. Specifically, we formulate STN identification problem as an integer linear programming (ILP) model, which can be actually solved by a relaxed linear programming algorithm and is flexible for handling various prior information without any restriction on the network structures. The numerical results on yeast MAPK signaling pathways demonstrate that the proposed ILP model is able to uncover STNs or pathways in an efficient and accurate manner. In particular, the prediction results are found to be in high agreement with current biological knowledge and available information in literature. In addition, the proposed model is simple to be interpreted and easy to be implemented even for a large-scale system.

  3. Kinetic Measurements for Enzyme Immobilization.

    Science.gov (United States)

    Cooney, Michael J

    2017-01-01

    Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes, with a focus on their reaction rates. The study of an enzyme's kinetics considers the various stages of activity, reveals the catalytic mechanism of this enzyme, correlates its value to assay conditions, and describes how a drug or a poison might inhibit the enzyme. Victor Henri initially reported that enzyme reactions were initiated by a bond between the enzyme and the substrate. By 1910, Michaelis and Menten were advancing their work by studying the kinetics of an enzyme saccharase which catalyzes the hydrolysis of sucrose into glucose and fructose. They published their analysis and ever since the Michaelis-Menten equation has been used as the standard to describe the kinetics of many enzymes. Unfortunately, soluble enzymes must generally be immobilized to be reused for long times in industrial reactors. In addition, other critical enzyme properties have to be improved like stability, activity, inhibition by reaction products, and selectivity towards nonnatural substrates. Immobilization is by far the chosen process to achieve these goals.Although the Michaelis-Menten approach has been regularly adapted to the analysis of immobilized enzyme activity, its applicability to the immobilized state is limited by the barriers the immobilization matrix places upon the measurement of compounds that are used to model enzyme kinetics. That being said, the estimated value of the Michaelis-Menten coefficients (e.g., V max, K M) can be used to evaluate effects of immobilization on enzyme activity in the immobilized state when applied in a controlled manner. In this review enzyme activity and kinetics are discussed in the context of the immobilized state, and a few novel protocols are presented that address some of the unique constraints imposed by the immobilization barrier.

  4. Transcript profiles uncover temporal and stress-induced changes of metabolic pathways in germinating sugar beet seeds

    Directory of Open Access Journals (Sweden)

    Windhövel Andrea

    2008-12-01

    Full Text Available Abstract Background With a cultivation area of 1.75 Mio ha and sugar yield of 16.7 Mio tons in 2006, sugar beet is a crop of great economic importance in Europe. The productivity of sugar beet is determined significantly by seed vigour and field emergence potential; however, little is known about the molecular mechanisms underlying these traits. Both traits exhibit large variations within sugar beet germplasm that have been difficult to ascribe to either environmental or genetic causes. Among potential targets for trait improvement, an enhancement of stress tolerance is considered because of the high negative influence of environmental stresses on trait parameters. Extending our knowledge of genetic and molecular determinants of sugar beet germination, stress response and adaptation mechanisms would facilitate the detection of new targets for breeding crop with an enhanced field emergence potential. Results To gain insight into the sugar beet germination we initiated an analysis of gene expression in a well emerging sugar beet hybrid showing high germination potential under various environmental conditions. A total of 2,784 ESTs representing 2,251 'unigenes' was generated from dry mature and germinating seeds. Analysis of the temporal expression of these genes during germination under non-stress conditions uncovered drastic transcriptional changes accompanying a shift from quiescent to metabolically active stages of the plant life cycle. Assay of germination under stressful conditions revealed 157 genes showing significantly different expression patterns in response to stress. As deduced from transcriptome data, stress adaptation mechanisms included an alteration in reserve mobilization pathways, an accumulation of the osmoprotectant glycine betaine, late embryogenesis abundant proteins and detoxification enzymes. The observed transcriptional changes are supposed to be regulated by ABA-dependent signal transduction pathway. Conclusion This study

  5. Mass-array芯片技术在葡萄糖-6-磷酸脱氢酶基因突变位点检测中的应用%Application of Mass-array gene chip to detect glucose-6-phosphate dehydrogenase gene mutations

    Institute of Scientific and Technical Information of China (English)

    陈瑶; 苏跃青; 周进福; 王旌; 赵红; 曾颖琳; 林庆颖; 林枫; 张洪华

    2015-01-01

    目的 探讨应用Mass-array基因芯片技术检测葡萄糖-6-磷酸脱氢酶(G6PD)基因突变位点的价值,并对其进行质量评价.方法 收集2006至2013年在福建省妇幼保健院新生儿疾病筛查中心进行G6PD筛查的婴儿,根据化学筛查结果分成2组:G6PD缺乏症患儿和正常儿童,随机抽取患儿和正常对照儿童各300例.采用基因分析工具(Genotyping Tools)与Mass-array Design软件,利用中国人群已报道的G6PD基因33个突变位点芯片,应用Mass-array基因技术检测G6PD基因突变位点,并通过DNASanger测序法验证基因芯片检测结果的准确性.结果 在300例G6PD患儿中,共检出单纯型单点突变9种:1376G>T、1388G>A、95A>G、1024C>T、392G>T、1360C>T、487G>A、517T>C、1365-13T>C;复合突变型7种:871G> A/1365-13T> C/1311C>T、1004C> A/1311C> T/1365-13T>C、1376G >T/1365-13T>C/1311C>T、1365-13T >C/1311C >T、1376G> T/1365-13T>C、95A> G/1365-13T> C/1311C>T、1388G> A/1365-13T>C;300名正常对照儿童中未检测到G6PD基因突变.进一步所有样本的Sanger DNA测序结果与基因芯片检测结果完全一致.结论 采用Massarray基因芯片技术检测G6PD基因突变方法是一种准确、高效的G6PD基因突变筛查方法.%Objective To develop the Mass-array gene chip to detect glucose-6-phosphate dehydrogenase (G6PD) gene mutations,and to evaluate its quality.Methods Randomly choosing the children who perform neonatal screening in Neonatal Screening Center of Fujian Maternity and Children Health Hospital from 2006 to 2013.Children were divided into control group and G6PD patient group.Using Genotyping Tools from Sequenom company and the software of Mass-array Assay Design to design the PCR amplification primer of 33 G6PD gene mutations which were well-known in Chinese.Then depending on Mass-array gene chip technology to detect glucose-6-phosphate dehydrogenase gene mutations.DNA Sanger sequencing was

  6. Structure Biology of Membrane Bound Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Dax [Johns Hopkins Univ., Baltimore, MD (United States). School of Medicine. Dept. of Physiology

    2016-11-30

    The overall goal of the proposed research is to understand the membrane-associated active processes catalyzed by an alkane $\\square$-hydroxylase (AlkB) from eubacterium Pseudomonase oleovorans. AlkB performs oxygenation of unactivated hydrocarbons found in crude oils. The enzymatic reaction involves energy-demanding steps in the membrane with the uses of structurally unknown metal active sites featuring a diiron [FeFe] center. At present, a critical barrier to understanding the membrane-associated reaction mechanism is the lack of structural information. The structural biology efforts have been challenged by technical difficulties commonly encountered in crystallization and structural determination of membrane proteins. The specific aims of the current budget cycle are to crystalize AlkB and initiate X-ray analysis to set the stage for structural determination. The long-term goals of our structural biology efforts are to provide an atomic description of AlkB structure, and to uncover the mechanisms of selective modification of hydrocarbons. The structural information will help elucidating how the unactivated C-H bonds of saturated hydrocarbons are oxidized to initiate biodegradation and biotransformation processes. The knowledge gained will be fundamental to biotechnological applications to biofuel transformation of non-edible oil feedstock. Renewable biodiesel is a promising energy carry that can be used to reduce fossil fuel dependency. The proposed research capitalizes on prior BES-supported efforts on over-expression and purification of AlkB to explore the inner workings of a bioenergy-relevant membrane-bound enzyme.

  7. Enzyme replacement in a human model of mucopolysaccharidosis IVA in vitro and its biodistribution in the cartilage of wild type mice.

    Science.gov (United States)

    Dvorak-Ewell, Melita; Wendt, Dan; Hague, Chuck; Christianson, Terri; Koppaka, Vish; Crippen, Danielle; Kakkis, Emil; Vellard, Michel

    2010-08-16

    Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active ( approximately 2 U/mg) and pure (>or=97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K(uptake) = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.

  8. Enzyme replacement in a human model of mucopolysaccharidosis IVA in vitro and its biodistribution in the cartilage of wild type mice.

    Directory of Open Access Journals (Sweden)

    Melita Dvorak-Ewell

    Full Text Available Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS, an enzyme that degrades keratan sulfate (KS. Currently no therapy for MPS IVA is available. We produced recombinant human (rhGALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active ( approximately 2 U/mg and pure (>or=97% rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K(uptake = 2.5 nM, thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for

  9. Nephro-protective effects of a ginger extract on cytosolic and mitochondrial enzymes against streptozotocin (STZ)-induced diabetic complications in rats.

    Science.gov (United States)

    Ramudu, Shanmugam Kondeti; Korivi, Mallikarjuna; Kesireddy, Nishanth; Lee, Li-Chen; Cheng, I-Shiung; Kuo, Chia-Hua; Kesireddy, Sathyavelu Reddy

    2011-04-30

    Diabetes is characterized by elevated blood glucose levels and disturbed homeostasis of metabolic enzymes in whole-body. This study aimed to investigate the effect of ginger administration on altered blood glucose levels, intra- and extra-mitochondrial enzymes and tissue injuries in streptozotocin (STZ)-induced diabetic rats. Wistar strain rats (n = 30) were equally divided into 5 groups: normal control (NC), ginger treated (Gt, 200 mg/kg b.w. orally/30 days), diabetic control (DC, 50 mg/kg b.w.), diabetic plus ginger treated (D + Gt) and diabetic plus glibenclamide treated (D + Gli) groups. We found highly elevated blood glucose levels in the diabetic group, and the glucose levels were significantly (P ginger administration. Activities of intra- and extra-mitochondrial enzymes such as glucose-6-phosphate dehydrogenase (G6PD), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) were significantly (P diabetic rats, while this was significantly reversed by 30 days of ginger treatment. We also observed consistent renal tissue damages in the diabetic rats; however, these injuries recovered in the ginger-treated diabetic rats as shown in histopathological studies. In this study, we demonstrated that an ethanolic extract of ginger could lower the blood glucose levels as well as improve activities of intra- and extra-mitochondrial enzymes in diabetic rats. Our results suggest that ginger extracts could be used as a nephro-protective supplement particularly to reverse diabetic-induced complications.

  10. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    Science.gov (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  11. Representations of God uncovered in a spirituality group of borderline inpatients.

    Science.gov (United States)

    Goodman, Geoff; Manierre, Amy

    2008-01-01

    We present aspects of a psychoanalytically-oriented, exploratory spirituality group for nine female psychiatric inpatients diagnosed with borderline personality disorder. Through drawings and group process, the patients uncovered and elaborated on their representations of God. Two patterns of representations were identified: (1) representations of a punitive, judgmental, rigid God that seemed directly to reflect and correspond with parental representations and (2) representations of a depersonified, inanimate, abstract God entailing aspects of idealization that seemed to compensate for parental representations. Interestingly, the second pattern was associated with comorbid narcissistic features in the patients. Those patients who presented punitive God representations were able to begin the process of re-creating these representations toward more benign or benevolent images in the context of this group, while those participants who presented depersonified God representations seemed unable to do so.

  12. Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

    Science.gov (United States)

    Dib, Julián R.; Wagenknecht, Martin; Farías, María E.; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions. PMID:26074886

  13. Genome-wide meta-analysis uncovers novel loci influencing circulating leptin levels

    DEFF Research Database (Denmark)

    Kilpeläinen, Tuomas O; Carli, Jayne F Martin; Skowronski, Alicja A

    2016-01-01

    Leptin is an adipocyte-secreted hormone, the circulating levels of which correlate closely with overall adiposity. Although rare mutations in the leptin (LEP) gene are well known to cause leptin deficiency and severe obesity, no common loci regulating circulating leptin levels have been uncovered....... Therefore, we performed a genome-wide association study (GWAS) of circulating leptin levels from 32,161 individuals and followed up loci reaching Pleptin levels in/near LEP, SLC32A1, GCKR, CCNL1 and FTO....... Although the association of the FTO obesity locus with leptin levels is abolished by adjustment for BMI, associations of the four other loci are independent of adiposity. The GCKR locus was found associated with multiple metabolic traits in previous GWAS and the CCNL1 locus with birth weight. Knockdown...

  14. Uncover the Aesthetic Simplicity Associated with Mass Transfer in Energy Materials

    Institute of Scientific and Technical Information of China (English)

    Jiang-Wei Li; Jia Li; Ke-Chun Wen

    2016-01-01

    Aesthetics, referred frequently to as a philosophical term, has played a starring role in forming and evolving a number of aspects of human society, including arts, politics, economics, ethics, etc. Indeed, exploring and investigating the aesthetic phenomena in the scientific field have aroused insightful research findings, which in turn has stimulated research interests in such a science-aesthetics field. In particular, better-evaluated aesthetic aspects of the materials field are expected to be uncovered upon the exceedingly-exposed fundamental breakthroughs in researching the basic structure and functionality of materials. In this report, we glimpse into the aesthetic simplicity of energy materials and comprehend specifically the mass transfer functionalities of key categories of energy materials through an intuitive and bottom-up approach. Our effort aspires to shed new lights on the functionality understanding and manipulation of functional materials in general.

  15. Health Detectives: Uncovering the Mysteries of Disease (LBNL Science at the Theater)

    Energy Technology Data Exchange (ETDEWEB)

    Bissell, Mina; Canaria, Christie; Celnicker, Susan; Karpen, Gary

    2012-04-23

    In this April 23, 2012 Science at the Theater event, Berkeley Lab scientists discuss how they uncover the mysteries of disease in unlikely places. Speakers and topics include: World-renowned cancer researcher Mina Bissell's pioneering research on the role of the cellular microenvironment in breast cancer has changed the conversation about the disease. How does DNA instability cause disease? To find out, Christie Canaria images neural networks to study disorders such as Huntington's disease. Fruit flies can tell us a lot about ourselves. Susan Celniker explores the fruit fly genome to learn how our genome works. DNA is not destiny. Gary Karpen explores how environmental factors shape genome function and disease through epigenetics.

  16. Systems-level approach to uncovering diffusive states and their transitions from single particle trajectories

    CERN Document Server

    Koo, Peter K

    2016-01-01

    The stochastic motions of a diffusing particle contain information concerning the particle's interactions with binding partners and with its local environment. However, accurate determination of the underlying diffusive properties, beyond normal diffusion, has remained challenging when analyzing particle trajectories on an individual basis. Here, we introduce the maximum likelihood estimator (MLE) for confined diffusion and fractional Brownian motion. We demonstrate that this MLE yields improved estimation over traditional mean square displacement analyses. We also introduce a model selection scheme (that we call mleBIC) that classifies individual trajectories to a given diffusion mode. We demonstrate the statistical limitations of classification via mleBIC using simulated data. To overcome these limitations, we introduce a new version of perturbation expectation-maximization (pEMv2), which simultaneously analyzes a collection of particle trajectories to uncover the system of interactions which give rise to u...

  17. Rhodiola-induced inhibition of adipogenesis involves antioxidant enzyme response associated with pentose phosphate pathway.

    Science.gov (United States)

    Lee, Ok-Hwan; Kwon, Young-In; Apostolidis, Emmanouil; Shetty, Kalidas; Kim, Young-Cheul

    2011-01-01

    The aim of this study was to investigate whether Rhodiola crenulata extract and tyrosol, a major bioactive phenolic compound present in Rhodiola, change the activities of endogenous antioxidant enzyme response (AER) and energy pathways linked to proline-mediated pentose phosphate pathway (PPP) during adipogenesis. Treatment with Rhodiola extracts inhibited the activities of proline dehydrogenase (PDH) and glucose-6-phosphate dehydrogenase (G6PDH) as well as lipid accumulation and reactive oxygen species (ROS) production. The inhibition of PDH and G6PDH activities by Rhodiola likely prevented proline oxidation required for critical ATP generation that is coupled to AER via the PPP, leading to inhibition of adipogenesis. Rhodiola extracts dose-dependently increased superoxide dismutase (SOD) activity, resulting in a reduced ROS level during adipogenesis. Moreover, the effects of tyrosol, a major bioactive compound in Rhodiola species, were directly correlated with all observed effects by Rhodiola extracts. These results indicate that the antiadipogenic effects of Rhodiola extracts can be attributed to a phenolic tyrosol that may potentially disrupt proline-mediated energy generation and AER via PPP, resulting in the suppression of adipogenesis and lipid accumulation. This further provides a biochemical rationale to identify the roles of phenolics that modulate the cellular redox environment and therefore have relevance for obesity management.

  18. Uncovering the role of p53 splice variants in human malignancy: a clinical perspective

    Directory of Open Access Journals (Sweden)

    Surget S

    2013-12-01

    Full Text Available Sylvanie Surget,1,2 Marie P Khoury,1,2 Jean-Christophe Bourdon1,21Dundee Cancer Centre, 2Jacqui Wood Cancer Centre, Ninewells Hospital, University of Dundee, Dundee, UKAbstract: Thirty-five years of research on p53 gave rise to more than 68,000 articles and reviews, but did not allow the uncovering of all the mysteries that this major tumor suppressor holds. How p53 handles the different signals to decide the appropriate cell fate in response to a stress and its implication in tumorigenesis and cancer progression remains unclear. Nevertheless, the uncovering of p53 isoforms has opened new perspectives in the cancer research field. Indeed, the human TP53 gene encodes not only one but at least twelve p53 protein isoforms, which are produced in normal tissues through alternative initiation of translation, usage of alternative promoters, and alternative splicing. In recent years, it became obvious that the different p53 isoforms play an important role in regulating cell fate in response to different stresses in normal cells by differentially regulating gene expression. In cancer cells, abnormal expression of p53 isoforms contributes actively to cancer formation and progression, regardless of TP53 mutation status. They can also be associated with response to treatment, depending on the cell context. The determination of p53 isoform expression and p53 mutation status helps to define different subtypes within a particular cancer type, which would have different responses to treatment. Thus, the understanding of the regulation of p53 isoform expression and their biological activities in relation to the cellular context would constitute an important step toward the improvement of the diagnostic, prognostic, and predictive values of p53 in cancer treatment. This review aims to summarize the involvement of p53 isoforms in cancer and to highlight novel potential therapeutic targets.Keywords: p53, isoforms, p63, p73, alternative splicing, cancer

  19. Enzyme Molecules in Solitary Confinement

    Directory of Open Access Journals (Sweden)

    Raphaela B. Liebherr

    2014-09-01

    Full Text Available Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  20. Enzyme molecules in solitary confinement.

    Science.gov (United States)

    Liebherr, Raphaela B; Gorris, Hans H

    2014-09-12

    Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  1. Heat Stable Enzymes from Thermophiles

    Science.gov (United States)

    1998-02-01

    ultrafiltration and microfiltration that might be suitable. These utilize hollow fiber membranes manufactured in such a manner that they are free of...words) Alkaline phosphatase is widely used in the military and civilian sectors . Commercially available enzyme from calf intestine is the weak link in...widely used enzymes with numerous uses in both the military and civilian sectors . The commercially available enzyme from calf intestine breaks down

  2. Enzyme therapeutics for systemic detoxification.

    Science.gov (United States)

    Liu, Yang; Li, Jie; Lu, Yunfeng

    2015-08-01

    Life relies on numerous biochemical processes working synergistically and correctly. Certain substances disrupt these processes, inducing living organism into an abnormal state termed intoxication. Managing intoxication usually requires interventions, which is referred as detoxification. Decades of development on detoxification reveals the potential of enzymes as ideal therapeutics and antidotes, because their high substrate specificity and catalytic efficiency are essential for clearing intoxicating substances without adverse effects. However, intrinsic shortcomings of enzymes including low stability and high immunogenicity are major hurdles, which could be overcome by delivering enzymes with specially designed nanocarriers. Extensive investigations on protein delivery indicate three types of enzyme-nanocarrier architectures that show more promise than others for systemic detoxification, including liposome-wrapped enzymes, polymer-enzyme conjugates, and polymer-encapsulated enzymes. This review highlights recent advances in these nano-architectures and discusses their applications in systemic detoxifications. Therapeutic potential of various enzymes as well as associated challenges in achieving effective delivery of therapeutic enzymes will also be discussed.

  3. Multi-enzyme Process Modeling

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia

    The subject of this thesis is to develop a methodological framework that can systematically guide mathematical model building for better understanding of multi-enzyme processes. In this way, opportunities for process improvements can be identified by analyzing simulations of either existing...... are affected (in a positive or negative way) by the presence of the other enzymes and compounds in the media. In this thesis the concept of multi-enzyme in-pot term is adopted for processes that are carried out by the combination of enzymes in a single reactor and implemented at pilot or industrial scale...

  4. Anestesia em paciente portador de deficiência de glicose-6-fosfato-desidrogenase: relato de caso Anestesia en paciente portador de deficiencia de glicosa-6-fosfato-desidrogenasa: relato de caso Anesthesia in glucose 6-phosphate dehydrogenase-deficient patient: case report

    Directory of Open Access Journals (Sweden)

    Múcio Paranhos de Abreu

    2002-11-01

    caso relatado, la anestesia subaracnóidea con bupivacaína asociada a anestesia venosa total con propofol, mostró que es una técnica segura en pacientes portadores de deficiencia de G6PD.BACKGROUND AND OBJECTIVES: Glucose 6-phosphate dehydrogenase (G6PD deficiency is a relatively common enzymopathy, but there are few publications relating such condition to anesthesia. This report aimed at presenting a case of a G6PD-deficient patient, submitted to Achilles tendon tenotomy under intravenous anesthesia associated to spinal block. CASE REPORT: Male patient, 9 years old, 48 kg, with G6PD deficiency and peripheral polineuropathy, submitted to Achilles tendon tenotomy under general intravenous anesthesia with midazolam, propofol and fentanyl, associated to spinal block with 0.5% hyperbaric bupivacaine. At surgery completion patient awakened relaxed, without pain or other complaints, had a good evolution and was discharged without intercurrences. CONCLUSIONS: According to the evolution of this case, spinal anesthesia with bupivacaine associated to total intravenous anesthesia with propofol has shown to be a safe technique for G6PD-deficient patients.

  5. 共转染小干扰RNA质粒联合抑制HeLa细胞葡萄糖-6-磷酸脱氢酶和转酮醇酶样基因-1表达%Combined inhibitory effect of co-transfected siRNA plasmids in Hela cells on the expressions of glucose-6-phosphate dehydrogenase and transketolase like-1

    Institute of Scientific and Technical Information of China (English)

    张德太; 杨文; 涂启明; 张科; 胡丽华

    2013-01-01

    目的 葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase, G6PD)及转酮醇酶样基因-1(transketolase like-1, TKTL-1)是肿瘤细胞磷酸戊糖代谢途径(pentose phosphate pathway, PPP)的2个重要关键调节酶.拟通过共转染小干扰RNA(small interfering RNA, siRNA)表达载体同时沉默人宫颈癌Hela G6PD及TKTL-1后,观察其对G6PD和TKTL1的联合抑制作用,旨在探讨共转染siRNA表达载体联合抑制PPP的效果及可行性.方法 分别构建靶向G6PD和TKTL1基因siRNA干扰质粒载体,通过酶切和测序鉴定siRNA干扰质粒载体构建成功后,单独或共转染HeLa细胞株,RT-PCR检测G6PD、TKTL1 mRNA表达并结合G6PD、转酮醇酶(transketolase, TKT)活性测定判断并比较共转染联合抑制Hela细胞G6PD、TKTL1表达的效果.结果 共转染靶向G6PD和TKTL1基因的siRNA干扰载体能显著下调G6PD和TKTL1基因表达水平[(0.96±0.05) vs (0.47±0.04),(0.98±0.05)vs(0.41±0.04), P<0.01].同时,2个酶的活性也显著下降(99.2%vs34.5%和99.8%vs40.1%, P<0.01).共转染抑制G6PD基因mRNA表达较单独转染靶向G6PD基因的siRNA干扰载体的效果有显著下降[(0.47±0.04 )vs(0.31±0.04), P<0.01)].结论 共转染siRNA干扰载体对人宫颈癌HeLa细胞PPP代谢通路进行联合抑制是一种有效的实验方案.

  6. Digestive Enzyme Replacement Therapy: Pancreatic Enzymes and Lactase.

    Science.gov (United States)

    Felicilda-Reynaldo, Rhea Faye D; Kenneally, Maria

    2016-01-01

    Maldigestion occurs when digestive enzymes are lacking to help break complex food components into absorbable nutrients within the gastrointestinal tract. Education is needed to help patients manage the intricacies of digestive enzyme replacement therapies and ensure their effectiveness in reducing symptoms of maldigestion.

  7. [The rise of enzyme engineering in China].

    Science.gov (United States)

    Li, Gaoxiang

    2015-06-01

    Enzyme engineering is an important part of the modern biotechnology. Industrial biocatalysis is considered the third wave of biotechnology following pharmaceutical and agricultural waves. In 25 years, China has made a mighty advances in enzyme engineering research. This review focuses on enzyme genomics, enzyme proteomics, biosynthesis, microbial conversion and biosensors in the Chinese enzyme engineering symposiums and advances in enzyme preparation industry in China.

  8. Deubiquitylating enzymes and disease

    Directory of Open Access Journals (Sweden)

    Baker Rohan T

    2008-10-01

    Full Text Available Abstract Deubiquitylating enzymes (DUBs can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin, including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80–90 DUBs in five different subfamilies, only a handful of which have been characterized with respect to the proteins that they interact with and deubiquitylate. Several other DUBs have been implicated in various disease processes in which they are changed by mutation, have altered expression levels, and/or form part of regulatory complexes. Specific examples of DUB involvement in various diseases are presented. While no specific drugs targeting DUBs have yet been described, sufficient functional and structural information has accumulated in some cases to allow their rapid development. Publication history Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com.

  9. Effect of high dietary copper on growth, antioxidant and lipid metabolism enzymes of juvenile larger yellow croaker Larimichthys croceus

    Directory of Open Access Journals (Sweden)

    Fanxing Meng

    2016-05-01

    Full Text Available A study was carried out to test the responses of juvenile larger yellow croaker Larimichthys croceus to high Cu intake. Experimental diets were formulated containing three levels of Cu: low Cu (3.67 mg/kg, middle Cu (13.65 mg/kg and high Cu (25.78 mg/kg, and each diet were fed to large yellow croaker in triplicate for 10 weeks. Final body weight, weight gain and feed intake were the lowest in high Cu group, but hepatosomatic index was the highest; Cu concentrations in the whole-body, muscle and liver of fish fed low Cu diet was the lowest; Liver superoxide dismutase, catalase and glutathione peroxidase activities in fish fed high Cu diet were lower than those in fish fed other diets; The higher content of liver thiobarbituric acid reactive substance content was found in high Cu group, followed by middle Cu group, and the lowest in low Cu group; Liver 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malic enzyme, isocitrate dehydrogenase and fatty acid synthase activities were the lowest in high Cu group, but lipoprotein lipase activity was the highest. This study indicated that high copper intake reduced growth of juvenile larger yellow croaker, inhibited activities of antioxidant enzymes and lipid synthetases, and led to energy mobilization.

  10. Expression, cellular localization, and involvement of the pentose phosphate pathway enzymes in the regulation of ram sperm capacitation.

    Science.gov (United States)

    Luna, C; Serrano, E; Domingo, J; Casao, A; Pérez-Pé, R; Cebrián-Pérez, J A; Muiño-Blanco, T

    2016-08-01

    Spermatozoa require substantially more ATP than other cells, not only for sustaining sperm motility but also for regulating protein phosphorylation during capacitation. In this study, we have reported for the first time the presence of the two key enzymes of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in ovine spermatozoa by indirect immunofluorescence, Western blotting, in-gel activity, and reverse transcription polymerase chain reaction analysis. We found that the activity of both enzymes significantly increased after in vitro capacitation in the presence of high-cAMP levels, with a concomitant increase in protein tyrosine phosphorylation and in the proportion of sperm-capacitated pattern assessed by the chlortetracycline staining. These results suggest that PPP is related with the progress of capacitation and that a relationship between calcium compartmentalization, protein tyrosine phosphorylation and PPP seems to exist. This is the first report that shows a connection between the PPP, cAMP/PKA signaling pathways and sperm capacitation. These findings can be of high-biological importance to improve our knowledge of the biochemical mechanisms involved in the acquisition of mammalian sperm functional competence and, ultimately, fertility.

  11. Computational enzyme design: transitioning from catalytic proteins to enzymes.

    Science.gov (United States)

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward.

  12. Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

    DEFF Research Database (Denmark)

    Biran, Suzan; Bach, Poul; Simonsen, Ole

    Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care...... particles. However, enzymes may loose a significant part of their activity over a time period of several weeks. Possible causes of inactivation of enzymes in a granule may be related to the release of hydrogen peroxide from the bleaching chemicals in a moisture-containing atmosphere, humidity, autolysis...... and humidity by bubbling nitrogen gas through their corresponding solutions. An enzyme column, acting as a plug-flow reactor, was exposed to known concentrations of H2O2 (g) and humidity in a thermally stabilized chamber. Samples were analyzed for adsorptive behavior and residual enzyme activity. Since...

  13. A chemical genetic screen uncovers a small molecule enhancer of the N-acylethanolamine degrading enzyme, fatty acid amide hydrolase, in Arabidopsis

    Science.gov (United States)

    Khan, Bibi Rafeiza; Faure, Lionel; Chapman, Kent D.; Blancaflor, Elison B.

    2017-01-01

    N-Acylethanolamines (NAEs) are a group of fatty acid amides that play signaling roles in diverse physiological processes in eukaryotes. Fatty acid amide hydrolase (FAAH) degrades NAE into ethanolamine and free fatty acid to terminate its signaling function. In animals, chemical inhibitors of FAAH have been used for therapeutic treatment of pain and as tools to probe deeper into biochemical properties of FAAH. In a chemical genetic screen for small molecules that dampened the inhibitory effect of N-lauroylethanolamine (NAE 12:0) on Arabidopsis thaliana seedling growth, we identified 6-(2-methoxyphenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3 H,6 H)-dione (or MDPD). MDPD alleviated the growth inhibitory effects of NAE 12:0, in part by enhancing the enzymatic activity of Arabidopsis FAAH (AtFAAH). In vitro, biochemical assays showed that MDPD enhanced the apparent Vmax of AtFAAH but did not alter the affinity of AtFAAH for its NAE substrates. Structural analogs of MDPD did not affect AtFAAH activity or dampen the inhibitory effect of NAE 12:0 on seedling growth indicating that MDPD is a specific synthetic chemical activator of AtFAAH. Collectively, our study demonstrates the feasibility of using an unbiased chemical genetic approach to identify new pharmacological tools for manipulating FAAH- and NAE-mediated physiological processes in plants. PMID:28112243

  14. Enzymic hydrolysis of chlorella cells

    Energy Technology Data Exchange (ETDEWEB)

    Khraptsova, G.I.; Tsaplina, I.A.; Burdenko, L.G.; Khoreva, S.L.; Loginova, L.G.

    1981-01-01

    Treatment of C. ellipsoidea, C. pyrenoidosa, and C. vulgaris with cellulolytic enzymes (from Aspergillus terreus) and pectofoetidin p10x (from A. foetidus) resulted in the degradation and lysis of the algae cells. The cells were more sensitive to cellulase than to pectinase. The combination of both enzymes produced a synergistic effect on cell lysis.

  15. Phage lytic enzymes: a history

    Institute of Scientific and Technical Information of China (English)

    David; Trudil

    2015-01-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters’ or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well(Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay(Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes–from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  16. An antioxidant nanozyme that uncovers the cytoprotective potential of vanadia nanowires

    Science.gov (United States)

    Vernekar, Amit A.; Sinha, Devanjan; Srivastava, Shubhi; Paramasivam, Prasath U.; D'Silva, Patrick; Mugesh, Govindasamy

    2014-11-01

    Nanomaterials with enzyme-like properties has attracted significant interest, although limited information is available on their biological activities in cells. Here we show that V2O5 nanowires (Vn) functionally mimic the antioxidant enzyme glutathione peroxidase by using cellular glutathione. Although bulk V2O5 is known to be toxic to the cells, the property is altered when converted into a nanomaterial form. The Vn nanozymes readily internalize into mammalian cells of multiple origin (kidney, neuronal, prostate, cervical) and exhibit robust enzyme-like activity by scavenging the reactive oxygen species when challenged against intrinsic and extrinsic oxidative stress. The Vn nanozymes fully restore the redox balance without perturbing the cellular antioxidant defense, thus providing an important cytoprotection for biomolecules against harmful oxidative damage. Based on our findings, we envision that biocompatible Vn nanowires can provide future therapeutic potential to prevent ageing, cardiac disorders and several neurological conditions, including Parkinson’s and Alzheimer’s disease.

  17. Statistical Mechanics of Allosteric Enzymes.

    Science.gov (United States)

    Einav, Tal; Mazutis, Linas; Phillips, Rob

    2016-07-07

    The concept of allostery in which macromolecules switch between two different conformations is a central theme in biological processes ranging from gene regulation to cell signaling to enzymology. Allosteric enzymes pervade metabolic processes, yet a simple and unified treatment of the effects of allostery in enzymes has been lacking. In this work, we take a step toward this goal by modeling allosteric enzymes and their interaction with two key molecular players-allosteric regulators and competitive inhibitors. We then apply this model to characterize existing data on enzyme activity, comment on how enzyme parameters (such as substrate binding affinity) can be experimentally tuned, and make novel predictions on how to control phenomena such as substrate inhibition.

  18. Moonlighting enzymes in parasitic protozoa.

    Science.gov (United States)

    Collingridge, Peter W; Brown, Robert W B; Ginger, Michael L

    2010-08-01

    Enzymes moonlight in a non-enzymatic capacity in a diverse variety of cellular processes. The discovery of these non-enzymatic functions is generally unexpected, and moonlighting enzymes are known in both prokaryotes and eukaryotes. Importantly, this unexpected multi-functionality indicates that caution might be needed on some occasions in interpreting phenotypes that result from the deletion or gene-silencing of some enzymes, including some of the best known enzymes from classic intermediary metabolism. Here, we provide an overview of enzyme moonlighting in parasitic protists. Unequivocal and putative examples of moonlighting are discussed, together with the possibility that the unusual biological characteristics of some parasites either limit opportunities for moonlighting to arise or perhaps contribute to the evolution of novel proteins with clear metabolic ancestry.

  19. Uncovering the Links between Prospective Teachers' Personal Responsibility, Academic Optimism, Hope, and Emotions about Teaching: A Mediation Analysis

    Science.gov (United States)

    Eren, Altay

    2014-01-01

    Prospective teachers' sense of personal responsibility has not been examined together with their academic optimism, hope, and emotions about teaching in a single study to date. However, to consider hope, academic optimism, and emotions about teaching together with personal responsibility is important to uncover the factors affecting…

  20. Water Balance Components in Covered and Uncovered Soil Growing Irrigated Muskmelon

    Directory of Open Access Journals (Sweden)

    Paulo Leonel Libardi

    2015-10-01

    Full Text Available ABSTRACT Knowledge of the terms (or processes of the soil water balance equation or simply the components of the soil water balance over the cycle of an agricultural crop is essential for soil and water management. Thus, the aim of this study was to analyze these components in a Cambissolo Háplico (Haplocambids growing muskmelon (Cucumis melo L. under drip irrigation, with covered and uncovered soil, in the municipality of Baraúna, State of Rio Grande do Norte, Brazil (05º 04’ 48” S, 37º 37’ 00” W. Muskmelon, variety AF-646, was cultivated in a flat experimental area (20 × 50 m. The crop was spaced at 2.00 m between rows and 0.35 m between plants, in a total of ten 50-m-long plant rows. At points corresponding to ⅓ and ⅔ of each plant row, four tensiometers (at a distance of 0.1 m from each other were set up at the depths of 0.1, 0.2, 0.3, and 0.4 m, adjacent to the irrigation line (0.1 m from the plant row, between two selected plants. Five random plant rows were mulched using dry leaves of banana (Musa sp. along the drip line, forming a 0.5-m-wide strip, which covered an area of 25 m2 per of plant row with covered soil. In the other five rows, there was no covering. Thus, the experiment consisted of two treatments, with 10 replicates, in four phenological stages: initial (7-22 DAS - days after sowing, growing (22-40 DAS, fruiting (40-58 DAS and maturation (58-70 DAS. Rainfall was measured with a rain gauge and water storage was estimated by the trapezoidal method, based on tensiometer readings and soil water retention curves. For soil water flux densities at 0.3 m, the tensiometers at the depths of 0.2, 0.3, and 0.4 m were considered; the tensiometer at 0.3 m was used to estimate soil water content from the soil water retention curve at this depth, and the other two to calculate the total potential gradient. Flux densities were calculated through use of the Darcy-Buckingham equation, with hydraulic conductivity determined by

  1. NAD(PH-hydrate dehydratase- a metabolic repair enzyme and its role in Bacillus subtilis stress adaptation.

    Directory of Open Access Journals (Sweden)

    Miroslava Petrovova

    Full Text Available BACKGROUND: One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR, which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase, which was recently assigned in vitro as an ADP/ATP-dependent NAD(PH-hydrate dehydratase and was demonstrated to belong to the SigB operon. METHODS AND RESULTS: We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin, citrate cycle (isocitrate dehydrogenase, malate dehydrogenase, glycolysis (phosphoglycerate kinase, and decomposition of Amadori products (fructosamine-6-phosphate deglycase. Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase were altered after ethanol stress. CONCLUSION: We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.

  2. Optimal response of key enzymes and uncoupling protein to cold in BAT depends on local T/sub 3/ generation

    Energy Technology Data Exchange (ETDEWEB)

    Bianco, A.C.; Silva, J.E.

    1987-09-01

    The authors have examined the activity of three lipogenic enzymes (malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), and acetyl coenzyme A (CoA) carboxylase), the activity of the mitochondrial FAD-dependent ..cap alpha..-glycerolphosphate dehydrogenase (..cap alpha..-GPD), and the mitochondrial concentration of uncoupling protein (UCP) in brown adipose tissue (BAT) of euthyroid and hypothyroid rats, both at room temperature and in response to acute cold stress. These enzymes and UCP are important for the thermogenic response of BAT in adaptation to cold. The basal level of the lipogenic enzymes was normal or slightly elevated in hypothyroid rats maintained at 23/sup 0/C, but the levels of ..cap alpha..-GPD and UCP were markedly reduced. Forty-eight hours at 4/sup 0/C resulted in an increase in the activity of G-6-PD, acetyl-CoA carboxylase, and ..cap alpha..-GPD and in the concentration of UCP both in euthyroid and hypothyroid animals, but the levels reached were invariably less in hypothyroid animals, indicating that thyroid hormone is necessary for a full metabolic response of BAT under maximal demands. Of all variables measured, the most affected was UCP followed by ..cap alpha..-GDP. Dose-response relationship analysis of the UCP response to T/sub 3/ indicated that the normalization of the response to cold requires saturation of the nuclear T/sub 3/ receptors. They concluded, therefore, that the activation of the BAT 5'-deiodinase induced by cold exposure is essential to provide the high levels of nuclear T/sub 3/ required for the full expression of BAT thermogenic potential.

  3. Uncovering Molecular Relaxation Processes with Nonlinear Spectroscopies in the Deep UV

    Science.gov (United States)

    West, Brantley Andrew

    Conical intersections mediate internal conversion dynamics that compete with even the fastest nuclear motions in molecular systems. Traditional kinetic models do not apply in this regime of commensurate electronic and nuclear motion because the surroundings do not maintain equilibrium throughout the relaxation process. This dissertation focuses on uncovering the physics associated with vibronic interactions at conical intersections. Of particular interest are coherent nuclear motions driven by steep excited state potential energy gradients. Technical advances have only recently made these dynamics accessible in many systems including DNA nucleobases and cyclic polyene molecules. Optical analogues of multidimensional NMR spectroscopies have recently yielded transformative insight in relaxation processes ranging from energy transfer in photosynthesis to bond making and breaking in liquids. Prior to the start of this research, such experiments had only been conducted at infrared and visible wavelengths. Applications in the ultraviolet were motivated by studies of numerous biological systems (e.g., DNA, proteins), but had been challenged by technical issues. The work presented in this dissertation combines pulse generation techniques developed in the optical physics community with spectroscopic techniques largely pioneered by physical chemists to implement two-dimensional ultraviolet spectroscopy (2DUV). This technique is applied at the shortest wavelengths and with the best signal-to-noise ratios reported to date. Sub-picosecond excited state deactivation processes provide photo stability to the DNA double helix. Vibrational energy transfer from the solute to surrounding solvent enables relaxation of the highly non-equilibrium ground state produced by fast internal conversion. In this dissertation, nonlinear spectroscopies carried out at cryogenic temperatures are used to uncover the particular nuclear modes in the solvent that primarily accept vibrational energy from

  4. Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

    OpenAIRE

    Biran, Suzan; Jensen, Anker Degn; Kiil, Søren; Bach, Poul; Simonsen, Ole

    2010-01-01

    Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care. However, incorporating enzymes in detergent formulations gives rise to numerous practical problems due to their incompatibility with and stability against various detergent components. In powdered...

  5. Type 2 diabetic rats on diet supplemented with chromium malate show improved glycometabolism, glycometabolism-related enzyme levels and lipid metabolism.

    Directory of Open Access Journals (Sweden)

    Weiwei Feng

    Full Text Available Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism in type 2 diabetic rats. Our results showed that fasting blood glucose, serum insulin level, insulin resistance index and C-peptide level in the high dose group had a significant downward trend when compared with the model group, chromium picolinate group and chromium trichloride group. The hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, Glut4, phosphor-AMPKβ1 and Akt levels in the high dose group were significantly higher than those of the model, chromium picolinate and chromium trichloride groups. Chromium malate in a high dose group can significantly increase high density lipoprotein cholesterol level while decreasing the total cholesterol, low density lipoprotein cholesterol and triglyceride levels when compared with chromium picolinate and chromium trichloride. The serum chromium content in chromium malate and chromium picolinate group is significantly higher than that of the chromium trichloride group. The results indicated that the curative effects of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism changes are better than those of chromium picolinate and chromium trichloride. Chromium malate contributes to glucose uptake and transport in order to improved glycometabolism and glycometabolism-related enzymes.

  6. Evaluation of 90-day Repeated Dose Oral Toxicity, Glycometabolism, Learning and Memory Ability, and Related Enzyme of Chromium Malate Supplementation in Sprague-Dawley Rats.

    Science.gov (United States)

    Feng, Weiwei; Wu, Huiyu; Li, Qian; Zhou, Zhaoxiang; Chen, Yao; Zhao, Ting; Feng, Yun; Mao, Guanghua; Li, Fang; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the 90-day oral toxicity of chromium malate in Sprague-Dawley rats. The present study inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, lipid metabolism, and learning and memory ability in metabolically healthy Sprague-Dawley rats. The results showed that all rats survived and pathological, toxic, feces, and urine changes were not observed. Chromium malate did not cause measurable damage on liver, brain, and kidney. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of normal rats in chromium malate groups had no significant change when compared with control group and chromium picolinate group under physiologically relevant conditions. The serum and organ content of Cr in chromium malate groups had no significant change compared with control group. No significant changes were found in morris water maze test and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and true choline esterase (TChE) activity. The results indicated that supplementation with chromium malate did not cause measurable toxicity and has no obvious effect on glycometabolism and related enzymes, learning and memory ability, and related enzymes and lipid metabolism of female and male rats. The results of this study suggest that chromium malate is safe for human consumption.

  7. Plant Phenotyping using Probabilistic Topic Models: Uncovering the Hyperspectral Language of Plants.

    Science.gov (United States)

    Wahabzada, Mirwaes; Mahlein, Anne-Katrin; Bauckhage, Christian; Steiner, Ulrike; Oerke, Erich-Christian; Kersting, Kristian

    2016-03-09

    Modern phenotyping and plant disease detection methods, based on optical sensors and information technology, provide promising approaches to plant research and precision farming. In particular, hyperspectral imaging have been found to reveal physiological and structural characteristics in plants and to allow for tracking physiological dynamics due to environmental effects. In this work, we present an approach to plant phenotyping that integrates non-invasive sensors, computer vision, as well as data mining techniques and allows for monitoring how plants respond to stress. To uncover latent hyperspectral characteristics of diseased plants reliably and in an easy-to-understand way, we "wordify" the hyperspectral images, i.e., we turn the images into a corpus of text documents. Then, we apply probabilistic topic models, a well-established natural language processing technique that identifies content and topics of documents. Based on recent regularized topic models, we demonstrate that one can track automatically the development of three foliar diseases of barley. We also present a visualization of the topics that provides plant scientists an intuitive tool for hyperspectral imaging. In short, our analysis and visualization of characteristic topics found during symptom development and disease progress reveal the hyperspectral language of plant diseases.

  8. Uncovering cryptic species diversity of a termite community in a West African savanna.

    Science.gov (United States)

    Hausberger, Barbara; Kimpel, Dorothea; van Neer, Abbo; Korb, Judith

    2011-12-01

    To uncover the termite species diversity of a natural African savanna ecosystem, we combined morphological analyses and sequencing of three gene fragments (cytochrome oxidase I, cytochrome oxidase II and 28SrDNA, total length about 2450 bp) to infer putative species from phylogenetic trees. We identified 18 putative species clusters with high support values and which we retrieved consistently. Samples from two genera (Ancistrotermes and Microcerotermes) were excluded from the mitochondrial phylogenetic analyses as they might represent nuclear mitochondrial sequences (NUMTs). In total, our data suggest a species richness of at least 20 species, all but one belonging to the Termitidae (higher termites), and among them the fungus-growing Macrotermitinae were most prevalent with at least nine putative species. Within the fungus-growers the most species-rich genus was Microtermes and its four putative species were all cryptic species. Their abundance in the samples suggests that they play an important ecological role which is completely unstudied also due to the lack of reliable identification means. Our study shows that morphological traits are unreliable means of species identification for several termite taxa. Yet reliable and consistent identification is necessary for studying the functional role of termites in ecosystem and global processes.

  9. The work is never ending: uncovering teamwork sustainability using realistic evaluation.

    Science.gov (United States)

    Frykman, Mandus; von Thiele Schwarz, Ulrica; Muntlin Athlin, Åsa; Hasson, Henna; Mazzocato, Pamela

    2017-03-20

    Purpose The purpose of this paper is to uncover the mechanisms influencing the sustainability of behavior changes following the implementation of teamwork. Design/methodology/approach Realistic evaluation was combined with a framework (DCOM®) based on applied behavior analysis to study the sustainability of behavior changes two and a half years after the initial implementation of teamwork at an emergency department. The DCOM® framework was used to categorize the mechanisms of behavior change interventions (BCIs) into the four categories of direction, competence, opportunity, and motivation. Non-participant observation and interview data were used. Findings The teamwork behaviors were not sustained. A substantial fallback in managerial activities in combination with a complex context contributed to reduced direction, opportunity, and motivation. Reduced direction made staff members unclear about how and why they should work in teams. Deterioration of opportunity was evident from the lack of problem-solving resources resulting in accumulated barriers to teamwork. Motivation in terms of management support and feedback was reduced. Practical implications The implementation of complex organizational changes in complex healthcare contexts requires continuous adaption and managerial activities well beyond the initial implementation period. Originality/value By integrating the DCOM® framework with realistic evaluation, this study responds to the call for theoretically based research on behavioral mechanisms that can explain how BCIs interact with context and how this interaction influences sustainability.

  10. Systematic Triple-Mutant Analysis Uncovers Functional Connectivity between Pathways Involved in Chromosome Regulation

    Directory of Open Access Journals (Sweden)

    James E. Haber

    2013-06-01

    Full Text Available Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed triple-mutant analysis (TMA. We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae, including ASF1 and CAC1, two histone chaperones. After subjecting asf1Δ cac1Δ to TMA, we found that the Swi/Snf Rdh54 protein compensates for the absence of Asf1 and Cac1. Rdh54 more strongly associates with the chromatin apparatus and the pericentromeric region in the double mutant. Moreover, Asf1 is responsible for the synthetic lethality observed in cac1Δ strains lacking the HIRA-like proteins. A similar TMA was carried out after deleting both CLB5 and CLB6, cyclins that regulate DNA replication, revealing a strong functional connection to chromosome segregation. This approach can reveal functional redundancies that cannot be uncovered through traditional double-mutant analyses.

  11. Uncovering secrets behind low-resistance planing craft hull forms through optimization

    Science.gov (United States)

    Mohamad Ayob, Ahmad F.; Ray, Tapabrata; Smith, Warren F.

    2011-11-01

    There has always been significant interest within the naval architectural research community to identify ship hull forms with low resistance. While numerous design optimization frameworks have been proposed over the years to support the activity, very little attention has been paid towards the process of gaining an understanding of 'what makes a good ship design superior?'. Furthermore, there have been limited attempts to identify computationally cheap indicators that can be used to distinguish between good and poor designs. A recent technique named discovery of innovative design principles, which is aimed at understanding the relationship between the design variables, is incorporated in this work. In this article, optimal high-speed planing craft hull forms with minimum calm-water resistance are identified through the use of three state-of-the-art optimization algorithms. Collections of such designs are then used to uncover insights into the underlying relationships between the variables. The importance of such relationships is further analysed to identify computationally cheap performance indicators that can be used in lieu of detailed calm-water resistance calculations. Such indicators are useful at the concept and preliminary design stages, where one needs to sieve efficiently through a number of candidate designs to identify the better ones for further analysis.

  12. DNA barcoding uncovers cryptic diversity in 50% of deep-sea Antarctic polychaetes.

    Science.gov (United States)

    Brasier, Madeleine J; Wiklund, Helena; Neal, Lenka; Jeffreys, Rachel; Linse, Katrin; Ruhl, Henry; Glover, Adrian G

    2016-11-01

    The Antarctic marine environment is a diverse ecosystem currently experiencing some of the fastest rates of climatic change. The documentation and management of these changes requires accurate estimates of species diversity. Recently, there has been an increased recognition of the abundance and importance of cryptic species, i.e. those that are morphologically identical but genetically distinct. This article presents the largest genetic investigation into the prevalence of cryptic polychaete species within the deep Antarctic benthos to date. We uncover cryptic diversity in 50% of the 15 morphospecies targeted through the comparison of mitochondrial DNA sequences, as well as 10 previously overlooked morphospecies, increasing the total species richness in the sample by 233%. Our ability to describe universal rules for the detection of cryptic species within polychaetes, or normalization to expected number of species based on genetic data is prevented by taxon-specific differences in phylogenetic outputs and genetic variation between and within potential cryptic species. These data provide the foundation for biogeographic and functional analysis that will provide insight into the drivers of species diversity and its role in ecosystem function.

  13. Unexpected novel relational links uncovered by extensive developmental profiling of nuclear receptor expression.

    Directory of Open Access Journals (Sweden)

    Stéphanie Bertrand

    2007-11-01

    Full Text Available Nuclear receptors (NRs are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily.

  14. MARS A Cosmic Stepping Stone Uncovering Humanity’s Cosmic Context

    CERN Document Server

    Nolan, Kevin

    2008-01-01

    The questions of our origin and cosmic abundance of life are among the most compelling facing humanity. We have determined much about the nature and origin of the Universe and our place in it, but with virtually all evidence of our origin long since gone from our world and an unimaginably vast Universe still to explore, defining answers are difficult to obtain. For all of the difficulties facing us however, the planet Mars may act as a ‘cosmic stepping stone’ in uncovering some of the answers. Although different today, the origin and early history of both Earth and Mars may have been similar enough to consider an origin to life on both. But because Mars’ planetary processes collapsed over three billion years ago – just as life was beginning to flourish on Earth – a significant and unique record of activity from that era perhaps relevant to the origin of life still resides there today. In recognition of this, both the US and Europe are currently engaged in one of the most ambitious programs of explor...

  15. Antisocial behaviour and psychopathy: uncovering the externalizing link in the P3 modulation.

    Science.gov (United States)

    Pasion, Rita; Fernandes, Carina; Pereira, Mariana R; Barbosa, Fernando

    2017-03-22

    In 2009, Gao and Raine's meta-analysis analysed P3 modulation over the antisocial spectrum. However, some questions remained open regarding the P3 modulation patterns across impulsive and violent manifestations of antisocial behaviour, phenotypic components of psychopathy, and P3 components. A systematic review of 36 studies was conducted (N=3514) to extend previous results and to address these unresolved questions. A clear link between decreased P3 amplitude and antisocial behaviour was found. In psychopathy, dimensional approaches become more informative than taxonomic models. Distinct etiological pathways of psychopathy were evidenced in cognitive tasks: impulsive-antisocial psychopathic traits mainly predicted blunted P3 amplitude, while interpersonal-affective psychopathic traits explained enhanced P3 amplitude. Supporting the low fear hypothesis, the interpersonal-affective traits were associated with reduced P3 amplitude in emotional-affective learning tasks. From the accumulated knowledge we propose a framework of P3 amplitude modulation that uncovers the externalizing link between psychopathy and antisocial behaviour. However, the main hypotheses are exploratory and call for more data before stablishing robust conclusions.

  16. Novel resistance functions uncovered using functional metagenomic investigations of resistance reservoirs

    Directory of Open Access Journals (Sweden)

    Erica C. Pehrsson

    2013-06-01

    Full Text Available Rates of infection with antibiotic-resistant bacteria have increased precipitously over the past several decades, with far-reaching healthcare and societal costs. Recent evidence has established a link between antibiotic resistance genes in human pathogens and those found in non-pathogenic, commensal, and environmental organisms, prompting deeper investigation of natural and human-associated reservoirs of antibiotic resistance. Functional metagenomic selections, in which shotgun-cloned DNA fragments are selected for their ability to confer survival to an indicator host, have been increasingly applied to the characterization of many antibiotic resistance reservoirs. These experiments have demonstrated that antibiotic resistance genes are highly diverse and widely distributed, many times bearing little to no similarity to known sequences. Through unbiased selections for survival to antibiotic exposure, functional metagenomics can improve annotations by reducing the discovery of false-positive resistance and by allowing for the identification of previously unrecognizable resistance genes. In this review, we summarize the novel resistance functions uncovered using functional metagenomic investigations of natural and human-impacted resistance reservoirs. Examples of novel antibiotic resistance genes include those highly divergent from known sequences, those for which sequence is entirely unable to predict resistance function, bifunctional resistance genes, and those with unconventional, atypical resistance mechanisms. Overcoming antibiotic resistance in the clinic will require a better understanding of existing resistance reservoirs and the dissemination networks that govern horizontal gene exchange, informing best practices to limit the spread of resistance-conferring genes to human pathogens.

  17. Uncovering Special Nuclear Materials by Low-energy Nuclear Reaction Imaging.

    Science.gov (United States)

    Rose, P B; Erickson, A S; Mayer, M; Nattress, J; Jovanovic, I

    2016-04-18

    Weapons-grade uranium and plutonium could be used as nuclear explosives with extreme destructive potential. The problem of their detection, especially in standard cargo containers during transit, has been described as "searching for a needle in a haystack" because of the inherently low rate of spontaneous emission of characteristic penetrating radiation and the ease of its shielding. Currently, the only practical approach for uncovering well-shielded special nuclear materials is by use of active interrogation using an external radiation source. However, the similarity of these materials to shielding and the required radiation doses that may exceed regulatory limits prevent this method from being widely used in practice. We introduce a low-dose active detection technique, referred to as low-energy nuclear reaction imaging, which exploits the physics of interactions of multi-MeV monoenergetic photons and neutrons to simultaneously measure the material's areal density and effective atomic number, while confirming the presence of fissionable materials by observing the beta-delayed neutron emission. For the first time, we demonstrate identification and imaging of uranium with this novel technique using a simple yet robust source, setting the stage for its wide adoption in security applications.

  18. Uncovering stem-cell heterogeneity in the microniche with label-free microfluidics

    Science.gov (United States)

    Sohn, Lydia L.

    2013-03-01

    Better suited for large number of cells from bulk tissue, traditional cell-screening techniques, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, they rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. We have developed a label-free, single-cell analysis microfluidic platform capable of quantifying cell-surface marker expression of functional organ stem cells directly isolated from their micro-anatomical niche. With this platform, we have screened single quiescent muscle stem (satellite) cells derived from single myofibers, and we have uncovered an important heterogeneity in the surface-marker expression of these cells. By sorting the screened cells with our microfluidic device, we have determined what this heterogeneity means in terms of muscle stem-cell functionality. For instance, we show that the levels of beta1-integrin can predict the differentiation capacity of quiescent satellite cells, and in contrast to recent literature, that some CXCR4 + cells are not myogenic. Our results provide the first direct demonstration of a microniche-specific variation in gene expression in stem cells of the same lineage. Overall, our label-free, single-cell analysis and cell-sorting platform could be extended to other systems involving rare-cell subsets. This work was funded by the W. M. Keck Foundation, NIH, and California Institute of Regenerative Medicine

  19. Genome-wide meta-analysis uncovers novel loci influencing circulating leptin levels

    Science.gov (United States)

    Kilpeläinen, Tuomas O.; Carli, Jayne F. Martin; Skowronski, Alicja A.; Sun, Qi; Kriebel, Jennifer; Feitosa, Mary F; Hedman, Åsa K.; Drong, Alexander W.; Hayes, James E.; Zhao, Jinghua; Pers, Tune H.; Schick, Ursula; Grarup, Niels; Kutalik, Zoltán; Trompet, Stella; Mangino, Massimo; Kristiansson, Kati; Beekman, Marian; Lyytikäinen, Leo-Pekka; Eriksson, Joel; Henneman, Peter; Lahti, Jari; Tanaka, Toshiko; Luan, Jian'an; Greco M, Fabiola Del; Pasko, Dorota; Renström, Frida; Willems, Sara M.; Mahajan, Anubha; Rose, Lynda M.; Guo, Xiuqing; Liu, Yongmei; Kleber, Marcus E.; Pérusse, Louis; Gaunt, Tom; Ahluwalia, Tarunveer S.; Ju Sung, Yun; Ramos, Yolande F.; Amin, Najaf; Amuzu, Antoinette; Barroso, Inês; Bellis, Claire; Blangero, John; Buckley, Brendan M.; Böhringer, Stefan; I Chen, Yii-Der; de Craen, Anton J. N.; Crosslin, David R.; Dale, Caroline E.; Dastani, Zari; Day, Felix R.; Deelen, Joris; Delgado, Graciela E.; Demirkan, Ayse; Finucane, Francis M.; Ford, Ian; Garcia, Melissa E.; Gieger, Christian; Gustafsson, Stefan; Hallmans, Göran; Hankinson, Susan E.; Havulinna, Aki S; Herder, Christian; Hernandez, Dena; Hicks, Andrew A.; Hunter, David J.; Illig, Thomas; Ingelsson, Erik; Ioan-Facsinay, Andreea; Jansson, John-Olov; Jenny, Nancy S.; Jørgensen, Marit E.; Jørgensen, Torben; Karlsson, Magnus; Koenig, Wolfgang; Kraft, Peter; Kwekkeboom, Joanneke; Laatikainen, Tiina; Ladwig, Karl-Heinz; LeDuc, Charles A.; Lowe, Gordon; Lu, Yingchang; Marques-Vidal, Pedro; Meisinger, Christa; Menni, Cristina; Morris, Andrew P.; Myers, Richard H.; Männistö, Satu; Nalls, Mike A.; Paternoster, Lavinia; Peters, Annette; Pradhan, Aruna D.; Rankinen, Tuomo; Rasmussen-Torvik, Laura J.; Rathmann, Wolfgang; Rice, Treva K.; Brent Richards, J; Ridker, Paul M.; Sattar, Naveed; Savage, David B.; Söderberg, Stefan; Timpson, Nicholas J.; Vandenput, Liesbeth; van Heemst, Diana; Uh, Hae-Won; Vohl, Marie-Claude; Walker, Mark; Wichmann, Heinz-Erich; Widén, Elisabeth; Wood, Andrew R.; Yao, Jie; Zeller, Tanja; Zhang, Yiying; Meulenbelt, Ingrid; Kloppenburg, Margreet; Astrup, Arne; Sørensen, Thorkild I. A.; Sarzynski, Mark A.; Rao, D. C.; Jousilahti, Pekka; Vartiainen, Erkki; Hofman, Albert; Rivadeneira, Fernando; Uitterlinden, André G.; Kajantie, Eero; Osmond, Clive; Palotie, Aarno; Eriksson, Johan G.; Heliövaara, Markku; Knekt, Paul B.; Koskinen, Seppo; Jula, Antti; Perola, Markus; Huupponen, Risto K.; Viikari, Jorma S.; Kähönen, Mika; Lehtimäki, Terho; Raitakari, Olli T.; Mellström, Dan; Lorentzon, Mattias; Casas, Juan P.; Bandinelli, Stefanie; März, Winfried; Isaacs, Aaron; van Dijk, Ko W.; van Duijn, Cornelia M.; Harris, Tamara B.; Bouchard, Claude; Allison, Matthew A.; Chasman, Daniel I.; Ohlsson, Claes; Lind, Lars; Scott, Robert A.; Langenberg, Claudia; Wareham, Nicholas J.; Ferrucci, Luigi; Frayling, Timothy M.; Pramstaller, Peter P.; Borecki, Ingrid B.; Waterworth, Dawn M.; Bergmann, Sven; Waeber, Gérard; Vollenweider, Peter; Vestergaard, Henrik; Hansen, Torben; Pedersen, Oluf; Hu, Frank B.; Eline Slagboom, P; Grallert, Harald; Spector, Tim D.; Jukema, J.W.; Klein, Robert J.; Schadt, Erik E; Franks, Paul W.; Lindgren, Cecilia M.; Leibel, Rudolph L.; Loos, Ruth J. F.

    2016-01-01

    Leptin is an adipocyte-secreted hormone, the circulating levels of which correlate closely with overall adiposity. Although rare mutations in the leptin (LEP) gene are well known to cause leptin deficiency and severe obesity, no common loci regulating circulating leptin levels have been uncovered. Therefore, we performed a genome-wide association study (GWAS) of circulating leptin levels from 32,161 individuals and followed up loci reaching P<10−6 in 19,979 additional individuals. We identify five loci robustly associated (P<5 × 10−8) with leptin levels in/near LEP, SLC32A1, GCKR, CCNL1 and FTO. Although the association of the FTO obesity locus with leptin levels is abolished by adjustment for BMI, associations of the four other loci are independent of adiposity. The GCKR locus was found associated with multiple metabolic traits in previous GWAS and the CCNL1 locus with birth weight. Knockdown experiments in mouse adipose tissue explants show convincing evidence for adipogenin, a regulator of adipocyte differentiation, as the novel causal gene in the SLC32A1 locus influencing leptin levels. Our findings provide novel insights into the regulation of leptin production by adipose tissue and open new avenues for examining the influence of variation in leptin levels on adiposity and metabolic health. PMID:26833098

  20. Gene Expression Deconvolution for Uncovering Molecular Signatures in Response to Therapy in Juvenile Idiopathic Arthritis.

    Directory of Open Access Journals (Sweden)

    Ang Cui

    Full Text Available Gene expression-based signatures help identify pathways relevant to diseases and treatments, but are challenging to construct when there is a diversity of disease mechanisms and treatments in patients with complex diseases. To overcome this challenge, we present a new application of an in silico gene expression deconvolution method, ISOpure-S1, and apply it to identify a common gene expression signature corresponding to response to treatment in 33 juvenile idiopathic arthritis (JIA patients. Using pre- and post-treatment gene expression profiles only, we found a gene expression signature that significantly correlated with a reduction in the number of joints with active arthritis, a measure of clinical outcome (Spearman rho = 0.44, p = 0.040, Bonferroni correction. This signature may be associated with a decrease in T-cells, monocytes, neutrophils and platelets. The products of most differentially expressed genes include known biomarkers for JIA such as major histocompatibility complexes and interleukins, as well as novel biomarkers including α-defensins. This method is readily applicable to expression datasets of other complex diseases to uncover shared mechanistic patterns in heterogeneous samples.

  1. CHANG-ES VIII: Uncovering Hidden AGN Activity in Radio Polarization

    CERN Document Server

    Irwin, Judith A; Damas-Segovia, A; Beck, Rainer; English, Jayanne; Heald, George; Henriksen, Richard N; Krause, Marita; Li, Jiang-Tao; Rand, Richard J; Wang, Q Daniel; Wiegert, Theresa; Kamieneski, Patrick; Paré, Dylan; Sullivan, Kendall

    2016-01-01

    We report on C-band (5 - 7 GHz) observations of the galaxy, NGC~2992, from the CHANG-ES sample. This galaxy displays an embedded nuclear double-lobed radio morphology within its spiral disk, as revealed in linearly polarized emission but {\\it not} in total intensity emission. The radio lobes are kpc-sized, similar to what has been observed in the past for other Seyfert galaxies, and show ordered magnetic fields. NGC~2992 has shown previous evidence for AGN-related activity, but not the linearly polarized radio features that we present here. We draw attention to this galaxy as the first clear example (and prototype) of bipolar radio outflow that is revealed in linearly polarized emission only. Such polarization observations, which are unobscured by dust, provide a new tool for uncovering hidden weak AGN activity which may otherwise be masked by brighter unpolarized emission within which it is embedded. The radio lobes observed in NGC~2992 are interacting with the surrounding interstellar medium and offer new o...

  2. Uncovering Special Nuclear Materials by Low-energy Nuclear Reaction Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Rose, Jr., P. B.; Erickson, A. S.; Mayer, Michael F.; Nattress, J.; Jovanovic, I

    2016-04-18

    Weapons-grade uranium and plutonium could be used as nuclear explosives with extreme destructive potential. The problem of their detection, especially in standard cargo containers during transit, has been described as “searching for a needle in a haystack” because of the inherently low rate of spontaneous emission of characteristic penetrating radiation and the ease of its shielding. Currently, the only practical approach for uncovering well-shielded special nuclear materials is by use of active interrogation using an external radiation source. However, the similarity of these materials to shielding and the required radiation doses that may exceed regulatory limits prevent this method from being widely used in practice. We introduce a low-dose active detection technique, referred to as low-energy nuclear reaction imaging, which exploits the physics of interactions of multi-MeV monoenergetic photons and neutrons to simultaneously measure the material’s areal density and effective atomic number, while confirming the presence of fissionable materials by observing the beta-delayed neutron emission. For the first time, we demonstrate identification and imaging of uranium with this novel technique using a simple yet robust source, setting the stage for its wide adoption in security applications.

  3. Methods uncovering usability issues in medication-related alerting functions: results from a systematic review.

    Science.gov (United States)

    Marcilly, Romaric; Vasseur, Francis; Ammenwerth, Elske; Beuscart-Zephir, Marie-Catherine

    2014-01-01

    This paper aims at listing the methods used to evaluate the usability of medication-related alerting functions and at knowing what type of usability issues those methods allow to detect. A sub-analysis of data from this systematic review has been performed. Methods applied in the included papers were collected. Then, included papers were sorted in four types of evaluation: "expert evaluation", "user- testing/simulation", "on site observation" and "impact studies". The types of usability issues (usability flaws, usage problems and negative outcomes) uncovered by those evaluations were analyzed. Results show that a large set of methods are used. The largest proportion of papers uses "on site observation" evaluation. This is the only evaluation type for which every kind of usability flaws, usage problems and outcomes are detected. It is somehow surprising that, in a usability systematic review, most of the papers included use a method that is not often presented as a usability method. Results are discussed about the opportunity to provide usability information collected after the implementation of the technology during their design process, i.e. before their implementation.

  4. Uncovering Special Nuclear Materials by Low-energy Nuclear Reaction Imaging

    Science.gov (United States)

    Rose, P. B.; Erickson, A. S.; Mayer, M.; Nattress, J.; Jovanovic, I.

    2016-04-01

    Weapons-grade uranium and plutonium could be used as nuclear explosives with extreme destructive potential. The problem of their detection, especially in standard cargo containers during transit, has been described as “searching for a needle in a haystack” because of the inherently low rate of spontaneous emission of characteristic penetrating radiation and the ease of its shielding. Currently, the only practical approach for uncovering well-shielded special nuclear materials is by use of active interrogation using an external radiation source. However, the similarity of these materials to shielding and the required radiation doses that may exceed regulatory limits prevent this method from being widely used in practice. We introduce a low-dose active detection technique, referred to as low-energy nuclear reaction imaging, which exploits the physics of interactions of multi-MeV monoenergetic photons and neutrons to simultaneously measure the material’s areal density and effective atomic number, while confirming the presence of fissionable materials by observing the beta-delayed neutron emission. For the first time, we demonstrate identification and imaging of uranium with this novel technique using a simple yet robust source, setting the stage for its wide adoption in security applications.

  5. Combining Novel Simulation Methods and Nucleation Theory to Uncover the Secrets of Gas Hydrates

    Energy Technology Data Exchange (ETDEWEB)

    Keyes, Thomas [Boston Univ., MA (United States). Dept. of Chemistry

    2016-04-14

    Conventional computer simulation methods fail for some of the most important problems. With the design and application of innovative algorithms, this project achieved a breakthrough for the case of systems undergoing first-order phase transitions. We gave a complete simulation protocol based upon a well optimized version of our "generalized replica exchange method". The transition of primary interest was gas hydrate formation, a process of significance for climate science and natural gas retrieval. Since hydrates consist of guest molecules in the cages of a water matrix, β ice, the freezing and melting of water was also studied. New information was uncovered about the transition pathways and thermodynamics. Some highlights are 1. the finding that in a very dilute solution without deep supercooling, representative of real-world conditions and very challenging to conventional algorithms, methane can act as a catalyst to drive the formation of large amounts of β ice with empty cages as metastable intermediates, which might be filled by additional methane in a mechanism for hydrate formation, and 2. illumination of the role of metastable cubic ice in water freezing, with determination of the surface tensions of the cubic, hexagonal, and β ices, and the free energy difference of cubic vs hexagonal ice. Work was begun on lipid systems, bilayers and nanoreactors promising for energy-related photoreductions, and targets for future research. Our methods yielded what is arguably the most complete description of the composite lipid/water phases and the transition pathways among them.

  6. Genome-wide meta-analysis uncovers novel loci influencing circulating leptin levels.

    Science.gov (United States)

    Kilpeläinen, Tuomas O; Carli, Jayne F Martin; Skowronski, Alicja A; Sun, Qi; Kriebel, Jennifer; Feitosa, Mary F; Hedman, Åsa K; Drong, Alexander W; Hayes, James E; Zhao, Jinghua; Pers, Tune H; Schick, Ursula; Grarup, Niels; Kutalik, Zoltán; Trompet, Stella; Mangino, Massimo; Kristiansson, Kati; Beekman, Marian; Lyytikäinen, Leo-Pekka; Eriksson, Joel; Henneman, Peter; Lahti, Jari; Tanaka, Toshiko; Luan, Jian'an; Del Greco M, Fabiola; Pasko, Dorota; Renström, Frida; Willems, Sara M; Mahajan, Anubha; Rose, Lynda M; Guo, Xiuqing; Liu, Yongmei; Kleber, Marcus E; Pérusse, Louis; Gaunt, Tom; Ahluwalia, Tarunveer S; Ju Sung, Yun; Ramos, Yolande F; Amin, Najaf; Amuzu, Antoinette; Barroso, Inês; Bellis, Claire; Blangero, John; Buckley, Brendan M; Böhringer, Stefan; I Chen, Yii-Der; de Craen, Anton J N; Crosslin, David R; Dale, Caroline E; Dastani, Zari; Day, Felix R; Deelen, Joris; Delgado, Graciela E; Demirkan, Ayse; Finucane, Francis M; Ford, Ian; Garcia, Melissa E; Gieger, Christian; Gustafsson, Stefan; Hallmans, Göran; Hankinson, Susan E; Havulinna, Aki S; Herder, Christian; Hernandez, Dena; Hicks, Andrew A; Hunter, David J; Illig, Thomas; Ingelsson, Erik; Ioan-Facsinay, Andreea; Jansson, John-Olov; Jenny, Nancy S; Jørgensen, Marit E; Jørgensen, Torben; Karlsson, Magnus; Koenig, Wolfgang; Kraft, Peter; Kwekkeboom, Joanneke; Laatikainen, Tiina; Ladwig, Karl-Heinz; LeDuc, Charles A; Lowe, Gordon; Lu, Yingchang; Marques-Vidal, Pedro; Meisinger, Christa; Menni, Cristina; Morris, Andrew P; Myers, Richard H; Männistö, Satu; Nalls, Mike A; Paternoster, Lavinia; Peters, Annette; Pradhan, Aruna D; Rankinen, Tuomo; Rasmussen-Torvik, Laura J; Rathmann, Wolfgang; Rice, Treva K; Brent Richards, J; Ridker, Paul M; Sattar, Naveed; Savage, David B; Söderberg, Stefan; Timpson, Nicholas J; Vandenput, Liesbeth; van Heemst, Diana; Uh, Hae-Won; Vohl, Marie-Claude; Walker, Mark; Wichmann, Heinz-Erich; Widén, Elisabeth; Wood, Andrew R; Yao, Jie; Zeller, Tanja; Zhang, Yiying; Meulenbelt, Ingrid; Kloppenburg, Margreet; Astrup, Arne; Sørensen, Thorkild I A; Sarzynski, Mark A; Rao, D C; Jousilahti, Pekka; Vartiainen, Erkki; Hofman, Albert; Rivadeneira, Fernando; Uitterlinden, André G; Kajantie, Eero; Osmond, Clive; Palotie, Aarno; Eriksson, Johan G; Heliövaara, Markku; Knekt, Paul B; Koskinen, Seppo; Jula, Antti; Perola, Markus; Huupponen, Risto K; Viikari, Jorma S; Kähönen, Mika; Lehtimäki, Terho; Raitakari, Olli T; Mellström, Dan; Lorentzon, Mattias; Casas, Juan P; Bandinelli, Stefanie; März, Winfried; Isaacs, Aaron; van Dijk, Ko W; van Duijn, Cornelia M; Harris, Tamara B; Bouchard, Claude; Allison, Matthew A; Chasman, Daniel I; Ohlsson, Claes; Lind, Lars; Scott, Robert A; Langenberg, Claudia; Wareham, Nicholas J; Ferrucci, Luigi; Frayling, Timothy M; Pramstaller, Peter P; Borecki, Ingrid B; Waterworth, Dawn M; Bergmann, Sven; Waeber, Gérard; Vollenweider, Peter; Vestergaard, Henrik; Hansen, Torben; Pedersen, Oluf; Hu, Frank B; Eline Slagboom, P; Grallert, Harald; Spector, Tim D; Jukema, J W; Klein, Robert J; Schadt, Erik E; Franks, Paul W; Lindgren, Cecilia M; Leibel, Rudolph L; Loos, Ruth J F

    2016-02-01

    Leptin is an adipocyte-secreted hormone, the circulating levels of which correlate closely with overall adiposity. Although rare mutations in the leptin (LEP) gene are well known to cause leptin deficiency and severe obesity, no common loci regulating circulating leptin levels have been uncovered. Therefore, we performed a genome-wide association study (GWAS) of circulating leptin levels from 32,161 individuals and followed up loci reaching P<10(-6) in 19,979 additional individuals. We identify five loci robustly associated (P<5 × 10(-8)) with leptin levels in/near LEP, SLC32A1, GCKR, CCNL1 and FTO. Although the association of the FTO obesity locus with leptin levels is abolished by adjustment for BMI, associations of the four other loci are independent of adiposity. The GCKR locus was found associated with multiple metabolic traits in previous GWAS and the CCNL1 locus with birth weight. Knockdown experiments in mouse adipose tissue explants show convincing evidence for adipogenin, a regulator of adipocyte differentiation, as the novel causal gene in the SLC32A1 locus influencing leptin levels. Our findings provide novel insights into the regulation of leptin production by adipose tissue and open new avenues for examining the influence of variation in leptin levels on adiposity and metabolic health.

  7. CHANG-ES - VIII. Uncovering hidden AGN activity in radio polarization

    Science.gov (United States)

    Irwin, Judith A.; Schmidt, Philip; Damas-Segovia, A.; Beck, Rainer; English, Jayanne; Heald, George; Henriksen, Richard N.; Krause, Marita; Li, Jiang-Tao; Rand, Richard J.; Wang, Q. Daniel; Wiegert, Theresa; Kamieneski, Patrick; Paré, Dylan; Sullivan, Kendall

    2017-01-01

    We report on C-band (5-7 GHz) observations of the galaxy, NGC 2992, from the Continuum Halos in Nearby Galaxies - an EVLA Survey (CHANG-ES) sample. This galaxy displays an embedded nuclear double-lobed radio morphology within its spiral disc, as revealed in linearly polarized emission but not in total intensity emission. The radio lobes are kpc-sized, similar to what has been observed in the past for other Seyfert galaxies, and show ordered magnetic fields. NGC 2992 has shown previous evidence for AGN-related activity, but not the linearly polarized radio features that we present here. We draw attention to this galaxy as the first clear example (and prototype) of bipolar radio outflow that is revealed in linearly polarized emission only. Such polarization observations, which are unobscured by dust, provide a new tool for uncovering hidden weak active galactic nucleus (AGN) activity which may otherwise be masked by brighter unpolarized emission within which it is embedded. The radio lobes observed in NGC 2992 are interacting with the surrounding interstellar medium (ISM) and offer new opportunities to investigate the interactions between nuclear outflows and the ISM in nearby galaxies. We also compare the radio emission with a new CHANDRA X-ray image of this galaxy. A new CHANG-ES image of NGC 3079 is also briefly shown as another example as to how much more obvious radio lobes appear in linear polarization as opposed to total intensity.

  8. The proteome and phosphoproteome of maize pollen uncovers fertility candidate proteins.

    Science.gov (United States)

    Chao, Qing; Gao, Zhi-Fang; Wang, Yue-Feng; Li, Zhe; Huang, Xia-He; Wang, Ying-Chun; Mei, Ying-Chang; Zhao, Biligen-Gaowa; Li, Liang; Jiang, Yu-Bo; Wang, Bai-Chen

    2016-06-01

    Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future.

  9. Uncovering Multiple Populations with Washington Photometry: I. The Globular Cluster NGC 1851

    CERN Document Server

    Cummings, Jeffrey D; Villanova, S; Carraro, G

    2014-01-01

    The analysis of multiple populations (MPs) in globular clusters (GCs) has become a forefront area of research in astronomy. Multiple red giant branches (RGBs), subgiant branches (SGBs), and even main sequences (MSs) have now been observed photometrically in many GCs. UV photometry has been crucial in discovering and analyzing these MPs, but the Johnson U and the Stromgren and Sloan u filters that have generally been used are relatively inefficient and very sensitive to reddening and atmospheric extinction. In contrast, the Washington C filter is much broader and redder than these competing UV filters. Here we investigate the use of the Washington system to uncover MPs using only a 1-meter telescope. Our analysis of the well-studied GC NGC 1851 finds that the C filter is both very efficient and effective at detecting its previously discovered MPs in the RGB and SGB. Remarkably, we have also detected an intrinsically broad MS best characterized by two distinct but heavily overlapping populations that cannot be ...

  10. Immunization with mixed peptides derived from glucose-6-phosphate isomerase induces rheumatoid arthritis in DBA/1 mice%葡萄糖-6-磷酸异构酶混合肽段诱导的 DBA/1小鼠类风湿性关节炎模型的建立

    Institute of Scientific and Technical Information of China (English)

    张雪娇; 刘嘉琳; 杨飞; 娄永富; 王强; 陈冬志; 孟明

    2016-01-01

    [ ABSTRACT] AIM:To establish an animal model of rheumatoid arthritis ( RA) in DBA/1 mice induced by im-munodominant mixed peptides derived from glucose-6-phosphate isomerase (GPI).METHODS: The DBA/1 mice were immunized with emulsified mixed peptide fragments of hGPI 325-339+hGPI469-483 or single peptide hGPI325-339 in com-plete Freund′s adjuvant by subcutaneous injection to induce the model of RA .Body weight , ankle joint symptom scores , the pathological change of the ankle joint , the levels of CD4 +T cells in the spleen and peripheral blood , the proportion of iNKT cells in the peripheral blood , and the levels of TNF-αand IL-6 in serum were detected to evaluate and analyze the model.RESULTS:The hind paw of the model mice appeared red swelling on the 8th day, and then aggravated gradually to the limbs.The red swelling reached peak on the 14th day, and then relieved gradually .Inflammation response dominated by lymphocytes and monocytes was observed in the ankle joint .The inflammatory effect of mixed peptides was more obvious than that of the single one (P<0.05).Compared with control group and the mice treated with single peptide , the weight gain was slow, the amount of CD4 +T cells in the peripheral blood and spleen were increased , the proportion of peripheral iNKT cells in the inflammatory peak was decreased (P<0.05), and the serum level of TNF-αwas increased significantly ( P<0.05) in the mice treated with mixed peptide fragments .CONCLUSION: The immunological characteristics of RA model induced by mixed GPI peptides in DBA/1 mice is closer to that in RA patients , especially in the immunopathology of iNKT cells.Therefore, this model can be used as a new tool for studying the mechanism and immunological intervention of RA.%目的:利用葡萄糖-6-磷酸异构酶( GPI)单一肽段及混合多肽片段免疫DBA/1小鼠,建立类风湿性关节炎( RA)模型,分析其主要评价指标及特点,为探讨RA的免疫机制及治疗提

  11. Clinical research of two cases of glutamine-fructose-6-phosphate transaminase 1-related limb-girdle congenital myasthenic syndrome%谷氨酰胺-果糖-6-磷酸转氨酶1相关性肢带型先天性肌无力综合征二例临床分析

    Institute of Scientific and Technical Information of China (English)

    张巍; 徐春晓; 孟令超; 吕鹤; 左越焕; 刘靖; 王朝霞; 袁云

    2015-01-01

    Objective To describe clinical,neurophysiological and pathological features in 2 patients with glutamine-fructose-6-phosphate transaminase 1 (GFPT1)-related limb-girdle congenital myasthenic syndrome.Methods We recruited two patients diagnosed as GFPT1-related limb-girdle congenital myasthenic syndrome in Peking University First Hospital in March and June 2014 respectively.Then we collected clinical,laboratory,neurophysiological,neuropathological and genetic data of the 2 patients to characterize the disease features.We also followed up the two patients to evaluate therapeutic effects.Results Case 1 was a sixteen years old boy complaining of exercise related fatigue for 10 years.Case 2 was a nine years old boy complaining of exercise related fatigue for 6 years.Both patients revealed mild proximal weakness during physical examinations.The level of serum creatine kinase was 224 IU/L in case 1 and within normal range in case 2.Repetitive nerve stimulation with 3 Hz at axillary nerve revealed decremental response of the main compound muscle action potential amplitude,which was 46.9% in case 1 and 17.5% in case 2.Anti-acetylcholine receptor antibody was not detected in both cases.Both of them responded well to oral pyridostigmine bromide.Muscle biopsies and GFPT1 gene analysis were performed in 2 cases.Muscle biopsy revealed massive tubular aggregates within muscle fibers in case 1 and type 1 fiber predoninance in case 2.GFPT1 showed 2 compound heterozygous mutations in patient 1 with p.Y367C and p.G564C,and in patient 2 with p.G26S and p.V291I respectively.Conclusions Childhood onset,fluctuating limb girdle weakness,decrement on low frequency repetitive nerve stimulation,tubular aggregates on muscle pathology could be considered as the diagnostic clues for GFPT1-related limb-girdle congenital myasthenic syndrome.Cholinesterase inhibitors therapy could be used as the first choice in this disease.%目的 观察2例谷氨酰胺-果糖-6-磷酸转氨酶1(GFPT1)相关

  12. Cytosolic NADPH homeostasis in glucose-starved procyclic Trypanosoma brucei relies on malic enzyme and the pentose phosphate pathway fed by gluconeogenic flux.

    Science.gov (United States)

    Allmann, Stefan; Morand, Pauline; Ebikeme, Charles; Gales, Lara; Biran, Marc; Hubert, Jane; Brennand, Ana; Mazet, Muriel; Franconi, Jean-Michel; Michels, Paul A M; Portais, Jean-Charles; Boshart, Michael; Bringaud, Frédéric

    2013-06-21

    All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/(RNAi)PGI double mutant when compared with the single mutants, and (iii) the (13)C enrichment of glycolytic and PPP intermediates from cells incubated with [U-(13)C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host.

  13. Photosynthetic fuel for heterologous enzymes

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

    2017-01-01

    , competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...... of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However...

  14. GRE Enzymes for Vector Analysis

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme data that were collected during the 2004-2006 EMAP-GRE program. These data were then used by Moorhead et al (2016) in their ecoenzyme vector...

  15. Controlled enzyme catalyzed heteropolysaccharide degradation

    DEFF Research Database (Denmark)

    Rasmussen, Louise Enggaard

    The work presented in this PhD thesis has provided a better understanding of the enzyme kinetics and quantitative phenomena of the hydrolysis of xylan substrates by selected pure enzyme preparations. Furthermore, the options for producing specific substituted xylooligosaccharides from selected...... substrates by specific xylanase treatment have been examined. The kinetics of the enzymatic degradation of water-extractable wheat arabinoxylan (WE-AX) during designed treatments with selected monocomponent enzymes was investigated by monitoring the release of xylose and arabinose. The results of different...... between -xylosidase and the α-L-arabinofuranosidases on the xylose release were low as compared to the effect of xylanase addition with β-xylosidase, which increased the xylose release by ~25 times in 30 minutes. At equimolar addition levels of the four enzymes, the xylanase activity was thus rate...

  16. Enzymes: principles and biotechnological applications.

    Science.gov (United States)

    Robinson, Peter K

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed.

  17. Time-resolved fluorescence observation of di-tyrosine formation in horseradish peroxidase upon ultrasound treatment leading to enzyme inactivation

    Science.gov (United States)

    Tsikrika, Konstantina; Lemos, M. Adília; Chu, Boon-Seang; Bremner, David H.; Hungerford, Graham

    2017-02-01

    The application of ultrasound to a solution can induce cavitional phenomena and generate high localised temperatures and pressures. These are dependent of the frequency used and have enabled ultrasound application in areas such as synthetic, green and food chemistry. High frequency (100 kHz to 1 MHz) in particular is promising in food chemistry as a means to inactivate enzymes, replacing the need to use periods of high temperature. A plant enzyme, horseradish peroxidase, was studied using time-resolved fluorescence techniques as a means to assess the effect of high frequency (378 kHz and 583 kHz) ultrasound treatment at equivalent acoustic powers. This uncovered the fluorescence emission from a newly formed species, attributed to the formation of di-tyrosine within the horseradish peroxidase structure caused by auto-oxidation, and linked to enzyme inactivation.

  18. Engineering Cellulase Enzymes for Bioenergy

    Science.gov (United States)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  19. Isothermal titration calorimetry uncovers substrate promiscuity of bicupin oxalate oxidase from Ceriporiopsis subvermispora

    Directory of Open Access Journals (Sweden)

    Hassan Rana

    2016-03-01

    Full Text Available Isothermal titration calorimetry (ITC may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx from Ceriporiopsis subvermispora (CsOxOx, a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔHapp. Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.

  20. Uncovering highly obfuscated plagiarism cases using fuzzy semantic-based similarity model

    Directory of Open Access Journals (Sweden)

    Salha M. Alzahrani

    2015-07-01

    Full Text Available Highly obfuscated plagiarism cases contain unseen and obfuscated texts, which pose difficulties when using existing plagiarism detection methods. A fuzzy semantic-based similarity model for uncovering obfuscated plagiarism is presented and compared with five state-of-the-art baselines. Semantic relatedness between words is studied based on the part-of-speech (POS tags and WordNet-based similarity measures. Fuzzy-based rules are introduced to assess the semantic distance between source and suspicious texts of short lengths, which implement the semantic relatedness between words as a membership function to a fuzzy set. In order to minimize the number of false positives and false negatives, a learning method that combines a permission threshold and a variation threshold is used to decide true plagiarism cases. The proposed model and the baselines are evaluated on 99,033 ground-truth annotated cases extracted from different datasets, including 11,621 (11.7% handmade paraphrases, 54,815 (55.4% artificial plagiarism cases, and 32,578 (32.9% plagiarism-free cases. We conduct extensive experimental verifications, including the study of the effects of different segmentations schemes and parameter settings. Results are assessed using precision, recall, F-measure and granularity on stratified 10-fold cross-validation data. The statistical analysis using paired t-tests shows that the proposed approach is statistically significant in comparison with the baselines, which demonstrates the competence of fuzzy semantic-based model to detect plagiarism cases beyond the literal plagiarism. Additionally, the analysis of variance (ANOVA statistical test shows the effectiveness of different segmentation schemes used with the proposed approach.

  1. Treatment of gastric outlet and duodenal obstructions with uncovered expandable metal stents

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate and evaluate the technical feasibility and clinical effectiveness of fluoroscopically guided peroral uncovered expandable metal stent placement to treat gastric outlet and duodenal obstructions. METHODS: Fifteen consecutive patients underwent peroral placement of WallstentTM Enteral Endoprosthesis to treat gastric outlet and duodenal obstructions (14 malignant, 1 benign). All procedures were completed under fluoroscopic guidance without endoscopic assistance. Follow-up was completed until the patients died or were lost, and the clinical outcomes were analyzed. RESULTS: The technique success rate was 100%, and the oral intake was maintained in 12 of 14 patients varying from 7 d to 270 d. Two patients remained unable to resume oral intake, although their stents were proven to be patent with the barium study. One patient with acute necrotizing pancreatitis underwent enteral stenting to treat intestinal obstruction, and nausea and vomiting disappeared. Ten patients died during the followup period, and their mean oral intake time was 50 d. No procedure-related complications occurred. Stent migration to the gastric antrum occurred in one patient 1 year after the procedure, a tumor grew at the proximal end of the stent in another patient 38 d post-stent insertion. CONCLUSION: Fluoroscopically guided peroral metal stent implantation is a safe and effective method to treat malignant gastrointestinal obstructions, and complications can be ignored based on our short-term study. Indications for this procedure should be discreetly considered because a few patients may not benefit from gastrointestinal insertion, but some benign gastrointestinal obstructions can be treated using this procedure.

  2. Uncovering phenotypes of poor-pitch singing: The Sung Performance Battery (SPB

    Directory of Open Access Journals (Sweden)

    Magdalena eBerkowska

    2013-10-01

    Full Text Available Singing is as natural as speaking for humans. Increasing evidence shows that the layman can carry a tune (e.g., when asked to sing a well-known song or to imitate single pitches, intervals and short melodies. Yet, important individual differences exist in the general population with regard to singing proficiency. Some individuals are particularly inaccurate or imprecise in producing or imitating pitch information (poor-pitch singers, thus showing a variety of singing phenotypes. Unfortunately, so far there is not a standard set of tasks for assessing singing proficiency in the general population, allowing to uncover and characterize individual profiles of poor-pitch singing. Different tasks and analysis methods are typically used in various experiments, making the comparison of the results across studies arduous. To fill this gap we propose here a new tool for assessing singing proficiency (the Sung Performance Battery, SPB. The SPB starts from the assessment of participants’ vocal range followed by five tasks: 1 single-pitch matching, 2 pitch-interval matching, 3 novel-melody matching, 4 singing from memory of familiar melodies (with lyrics and on a syllable, and 5 singing of familiar melodies (with lyrics and on a syllable at a slow tempo indicated by a metronome. Data analysis via acoustical methods provides objective measures of pitch accuracy and precision in terms of absolute and relative pitch. The SPB has been tested in a group of 50 occasional singers. The results indicate that the battery is useful for characterizing proficient singing and for detecting cases of inaccurate and/or imprecise singing.

  3. Quick determination of gas pressure before uncovering coal in cross-cuts and shafts

    Institute of Scientific and Technical Information of China (English)

    JIANG Cheng-lin; DENG Su-hua; ZHANG Chao-jie; CHENG Song-li; LV Shu-wen; WANG Chen; LI Xiao-wei; CHEN Yu-jia; XIE Qing-xue; LIU Ying; TANG Jun; YANG Fei-long; WANG Fa-kai

    2008-01-01

    The determination of gas pressure before uncovering coal in cross-cuts and in shafts is one of the important steps in predicting coal and gas outbursts. However, the time spent for testing gas pressure is, at present, very long, seriously affecting the application of outburst prediction techniques in opening coal seams in cross-cuts and shafts. In order to reduce the time needed in gas pressure tests and to improve the accuracy of tests, we analyzed the process of gas pressure tests and examined the effect of the length of boreholes in coal seams in tests. The result shows that 1) the shorter the borehole, the easier the real pressure value of gas can be obtained and 2) the main factors affecting the time spent in gas pressure tests are the length of the borehole in coal seams,the gas emission time after the borehole has been formed and the quality of the borehole-sealing. The longer the length of the borehole, the longer the gas emission time and the larger the pressure-relief circle formed around the borehole, the longer the time needed for pressure tests. By controlling the length of the borehole in a test case in the Huainan mining area, and adopting a quick sealing technique using a sticky liquid method, the sealing quality was clearly improved and the gas emission time as well as the amount of gas discharged greatly decreased. Before the method described, the time required for the gas pressure to increase during the pressure test process, was more than 10 days. With our new method the required time is only 5 hours. In addition, the accuracy of the gas pressure test is greatly improved.

  4. Uncovering multiple populations with washington photometry. I. The globular cluster NGC 1851

    Energy Technology Data Exchange (ETDEWEB)

    Cummings, Jeffrey D.; Geisler, D.; Villanova, S. [Departamento de Astronomía, Casilla 160-C, Universidad de Concepción (Chile); Carraro, G. [ESO, Alonso de Cordova 3107, Casilla 19001, Santiago de Chile (Chile)

    2014-08-01

    The analysis of multiple populations (MPs) in globular clusters (GCs) has become a forefront area of research in astronomy. Multiple red giant branches (RGBs), subgiant branches (SGBs), and even main sequences (MSs) have now been observed photometrically in many GCs, while broad abundance distributions of certain elements have been detected spectroscopically in most, if not all, GCs. UV photometry has been crucial in discovering and analyzing these MPs, but the Johnson U and the Stromgren and Sloan u filters that have generally been used are relatively inefficient and very sensitive to reddening and atmospheric extinction. In contrast, the Washington C filter is much broader and redder than these competing UV filters, making it far more efficient at detecting MPs and much less sensitive to reddening and extinction. Here, we investigate the use of the Washington system to uncover MPs using only a 1 m telescope. Our analysis of the well-studied GC NGC 1851 finds that the C filter is both very efficient and effective at detecting its previously discovered MPs in the RGB and SGB. Remarkably, we have also detected an intrinsically broad MS best characterized by two distinct but heavily overlapping populations that cannot be explained by binaries, field stars, or photometric errors. The MS distribution is in very good agreement with that seen on the RGB, with ∼30% of the stars belonging to the second population. There is also evidence for two sequences in the red horizontal branch, but this appears to be unrelated to the MPs in this cluster. Neither of these latter phenomena have been observed previously in this cluster. The redder MS stars are also more centrally concentrated than the blue MS. This is the first time MPs in an MS have been discovered from the ground, and using only a 1 m telescope. The Washington system thus proves to be a very powerful tool for investigating MPs, and holds particular promise for extragalactic objects where photons are limited.

  5. Uncovering Multiple Populations with Washington Photometry. I. The Globular Cluster NGC 1851

    Science.gov (United States)

    Cummings, Jeffrey D.; Geisler, D.; Villanova, S.; Carraro, G.

    2014-08-01

    The analysis of multiple populations (MPs) in globular clusters (GCs) has become a forefront area of research in astronomy. Multiple red giant branches (RGBs), subgiant branches (SGBs), and even main sequences (MSs) have now been observed photometrically in many GCs, while broad abundance distributions of certain elements have been detected spectroscopically in most, if not all, GCs. UV photometry has been crucial in discovering and analyzing these MPs, but the Johnson U and the Stromgren and Sloan u filters that have generally been used are relatively inefficient and very sensitive to reddening and atmospheric extinction. In contrast, the Washington C filter is much broader and redder than these competing UV filters, making it far more efficient at detecting MPs and much less sensitive to reddening and extinction. Here, we investigate the use of the Washington system to uncover MPs using only a 1 m telescope. Our analysis of the well-studied GC NGC 1851 finds that the C filter is both very efficient and effective at detecting its previously discovered MPs in the RGB and SGB. Remarkably, we have also detected an intrinsically broad MS best characterized by two distinct but heavily overlapping populations that cannot be explained by binaries, field stars, or photometric errors. The MS distribution is in very good agreement with that seen on the RGB, with ~30% of the stars belonging to the second population. There is also evidence for two sequences in the red horizontal branch, but this appears to be unrelated to the MPs in this cluster. Neither of these latter phenomena have been observed previously in this cluster. The redder MS stars are also more centrally concentrated than the blue MS. This is the first time MPs in an MS have been discovered from the ground, and using only a 1 m telescope. The Washington system thus proves to be a very powerful tool for investigating MPs, and holds particular promise for extragalactic objects where photons are limited.

  6. Clinical analysis of ABO hemolytic disease in newborn with giucose-6-phosphate dehydrogenase deficiency.%新生儿ABO溶血病并红细胞葡萄糖-6-磷酸脱氢酶缺乏症临床对比分析

    Institute of Scientific and Technical Information of China (English)

    黄世荣; 段捷华

    2010-01-01

    目的 探讨新生儿ABO溶血病、红细胞葡萄糖-6-磷酸脱氢酶(G-6-PD)缺乏症及两者合并患儿的临床特点.方法 对160例新生儿ABO溶血病(ABO组)、219例G-6-PD缺乏症(G6PD组)、52例新生儿ABO溶血病并G-6-PD缺乏症(ABO+G617D组)3组临床相关指标进行对比分析.结果 G6PD组血红蛋白[(159.7±24.9)g/L]高于ABO组[(150.2±23.0)g/L]和ABO+G6PD组[(149.2±22.8)g/L],差异均有统计学意义(P均<0.01);血清总胆红紊高于ABO组[(419.0±152.9)μmol/L与(355.4±113.2)μmol/L],差异有统计学意义(P<0.01);黄疸消退时间较ABO组长[(9.4±2.3)d与(8.1±2.2)d],差异有统计学意义(P<0.01).ABO+G6PD组黄疸消退时间[(12.0±2.7)d]、光疗时间[(43.2±16.0)h]、光疗次数[(3.5±1.2)次]均长或多于ABO组[(8.1.4-2.2)d、(36.1 4-15.9)h、(2.6±1.2)次]及其G6PD组[(9.4±2.3)d、(37.6±17.3)h、(2.8 4-1.3)次],差异均有统计学意义(P均<0.05).G6PD组胆红素脑病(16.O%)、低钙血症发生率(32.9%)高于ABO组(6.9%、20.0%),差异有统计学意义(P<0.05);而其贫血发生率(23.3%)则低于ABO组(40.0%)及其ABO+G6PD组(51.9%),差异有统计学意义(P<0.01).结论 新生儿ABO溶血病并G-6-PD缺乏症时,黄疸出现时间、黄疸程度、胆红素脑病发生率与新生儿ABO溶血病、G-6-PD缺乏症差异无显著性,但黄疸消退时间更长,黄疸更易反复.G-6-PD缺乏症与新生儿ABO溶血痛相比,黄疸程度更重,退黄时间更长,更易发生胆红素脑病,但贫血发生率更低.%Objective To explore the clinical features of ABO hemolytic disease in newboms,red blood cell glucose-6-phosphate dehydrogenase(G-6-PD) deficiency and the combined. Methods In the study, 160 cases of ABO hemolytic disease in newborn (ABO group) ,219 cases of G-6-PD deficiency(G6PD group) ,52 cases of the combined(ABO + G6PD group). The three groups were analyzed. Results The hemoglobin in the G6PD group ( (159. 7 ± 24.9) g/L) was significantly higher than in the ABO group ((150. 2

  7. Compounds from Terminalia mantaly L. (Combretaceae Stem Bark Exhibit Potent Inhibition against Some Pathogenic Yeasts and Enzymes of Metabolic Significance

    Directory of Open Access Journals (Sweden)

    Marthe Aimée Tchuente Tchuenmogne

    2017-01-01

    Full Text Available Background: Pathogenic yeasts resistance to current drugs emphasizes the need for new, safe, and cost-effective drugs. Also, new inhibitors are needed to control the effects of enzymes that are implicated in metabolic dysfunctions such as cancer, obesity, and epilepsy. Methods: The anti-yeast extract from Terminalia mantaly (Combretaceae was fractionated and the structures of the isolated compounds established by means of spectroscopic analysis and comparison with literature data. Activity was assessed against Candida albicans, C. parapsilosis and C. krusei using the microdilution method, and against four enzymes of metabolic significance: glucose-6-phosphate dehydrogenase, human erythrocyte carbonic anhydrase I and II, and glutathione S-transferase. Results: Seven compounds, 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside; 3-O-methylellagic acid; arjungenin or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid; arjunglucoside or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid glucopyranoside; 2α,3α,24-trihydroxyolean-11,13(18-dien-28-oïc acid; stigmasterol; and stigmasterol 3-O-β-d-glucopyranoside were isolated from the extract. Among those, 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside, 3-O-methylellagic acid, and arjunglucoside showed anti-yeast activity comparable to that of reference fluconazole with minimal inhibitory concentrations (MIC below 32 µg/mL. Besides, Arjunglucoside potently inhibited the tested enzymes with 50% inhibitory concentrations (IC50 below 4 µM and inhibitory constant (Ki <3 µM. Conclusions: The results achieved indicate that further SAR studies will likely identify potent hit derivatives that should subsequently enter the drug development pipeline.

  8. Flaxseed Protects Against Diabetes-Induced Glucotoxicity by Modulating Pentose Phosphate Pathway and Glutathione-Dependent Enzyme Activities in Rats.

    Science.gov (United States)

    Gök, Müslüm; Ulusu, Nuray N; Tarhan, Nilay; Tufan, Can; Ozansoy, Gülgün; Arı, Nuray; Karasu, Çimen

    2016-01-01

    This study investigated the effects of flaxseed (Linum usitatissimum L.) intake on general metabolism, pentose phosphate pathway (PPP) and glutathione-dependent enzymes in diabetic rats. Diabetes was induced by streptozotocin injection (40 mg/kg, i.p.) and the enzyme activities were determined spectrophotometrically. Diabetic and control rats were divided in two subgroups, one untreated, and one treated with flaxseed (0.714 g/kg body weight/day; orally) for 12 weeks. Flaxseed ameliorated decreased body weight (p < .05) and increased blood glucose (p < .001), triglyceride (p < .001), ALT (p < .001) and AST (p < .001) in diabetic rats. Diabetes resulted in increased glucose-6-phosphate dehydrogenase (G6PD) (p < .05) and decreased glutathione-S-transferase (GST) (p < .01), but unchanged 6-phosphogluconate dehydrogenase (6PGD) and glutathione reductase (GR) in the brain of rats. These alterations were partially improved by flaxseed in comparison to diabetic untreated group (p < .05). G6PD, 6PGD, GR were elevated (p < .001), while GST unchanged in the lung of diabetic untreated group compared to control. Flaxseed partially prevented the increase in 6PGD (p < .05) and GR (p < .01), but unaffected G6PD in the lung of diabetic rats. G6PD (p < .001), 6PGD (p < .05), GR (p < .001) were augmented, while GST showed a significant (p < .001) depletion in the pancreas of diabetic untreated rats compared to control. Diabetic alterations observed in pancreatic enzyme activities were significantly prevented by flaxseed. Furthermore, a remarkable decrease in 6PGD (p < .001) and an increase in G6PD (threefold of control) were found in the lens of diabetic untreated group that were completely prevented by flaxseed (p < .001). Flaxseed has beneficial effects against diabetes-induced glucotoxicity by modulating G6PD, 6PGD, GR and GST activities in tissues.

  9. Heavy enzymes--experimental and computational insights in enzyme dynamics.

    Science.gov (United States)

    Swiderek, Katarzyna; Ruiz-Pernía, J Javier; Moliner, Vicent; Tuñón, Iñaki

    2014-08-01

    The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory.

  10. The Kinetics of Enzyme Mixtures

    Directory of Open Access Journals (Sweden)

    Simon Brown

    2014-03-01

    Full Text Available Even purified enzyme preparations are often heterogeneous. For example, preparations of aspartate aminotransferase or cytochrome oxidase can consist of several different forms of the enzyme. For this reason we consider how different the kinetics of the reactions catalysed by a mixture of forms of an enzyme must be to provide some indication of the characteristics of the species present. Based on the standard Michaelis-Menten model, we show that if the Michaelis constants (Km of two isoforms differ by a factor of at least 20 the steady-state kinetics can be used to characterise the mixture. However, even if heterogeneity is reflected in the kinetic data, the proportions of the different forms of the enzyme cannot be estimated from the kinetic data alone. Consequently, the heterogeneity of enzyme preparations is rarely reflected in measurements of their steady-state kinetics unless the species present have significantly different kinetic properties. This has two implications: (1 it is difficult, but not impossible, to detect molecular heterogeneity using kinetic data and (2 even when it is possible, a considerable quantity of high quality data is required.

  11. trans-10,cis-12 Conjugated linoleic acid inhibits lipoprotein lipase but increases the activity of lipogenic enzymes in adipose tissue from hamsters fed an atherogenic diet.

    Science.gov (United States)

    Zabala, Amaia; Churruca, Itziar; Fernández-Quintela, Alfredo; Rodríguez, Víctor M; Macarulla, M Teresa; Martínez, J Alfredo; Portillo, María P

    2006-06-01

    The aim of the present work was to investigate the effects of trans-10,cis-12 conjugated linoleic acid (CLA) on the activity and expression of lipogenic enzymes and lipoprotein lipase (LPL), as well as on the expression of transcriptional factors controlling these enzymes, in adipose tissue from hamsters, and to evaluate the involvement of these changes in the body fat-reducing effect of this CLA isomer. Thirty male hamsters were divided into three groups and fed atherogenic diets supplemented with 0 (linoleic group), 5 or 10 g trans-10,cis-12 CLA/kg diet, for 6 weeks. Body and adipose tissue weights, food intake and serum insulin were measured. Total and heparin-releasable LPL and lipogenic enzyme activities (acetyl-CoA carboxylase (ACC); fatty acid synthase (FAS); glucose-6-phosphate dehydrogenase (G6PDH); and malic enzyme (ME)) were assessed. ACC, FAS, LPL, sterol regulatory element-binding proteins (SREBP-1a), SREBP-1c and PPARgamma mRNA levels were also determined by real-time PCR. CLA did not modify food intake, body weight and serum insulin level. CLA feeding reduced adipose tissue weight, LPL activity and expression, and increased lipogenic enzyme activities, despite a significant reduction in ACC and FAS mRNA levels. The expression of the three transcriptional factors analysed (SREBP-1a, SREBP-1c and PPARgamma) was also reduced. These results appear to provide a framework for partially understanding the reduction in body fat induced by CLA. Inhibition of LPL activity seems to be an important mechanism underlying body fat reduction in hamsters. Further research is needed to better characterize the effects of CLA on lipogenesis and the role of these effects in CLA action.

  12. Enzymes in CO2 Capture

    DEFF Research Database (Denmark)

    Fosbøl, Philip Loldrup; Gladis, Arne; Thomsen, Kaj

    of carbon capture is the application of enzymes for acceleration of typically slow ternary amines or inorganic carbonates. There is a hidden potential to revive currently infeasible amines which have an interesting low energy consumption for regeneration but too slow kinetics for viable CO2 capture. The aim......The enzyme Carbonic Anhydrase (CA) can accelerate the absorption rate of CO2 into aqueous solutions by several-fold. It exist in almost all living organisms and catalyses different important processes like CO2 transport, respiration and the acid-base balances. A new technology in the field...... of this work is to discuss the measurements of kinetic properties for CA promoted CO2 capture solvent systems. The development of a rate-based model for enzymes will be discussed showing the principles of implementation and the results on using a well-known ternary amine for CO2 capture. Conclusions...

  13. Neonatal hyperglycemia induces oxidative stress in the rat brain: the role of pentose phosphate pathway enzymes and NADPH oxidase.

    Science.gov (United States)

    Rosa, Andrea Pereira; Jacques, Carlos Eduardo Dias; de Souza, Laila Oliveira; Bitencourt, Fernanda; Mazzola, Priscila Nicolao; Coelho, Juliana Gonzales; Mescka, Caroline Paula; Dutra-Filho, Carlos Severo

    2015-05-01

    Recently, the consequences of diabetes on the central nervous system (CNS) have received great attention. However, the mechanisms by which hyperglycemia affects the central nervous system remain poorly understood. In addition, recent studies have shown that hyperglycemia induces oxidative damage in the adult rat brain. In this regard, no study has assessed oxidative stress as a possible mechanism that affects the brain normal function in neonatal hyperglycemic rats. Thus, the present study aimed to investigate whether neonatal hyperglycemia elicits oxidative stress in the brain of neonate rats subjected to a streptozotocin-induced neonatal hyperglycemia model (5-day-old rats). The activities of glucose-6-phosphate-dehydrogenase (G6PD), 6-phosphogluconate-dehydrogenase (6-PGD), NADPH oxidase (Nox), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), the production of superoxide anion, the thiobarbituric acid-reactive substances (TBA-RS), and the protein carbonyl content were measured. Neonatal hyperglycemic rats presented increased activities of G6PD, 6PGD, and Nox, which altogether may be responsible for the enhanced production of superoxide radical anion that was observed. The enhanced antioxidant enzyme activities (SOD, CAT, and GSHPx) that were observed in neonatal hyperglycemic rats, which may be caused by a rebound effect of oxidative stress, were not able to hinder the observed lipid peroxidation (TBA-RS) and protein damage in the brain. Consequently, these results suggest that oxidative stress could represent a mechanism that explains the harmful effects of neonatal hyperglycemia on the CNS.

  14. Intermittent Photoperiod Schedule does not Influence Brain and Serum Melatonin and Selected Serum Antioxidant Enzymes Activity in Broiler Chickens

    Directory of Open Access Journals (Sweden)

    Mosleh N

    2016-07-01

    Full Text Available This study evaluated the effect of the intermittent light program on serum and brain melatonin concentrations, antioxidant enzyme activities, and homocysteine concentration in broiler chickens. A total of 60 one-day-old broiler chickens (Cobb 500 were distributed in three light-proof controlled rooms (20 chicks per room. All birds were reared in continuous light until 3 days of age. Then, chicks were treated as follows: 1 intermittent lighting program (1L: 3D cycles, 2 continuous lighting program (24L, and 3 nonintermittent restricted lighting program (8L: 16D. At day 42 of age, sera and brains were collected from all chicks of each group (at the end of the dark phase for groups 1 and 3. Serum and brain melatonin concentrations were not affected by the different photoperiod schedules. Serum glutathione reductase, glucose-6-phosphate dehydrogenase, superoxide dismutase, and glutathione peroxidase activity as well as homocysteine concentration were also similar between different treatment groups. In conclusion, intermittent lighting program during the rearing period does not affect serum and brain melatonin levels as well as antioxidant status at the end of the dark phase in broilers.

  15. Differential SAGE analysis in Arabidopsis uncovers increased transcriptome complexity in response to low temperature

    Directory of Open Access Journals (Sweden)

    Parkin Isobel AP

    2008-09-01

    Full Text Available Abstract Background Abiotic stress, including low temperature, limits the productivity and geographical distribution of plants, which has led to significant interest in understanding the complex processes that allow plants to adapt to such stresses. The wide range of physiological, biochemical and molecular changes that occur in plants exposed to low temperature require a robust global approach to studying the response. We have employed Serial Analysis of Gene Expression (SAGE to uncover changes in the transcriptome of Arabidopsis thaliana over a time course of low temperature stress. Results Five SAGE libraries were generated from A. thaliana leaf tissue collected at time points ranging from 30 minutes to one week of low temperature treatment (4°C. Over 240,000 high quality SAGE tags, corresponding to 16,629 annotated genes, provided a comprehensive survey of changes in the transcriptome in response to low temperature, from perception of the stress to acquisition of freezing tolerance. Interpretation of these data was facilitated by representing the SAGE data by gene identifier, allowing more robust statistical analysis, cross-platform comparisons and the identification of genes sharing common expression profiles. Simultaneous statistical calculations across all five libraries identified 920 low temperature responsive genes, only 24% of which overlapped with previous global expression analysis performed using microarrays, although similar functional categories were affected. Clustering of the differentially regulated genes facilitated the identification of novel loci correlated with the development of freezing tolerance. Analysis of their promoter sequences revealed subsets of genes that were independent of CBF and ABA regulation and could provide a mechanism for elucidating complementary signalling pathways. The SAGE data emphasised the complexity of the plant response, with alternate pre-mRNA processing events increasing at low temperatures

  16. Heterologous Expression, Purification and Characterization of Thermo-stable 6-phosphate-β-glucosidase TteBglB%热稳定6-磷酸-β-葡萄糖苷酶TteBglB异源表达、分离纯化及酶学性质分析

    Institute of Scientific and Technical Information of China (English)

    刘晴; 张宇微; 张宇宏; 徐欣欣; 张伟; 刘波; 顾青

    2014-01-01

    6⁃phosphate⁃β⁃glucosidase (EC 3.2.1.86), an enzyme catalyzes 6⁃phosphate⁃glucoside compounds (such as 6⁃phosphate⁃cellobiose, 6⁃phosphate⁃cellulose oligosaccharides ) to produce 6⁃phosphate⁃glucose, plays an important role in carbon utilization for microbes. Thermoanaerobacter tengcongensis MB4, a strain of thermophilic anaerobic microorganism, is an excellent resource for thermo⁃stable protein. In this study, gene TtebglB was isolated from this strain and then heterologously expressed in E. coli BL21 ( DE3 ) . The optimum pH and temperature of TteBglB were pH 6.0 and 70℃, respectively, and the enzyme kept stable at a pH 4~10 at 70℃. Additionally, it was also confirmed that TteBglB belonged to glycosyl hydrolase family 1 ( GH1) and could act without Mn2+, Ni2+, Co2+, Fe2+ and other divalent metal ions. Using pNPβG6P as substrate, the values of Km , Kcat , and Vmax for this enzyme was 0.054 mmol/L, 81.47/min and 0.003 992 mmol/min, respectively, and specific activity of the enzyme reached to 18�093 U/mg.%6⁃磷酸⁃β⁃葡萄糖苷酶( EC 3.2.1.86)催化6⁃磷酸-葡萄糖苷类化合物(如6⁃磷酸-纤维二糖、6⁃磷酸-纤维素寡糖)产生6⁃磷酸-葡萄糖而使纤维素完全分解,在微生物碳源利用过程中起重要作用。腾冲嗜热厌氧杆菌是一株嗜热厌氧微生物,提供了丰富的热稳定性蛋白基因资源。从该菌株中克隆编码6⁃磷酸⁃β⁃葡萄糖苷酶的基因TtebglB,并在E.coli BL21(DE3)进行了异源表达。结果表明,TteBglB催化反应的最适pH为6.0、最适温度为70℃,在pH 4~10或70℃时有着良好的稳定性;同时证实,TteBglB属于糖基水解酶家族1( GH1)成员,可不依赖Mn2+、Ni2+、Co2+或Fe2+等二价金属离子而发挥催化作用。以pNPβG6P为底物时,催化反应的Km为0.054 mmol/L,Kcat为81.47/min,Vmax为0.003992 mmol/min,酶比活为18.093 U/mg。

  17. Taking the Mystery Out of Enzymes.

    Science.gov (United States)

    DeYoung, H. Garrett

    1984-01-01

    Discusses structure and function of enzymes, design of new enzymes and enzyme substitutes, and enzyme uses in industry, medicine, and wastewater treatment. The latter is a low-cost method which can remove as much as 99 percent of toxic substances found in many industrial wastewater streams. (JN)

  18. Teaching and Learning on Enzymes: The Need for New didactic tools

    Directory of Open Access Journals (Sweden)

    M.L. Luiele

    2005-07-01

    Full Text Available Enzymes  are biological catalysts essential  for vital  chemical reactions  in the  cell.  The proper  under- standing  of enzyme  functioning  is an  important step  to  learn  more about life, the  subject  study  of biology. However, without  the opportunity to use a laboratory, it is difficult to the student to visualize the enzyme function.  Our project  is based in the production of a didactic  tool in a CD-rom media to teach enzymes.  In this way, we intend  to teach enzymology in a easy, playful, and more comprehensive way than  usually  is done by lectures.  The CD-room will present three  main subjects:  (i Theoretical aspects of enzymes; (ii Experimental and Interactive; and (iii Applications.  The first part  will bring short  texts  on the  structure and  function  of enzymes as well as some of their  history.   There  will be also a interactive show of some structures of enzymes,  collected  at  the  Protein Data  Bank,  where important  residues  and  location  of the  active  site will be shown in evidence.   In the  second part  of the CD-rom,  the student will be able to choose from different conditions  (substrate or concentration, pH, and temperature to visualize the kinetics  of a reaction  in a virtual  spctrophotometer where the changes  in absorbance  with  time  will be based  on actual  experiments done at the laboratory. The kinetic data  bank  includes reaction with  chymotrypsin, trypsin, glucose-6-phosphate dehydrogenase, and alkaline phosphatase. From the experiments it will be possible to show how the rate  of a reaction is measured,  and how the kinetic constants can be obtained. The interactive part will show schematics where the  conditions  can be changed  and  a cartoon  corresponding  to the  situation will be displayed and will change according to the movement of a cursor.  Part III will describe some applications of

  19. Insolubilized enzymes for food synthesis

    Science.gov (United States)

    Marshall, D. L.

    1972-01-01

    Cellulose matrix with numerous enzyme-coated silica particles of colloidal size permanently bound at various sites within matrix was produced that has high activity and possesses requisite physical characteristics for filtration or column operations. Product also allows coupling step in synthesis of edible food to proceed under mild conditions.

  20. Kathepsine C : Een allosterisch enzyme

    NARCIS (Netherlands)

    Gorter, Jeannette

    1969-01-01

    In chapter I an introduction into allosteric systems is given. In chapter II is a detailed method is described for the applica of Gly-Phe--p. nitroanilide (GPNA) as a substrate for the activity assay of the lysosomal enzyme cathepsin C. It is an allosteric which is activated by Cl-, Br-, 1-, CNS-, N

  1. Rapid-Equilibrium Enzyme Kinetics

    Science.gov (United States)

    Alberty, Robert A.

    2008-01-01

    Rapid-equilibrium rate equations for enzyme-catalyzed reactions are especially useful because if experimental data can be fit by these simpler rate equations, the Michaelis constants can be interpreted as equilibrium constants. However, for some reactions it is necessary to use the more complicated steady-state rate equations. Thermodynamics is…

  2. Enzyme nanoassemblies for biomass conversion

    Science.gov (United States)

    Biomass represents a vast resource for the production of the world’s fuel and chemical feedstock needs. The use of enzymes to effect these bioconversions offers an alternative that is potentially more specific and environmentally-friendly than harsher chemical methodologies. Some species of anaero...

  3. Silica-Immobilized Enzyme Reactors

    Science.gov (United States)

    2007-08-01

    immobilized artificial membrane chromatography and lysophospholipid micellar electrokinetic chromatography . J. Chromatogr. A 1998, 810, 95-103. 50...Journal of Liquid Chromatography and Related Technologies. Air Force Research Laboratory Materials and Manufacturing Directorate Airbase...immobilized enzyme reactors (IMERs) can also be integrated directly to further analytical methods such as liquid chromatography or mass spectrometry.[6] In

  4. Enzyme recovery using reversed micelles.

    NARCIS (Netherlands)

    Dekker, M.

    1990-01-01

    The objective of this study was to develop a liquid-liquid extraction process for the recovery of extracellular enzymes. The potentials of reaching this goal by using reversed micelles in an organic solvent have been investigated.Reversed micelles are aggregates of surfactant molecules containing an

  5. The enzymes associated with denitrification

    Science.gov (United States)

    Hochstein, L. I.; Tomlinson, G. A.

    1988-01-01

    The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes. This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases.

  6. [Effects of waterlogging on the growth and energy-metabolic enzyme activities of different tree species].

    Science.gov (United States)

    Wang, Gui-Bin; Cao, Fu-Liang; Zhang, Xiao-Yan; Zhang, Wang-Xiang

    2010-03-01

    Aimed to understand the waterlogging tolerance and adaptation mechanisms of different tree species, a simulated field experiment was conducted to study the growth and energy-metabolic enzyme activities of one-year-old seedlings of Taxodium distichum, Carya illinoensis, and Sapium sebiferum. Three treatments were installed, i. e., CK, waterlogging, and flooding, with the treatment duration being 60 days. Under waterlogging and flooding, the relative growth of test tree species was in the order of T. distichum > C. illinoensis > S. sebiferum, indicating that T. distichum had the strongest tolerance against waterlogging and flooding, while S. sebiferum had the weakest one. Also under waterlogging and flooding, the root/crown ratio of the three tree species increased significantly, suggesting that more photosynthates were allocated in roots, and the lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) activities of the tree species also had a significant increase. Among the test tree species, T. distichum had the lowest increment of LDH and ADH activities under waterlogging and flooding, but the increment could maintain at a higher level in the treatment duration, while for C. illinoensis and S. sebiferum, the increment was larger during the initial and medium period, but declined rapidly during the later period of treatment. The malate dehydrogenase (MDH), phosphohexose (HPI), and glucose-6-phosphate dehydrogenase (G6PDH) -6-phosphogluconate dehydrogenase (6PGDH) activities of the tree species under waterlogging and flooding had a significant decrease, and the decrement was the largest for T. distichum, being 35.6% for MDH, 21.0% for HPI, and 22.7% for G6PDH - 6PGDH under flooding. It was suggested that under waterlogging and flooding, the tree species with strong waterlogging tolerance had a higher ability to maintain energy-metabolic balance, and thus, its growth could be maintained at a certain level.

  7. Virulence-Associated Enzymes of Cryptococcus neoformans

    OpenAIRE

    2015-01-01

    Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review f...

  8. Thermodynamics of Enzyme-Catalyzed Reactions Database

    Science.gov (United States)

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  9. Evaluation of the Reproductive Toxicity, Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism of Chromium Malate Supplementation in Sprague-Dawley Rats.

    Science.gov (United States)

    Feng, Weiwei; Zhang, Weijie; Zhao, Ting; Mao, Guanghua; Wang, Wei; Wu, Xueshan; Zhou, Zhaoxiang; Huang, Jing; Bao, Yongtuan; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the reproductive toxicity of chromium malate in Sprague-Dawley rats and then inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, and lipid metabolism. The results showed that no pathological, toxic feces and urine changes were observed in clinical signs of parental and fetal rats in chromium malate groups. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of chromium malate groups have no significant change compared with control group and chromium picolinate group. The serum and organ contents of Cr in chromium malate groups have no significant change when compared with control group. No measurable damage on liver, brain, kidney, and testis/uterus of chromium malate groups was found. No significant change in body mass, absolute and relative organ weights, and hematological and biochemical changes of rats were observed compared with the control and chromium picolinate groups. The results indicated that supplements with chromium malate does not cause obvious damage and has no obvious effect on glycometabolism, glycometabolism-related enzyme, and lipid metabolism on female and male rats. The results of this study suggested that chromium malate is safe for human consumption and has the potential for application as a functional food ingredient and dietary supplement.

  10. 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.

  11. Engineering cytochrome p450 enzymes.

    Science.gov (United States)

    Gillam, Elizabeth M J

    2008-01-01

    The last 20 years have seen the widespread and routine application of methods in molecular biology such as molecular cloning, recombinant protein expression, and the polymerase chain reaction. This has had implications not only for the study of toxicological mechanisms but also for the exploitation of enzymes involved in xenobiotic clearance. The engineering of P450s has been performed with several purposes. The first and most fundamental has been to enable successful recombinant expression in host systems such as bacteria. This in turn has led to efforts to solubilize the proteins as a prerequisite to crystallization and structure determination. Lagging behind has been the engineering of enzyme activity, hampered in part by our still-meager comprehension of fundamental structure-function relationships in P450s. However, the emerging technique of directed evolution holds promise in delivering both engineered enzymes for use in biocatalysis and incidental improvements in our understanding of sequence-structure and sequence-function relationships, provided that data mining can extract the fundamental correlations underpinning the data. From the very first studies on recombinant P450s, efforts were directed toward constructing fusions between P450s and redox partners in the hope of generating more efficient enzymes. While this aim has been allowed to lie fallow for some time, this area merits further investigation as does the development of surface-displayed P450 systems for biocatalytic and biosensor applications. The final application of engineered P450s will require other aspects of their biology to be addressed, such as tolerance to heat, solvents, and high substrate and product concentrations. The most important application of these enzymes in toxicology in the near future is likely to be the biocatalytic generation of drug metabolites for the pharmaceutical industry. Further tailoring will be necessary for specific toxicological applications, such as in

  12. Enzymes involved in triglyceride hydrolysis.

    Science.gov (United States)

    Taskinen, M R; Kuusi, T

    1987-08-01

    The lipolytic enzymes LPL and HL play important roles in the metabolism of lipoproteins and participate in lipoprotein interconversions. LPL was originally recognized to be the key enzyme in the hydrolysis of chylomicrons and triglyceride, but it also turned out to be one determinant of HDL concentration in plasma. When LPL activity is high, chylomicrons and VLDL are rapidly removed from circulation and a concomitant rise of the HDL2 occurs. In contrast, low LPL activity impedes the removal of triglyceride-rich particles, resulting in the elevation of serum triglycerides and a decrease of HDL (HDL2). Concordant changes of this kind in LPL and HDL2 are induced by many physiological and pathological perturbations. Finally, the operation of LPL is also essential for the conversion of VLDL to LDL. This apparently clear-cut role of LPL in lipoprotein interconversions is contrasted with the enigmatic actions of HL. The enzyme was originally thought to participate in the catalyses of chylomicron and VLDL remnants generated in the LPL reaction. However, substantial in vitro and in vivo data indicate that HL is a key enzyme in the degradation of plasma HDL (HDL2) in a manner which opposes LPL. A scheme is presented for the complementary actions of the two enzymes in plasma HDL metabolism. In addition, recent studies have attributed a role to HL in the catabolism of triglyceride-rich lipoproteins, particularly those containing apo E. However, this function becomes clinically important only under conditions where the capacity of the LPL-mediated removal system is exceeded. Such a situation may arise when the input of triglyceride-rich particles (chylomicrons and/or VLDL) is excessive or LPL activity is decreased or absent.

  13. Screen for soil fungi highly resistant to dichloroaniline uncovers mostly Fusarium species.

    Science.gov (United States)

    Chan Ho Tong, Laetitia; Dairou, Julien; Bui, Linh-Chi; Bouillon, Julien; Rodrigues-Lima, Fernando; Dupret, Jean-Marie; Silar, Philippe

    2015-08-01

    Arylamines are frequent pollutants in soils. Fungi have proven to be efficient in detoxifying these chemicals by acetylating them using arylamine N-acetyl transferase enzymes. Here, we selected from natural soils fungi highly resistant to 3,4-dichloroaniline (DCA). Fusarium species were the most frequently isolated species, especially Fusarium solani. The sequenced strain of F. solani contains five NAT genes, as did all the DCA-resistant isolates. RT-PCR analysis showed that the five genes were expressed in F. solani. Expression of the F. solani genes in Podospora anserina and analysis of acetylation directly in F. solani showed that only the NhNAT2B gene conferred significant resistance to DCA and that F. solani likely uses pathways different from acetylation to resist high doses of DCA, as observed previously for Trichoderma.

  14. Uncovering the SUMOylation and ubiquitylation crosstalk in human cells using sequential peptide immunopurification

    Science.gov (United States)

    Lamoliatte, Frédéric; McManus, Francis P.; Maarifi, Ghizlane; Chelbi-Alix, Mounira K.; Thibault, Pierre

    2017-01-01

    Crosstalk between the SUMO and ubiquitin pathways has recently been reported. However, no approach currently exists to determine the interrelationship between these modifications. Here, we report an optimized immunoaffinity method that permits the study of both protein ubiquitylation and SUMOylation from a single sample. This method enables the unprecedented identification of 10,388 SUMO sites in HEK293 cells. The sequential use of SUMO and ubiquitin remnant immunoaffinity purification facilitates the dynamic profiling of SUMOylated and ubiquitylated proteins in HEK293 cells treated with the proteasome inhibitor MG132. Quantitative proteomic analyses reveals crosstalk between substrates that control protein degradation, and highlights co-regulation of SUMOylation and ubiquitylation levels on deubiquitinase enzymes and the SUMOylation of proteasome subunits. The SUMOylation of the proteasome affects its recruitment to promyelocytic leukemia protein (PML) nuclear bodies, and PML lacking the SUMO interacting motif fails to colocalize with SUMOylated proteasome further demonstrating that this motif is required for PML catabolism. PMID:28098164

  15. Uncovering the Molecular Mechanism of Actions between Pharmaceuticals and Proteins on the AD Network.

    Directory of Open Access Journals (Sweden)

    Shujuan Cao

    Full Text Available This study begins with constructing the mini metabolic networks (MMNs of beta amyloid (Aβ and acetylcholine (ACh which stimulate the Alzheimer's Disease (AD. Then we generate the AD network by incorporating MMNs of Aβ and ACh, and other MMNs of stimuli of AD. The panel of proteins contains 49 enzymes/receptors on the AD network which have the 3D-structure in PDB. The panel of drugs is formed by 5 AD drugs and 5 AD nutraceutical drugs, and 20 non-AD drugs. All of these complexes formed by these 30 drugs and 49 proteins are transformed into dyadic arrays. Utilizing the prior knowledge learned from the drug panel, we propose a statistical classification (dry-lab. According to the wet-lab for the complex of amiloride and insulin degrading enzyme, and the complex of amiloride and neutral endopeptidase, we are confident that this dry-lab is reliable. As the consequences of the dry-lab, we discover many interesting implications. Especially, we show that possible causes of Tacrine, donepezil, galantamine and huperzine A cannot improve the level of ACh which is against to their original design purpose but they still prevent AD to be worse as Aβ deposition appeared. On the other hand, we recommend Miglitol and Atenolol as the safe and potent drugs to improve the level of ACh before Aβ deposition appearing. Moreover, some nutrients such as NADH and Vitamin E should be controlled because they may harm health if being used in wrong way and wrong time. Anyway, the insights shown in this study are valuable to be developed further.

  16. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  17. No Place to Hide: Missing Primitive Stars Outside Milky Way Uncovered

    Science.gov (United States)

    2010-02-01

    After years of successful concealment, the most primitive stars outside our Milky Way galaxy have finally been unmasked. New observations using ESO's Very Large Telescope have been used to solve an important astrophysical puzzle concerning the oldest stars in our galactic neighbourhood - which is crucial for our understanding of the earliest stars in the Universe. "We have, in effect, found a flaw in the forensic methods used until now," says Else Starkenburg, lead author of the paper reporting the study. "Our improved approach allows us to uncover the primitive stars hidden among all the other, more common stars." Primitive stars are thought to have formed from material forged shortly after the Big Bang, 13.7 billion years ago. They typically have less than one thousandth the amount of chemical elements heavier than hydrogen and helium found in the Sun and are called "extremely metal-poor stars" [1]. They belong to one of the first generations of stars in the nearby Universe. Such stars are extremely rare and mainly observed in the Milky Way. Cosmologists think that larger galaxies like the Milky Way formed from the merger of smaller galaxies. Our Milky Way's population of extremely metal-poor or "primitive" stars should already have been present in the dwarf galaxies from which it formed, and similar populations should be present in other dwarf galaxies. "So far, evidence for them has been scarce," says co-author Giuseppina Battaglia. "Large surveys conducted in the last few years kept showing that the most ancient populations of stars in the Milky Way and dwarf galaxies did not match, which was not at all expected from cosmological models." Element abundances are measured from spectra, which provide the chemical fingerprints of stars [2]. The Dwarf galaxies Abundances and Radial-velocities Team [3] used the FLAMES instrument on ESO's Very Large Telescope to measure the spectra of over 2000 individual giant stars in four of our galactic neighbours, the Fornax

  18. Enzyme technology: Key to selective biorefining

    DEFF Research Database (Denmark)

    Meyer, Anne S.

    2014-01-01

    to the reaction is a unique trait of enzyme catalysis. Since enzyme selectivity means that a specific reaction is catalysed between particular species to produce definite products, enzymes are particularly fit for converting specific compounds in mixed biomass streams. Since enzymes are protein molecules...... their rational use in biorefinery processes requires an understanding of the basic features of enzymes and reaction traits with respect to specificity, kinetics, reaction optima, stability and structure-function relations – we are now at a stage where it is possible to use nature’s enzyme structures as starting...... point and then improve the functional traits by targeted mutation of the protein. The talk will display some of our recent hypotheses related to enzyme action, recently obtained results within knowledge-based enzyme improvements as well as cast light on research methods used in optimizing enzyme...

  19. Partially uncovered Cheatham platinum-covered stent to treat complex aortic coarctation associated with aortic wall aneurysm.

    Science.gov (United States)

    Butera, Gianfranco; Piazza, Luciane

    2015-04-01

    Percutaneous treatment of aortic coarctation is a widely used option. Covered stents have increased the profile of efficacy and safety of this procedure. Here we report on a 32-year-old woman with significant aortic recoarctation associated with aortic wall aneurysm and close proximity of both lesions to the origin of both the subclavian arteries. It was decided to manually and partially uncover the proximal part of the stent to have a hybrid stent that could act as a bare stent at the level of the origin of the subclavian arteries and as a covered stent at the level of the aneurysm.

  20. Effects of sodium-orthovanadate and Trigonella foenum-graecum seeds on hepatic and renal lipogenic enzymes and lipid profile during alloxan diabetes

    Indian Academy of Sciences (India)

    Umesh C S Yadav; K Moorthy; Najma Z Baquer

    2004-03-01

    Sodium-orthovanadate (SOV) and seed powder of Trigonella foenum graecum Linn. (common name: fenugreek, family: Fabaceae) (TSP) besides being potential hypoglycemic agents have also been shown to ameliorate altered lipid metabolism during diabetes. This study evaluates the short-term effect of oral administration of SOV and TSP separately and in concert (for 21 days) on total lipid profile and lipogenic enzymes in tissues of alloxan diabetic rats. Diabetic rats showed 4-fold increase in blood glucose. The level of total lipids, triglycerides and total cholesterol in blood serum increased significantly during diabetes. During diabetes the level of total lipids increased significantly ( < 0.001) in liver and in kidney by 48% and 55%, respectively, compared to control. Triglycerides level increased by 32% ( < 0.01) in liver and by 51% ( < 0.005) in kidney, respectively, compared to control. Total cholesterol level also increased significantly in both liver and kidney ( < 0.01 and P < 0.001, respectively). The activities of NADP-linked enzymes; namely glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and the activities of lipogenic enzymes namely ATP-citrate lyase (ATP-CL) and fatty acid synthase (FAS) were decreased significantly in liver and increased in kidney during diabetes as compared to control. SOV and TSP administration to diabetic animals prevented the development of hyperglycemia and alteration in lipid profile in plasma and tissues and maintained it near normal. Maximum prevention was observed in the combined treatment with lower dose of SOV (0.2%) after 21 days. We are presenting for the first time effectiveness of combined treatment of SOV and TSP in amelioration of altered lipid metabolism during experimental type-I diabetes.

  1. Finding homes for orphan enzymes

    Directory of Open Access Journals (Sweden)

    Frank M. Raushel

    2016-12-01

    Full Text Available The rate at which new genes are being sequenced greatly exceeds our ability to correctly annotate the functional properties of the corresponding proteins. Annotations based primarily on sequence identity to experimentally characterized proteins are often misleading because closely related sequences may have different functions, while highly divergent sequences may have identical functions. Our understanding of the principles that dictate the catalytic properties of enzymes, based on protein sequence alone, is often insufficient to correctly annotate proteins of unknown function. To address these problems, we are working to develop a comprehensive strategy for the functional annotation of newly sequenced genes using a combination of structural biology, bioinformatics, computational biology, and molecular enzymology. The power of this multidisciplinary approach for discovering new reactions catalyzed by uncharacterized enzymes has been tested using the amidohydrolase superfamily as a model system.

  2. Enzyme dynamics from NMR spectroscopy.

    Science.gov (United States)

    Palmer, Arthur G

    2015-02-17

    CONSPECTUS: Biological activities of enzymes, including regulation or coordination of mechanistic stages preceding or following the chemical step, may depend upon kinetic or equilibrium changes in protein conformations. Exchange of more open or flexible conformational states with more closed or constrained states can influence inhibition, allosteric regulation, substrate recognition, formation of the Michaelis complex, side reactions, and product release. NMR spectroscopy has long been applied to the study of conformational dynamic processes in enzymes because these phenomena can be characterized over multiple time scales with atomic site resolution. Laboratory-frame spin-relaxation measurements, sensitive to reorientational motions on picosecond-nanosecond time scales, and rotating-frame relaxation-dispersion measurements, sensitive to chemical exchange processes on microsecond-millisecond time scales, provide information on both conformational distributions and kinetics. This Account reviews NMR spin relaxation studies of the enzymes ribonuclease HI from mesophilic (Escherichia coli) and thermophilic (Thermus thermophilus) bacteria, E. coli AlkB, and Saccharomyces cerevisiae triosephosphate isomerase to illustrate the contributions of conformational flexibility and dynamics to diverse steps in enzyme mechanism. Spin relaxation measurements and molecular dynamics (MD) simulations of the bacterial ribonuclease H enzymes show that the handle region, one of three loop regions that interact with substrates, interconverts between two conformations. Comparison of these conformations with the structure of the complex between Homo sapiens ribonuclease H and a DNA:RNA substrate suggests that the more closed state is inhibitory to binding. The large population of the closed conformation in T. thermophilus ribonuclease H contributes to the increased Michaelis constant compared with the E. coli enzyme. NMR spin relaxation and fluorescence spectroscopy have characterized a

  3. Substrate mediated enzyme prodrug therapy.

    Directory of Open Access Journals (Sweden)

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  4. Characterization of CIM monoliths as enzyme reactors.

    Science.gov (United States)

    Vodopivec, Martina; Podgornik, Ales; Berovic, Marin; Strancar, Ales

    2003-09-25

    The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis-Menten constant K(m) and the turnover number k(3) of the immobilized enzymes were found to be flow-unaffected. Furthermore, the K(m) values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.

  5. Lignolytic Enzymes Production from Selected Mushrooms

    Directory of Open Access Journals (Sweden)

    H.M. Shantaveera Swamy

    2015-06-01

    Full Text Available In this paper, ligninase enzymes produced by selected mushrooms have been reported. We collected mushrooms from Western Ghats, most of them were edible food. Thirty samples isolated were tested using a plate assay through direct agar plate assay by using ABTS, decolourisation containing the fifteen isolates were able to decolourise the dye, indicating a lignin-degrading ability. Spectrophotometric enzyme assays from all selected isolates were carried out to examine the production of Ligninolytic enzymes (Laccase, lignin peroxidase and manganese peroxidase. Ten selected isolates produced all three kinds of enzymes tested. Lignolytic enzymes are groups of enzymes these are actively involved in bioremediation.

  6. Meiotic double-strand breaks uncover and protect against mitotic errors in the C. elegans germline.

    Science.gov (United States)

    Stevens, Deanna; Oegema, Karen; Desai, Arshad

    2013-12-01

    In sexually reproducing multicellular organisms, genetic information is propagated via the germline, the specialized tissue that generates haploid gametes. The C. elegans germline generates gametes in an assembly line-like process-mitotic divisions under the control of the stem cell niche produce nuclei that, upon leaving the niche, enter into meiosis and progress through meiotic prophase [1]. Here, we characterize the effects of perturbing cell division in the mitotic region of the C. elegans germline. We show that mitotic errors result in a spindle checkpoint-dependent cell-cycle delay, but defective nuclei are eventually formed and enter meiosis. These defective nuclei are eliminated by programmed cell death during meiotic prophase. The cell death-based removal of defective nuclei does not require the spindle checkpoint but instead depends on the DNA damage checkpoint. Removal of nuclei resulting from errors in mitosis also requires Spo11, the enzyme that creates double-strand breaks to initiate meiotic recombination. Consistent with this, double-strand breaks are increased in number and persist longer in germlines with mitotic defects. These findings reveal that the process of initiating meiotic recombination inherently selects against nuclei with abnormal chromosomal content generated by mitotic errors, thereby ensuring the genomic integrity of gametes.

  7. RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

    Science.gov (United States)

    Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

    2016-01-01

    Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

  8. Contribution of the Nurses’ Health Studies to Uncovering Risk Factors for Type 2 Diabetes: Diet, Lifestyle, Biomarkers, and Genetics

    Science.gov (United States)

    Ley, Sylvia H.; Ardisson Korat, Andres V.; Sun, Qi; Tobias, Deirdre K.; Zhang, Cuilin; Qi, Lu; Willett, Walter C.; Manson, JoAnn E.

    2016-01-01

    Objectives. To review the contribution of the Nurses’ Health Study (NHS) and the NHS II to addressing hypotheses regarding risk factors for type 2 diabetes. Methods. We carried out a narrative review of 1976 to 2016 NHS and NHS II publications. Results. The NHS and NHS II have uncovered important roles in type 2 diabetes for individual nutrients, foods, dietary patterns, and physical activity independent of excess body weight. Up to 90% of type 2 diabetes cases are potentially preventable if individuals follow a healthy diet and lifestyle. The NHS investigations have also identified novel biomarkers for diabetes, including adipokines, inflammatory cytokines, nutrition metabolites, and environmental pollutants, offering new insights into the pathophysiology of the disease. Global collaborative efforts have uncovered many common genetic variants associated with type 2 diabetes and improved our understanding of gene–environment interactions. Continued efforts to identify epigenetic, metagenomic, and metabolomic risk factors for type 2 diabetes have the potential to reveal new pathways and improve prediction and prevention. Conclusions. Over the past several decades, the NHS and NHS II have made major contributions to public health recommendations and strategies designed to reduce the global burden of diabetes. PMID:27459454

  9. A DNA tweezer-actuated enzyme nanoreactor.

    Science.gov (United States)

    Liu, Minghui; Fu, Jinglin; Hejesen, Christian; Yang, Yuhe; Woodbury, Neal W; Gothelf, Kurt; Liu, Yan; Yan, Hao

    2013-01-01

    The functions of regulatory enzymes are essential to modulating cellular pathways. Here we report a tweezer-like DNA nanodevice to actuate the activity of an enzyme/cofactor pair. A dehydrogenase and NAD(+) cofactor are attached to different arms of the DNA tweezer structure and actuation of enzymatic function is achieved by switching the tweezers between open and closed states. The enzyme/cofactor pair is spatially separated in the open state with inhibited enzyme function, whereas in the closed state, enzyme is activated by the close proximity of the two molecules. The conformational state of the DNA tweezer is controlled by the addition of specific oligonucleotides that serve as the thermodynamic driver (fuel) to trigger the change. Using this approach, several cycles of externally controlled enzyme inhibition and activation are successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.

  10. Editorial: Special Issue — Enzyme Immobilization

    Directory of Open Access Journals (Sweden)

    Roberto Fernandez-Lafuente

    2014-12-01

    Full Text Available Immobilization of enzymes and proteins is a requirement for many industrial enzyme applications, as this facilitates enzyme recovery and reuse. Bearing in mind this necessity, the coupling of immobilization to the improvement of other enzyme features has been pursued by many researchers, and nowadays immobilization is recognized as a tool to improve not only stability, but also enzyme selectivity, specificity, resistance to inhibition or chemical modifiers, etc. To achieve these overall improvements of enzymes’ properties via immobilization, it is necessary to both develop new immobilization systems suitable for these purposes, and to achieve a deeper knowledge of the mechanisms of interaction between enzymes and activated solids. That way, immobilization of enzymes, far being an old-fashioned methodology to just reuse these expensive biocatalysts, is a tool of continuous interest that requires a continuous effort to be exploited in all its potential. This special issue collects 23 papers reporting advances in the field of immobilization of enzymes.[...

  11. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...

  12. Activation of thiamin diphosphate in enzymes.

    Science.gov (United States)

    Hübner, G; Tittmann, K; Killenberg-Jabs, M; Schäffner, J; Spinka, M; Neef, H; Kern, D; Kern, G; Schneider, G; Wikner, C; Ghisla, S

    1998-06-29

    Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

  13. Moving college students to a better understanding of substrate specificity of enzymes through utilizing multimedia pre-training and an interactive enzyme model

    Science.gov (United States)

    Saleh, Mounir R.

    Scientists' progress in understanding enzyme specificity uncovered a complex natural phenomenon. However, not all of the currently available biology textbooks seem to be up to date on this progress. Students' understanding of how enzymes work is a core requirement in biochemistry and biology tertiary education. Nevertheless, current pre-college science education does not provide students with enough biochemical background to enable them to understand complex material such as this. To bridge this gap, a multimedia pre-training presentation was prepared to fuel the learner's prior knowledge with discrete facts necessary to understand the presented concept. This treatment is also known to manage intrinsic cognitive load during the learning process. An interactive instructional enzyme model was also built to motivate students to learn about substrate specificity of enzymes. Upon testing the effect of this combined treatment on 111 college students, desirable learning outcomes were found in terms of cognitive load, motivation, and achievement. The multimedia pre-training group reported significantly less intrinsic cognitive load, higher motivation, and demonstrated higher transfer performance than the control and post-training groups. In this study, a statistical mediation model is also proposed to explain how cognitive load and motivation work in concert to foster learning from multimedia pre-training. This type of research goes beyond simple forms of "what works" to a deeper understanding of "how it works", thus enabling informed decisions for multimedia instructional design. Multimedia learning plays multiple roles in science education. Therefore, science learners would be some of the first to benefit from improving multimedia instructional design. Accordingly, complex scientific phenomena can be introduced to college students in a motivating, informative, and cognitively efficient learning environment.

  14. Tracking dynamics of plant biomass composting by changes in substrate structure, microbial community, and enzyme activity

    Directory of Open Access Journals (Sweden)

    Wei Hui

    2012-04-01

    Full Text Available Abstract Background Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars. However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process. Results In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera wood-chips and mown lawn grass clippings (85:15 in dry-weight and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed. Conclusion The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP and solid-state fermentation for the production of cellulolytic enzymes and biofuels.

  15. Determining Enzyme Activity by Radial Diffusion

    Science.gov (United States)

    Davis, Bill D.

    1977-01-01

    Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

  16. 21 CFR 864.4400 - Enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme preparations. 864.4400 Section 864.4400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the...

  17. Metabolite profiling uncovers plasmid-induced cobalt limitation under methylotrophic growth conditions.

    Directory of Open Access Journals (Sweden)

    Patrick Kiefer

    Full Text Available BACKGROUND: The introduction and maintenance of plasmids in cells is often associated with a reduction of growth rate. The reason for this growth reduction is unclear in many cases. METHODOLOGY/PRINCIPAL FINDINGS: We observed a surprisingly large reduction in growth rate of about 50% of Methylobacterium extorquens AM1 during methylotrophic growth in the presence of a plasmid, pCM80 expressing the tetA gene, relative to the wild-type. A less pronounced growth delay during growth under non-methylotrophic growth conditions was observed; this suggested an inhibition of one-carbon metabolism rather than a general growth inhibition or metabolic burden. Metabolome analyses revealed an increase in pool sizes of ethylmalonyl-CoA and methylmalonyl-CoA of more than 6- and 35-fold, respectively, relative to wild type, suggesting a strongly reduced conversion of these central intermediates, which are essential for glyoxylate regeneration in this model methylotroph. Similar results were found for M. extorquens AM1 pCM160 which confers kanamycin resistance. These intermediates of the ethylmalonyl-CoA pathway have in common their conversion by coenzyme B(12-dependent mutases, which have cobalt as a central ligand. The one-carbon metabolism-related growth delay was restored by providing higher cobalt concentrations, by heterologous expression of isocitrate lyase as an alternative path for glyoxylate regeneration, or by identification and overproduction of proteins involved in cobalt import. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the introduction of the plasmids leads to an apparent inhibition of the cobalt-dependent enzymes of the ethylmalonyl-CoA pathway. Possible explanations are presented and point to a limited cobalt concentration in the cell as a consequence of the antibiotic stress.

  18. ARABIDOPSIS THALIANA HOMEOBOX25 uncovers a role for Gibberellins in seed longevity.

    Science.gov (United States)

    Bueso, Eduardo; Muñoz-Bertomeu, Jesús; Campos, Francisco; Brunaud, Veronique; Martínez, Liliam; Sayas, Enric; Ballester, Patricia; Yenush, Lynne; Serrano, Ramón

    2014-02-01

    Seed longevity is crucial for agriculture and plant genetic diversity, but it is limited by cellular damage during storage. Seeds are protected against aging by cellular defenses and by structures such as the seed coat. We have screened an activation-tagging mutant collection of Arabidopsis (Arabidopsis thaliana) and selected four dominant mutants with improved seed longevity (isl1-1D to isl4-1D) under both natural and accelerated aging conditions. In the isl1-1D mutant, characterized in this work, overexpression of the transcription factor ARABIDOPSIS THALIANA HOMEOBOX25 (ATHB25; At5g65410) increases the expression of GIBBERELLIC ACID3-OXIDASE2, encoding a gibberellin (GA) biosynthetic enzyme, and the levels of GA1 and GA4 are higher (3.2- and 1.4-fold, respectively) in the mutant than in the wild type. The morphological and seed longevity phenotypes of the athb25-1D mutant were recapitulated in transgenic plants with moderate (4- to 6-fold) overexpression of ATHB25. Simultaneous knockdown of ATHB25, ATHB22, and ATHB31 expression decreases seed longevity, as does loss of ATHB25 and ATHB22 function in a double mutant line. Seeds from wild-type plants treated with GA and from a quintuple DELLA mutant (with constitutive GA signaling) are more tolerant to aging, providing additional evidence for a role of GA in seed longevity. A correlation was observed in several genotypes between seed longevity and mucilage formation at the seed surface, suggesting that GA may act by reinforcing the seed coat. This mechanism was supported by the observation of a maternal effect in reciprocal crosses between the wild type and the athb25-1D mutant.

  19. Curious cases of the enzymes

    OpenAIRE

    Ulusu Nuriye Nuray

    2015-01-01

    J Med Biochem 2015; 34 (3) DOI: 10.2478/jomb-2014-0045 UDK 577. 1 : 61 ISSN 1452-8258 J Med Biochem 34: 271–281, 2015 Review article Pregledni ~lanak CURIOUS CASES OF THE ENZYMES NEOBI^NA ISTORIJA ENZIMA Nuriye Nuray Ulusu Koç University, School of Medicine, Sariyer-Istanbul, Turkey Address for correspondence: N. Nuray Ulusu, PhD Koç University School of Medicine Professor of Biochemistry Rumelifeneri Yolu Sarıyer-Istanbul – Turkey Phone: +90 (212)...

  20. Enzyme Analysis to Determine Glucose Content

    Science.gov (United States)

    Carpenter, Charles; Ward, Robert E.

    Enzyme analysis is used for many purposes in food science and technology. Enzyme activity is used to indicate adequate processing, to assess enzyme preparations, and to measure constituents of foods that are enzyme substrates. In this experiment, the glucose content of corn syrup solids is determined using the enzymes, glucose oxidase and peroxidase. Glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide (H2O2), which then reacts with a dye in the presence of peroxidase to give a stable colored product.