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Sample records for metabolic enzyme activities

  1. Chemoprotective activity of boldine: modulation of drug-metabolizing enzymes.

    Science.gov (United States)

    Kubínová, R; Machala, M; Minksová, K; Neca, J; Suchý, V

    2001-03-01

    Possible chemoprotective effects of the naturally occurring alkaloid boldine, a major alkaloid of boldo (Peumus boldus Mol.) leaves and bark, including in vitro modulations of drug-metabolizing enzymes in mouse hepatoma Hepa-1 cell line and mouse hepatic microsomes, were investigated. Boldine manifested inhibition activity on hepatic microsomal CYP1A-dependent 7-ethoxyresorufin O-deethylase and CYP3A-dependent testosterone 6 beta-hydroxylase activities and stimulated glutathione S-transferase activity in Hepa-1 cells. In addition to the known antioxidant activity, boldine could decrease the metabolic activation of other xenobiotics including chemical mutagens.

  2. Inhibitors of Testosterone Biosynthetic and Metabolic Activation Enzymes

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    Leping Ye

    2011-12-01

    Full Text Available The Leydig cells of the testis have the capacity to biosynthesize testosterone from cholesterol. Testosterone and its metabolically activated product dihydrotestosterone are critical for the development of male reproductive system and spermatogenesis. At least four steroidogenic enzymes are involved in testosterone biosynthesis: Cholesterol side chain cleavage enzyme (CYP11A1 for the conversion of cholesterol into pregnenolone within the mitochondria, 3β-hydroxysteroid dehydrogenase (HSD3B, for the conversion of pregnenolone into progesterone, 17α-hydroxylase/17,20-lyase (CYP17A1 for the conversion of progesterone into androstenedione and 17β-hydroxysteroid dehydrogenase (HSD17B3 for the formation of testosterone from androstenedione. Testosterone is also metabolically activated into more potent androgen dihydrotestosterone by two isoforms 5α-reductase 1 (SRD5A1 and 2 (SRD5A2 in Leydig cells and peripheral tissues. Many endocrine disruptors act as antiandrogens via directly inhibiting one or more enzymes for testosterone biosynthesis and metabolic activation. These chemicals include industrial materials (perfluoroalkyl compounds, phthalates, bisphenol A and benzophenone and pesticides/biocides (methoxychlor, organotins, 1,2-dibromo-3-chloropropane and prochloraz and plant constituents (genistein and gossypol. This paper reviews these endocrine disruptors targeting steroidogenic enzymes.

  3. In vivo enzyme activity in inborn errors of metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D. (Clinical Research Centre, Harrow (England))

    1990-08-01

    Low-dose continuous infusions of (2H5)phenylalanine, (1-13C)propionate, and (1-13C)leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD.

  4. [Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

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    Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

    2015-09-01

    Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

  5. Novel TPP-riboswitch activators bypass metabolic enzyme dependency

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    Günter eMayer

    2014-07-01

    Full Text Available Riboswitches are conserved regions within mRNA molecules that bind specific metabolites and regulate gene expression. TPP-riboswitches, which respond to thiamine pyrophosphate (TPP, are involved in the regulation of thiamine metabolism in numerous bacteria. As these regulatory RNAs are often modulating essential biosynthesis pathways they have become increasingly interesting as promising antibacterial targets. Here, we describe thiamine analogs containing a central 1,2,3-triazole group to induce repression of thiM-riboswitch dependent gene expression in different E. coli strains. Additionally, we show that compound activation is dependent on proteins involved in the metabolic pathways of thiamine uptake and synthesis. The most promising molecule, triazolethiamine (TT, shows concentration dependent reporter gene repression that is dependent on the presence of thiamine kinase ThiK, whereas the effect of pyrithiamine (PT, a known TPP-riboswitch modulator, is ThiK independent. We further show that this dependence can be bypassed by triazolethiamine-derivatives that bear phosphate-mimicking moieties. As triazolethiamine reveals superior activity compared to pyrithiamine, it represents a very promising starting point for developing novel antibacterial compounds that target TPP-riboswitches. Riboswitch-targeting compounds engage diverse endogenous mechanisms to attain in vivo activity. These findings are of importance for the understanding of compounds that require metabolic activation to achieve effective riboswitch modulation and they enable the design of novel compound generations that are independent of endogenous activation mechanisms.

  6. Novel TPP-riboswitch activators bypass metabolic enzyme dependency

    Science.gov (United States)

    Mayer, Günter; Lünse, Christina; Suckling, Colin; Scott, Fraser

    2014-07-01

    Riboswitches are conserved regions within mRNA molecules that bind specific metabolites and regulate gene expression. TPP-riboswitches, which respond to thiamine pyrophosphate (TPP), are involved in the regulation of thiamine metabolism in numerous bacteria. As these regulatory RNAs are often modulating essential biosynthesis pathways they have become increasingly interesting as promising antibacterial targets. Here, we describe thiamine analogs containing a central 1,2,3-triazole group to induce repression of thiM-riboswitch dependent gene expression in different E. coli strains. Additionally, we show that compound activation is dependent on proteins involved in the metabolic pathways of thiamine uptake and synthesis. The most promising molecule, triazolethiamine (TT), shows concentration dependent reporter gene repression that is dependent on the presence of thiamine kinase ThiK, whereas the effect of pyrithiamine (PT), a known TPP-riboswitch modulator, is ThiK independent. We further show that this dependence can be bypassed by triazolethiamine-derivatives that bear phosphate-mimicking moieties. As triazolethiamine reveals superior activity compared to pyrithiamine, it represents a very promising starting point for developing novel antibacterial compounds that target TPP-riboswitches. Riboswitch-targeting compounds engage diverse endogenous mechanisms to attain in vivo activity. These findings are of importance for the understanding of compounds that require metabolic activation to achieve effective riboswitch modulation and they enable the design of novel compound generations that are independent of endogenous activation mechanisms.

  7. Altered Activities of Antioxidant Enzymes in Patients with Metabolic Syndrome

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    Lucie Vávrová

    2013-02-01

    Full Text Available Objective: In the pathogenesis of the metabolic syndrome (MetS, an increase of oxidative stress could play an important role which is closely linked with insulin resistance, endothelial dysfunction, and chronic inflammation. The aim of our study was to assess several parameters of the antioxidant status in MetS. Methods: 40 subjects with MetS and 40 age- and sex-matched volunteers without MetS were examined for activities of superoxide dismutase (CuZnSOD, catalase (CAT, glutathione peroxidase 1 (GPX1, glutathione reductase (GR, paraoxonase1 (PON1, concentrations of reduced glutathione (GSH, and conjugated dienes in low-density lipoprotein (CD-LDL. Results: Subjects with MetS had higher activities of CuZnSOD (p Conclusions: Our results implicated an increased oxidative stress in MetS and a decreased antioxidative defense that correlated with some laboratory (triglycerides, high-density lipoprotein cholesterol (HDL-C and clinical (waist circumference, blood pressure components of MetS.

  8. Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

    Science.gov (United States)

    Meyer, Pablo; Cecchi, Guillermo; Stolovitzky, Gustavo

    2014-12-14

    Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially. We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth. We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

  9. Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

    Science.gov (United States)

    Pavelka, S

    2014-01-01

    We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between

  10. changes in activities of enzymes of glutamate metabolism in rat ...

    African Journals Online (AJOL)

    of accumulation of acetylcholine. The signs of toxicity ... medulla. Eievated GAD activity in specific brain regions suggests the involvement of. GABAergic ... neurotransmitter balance. since GABA agonists and benzodiazipines are able IO.

  11. Determination of the activity signature of key carbohydrate metabolism enzymes in phenolic-rich grapevine tissues

    DEFF Research Database (Denmark)

    Covington, Elizabeth Dunn; Roitsch, Thomas Georg; Dermastia, Marina

    2016-01-01

    assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf....... As a case study we applied the protocol to grapevine leaf samples infected with plant pathogenic bacteria 'Candidatus Phytoplasma solani', known to alter carbohydrate metabolism in grapevine. The described adaptations may be useful for determination of metabolic fingerprints for physiological phenotyping...

  12. Differences in activities of the enzymes of nucleotide metabolism and its implications for cardiac xenotransplantation.

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    Yuen, A H Y; Khalpey, Z; Lavitrano, M; McGregor, C G A; Kalsi, K K; Yacoub, M H; Smolenski, R T

    2006-01-01

    Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5' nucleotidease (E5' N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.

  13. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

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    Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

    2000-01-01

    Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

  14. Fluvoxamine alters the activity of energy metabolism enzymes in the brain

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    Gabriela K. Ferreira

    2014-09-01

    Full Text Available Objective: Several studies support the hypothesis that metabolism impairment is involved in the pathophysiology of depression and that some antidepressants act by modulating brain energy metabolism. Thus, we evaluated the activity of Krebs cycle enzymes, the mitochondrial respiratory chain, and creatine kinase in the brain of rats subjected to prolonged administration of fluvoxamine. Methods: Wistar rats received daily administration of fluvoxamine in saline (10, 30, and 60 mg/kg for 14 days. Twelve hours after the last administration, rats were killed by decapitation and the prefrontal cortex, cerebral cortex, hippocampus, striatum, and cerebellum were rapidly isolated. Results: The activities of citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV were decreased after prolonged administration of fluvoxamine in rats. However, the activities of complex II, succinate dehydrogenase, and creatine kinase were increased. Conclusions: Alterations in activity of energy metabolism enzymes were observed in most brain areas analyzed. Thus, we suggest that the decrease in citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV can be related to adverse effects of pharmacotherapy, but long-term molecular adaptations cannot be ruled out. In addition, we demonstrated that these changes varied according to brain structure or biochemical analysis and were not dose-dependent.

  15. Evolution of a new chlorophyll metabolic pathway driven by the dynamic changes in enzyme promiscuous activity.

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    Ito, Hisashi; Tanaka, Ayumi

    2014-03-01

    Organisms generate an enormous number of metabolites; however, the mechanisms by which a new metabolic pathway is acquired are unknown. To elucidate the importance of promiscuous enzyme activity for pathway evolution, the catalytic and substrate specificities of Chl biosynthetic enzymes were examined. In green plants, Chl a and Chl b are interconverted by the Chl cycle: Chl a is hydroxylated to 7-hydroxymethyl chlorophyll a followed by the conversion to Chl b, and both reactions are catalyzed by chlorophyllide a oxygenase. Chl b is reduced to 7-hydroxymethyl chlorophyll a by Chl b reductase and then converted to Chl a by 7-hydroxymethyl chlorophyll a reductase (HCAR). A phylogenetic analysis indicated that HCAR evolved from cyanobacterial 3,8-divinyl chlorophyllide reductase (DVR), which is responsible for the reduction of an 8-vinyl group in the Chl biosynthetic pathway. In addition to vinyl reductase activity, cyanobacterial DVR also has Chl b reductase and HCAR activities; consequently, three of the four reactions of the Chl cycle already existed in cyanobacteria, the progenitor of the chloroplast. During the evolution of cyanobacterial DVR to HCAR, the HCAR activity, a promiscuous reaction of cyanobacterial DVR, became the primary reaction. Moreover, the primary reaction (vinyl reductase activity) and some disadvantageous reactions were lost, but the neutral promiscuous reaction (NADH dehydrogenase) was retained in both DVR and HCAR. We also show that a portion of the Chl c biosynthetic pathway already existed in cyanobacteria. We discuss the importance of dynamic changes in promiscuous activity and of the latent pathways for metabolic evolution.

  16. Iminosugar inhibitors of carbohydrate-active enzymes that underpin cereal grain germination and endosperm metabolism

    DEFF Research Database (Denmark)

    Andriotis, Vasilios M. E.; Rejzek, Martin; Rugen, Michael D.;

    2016-01-01

    limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate......-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying...... the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se....

  17. [Important application of intestinal transporters and metabolism enzymes on gastrointestinal disposal of active ingredients of Chinese materia medica].

    Science.gov (United States)

    Bi, Xiaolin; Du, Qiu; Di, Liuqing

    2010-02-01

    Oral drug bioavailability depends on gastrointestinal absorption, intestinal transporters and metabolism enzymes are the important factors in drug gastrointestinal absorption and they can also be induced or inhibited by the active ingredients of Chinese materia medica. This article presents important application of intestinal transporters and metabolism enzymes on gastrointestinal disposal of the active ingredients of Chinese materia medica, and points out the importance of research on transport and metabolism of the active ingredients of Chinese materia medica in Chinese extract and Chinese medicinal formulae.

  18. Can bioactive compounds of Crocus sativus L. influence the metabolic activity of selected CYP enzymes in the rat?

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    Dovrtělová, G; Nosková, K; Juřica, J; Turjap, M; Zendulka, O

    2015-01-01

    Safranal and crocin are biologically active compounds isolated from Crocus sativus L., commonly known as saffron. Clinical trials confirm that saffron has antidepressant effect, thus being a potential valuable alternative in the treatment of depression. The aim of the present study was to determine, whether systemic administration of safranal and crocin can influence the metabolic activity of CYP3A, CYP2C11, CYP2B, and CYP2A in rat liver microsomes (RLM). The experiments were carried out on male Wistar albino rats intragastrically administered with safranal (4, 20, and 100 mg/kg/day) or with intraperitoneal injections of crocin (4, 20, and 100 mg/kg/day). Our results demonstrate the ability of safranal and crocin to increase the total protein content and to change the metabolic activity of several CYP enzymes assessed as CYP specific hydroxylations of testosterone in RLM. Crocin significantly decreased the metabolic activity of all selected CYP enzymes, while safranal significantly increased the metabolic activity of CYP2B, CYP2C11 and CYP3A enzymes. Therefore, both substances could increase the risk of interactions with co-administered substances metabolized by cytochrome P450 enzymes.

  19. [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.

  20. Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue

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    Sofía Arriarán

    2015-11-01

    Full Text Available Background and Objectives. White adipose tissue (WAT shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism.Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities.Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males’ subcutaneous WAT.Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole.

  1. Turtles (Chelodina longicollis) regulate muscle metabolic enzyme activity in response to seasonal variation in body temperature.

    Science.gov (United States)

    Seebacher, F; Sparrow, J; Thompson, M B

    2004-04-01

    Fluctuations in the thermal environment may elicit different responses in animals: migration to climatically different areas, regulation of body temperature, modification of biochemical reaction rates, or assuming a state of dormancy. Many ectothermic reptiles are active over a range of body temperatures that vary seasonally. Here we test the hypothesis that metabolic enzyme activity acclimatises seasonally in freshwater turtles (Chelodina longicollis) in addition to, or instead of, behavioural regulation of body temperatures. We measured body temperatures in free-ranging turtles (n = 3) by radiotelemetry, and we assayed phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome c oxidase (CCO) activities in early autumn (March, n = 10 turtles), late autumn (May, n = 7) and mid-winter (July, n = 7) over a range of assay temperatures (10 degrees C, 15 degrees C, 20 degrees C, 25 degrees C). Body temperatures were either not different from, or higher than expected from a theoretical null-distribution of a randomly moving animal. Field body temperatures at any season were lower, however, than expected from animals that maximised their sun exposure. Turtles maintained constant PFK, LDH and CCO activities in different months, despite body temperature differences of nearly 13.0 degrees C between March (average daily body temperature = 24.4 degrees C) and July (average = 11.4 degrees C). CS activity did not vary between March and May (average daily body temperature = 20.2 degrees C), but it decreased in July. Thus C. longicollis use a combination of behavioural thermoregulation and biochemical acclimatisation in response to seasonally changing thermal conditions. Ectothermic reptiles were often thought not to acclimatise biochemically, and our results show that behavioural attainment of a preferred body temperature is not mandatory for activity or physiological performance in turtles.

  2. [Effect of hydroxylated pyrimidine derivatives on activities of thiamine-dependent enzymes and some parameters of lipid metabolism in mice].

    Science.gov (United States)

    Oparin, D A; Gorenshteĭn, B I; Karaedova, L M; Naruta, E E; Zabrodskaia, S V; Rudiak, T V; Akat'ev, V E; Larin, F S

    1997-01-01

    It has been found that hydroxylated pyrimidine derivatives actively participate in metabolic proceeds related to functioning of vitamin B1-dependent enzymes (transketolase, 2-oxo acid dehydrogenase). Hydroxypyrimidines also induce a significant increase in the levels of total lipids and cholesterol in the mice liver, not changing the phospholipid content.

  3. 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.

  4. Effects of Curcuma xanthorrhiza Extracts and Their Constituents on Phase II Drug-metabolizing Enzymes Activity

    Science.gov (United States)

    Salleh, Nurul Afifah Mohd; Ismail, Sabariah; Ab Halim, Mohd Rohaimi

    2016-01-01

    Background: Curcuma xanthorrhiza is a native Indonesian plant and traditionally utilized for a range of illness including liver damage, hypertension, diabetes, and cancer. Objective: The study determined the effects of C. xanthorrhiza extracts (ethanol and aqueous) and their constituents (curcumene and xanthorrhizol) on UDP-glucuronosyltransferase (UGT) and glutathione transferase (GST) activities. Materials and Methods: The inhibition studies were evaluated both in rat liver microsomes and in human recombinant UGT1A1 and UGT2B7 enzymes. p-nitrophenol and beetle luciferin were used as the probe substrates for UGT assay while 1-chloro-2,4-dinitrobenzene as the probe for GST assay. The concentrations of extracts studied ranged from 0.1 to 1000 μg/mL while for constituents ranged from 0.01 to 500 μM. Results: In rat liver microsomes, UGT activity was inhibited by the ethanol extract (IC50 =279.74 ± 16.33 μg/mL). Both UGT1A1 and UGT2B7 were inhibited by the ethanol and aqueous extracts with IC50 values ranging between 9.59–22.76 μg/mL and 110.71–526.65 μg/Ml, respectively. Rat liver GST and human GST Pi-1 were inhibited by ethanol and aqueous extracts, respectively (IC50 =255.00 ± 13.06 μg/mL and 580.80 ± 18.56 μg/mL). Xanthorrhizol was the better inhibitor of UGT1A1 (IC50 11.30 ± 0.27 μM) as compared to UGT2B7 while curcumene did not show any inhibition. For GST, both constituents did not show any inhibition. Conclusion: These findings suggest that C. xanthorrhiza have the potential to cause herb-drug interaction with drugs that are primarily metabolized by UGT and GST enzymes. SUMMARY Findings from this study would suggest which of Curcuma xanthorrhiza extracts and constituents that would have potential interactions with drugs which are highly metabolized by UGT and GST enzymes. Further clinical studies can then be designed if needed to evaluate the in vivo pharmacokinetic relevance of these interactions Abbreviations Used: BSA: Bovine serum albumin

  5. Metabolic enzyme activities of abyssal and hadal fishes: pressure effects and a re-evaluation of depth-related changes

    Science.gov (United States)

    Gerringer, M. E.; Drazen, J. C.; Yancey, P. H.

    2017-07-01

    Metabolic enzyme activities of muscle tissue have been useful and widely-applied indicators of whole animal metabolic capacity, particularly in inaccessible systems such as the deep sea. Previous studies have been conducted at atmospheric pressure, regardless of organism habitat depth. However, maximum reaction rates of some of these enzymes are pressure dependent, complicating the use of metabolic enzyme activities as proxies of metabolic rates. Here, we show pressure-related rate changes in lactate and malate dehydrogenase (LDH, MDH) and pyruvate kinase (PK) in six fish species (2 hadal, 2 abyssal, 2 shallow). LDH maximal reaction rates decreased with pressure for the two shallow species, but, in contrast to previous findings, it increased for the four deep species, suggesting evolutionary changes in LDH reaction volumes. MDH maximal reaction rates increased with pressure in all species (up to 51±10% at 60 MPa), including the tide pool snailfish, Liparis florae (activity increase at 60 MPa 44±9%), suggesting an inherent negative volume change of the reaction. PK was inhibited by pressure in all species tested, including the hadal liparids (up to 34±3% at 60 MPa), suggesting a positive volume change during the reaction. The addition of 400 mM TMAO counteracted this inhibition at both 0.5 and 2.0 mM ADP concentrations for the hadal liparid, Notoliparis kermadecensis. We revisit depth-related trends in metabolic enzyme activities according to these pressure-related rate changes and new data from seven abyssal and hadal species from the Kermadec and Mariana trenches. Results show that, with abyssal and hadal species, pressure-related rate changes are another variable to be considered in the use of enzyme activities as proxies for metabolic rate, in addition to factors such as temperature and body mass. Intraspecific increases in tricarboxylic acid cycle enzymes with depth of capture, independent of body mass, in two hadal snailfishes suggest improved nutritional

  6. The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid synthesizing enzymes.

    Science.gov (United States)

    Gorné, Lucas D; Acosta-Rodríguez, Victoria A; Pasquaré, Susana J; Salvador, Gabriela A; Giusto, Norma M; Guido, Mario Eduardo

    2015-02-01

    The circadian system involves central and peripheral oscillators regulating temporally biochemical processes including lipid metabolism; their disruption leads to severe metabolic diseases (obesity, diabetes, etc). Here, we investigated the temporal regulation of glycerophospholipid (GPL) synthesis in mouse liver, a well-known peripheral oscillator. Mice were synchronized to a 12:12 h light-dark (LD) cycle and then released to constant darkness with food ad libitum. Livers collected at different times exhibited a daily rhythmicity in some individual GPL content with highest levels during the subjective day. The activity of GPL-synthesizing/remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1/lipin) and lysophospholipid acyltransferases (LPLATs) also displayed significant variations, with higher levels during the subjective day and at dusk. We evaluated the temporal regulation of expression and activity of phosphatidylcholine (PC) synthesizing enzymes. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as a key regulatory enzyme or through the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. The PC/PE content ratio exhibited a daily variation with lowest levels at night, while ChoKα and PEMT mRNA expression displayed maximal levels at nocturnal phases. Our results demonstrate that mouse liver GPL metabolism oscillates rhythmically with a precise temporal control in the expression and/or activity of specific enzymes.

  7. Effects of Cu on metabolisms and enzyme activities of microbial communities in the process of composting.

    Science.gov (United States)

    Guo, Xingliang; Gu, Jie; Gao, Hua; Qin, Qingjun; Chen, Zhixue; Shao, Li; Chen, Lin; Li, Hailong; Zhang, Weijuan; Chen, Shengnan; Liu, Jiang

    2012-03-01

    With the compost matrix of pig manure, wheat straw, and spent mushroom substrate, and then inoculated with the Compound Microbe Preparation, the study investigated the effects of the heavy metal Cu on the process of composting. Biolog EcoPlate™ test revealed that at a low content, Cu could improve the capacities of microbial communities to transform and exploit carbon sources in the form of polymer, thus speeding up the decomposition of agricultural wastes, and at a high content, Cu presented inhibiting effect on microbial communities to exploit complex macromolecular carbon sources, thus extending the decomposition of agricultural wastes. Enzyme activity testing showed that at a low content, Cu presented enzyme activity-activating effect at the early period of composting and inhibiting effect in the late period of composting, and at a high content, Cu presented enzyme activity-inhibiting effects through the process of composting.

  8. 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.

  9. 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.

  10. Variation in metabolic enzyme activity of persistent Haemophilus influenzae in respiratory tracts of patients with cystic fibrosis.

    OpenAIRE

    Möller, L V; Grasselier, H; Dankert, J.; van Alphen, L

    1996-01-01

    Haemophilus influenzae organisms were isolated from sputum specimens prospectively collected from 40 patients with cystic fibrosis during 2 years to study variations in the metabolic enzyme activities of persistent H. influenzae strains as determined by biotyping. In total, 97 distinct H. influenzae strains without variations in their major outer membrane protein (MOMP) patterns and 73 MOMP variants derived from 30 of these distinct strains were obtained. Twelve distinct strains and 42 MOMP v...

  11. Effect of graded Nrf2 activation on phase-I and -II drug metabolizing enzymes and transporters in mouse liver.

    Directory of Open Access Journals (Sweden)

    Kai Connie Wu

    Full Text Available Nuclear factor erythroid 2-related factor 2 (Nrf2 is a transcription factor that induces a battery of cytoprotective genes in response to oxidative/electrophilic stress. Kelch-like ECH associating protein 1 (Keap1 sequesters Nrf2 in the cytosol. The purpose of this study was to investigate the role of Nrf2 in regulating the mRNA of genes encoding drug metabolizing enzymes and xenobiotic transporters. Microarray analysis was performed in livers of Nrf2-null, wild-type, Keap1-knockdown mice with increased Nrf2 activation, and Keap1-hepatocyte knockout mice with maximum Nrf2 activation. In general, Nrf2 did not have a marked effect on uptake transporters, but the mRNAs of organic anion transporting polypeptide 1a1, sodium taurocholate cotransporting polypeptide, and organic anion transporter 2 were decreased with Nrf2 activation. The effect of Nrf2 on cytochrome P450 (Cyp genes was minimal, with only Cyp2a5, Cyp2c50, Cyp2c54, and Cyp2g1 increased, and Cyp2u1 decreased with enhanced Nrf2 activation. However, Nrf2 increased mRNA of many other phase-I enzymes, such as aldo-keto reductases, carbonyl reductases, and aldehyde dehydrogenase 1. Many genes involved in phase-II drug metabolism were induced by Nrf2, including glutathione S-transferases, UDP- glucuronosyltransferases, and UDP-glucuronic acid synthesis enzymes. Efflux transporters, such as multidrug resistance-associated proteins, breast cancer resistant protein, as well as ATP-binding cassette g5 and g8 were induced by Nrf2. In conclusion, Nrf2 markedly alters hepatic mRNA of a large number of drug metabolizing enzymes and xenobiotic transporters, and thus Nrf2 plays a central role in xenobiotic metabolism and detoxification.

  12. [Effects of rutin on the activity of antioxidant enzymes and xenobiotic-metabolizing enzymes in liver of rats fed diets with different level of fat].

    Science.gov (United States)

    Aksenov, I V; Trusov, N V; Avren'eva, L I; Guseva, G V; Lashneva, N V; Kravchenko, L V; Tutel'ian, V A

    2014-01-01

    The study has been carried out on 6 groups of male Wistar rats, which received semi-synthetic diets within 28 days. Rats of 1st and 4th group received fat-free diet, 2nid.and 5th - diet containing standard amount of fat (10% by weight, 26% by caloric content; lard/sunflower oil - 1/1); 3rd and 6th group - a high-fat diet (30% by weight, 56% by caloric content). During the last 14 days of the experiment rats received rutin in the dose of 40 mg/kg b.w. AOA, MDA level and the activity of paraoxonase I have been evaluated in blood serum. In rat liver along with the parameters of the antioxidant status (MDA level, activity of paraoxonase 1, quinone reductase, heme oxygenase-1) the activity of xenobiotic-metabolizing enzymes (XME) (CYP1A1, CYP1A2, CYP3A1, CYP2B1, UDP-glucuronosyl transferase and glutathione transferase) and the activity of lysosomal enzymes (arylsulfatase A and B, β-galactosidase and β-glucuronidase) have been investigated. Elevation of the activity of antioxidant enzymes and XME in liver with the increase of diet fat content has been-noted. Rutin admihistration had no effect onparamete6rs of antioxidant status and decreased unsedimentable activity of lysosomal enzymes that did not depend on fat content in the diet. Rutin receiving increased the activity of all studied XME in rats fed standard diet, but practically did not effect on their activity in rats fed by fat-free and high-fat diets. Thus, rutin in pharmacological dose has no effect on the activity of antioxidant enzymes that doesn't depend on the level of fat in the diet, while the decrease or increase of diet fat content modulates (weakens) the influence of rutin on the XME activity.

  13. Thyme (Thymus vulgaris L.) leaves and its constituents increase the activities of xenobiotic-metabolizing enzymes in mouse liver.

    Science.gov (United States)

    Sasaki, Keiko; Wada, Keiji; Tanaka, Yoshiko; Yoshimura, Teruki; Matuoka, Koozi; Anno, Takahiko

    2005-01-01

    The effects of thyme (Thymus vulgaris L.) leaves and its phenolic compounds, thymol and carvacrol, on the activities of xenobiotic-metabolizing enzymes, i.e., phase I enzymes such as 7-ethoxycoumarin O-deethylase (ECOD) and phase II enzymes such as glutathione S-transferase (GST) and quinone reductase (QR), were investigated. Mice were fed with a diet containing thyme (0.5% or 2.0%) or treated orally with thymol (50-200 mg/kg) or carvacrol (50-200 mg/kg) once a day for 7 successive days, and then the enzyme activities in the livers were analyzed. Dietary administration of 2% thyme caused slightly but significantly higher ECOD, GST, and QR activities by 1.1-1.4-fold. Thymol (200 mg/kg) treatment resulted in significantly higher ECOD, GST, and QR activities by 1.3-1.9-fold, and carvacrol (200 mg/kg) treatment caused significantly higher ECOD, GST, and QR activities by 1.3-1.7-fold. Thymol-treated animals had significantly higher protein levels of GST alpha and GST micro, and carvacrol-treated animals had significantly higher levels of GST micro. These results imply that thyme contains bifunctional inducers (i.e., substances capable of inducing both phase I and phase II enzymes) and that thymol and carvacrol may account for the effects of thyme.

  14. Orphan enzymes in ether lipid metabolism.

    Science.gov (United States)

    Watschinger, Katrin; Werner, Ernst R

    2013-01-01

    Ether lipids are an emerging class of lipids which have so far not been investigated and understood in every detail. They have important roles as membrane components of e.g. lens, brain and testis, and as mediators such as platelet-activating factor. The metabolic enzymes for biosynthesis and degradation have been investigated to some extent. As most involved enzymes are integral membrane proteins they are tricky to handle in biochemical protocols. The sequence of some ether lipid metabolising enzymes has only recently been reported and other sequences still remain obscure. Defined enzymes without assigned sequence are known as orphan enzymes. One of these enzymes with uncharacterised sequence is plasmanylethanolamine desaturase, a key enzyme for the biosynthesis of one of the most abundant phospholipids in our body, the plasmalogens. This review aims to briefly summarise known functions of ether lipids, give an overview on their metabolism including the most prominent members, platelet-activating factor and the plasmalogens. A special focus is set on the description of orphan enzymes in ether lipid metabolism and on the successful strategies how four previous orphans have recently been assigned a sequence. Only one of these four was characterised by classical protein purification and sequencing, whereas the other three required alternative strategies such as bioinformatic candidate gene selection and recombinant expression or development of an inhibitor and multidimensional metabolic profiling.

  15. Metabolic enzyme activities and drug excretion in the small intestine and in the liver in the rat.

    Science.gov (United States)

    Almási, A; Bojcsev, Sz; Fischer, T; Simon, H; Perjési, P; Fischer, Emil

    2013-12-01

    The aim of these experiments was the investigation of the correlation between the metabolic enzyme activities and the intestinal and hepatic excretion of p-nitrophenol (PNP) and its metabolites (PNP-glucuronide: PNP-G and PNP-sulfate: PNP-S) in the same group of rats (n = 10). A jejunal loop was perfused with isotonic medium containing PNP in a concentration of 500 μM. The samples were obtained from the luminal perfusion medium and from the bile. For enzyme assays tissue samples were obtained from the liver and jejunum at the end of experiments. Significant differences were calculated by the Student's t-test. The activity of UDP-glucuronyltransferase and sulfotransferase was about three times higher in the liver than in the small intestine. The activity of the ß-glucuronidase was about six times higher, the activity of the arylsulfatase was approximately seven times greater in the liver than in the jejunum. No significant difference was found between the luminal appearance and the biliary excretion of PNP-G. Contrary to these findings, the biliary excretion of PNP-S was significantly higher than the luminal appearance of PNP-sulfate. It can be concluded that no direct correlation exists between the activity of metabolic enzymes and the excretion rate of PNP-metabolites in the liver and in the jejunal segment of the small intestine.

  16. [Effect of Low-Intensity 900 MHz Frequency Electromagnetic Radiation on Rat Brain Enzyme Activities Linked to Energy Metabolism].

    Science.gov (United States)

    Petrosyan, M S; Nersesova, L S; Gazaryants, M G; Meliksetyan, G O; Malakyan, M G; Bajinyan, S A; Akopian, J I

    2015-01-01

    The research deals with the effect of low-intensity 900 MHz frequency electromagnetic radiation (EMR), power density 25 μW/cm2, on the following rat brain and blood serum enzyme activities: creatine kinase (CK), playing a central role in the process of storing and distributing the cell energy, as well as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) that play a key role in providing the conjunction of carbohydrate and amino acid metabolism. The comparative analysis of the changes in the enzyme activity studied at different times following the two-hour single, as well as fractional, radiation equivalent of the total time showed that the most radiosensitive enzyme is the brain creatine kinase, which may then be recommended as a marker of the radio frequency radiation impact. According to the analysis of the changing dynamics of the CK, ALT and AST activity level, with time these changes acquire the adaptive character and are directed to compensate the damaged cell energy metabolism.

  17. 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.

  18. Remarkable reproducibility of enzyme activity profiles in tomato fruits grown under contrasting environments provides a roadmap for studies of fruit metabolism.

    Science.gov (United States)

    Biais, Benot; Bénard, Camille; Beauvoit, Bertrand; Colombié, Sophie; Prodhomme, Duyên; Ménard, Guillaume; Bernillon, Stéphane; Gehl, Bernadette; Gautier, Hélène; Ballias, Patricia; Mazat, Jean-Pierre; Sweetlove, Lee; Génard, Michel; Gibon, Yves

    2014-03-01

    To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum 'Moneymaker') plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels.

  19. Somatostatin modulates insulin-degrading-enzyme metabolism: implications for the regulation of microglia activity in AD.

    Directory of Open Access Journals (Sweden)

    Grazia Tundo

    Full Text Available The deposition of β-amyloid (Aβ into senile plaques and the impairment of somatostatin-mediated neurotransmission are key pathological events in the onset of Alzheimer's disease (AD. Insulin-degrading-enzyme (IDE is one of the main extracellular protease targeting Aβ, and thus it represents an interesting pharmacological target for AD therapy. We show that the active form of somatostatin-14 regulates IDE activity by affecting its expression and secretion in microglia cells. A similar effect can also be observed when adding octreotide. Following a previous observation where somatostatin directly interacts with IDE, here we demonstrate that somatostatin regulates Aβ catabolism by modulating IDE proteolytic activity in IDE gene-silencing experiments. As a whole, these data indicate the relevant role played by somatostatin and, potentially, by analogue octreotide, in preventing Aβ accumulation by partially restoring IDE activity.

  20. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

    Science.gov (United States)

    Smith, M Ryan; Vayalil, Praveen K; Zhou, Fen; Benavides, Gloria A; Beggs, Reena R; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

    2016-08-01

    Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study

  1. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

    Directory of Open Access Journals (Sweden)

    M. Ryan Smith

    2016-08-01

    Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

  2. The Effect of Different Nitrogen Form on Key Enzyme Activity of Sugarbeet (Vulgaris L.) Carbon and Nitrogen Metabolism

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    This article analyses the effect of the proportion of the different nitrogen forms on key enzyme activity of carbon and nitrogen metabolism under the condition of nutritional water while Tian Yan-7 was used as experimental material. The result showed that nitrate reductase(NR) activity in the leaves gradually enhanced with the increase of NO-3. No matter in root or leaves ,glutamina synthetase (GS) activity first enhanced with increasing NH4+ when NH4+ was lower than that of NO-3 ,and GS activity was the highest when NH4+and NO3-was equal ,then GS activity declined with NH4+ increasing further. In the anaphase of growth ,synthetic activity in root of sucrose synthetase(SS) in the mixed NH4+ and NO3- was obviously highr than or NO3- alone. Both of the root and sugar yields were the highest when the proportion of NH4+ and NO3- was 1: 1.

  3. Phenolic Compounds from Olea europaea L. Possess Antioxidant Activity and Inhibit Carbohydrate Metabolizing Enzymes In Vitro

    Directory of Open Access Journals (Sweden)

    Nadia Dekdouk

    2015-01-01

    Full Text Available Phenolic composition and biological activities of fruit extracts from Italian and Algerian Olea europaea L. cultivars were studied. Total phenolic and tannin contents were quantified in the extracts. Moreover 14 different phenolic compounds were identified, and their profiles showed remarkable quantitative differences among analysed extracts. Moreover antioxidant and enzymatic inhibition activities were studied. Three complementary assays were used to measure their antioxidant activities and consequently Relative Antioxidant Capacity Index (RACI was used to compare and easily describe obtained results. Results showed that Chemlal, between Algerian cultivars, and Coratina, among Italian ones, had the highest RACI values. On the other hand all extracts and the most abundant phenolics were tested for their efficiency to inhibit α-amylase and α-glucosidase enzymes. Leccino, among all analysed cultivars, and luteolin, among identified phenolic compounds, were found to be the best inhibitors of α-amylase and α-glucosidase enzymes. Results demonstrated that Olea europaea fruit extracts can represent an important natural source with high antioxidant potential and significant α-amylase and α-glucosidase inhibitory effects.

  4. Phenolic Compounds from Olea europaea L. Possess Antioxidant Activity and Inhibit Carbohydrate Metabolizing Enzymes In Vitro

    Science.gov (United States)

    Dekdouk, Nadia; Malafronte, Nicola; Russo, Daniela; Faraone, Immacolata; De Tommasi, Nunziatina; Ameddah, Souad; Severino, Lorella; Milella, Luigi

    2015-01-01

    Phenolic composition and biological activities of fruit extracts from Italian and Algerian Olea europaea L. cultivars were studied. Total phenolic and tannin contents were quantified in the extracts. Moreover 14 different phenolic compounds were identified, and their profiles showed remarkable quantitative differences among analysed extracts. Moreover antioxidant and enzymatic inhibition activities were studied. Three complementary assays were used to measure their antioxidant activities and consequently Relative Antioxidant Capacity Index (RACI) was used to compare and easily describe obtained results. Results showed that Chemlal, between Algerian cultivars, and Coratina, among Italian ones, had the highest RACI values. On the other hand all extracts and the most abundant phenolics were tested for their efficiency to inhibit α-amylase and α-glucosidase enzymes. Leccino, among all analysed cultivars, and luteolin, among identified phenolic compounds, were found to be the best inhibitors of α-amylase and α-glucosidase enzymes. Results demonstrated that Olea europaea fruit extracts can represent an important natural source with high antioxidant potential and significant α-amylase and α-glucosidase inhibitory effects. PMID:26557862

  5. Changes in the activities of starch metabolism enzymes in rice grains in response to elevated CO2 concentration

    Science.gov (United States)

    Xie, Li-Yong; Lin, Er-Da; Zhao, Hong-Liang; Feng, Yong-Xiang

    2016-05-01

    The global atmospheric CO2 concentration is currently (2012) 393.1 μmol mol-1, an increase of approximately 42 % over pre-industrial levels. In order to understand the responses of metabolic enzymes to elevated CO2 concentrations, an experiment was conducted using the Free Air CO2 Enrichment (FACE )system. Two conventional japonica rice varieties ( Oryza sativa L. ssp. japonica) grown in North China, Songjing 9 and Daohuaxiang 2, were used in this study. The activities of ADPG pyrophosphorylase, soluble and granule-bound starch synthases, and soluble and granule-bound starch branching enzymes were measured in rice grains, and the effects of elevated CO2 on the amylose and protein contents of the grains were analyzed. The results showed that elevated CO2 levels significantly increased the activity of ADPG pyrophosphorylase at day 8, 24, and 40 after flower, with maximum increases of 56.67 % for Songjing 9 and 21.31 % for Daohuaxiang 2. Similarly, the activities of starch synthesis enzymes increased significantly from the day 24 after flower to the day 40 after flower, with maximum increases of 36.81 % for Songjing 9 and 66.67 % for Daohuaxiang 2 in soluble starch synthase (SSS), and 25.00 % for Songjing 9 and 36.44 % for Daohuaxiang 2 in granule-bound starch synthase (GBSS), respectively. The elevated CO2 concentration significantly increased the activity of soluble starch branching enzyme (SSBE) at day 16, 32, and 40 after flower, and also significantly increased the activity of granule-bound starch branching enzyme (GBSBE) at day 8, 32, and 40 after flower. The elevated CO2 concentration increased the peak values of enzyme activity, and the timing of the activity peaks for SSS and GBSBE were earlier in Songjing 9 than in Daohuaxiang 2. There were obvious differences in developmental stages between the two varieties of rice, which indicated that the elevated CO2 concentration increased enzyme activity expression and starch synthesis, affecting the final contents

  6. Blood selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese

    Science.gov (United States)

    Franson, J. Christian; Hoffman, David J.; Schmutz, Joel A.

    2002-01-01

    In 1998, we collected blood samples from 63 emperor geese (Chen canagica) on their breeding grounds on the Yukon-Kuskokwim Delta (YKD) in western Alaska, USA. We studied the relationship between selenium concentrations in whole blood and the activities of glutathione peroxidase and glutathione reductase in plasma. Experimental studies have shown that plasma activities of these enzymes are useful biomarkers of selenium-induced oxidative stress, but little information is available on their relationship to selenium in the blood of wild birds. Adult female emperor geese incubating their eggs in mid-June had a higher mean concentration of selenium in their blood and a greater activity of glutathione peroxidase in their plasma than adult geese or goslings that were sampled during the adult flight feathermolting period in late July and early August. Glutathione peroxidase activity was positively correlated with the concentration of selenium in the blood of emperor geese, and the rate of increase relative to selenium was greater in goslings than in adults. The activity of glutathione reductase was greatest in the plasma of goslings and was greater in molting adults than incubating females but was not significantly correlated with selenium in the blood of adults or goslings. Incubating female emperor geese had high selenium concentrations in their blood, accompanied by increased glutathione peroxidase activity consistent with early oxidative stress. These findings indicate that further study of the effects of selenium exposure, particularly on reproductive success, is warranted in this species.

  7. Blood selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese

    Science.gov (United States)

    Franson, J.C.; Hoffman, D.J.; Schmutz, J.A.

    2002-01-01

    In 1998, we collected blood samples from 63 emperor geese (Chen canagica) on their breeding grounds on the Yukon-Kuskokwim Delta (YKD) in western Alaska, USA. We studied the relationship between selenium concentrations in whole blood and the activities of glutathione peroxidase and glutathione reductase in plasma. Experimental studies have shown that plasma activities of these enzymes are useful biomarkers of selenium-induced oxidative stress, but little information is available on their relationship to selenium in the blood of wild birds. Adult female emperor geese incubating their eggs in mid-June had a higher mean concentration of selenium in their blood and a greater activity of glutathione peroxidase in their plasma than adult geese or goslings that were sampled during the adult flight feathermolting period in late July and early August. Glutathione peroxidase activity was positively correlated with the concentration of selenium in the blood of emperor geese, and the rate of increase relative to selenium was greater in goslings than in adults. The activity of glutathione reductase was greatest in the plasma of goslings and was greater in molting adults than incubating females but was not significantly correlated with selenium in the blood of adults or goslings. Incubating female emperor geese had high selenium concentrations in their blood, accompanied by increased glutathione peroxidase activity consistent with early oxidative stress. These findings indicate that further study of the effects of selenium exposure, particularly on reproductive success, is warranted in this species.

  8. [Impacts of root-zone hypoxia stress on muskmelon growth, its root respiratory metabolism, and antioxidative enzyme activities].

    Science.gov (United States)

    Liu, Yi-Ling; Li, Tian-Lai; Sun, Zhou-Ping; Chen, Ya-Dong

    2010-06-01

    By using aeroponics culture system, this paper studied the impacts of root-zone hypoxia (10% O2 and 5% O2) stress on the plant growth, root respiratory metabolism, and antioxidative enzyme activities of muskmelon at its fruit development stage. Root-zone hypoxia stress inhibited the plant growth of muskmelon, resulting in the decrease of plant height, root length, and fresh and dry biomass. Comparing with the control (21% O2), hypoxia stress reduced the root respiration rate and malate dehydrogenase (MDH) activity significantly, and the impact of 5% O2 stress was more serious than that of 10% O2 stress. Under hypoxic conditions, the lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities and the malondialdehyde (MDA) content were significantly higher than the control. The increment of antioxidative enzyme activities under 10% O2 stress was significantly higher than that under 5% O2 stress, while the MDA content was higher under 5% O2 stress than under 10% O2 stress, suggesting that when the root-zone oxygen concentration was below 10%, the aerobic respiration of muskmelon at its fruit development stage was obviously inhibited while the anaerobic respiration was accelerated, and the root antioxidative enzymes induced defense reaction. With the increasing duration of hypoxic stress, the lipid peroxidation would be aggravated, resulting in the damages on muskmelon roots, inhibition of plant growth, and decrease of fruit yield and quality.

  9. Analysis of some enzymes activities of hydrogen sulfide metabolism in plants.

    Science.gov (United States)

    Li, Zhong-Guang

    2015-01-01

    Hydrogen sulfide (H2S) which is considered as a novel gasotransmitter after reactive oxygen species and nitric oxide in plants has dual character, that is, toxicity that inhibits cytochrome oxidase at high concentration and as signal molecule which is involved in plant growth, development, and the acquisition of tolerance to adverse environments such as extreme temperature, drought, salt, and heavy metal stress at low concentration. Therefore, H2S homeostasis is very important in plant cells. The level of H2S in plant cells is regulated by its synthetic and degradative enzymes, L-/D-cysteine desulfhydrase (L-/D-DES), sulfite reductase (SiR), and cyanoalanine synthase (CAS), which are responsible for H2S synthesis, while cysteine synthase (CS) takes charge of the degradation of H2S, but its reverse reaction also can produce H2S. Here, after crude enzyme is extracted from plant tissues, the activities of L-/D-DES, SiR, CAS, and CS are measured by spectrophotometry, the aim is to further understand homeostasis of H2S in plant cells and its potential mechanisms.

  10. Mechanistic insights into the regulation of metabolic enzymes by acetylation

    Science.gov (United States)

    2012-01-01

    The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

  11. Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses

    Institute of Scientific and Technical Information of China (English)

    LU Shao-wei; LI Tian-lai; JIANG Jing

    2009-01-01

    Changes in sucrose metabolism in response to salt (NaCl) and water (polyethylene glycol,PEG6000) iso-osmotic stresses were measured in tomato cultivar Liaoyuan Duoli (Solanum lycopersicum L.)and the objective was to provide a new evidence for the relationship between salt and osmotic stresses.The carbohydrate contents,as well as sucrose metabolizing enzymes activities and transcript levels were determined.The results indicated that soluble sugar and hexoses accumulated to higher levels and the contents of sucrose and starch were lower in mature fruit under the two stress treatments.Salt and water stresses can enhance the invertase and sucrose synthase activities of tomato fruit in a long period of time (45-60 days after anthesis),and elevate the expression of soluble acid invertase mRNA.It showed that two different stresses could also regulate the soluble acid invertase activity by controlling its gene expression.The activity of sucrose synthase was linked to the changes in soluble sugar levels but not with transcript levels.The effects of salt and water stress treatments on sucrose phosphate synthase activities were weak.

  12. Effects of phenol on metabolic activities and transcription profiles of cytochrome P450 enzymes in Chironomus kiinensis larvae.

    Science.gov (United States)

    Cao, C W; Sun, L L; Niu, F; Liu, P; Chu, D; Wang, Z Y

    2016-02-01

    Phenol, also known as carbolic acid or phenic acid, is a priority pollutant in aquatic ecosystems. The present study has investigated metabolic activities and transcription profiles of cytochrome P450 enzymes in Chironomus kiinensis under phenol stress. Exposure of C. kiinensis larvae to three sublethal doses of phenol (1, 10 and 100 µM) inhibited cytochrome P450 enzyme activity during the 96 h exposure period. The P450 activity measured after the 24 h exposure to phenol stress could be used to assess the level (low or high) of phenol contamination in the environment. To investigate the potential of cytochrome P450 genes as molecular biomarkers to monitor phenol contamination, the cDNA of ten CYP6 genes from the transcriptome of C. kiinensis were identified and sequenced. The open reading frames of the CYP6 genes ranged from 1266 to 1587 bp, encoding deduced polypeptides composed of between 421 and 528 amino acids, with predicted molecular masses from 49.01 to 61.94 kDa and isoelectric points (PI) from 6.01 to 8.89. Among the CYP6 genes, the mRNA expression levels of the CYP6EW3, CYP6EV9, CYP6FV1 and CYP6FV2 genes significantly altered in response to phenol exposure; therefore, these genes could potentially serve as biomarkers in the environment. This study shows that P450 activity combined with one or multiple CYP6 genes could be used to monitor phenol pollution.

  13. Interspecific metabolic diversity of root-colonizing endophytic fungi revealed by enzyme activity tests.

    Science.gov (United States)

    Knapp, Dániel G; Kovács, Gábor M

    2016-12-01

    Although dark septate endophytes (DSE) represent a worldwide dispersed form group of root-colonizing endophytic fungi, our knowledge on their role in ecosystem functioning is far limited. In this study, we aimed to test if functional diversity exists among DSE fungi representing different lineages of root endophytic fungal community of semiarid sandy grasslands. To address this question and to gain general information on function of DSE fungi, we adopted api-ZYM and BioLog FF assays to study those non-sporulating filamentous fungi and characterized the metabolic activity of 15 different DSE species. Although there were striking differences among the species, all of the substrates tested were utilized by the DSE fungi. When endophytes characteristic to grasses and non-grass host plants were separately considered, we found that the whole substrate repertoire was used by both groups. This might illustrate the complementary functional diversity of the communities root endophytic plant-associated fungi. The broad spectra of substrates utilized by these root endophytes illustrate the functional importance of their diversity, which can play role not only in nutrient mobilization and uptake of plants from with nutrient poor soils, but also in general plant performance and ecosystem functioning.

  14. High Temperature at Grain-filling Stage Affects Nitrogen Metabolism Enzyme Activities in Grains and Grain Nutritional Quality in Rice

    Institute of Scientific and Technical Information of China (English)

    LIANG Cheng-gang; CHEN Li-ping; WANG Yan; LIU Jia; Xu Guang-li; LI Tian

    2011-01-01

    Rice plants would more frequently suffer from high temperature (HT) stress at the grain-filling stage in future.A japonica rice variety Koshihikari and an indica rice variety IR72 were used to study the effect of high temperature on dynamic changes of glutamine synthetase (GS) activity,glutamate synthase (GOGAT) activity,glutamic oxalo-acetic transminase (GOT) activity,glutamate pyruvate transminase (GPT) activity in grains and grain nutritional quality at the grain-filling stage.Under HT,the activities of GOGAT,GOT,GPT and soluble protein content in grains significantly increased,whereas GS activity significantly decreased at the grain-filling stage.In addition to the increase of protein and amino acids contents,it was suggested that GOGAT,GOT and GPT in grains played important roles in nitrogen metabolism at the grain-filling stage.Since the decrease of GS activity in grains did not influence the accumulations of amino acids and protein,it is implied that GS might not be the key enzyme in regulating glutamine content in grains.

  15. High Temperature at Grain-filling Stage Affects Nitrogen Metabolism Enzyme Activities in Grains and Grain Nutritional Quality in Rice

    Directory of Open Access Journals (Sweden)

    Cheng-gang LIANG

    2011-09-01

    Full Text Available Rice plants would more frequently suffer from high temperature (HT stress at the grain-filling stage in future. A japonica rice variety Koshihikari and an indica rice variety IR72 were used to study the effect of high temperature on dynamic changes of glutamine synthetase (GS activity, glutamate synthase (GOGAT activity, glutamic oxalo-acetic transminase (GOT activity, glutamate pyruvate transminase (GPT activity in grains and grain nutritional quality at the grain-filling stage. Under HT, the activities of GOGAT, GOT, GPT and soluble protein content in grains significantly increased, whereas GS activity significantly decreased at the grain-filling stage. In addition to the increase of protein and amino acids contents, it was suggested that GOGAT, GOT and GPT in grains played important roles in nitrogen metabolism at the grain-filling stage. Since the decrease of GS activity in grains did not influence the accumulations of amino acids and protein, it is implied that GS might not be the key enzyme in regulating glutamine content in grains.

  16. Dependence of fructooligosaccharide content on activity of fructooligosaccharide-metabolizing enzymes in yacon (Smallanthus sonchifolius) tuberous roots during storage.

    Science.gov (United States)

    Narai-Kanayama, A; Tokita, N; Aso, K

    2007-08-01

    Tuberous roots of yacon (Smallanthus sonchifolius) accumulate about 10%, on a fresh weight basis, of inulin-type fructooligosacharides (FOSs), known as a food ingredient with various healthy benefits. However, we have a great difficulty to ensure these benefits because FOSs with a lower degree of polymerization (DP) decreased remarkably, and fructose increased when the tuberous roots were stored after harvesting even under previously recommended storage conditions of low temperature with high humidity. In the present study, to elucidate the involvement of FOS-metabolizing enzymes in FOS reduction during storage at 90% relative humidity and 8 degrees C, we extracted a crude protein from yacon tuberous roots and measured the activities of invertase (beta-fructofuranosidase, EC 3.2.1.26), sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan 1-exohydrolase (1-FEH, EC 3.2.1.80). The enzyme activities acting on sucrose, both invertase and 1-SST, were weakened after storage for a month. In addition, the activity of 1-FEH acting on short FOSs such as 1-kestose (GF(2)) and 1-nystose (GF(3)) was higher than that of 1-FFT. These results suggest that the continuous decline in FOSs of low DP during storage was dependent mainly on the 1-FEH activity. On the other hand, FOSs with a DP of >or= 9 only slightly decreased in stored yacon tuberous roots during storage, though distinct 1-FEH activity was observed in vitro toward a high-DP inulin-type substrate, indicating that highly polymerized FOSs content was unlikely to be closely connected with the 1-FEH activity.

  17. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    Science.gov (United States)

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

    2014-10-01

    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  18. Electrical stimulation affects metabolic enzyme phosphorylation, protease activation and meat tenderization in beef

    DEFF Research Database (Denmark)

    Li, C.B.; Li, J.; Zhou, G.H.

    2012-01-01

    The objective of this study was to investigate the response of sarcoplasmic proteins in bovine longissimus muscle to low-voltage electrical stimulation (ES, 80 V, 35 s) after dressing and its contribution to meat tenderization at early postmortem time. Proteome analysis showed that ES resulted...... muscles up to 24 h. Immunohistochemistry and transmission electron microscopy further indicated that lysosomal enzymes were released at early postmortem time. ES also induced ultrastructural disruption of sarcomeres. In addition, ES accelerated (P

  19. Simple and robust determination of the activity signature of key carbohydrate metabolism enzymes for physiological phenotyping in model and crop plants

    DEFF Research Database (Denmark)

    Jammer, Alexandra; Gasperl, Anna; Luschin-Ebengreuth, Nora;

    2015-01-01

    The analysis of physiological parameters is important to understand the link between plant phenotypes and their genetic bases, and therefore is needed as an important element in the analysis of model and crop plants. The activities of enzymes involved in primary carbohydrate metabolism have been...... shown to be strongly associated with growth performance, crop yield, and quality, as well as stress responses. A simple, fast, and cost-effective method to determine activities for 13 key enzymes involved in carbohydrate metabolism has been established, mainly based on coupled spectrophotometric kinetic...

  20. Final Project Report - Coupled Biogeochemical Process Evaluation for Conceptualizing Trichloriethylene Co-Metabolism: Co-Metabolic Enzyme Activity Probes and Modeling Co-Metabolism and Attenuation

    Energy Technology Data Exchange (ETDEWEB)

    Starr, Robert C; Orr, Brennon R; Lee, M Hope; Delwiche, Mark

    2010-02-26

    Trichloroethene (TCE) (also known as trichloroethylene) is a common contaminant in groundwater. TCE is regulated in drinking water at a concentration of 5 µg/L, and a small mass of TCE has the potential to contaminant large volumes of water. The physical and chemical characteristics of TCE allow it to migrate quickly in most subsurface environments, and thus large plumes of contaminated groundwater can form from a single release. The migration and persistence of TCE in groundwater can be limited by biodegradation. TCE can be biodegraded via different processes under either anaerobic or aerobic conditions. Anaerobic biodegradation is widely recognized, but aerobic degradation is less well recognized. Under aerobic conditions, TCE can be oxidized to non hazardous conditions via cometabolic pathways. This study applied enzyme activity probes to demonstrate that cometabolic degradation of TCE occurs in aerobic groundwater at several locations, used laboratory microcosm studies to determine aerobic degradation rates, and extrapolated lab-measured rates to in situ rates based on concentrations of microorganisms with active enzymes involved in cometabolic TCE degradation. Microcosms were constructed using basalt chips that were inoculated with microorganisms to groundwater at the Idaho National Laboratory Test Area North TCE plume by filling a set of Flow-Through In Situ Reactors (FTISRs) with chips and placing the FTISRs into the open interval of a well for several months. A parametric study was performed to evaluate predicted degradation rates and concentration trends using a competitive inhibition kinetic model, which accounts for competition for enzyme active sites by both a growth substrate and a cometabolic substrate. The competitive inhibition kinetic expression was programmed for use in the RT3D reactive transport package. Simulations of TCE plume evolution using both competitive inhibition kinetics and first order decay were performed.

  1. Relation between iron metabolism and antioxidants enzymes and δ-ALA-D activity in rats experimentally infected by Fasciola hepatica.

    Science.gov (United States)

    Bottari, Nathieli B; Mendes, Ricardo E; Baldissera, Matheus D; Bochi, Guilherme V; Moresco, Rafael N; Leal, Marta L R; Morsch, Vera M; Schetinger, Maria R C; Christ, Ricardo; Gheller, Larissa; Marques, Éder J; Da Silva, Aleksandro S

    2016-06-01

    The aim of this study was to evaluate the iron metabolism in serum, as well as antioxidant enzymes, in addition to the Delta-Aminolevulinic Acid Dehydratase (δ-ALA-D) activity in the liver of rats experimentally infected by Fasciola hepatica. Thirty male adult rats (Wistar) specific pathogen free were divided into four groups: two uninfected group (CTRL 1 and CTRL 2) with five animals each and two infected groups (INF 1 and INF 2) with 10 animals each. Infection was performed orally with 20 metacercariae at day 1. On day 15 (CTRL 1 and INF 1 groups) and 87 PI (CTRL 2 and INF 2 groups) blood and bone marrow were collected and the animals were subsequently euthanized for liver sampling. Blood was allocated in tubes without anticoagulant for serum acquisition to measure iron, transferrin and unsaturated iron binding capacity (UIBC). δ-ALA-D, superoxide dismutase (SOD), and catalase (CAT) activities were measured in the liver. A decrease in iron, transferrin and UIBC levels was observed in all infected animals compared to control groups (P iron accumulation was observed in bone marrow of infected mice. Infected animals showed an increase in δ-ALA-D activity at 87 post-infection (PI) (INF 2) as well as in SOD activity at days 15 (INF 1) and 87 PI (INF 2). On the other hand, CAT activity was reduced in rats infected by F. hepatica during acute and chronic phase of fasciolosis (INF 1 and INF 2 groups), when moderate (acute) and severe necrosis in the liver histopathology were observed. These results may suggest that oxidative damage to tissues along with antioxidant mechanisms might have taken part in fasciolosis pathogenesis and are also involved in iron deficiency associated to changes in δ-ALA-D activity during chronic phase of disease.

  2. Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins.

    Science.gov (United States)

    Castello, Alfredo; Hentze, Matthias W; Preiss, Thomas

    2015-12-01

    In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial cellular fate changes, cell differentiation, or malignant transformation are now known to involve 'metabolic remodeling' with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation ('moonlighting') and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme-RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future.

  3. Effects of Increased Night Temperature on Cellulose Synthesis and the Activity of Sucrose Metabolism Enzymes in Cotton Fiber

    Institute of Scientific and Technical Information of China (English)

    TIAN Jing-shan; HU Yuan-yuan; GAN Xiu-xia; ZHANG Ya-li; HU Xiao-bing; GOU Ling; LUO Hong-hai; ZHANG Wang-feng

    2013-01-01

    Temperature is one of the key factors that influence cotton fiber synthesis at the late growth stage of cotton. In this paper, using two early-maturing cotton varieties as experimental materials, night temperature increase was stimulated in the field using far-infrared quartz tubes set in semi-mobile incubators and compared with the normal night temperatures (control) in order to investigate the effects of night temperature on the cotton fiber cellulose synthesis during secondary wall thickening. The results showed that the activity of sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) quickly increased and remained constant during the development of cotton fiber, while the activity of acid invertase (AI) and alkaline invertase (NI) decreased, increased night temperatures prompted the rapid transformation of sugar, and all the available sucrose fully converted into cellulose. With night temperature increasing treatment, an increase in SuSy activity and concentration of sucrose indicate more sucrose converted into UDPG (uridin diphosphate-glucose) during the early and late stages of cotton fiber development. Furthermore, SPS activity and the increased concentration of fructose accelerated fructose degradation and reduced the inhibition of fructose to SuSy; maintaining higher value of allocation proportion of invertase and sucrose during the early development stages of cotton fiber, which was propitious to supply a greater carbon source and energy for cellulose synthesis. Therefore, the minimum temperature in the nightime was a major factor correlated with the activity of sucrose metabolism enzymes in cotton fiber. Consequently, soluble sugar transformation and cellulose accumulation were closely associated with the minimum night temperature.

  4. Light Activation of Rubisco by Rubisco Activase and Thylakoid Membranes : PROTEINS, ENZYMES AND METABOLISM

    OpenAIRE

    William J., Campbell; William L., OGREN; Photosynthesis Research Unit, Agricultural Research Service, United States Department of Agriculture

    1992-01-01

    A reconstituted system comprising ribulose bisphosphate carboxylase/oxygenase (rubisco), rubisco activase, washed thylakoid membranes, and ATP was used to demonstrate a light-dependent stimulation of rubisco activation. ATP, ribulose bisphosphate, H^+ , and Mg^ concentrations are normally light-dependent variables in the chloroplast but were maintained at pre-determined levels. Results indicated that rubisco activase and washed thylakoid membranes are sufficient to catalyze light stimulation ...

  5. Allosteric interactions coordinate catalytic activity between successive metabolic enzymes in the tryptophan synthase bienzyme complex.

    Science.gov (United States)

    Brzović, P S; Ngo, K; Dunn, M F

    1992-04-21

    Tryptophan synthase from enteric bacteria is an alpha 2 beta 2 bienzyme complex that catalyzes the final two reactions in the biosynthesis of L-tryptophan (L-Trp) from 3-indole-D-glycerol 3'-phosphate (IGP) and L-serine (L-Ser). The bienzyme complex exhibits reciprocal ligand-mediated allosteric interactions between the heterologous subunits [Houben, K., & Dunn, M. F. (1990) Biochemistry 29, 2421-2429], but the relationship between allostery and catalysis had not been completely defined. We have utilized rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy to study the relationship between allostery and catalysis in the alpha beta-reaction catalyzed by the bienzyme complex from Salmonella typhimurium. The pre-steady-state spectral changes that occur when L-Ser and IGP are mixed simultaneously with the alpha 2 beta 2 complex show that IGP binding to the alpha-site accelerates the formation of alpha-aminoacrylate [E(A-A)] from L-Ser at the beta-site. Through the use of L-Ser analogues, we show herein that the formation of the E(A-A) intermediate is the chemical signal which triggers the conformational transition that activates the alpha-subunit. beta-subunit ligands, such as L-Trp, that react to form covalent intermediates at the beta-site, but are incapable of E(A-A) formation, do not stimulate the activity of the alpha-subunit. Titration experiments show that the affinity of G3P and GP at the alpha-site is dependent upon the nature of the chemical intermediate present at the beta-active site.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Mutagenicity and Induction of Drug—metabolizing Enzyme Activity by LPG Combustion Particulates in Rats

    Institute of Scientific and Technical Information of China (English)

    YINXue-Jun; LIUJun-Zhuo; 等

    1994-01-01

    Methylene chloride extracts of particulates from liquefied petroleum gas(LPG)combustion appliance were studied by using Ames test,micronucleus test and inducibility of pulmonary and hepatic aryl hydrocarbon hydroxylase(AHH)and glutathione S-transferase(GST)in rats.The extracts showed mutagenicity for Salmonella typhimurium strain TA98 and its derivatives TA98NR and TA98/1,8-DNP6 with or without S9 mix.The revertants in strains TA98NR and TA98/1,8-DNP6 were less than 40%and 50% of than in strain TA98 without S9 mix,respectively.Positive results were obtained in mouse bone marrow micronucleus assay.Intratracheal instillation of the extracts led to increase in pulmonary(but not hepatic)AHH and GST activities in rats.In was seen that AHH was more sensitive than GST to induction by the extracts.

  7. Metabolic Enzymes of Cocaine Metabolite Benzoylecgonine.

    Science.gov (United States)

    Chen, Xiabin; Zheng, Xirong; Zhan, Max; Zhou, Ziyuan; Zhan, Chang-Guo; Zheng, Fang

    2016-08-19

    Cocaine is one of the most addictive drugs without a U.S. Food and Drug Administration (FDA)-approved medication. Enzyme therapy using an efficient cocaine-metabolizing enzyme is recognized as the most promising approach to cocaine overdose treatment. The actual enzyme, known as RBP-8000, under current clinical development for cocaine overdose treatment is our previously designed T172R/G173Q mutant of bacterial cocaine esterase (CocE). The T172R/G173Q mutant is effective in hydrolyzing cocaine but inactive against benzoylecgonine (a major, biologically active metabolite of cocaine). Unlike cocaine itself, benzoylecgonine has an unusually stable zwitterion structure resistant to further hydrolysis in the body and environment. In fact, benzoylecgonine can last in the body for a very long time (a few days) and, thus, is responsible for the long-term toxicity of cocaine and a commonly used marker for drug addiction diagnosis in pre-employment drug tests. Because CocE and its mutants are all active against cocaine and inactive against benzoylecgonine, one might simply assume that other enzymes that are active against cocaine are also inactive against benzoylecgonine. Here, through combined computational modeling and experimental studies, we demonstrate for the first time that human butyrylcholinesterase (BChE) is actually active against benzoylecgonine, and that a rationally designed BChE mutant can not only more efficiently accelerate cocaine hydrolysis but also significantly hydrolyze benzoylecgonine in vitro and in vivo. This sets the stage for advanced studies to design more efficient mutant enzymes valuable for the development of an ideal cocaine overdose enzyme therapy and for benzoylecgonine detoxification in the environment.

  8. Effects of Curcumin Analogue, 2, 6-Bis (2, 5-Dimethoxybenzylidene Cyclohexanone (BDMC33 on the Activities of Drug-Metabolizing Enzymes in Cultured Caco-2 Cell Model

    Directory of Open Access Journals (Sweden)

    Ndatsu Yakubu

    2016-01-01

    Full Text Available Poor systemic delivery of curcumin outside the gut due to its rapid metabolism has severely limited its application to many chronic diseases. Previously, our research group synthesized curcumin analogues 2, 6-bis (2, 5-dimethoxybenzylidene cyclohexanone (BDMC33 that has potent anti-inflammatory activities. Therefore, the aim of this study is to evaluate the effects of curcumin analog (BDMC33 on the activities of drug metabolizing enzymes in Caco-2 cells, which was compared with that of curcumin and 3-(2-Fluoro-benzylidene-5-(2-fluorocyclohexylmethylene-piperidin-4-one (EF-24. BDMC-33 was synthesized through the appropriate reaction of the aromatic aldehyde with cyclohexanone, under base catalyzed aldol condensation, at the ratio of ketone: aldehyde (1:2. Activity of drug metabolizing enzymes such as NADPH-cytochrome p450 reductase (CPR, UDP-glucuronosyltransferase (UGT, glutathione-S-transferase (GST and Sulfotransferase (SULT in Caco-2 cells were evaluated upon exposure to 50µM of BDMC33, curcumin, and EF-24, separately, for 4 hours. The BDMC33, EF-24, and curcumin treatments did not affect the activities of UGT, GST, SULT, and CPR in respect to their controls (29.45, 27.18, 23.64 and 2.08µmol/mg, respectively, at all periods of incubation. Hence, BDMC33 was able to maintain the activities of both phases I and II drug metabolizing enzymes, and therefore it could be a potential lead, anti-inflammatory agents.

  9. Excess nickel modulates activities of carbohydrate metabolizing enzymes and induces accumulation of sugars by upregulating acid invertase and sucrose synthase in rice seedlings.

    Science.gov (United States)

    Mishra, Pallavi; Dubey, R S

    2013-02-01

    The effects of increasing concentrations of nickel sulfate, NiSO(4) (200 and 400 μM) in the growth medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism were examined in seedlings of the two Indica rice cvs. Malviya-36 and Pant-12. During a 5-20 day growth period of seedlings in sand cultures, with Ni treatment, no definite pattern of alteration in starch level could be observed in the seedlings. In both roots and shoots of the seedlings Ni treatment led to a significant decrease in activities of starch degrading enzymes α-amylase, β-amylase, whereas starch phosphorylase activity increased. The contents of reducing, non-reducing, and total sugars increased in Ni-treated rice seedlings with a concomitant increase in the activities of sucrose degrading enzymes acid invertase and sucrose synthase. However, the activity of sucrose synthesizing enzyme sucrose phosphate synthase declined. These results suggest that Ni toxicity in rice seedlings causes marked perturbation in metabolism of carbohydrates leading to increased accumulation of soluble sugars. Such perturbation could serve as a limiting factor for growth of rice seedlings in Ni polluted environments and accumulating soluble sugars could serve as compatible solutes in the cells under Ni toxicity conditions.

  10. Schisandra chinensis regulates drug metabolizing enzymes and drug transporters via activation of Nrf2-mediated signaling pathway

    Directory of Open Access Journals (Sweden)

    He JL

    2014-12-01

    Full Text Available Jin-Lian He,1 Zhi-Wei Zhou,2,3 Juan-Juan Yin,2 Chang-Qiang He,1 Shu-Feng Zhou,2,3 Yang Yu1 1College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China Abstract: Drug metabolizing enzymes (DMEs and drug transporters are regulated via epigenetic, transcriptional, posttranscriptional, and translational and posttranslational modifications. Phase I and II DMEs and drug transporters play an important role in the disposition and detoxification of a large number of endogenous and exogenous compounds. The nuclear factor (erythroid-derived 2-like 2 (Nrf2 is a critical regulator of a variety of important cytoprotective genes that are involved in disposition and detoxification of xenobiotics. Schisandra chinensis (SC is a commonly used traditional Chinese herbal medicine that has been primarily used to protect the liver because of its potent antioxidative and anti-inflammatory activities. SC can modulate some DMEs and drug transporters, but the underlying mechanisms are unclear. In this study, we aimed to explore the role of Nrf2 in the regulatory effect of SC extract (SCE on selected DMEs and drug transporters in human hepatocellular liver carcinoma cell line (HepG2 cells. The results showed that SCE, schisandrin A, and schisandrin B significantly increased the expression of NAD(PH: Nicotinamide Adenine Dinucleotide Phosphate-oxidase or:quinone oxidoreductase 1, heme oxygenase-1, glutamate–cysteine ligase, and glutathione S-transferase A4 at both transcriptional and posttranscriptional levels. Incubation of HepG2 cells with SCE resulted in a significant

  11. Seasonal-dependent variations in metabolic status of spermatozoa and antioxidant enzyme activity in the reproductive tract fluids of wild boar/domestic pig hybrids.

    Science.gov (United States)

    Dziekońska, A; Fraser, L; Koziorowska-Gilun, M; Strzezek, J; Koziorowski, M; Kordan, W

    2014-01-01

    This study investigated seasonal changes in the metabolic performance of spermatozoa and activity of the antioxidant enzymes in the seminal plasma of three wild boar/domestic pigs (aged 1.5 to 2.5 years) and the activity of the antioxidant enzymes in fluids of the cauda epididymidis and vesicular glands from 16 wild boar/domestic pig hybrids (aged 1 to 3 years). Parameters of the sperm metabolic activity, such as total motility, mitochondrial functions, and measurements of oxygen uptake, ATP content and L-lactate production, were analyzed during the spring-summer and autumn-winter periods. Besides these sperm metabolic parameters, the sperm membrane integrity was also assessed. Total protein content and activity of the antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were measured in the reproductive tract fluids. There were no marked significant differences (P > 0.05) between the seasonal periods in terms of sperm motility, mitochondrial function and oxygen uptake; however, spermatozoa collected during the autumn-winter period exhibited higher (P pig hybrids.

  12. Effects of mechanical damage and herbivore wounding on H2O2 metabolism and antioxidant enzyme activities in hybrid poplar leaves

    Institute of Scientific and Technical Information of China (English)

    AN Yu; SHEN Ying-bai; ZHANG Zhi-xiang

    2009-01-01

    The changes of hydrogen peroxide (H2O2) metabolism and antioxidant enzyme activities in a hybrid poplar (Populus simonii ×P. pyramidalis ‘Opera 8277') in response to mechanical damage (MD) and herbivore wounding (HW) were investigated to determine whether H2O2 could function as the secondary messenger in the signaling of systemic resistance. Results show that H2O2 was generated in wounded leaves through MD and HW treatments and systemically in unwounded leaves around the wounded leaves. The activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were also enhanced. However, the H2O2 accumulation and antioxidant enzyme activities were inhibited in MD leaves through the pretreatment with DPI (which is a specific inhibitor of NADPH oxidase). The results of this study suggest that H2O2 could be systemically induced by MD and HW treatments, and H2O2 metabolism was closely related to the change in SOD, APX and CAT activities. A high level of antioxidant enzymes could decrease membrane lipid peroxidation levels and effectively induce plant defense responses.

  13. Structure-activity relationship and substrate-dependent phenomena in effects of ginsenosides on activities of drug-metabolizing P450 enzymes.

    Directory of Open Access Journals (Sweden)

    Miao Hao

    Full Text Available Ginseng, a traditional herbal medicine, may interact with several co-administered drugs in clinical settings, and ginsenosides, the major active components of ginseng, may be responsible for these ginseng-drug interactions (GDIs. Results from previous studies on ginsenosides' effects on human drug-metabolizing P450 enzymes are inconsistent and confusing. Herein, we first evaluated the inhibitory effects of fifteen ginsenosides and sapogenins on human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes by using commercially available fluorescent probes. The structure-activity relationship of their effects on the P450s was also explored and a pharmacophore model was established for CYP3A4. Moreover, substrate-dependent phenomena were found in ginsenosides' effects on CYP3A4 when another fluorescent probe was used, and were further confirmed in tests with conventional drug probes and human liver microsomes. These substrate-dependent effects of the ginsenosides may provide an explanation for the inconsistent results obtained in previous GDI reports.

  14. The activity of the endocannabinoid metabolising enzyme fatty acid amide hydrolase in subcutaneous adipocytes correlates with BMI in metabolically healthy humans

    Directory of Open Access Journals (Sweden)

    Alexander Stephen PH

    2011-08-01

    Full Text Available Abstract Background The endocannabinoid system (ECS is a ubiquitously expressed signalling system, with involvement in lipid metabolism and obesity. There are reported changes in obesity of blood concentrations of the endocannabinoids anandamide (AEA and 2-arachidonoylglcyerol (2-AG, and of adipose tissue expression levels of the two key catabolic enzymes of the ECS, fatty acid amide hydrolase (FAAH and monoacylglycerol lipase (MGL. Surprisingly, however, the activities of these enzymes have not been assayed in conditions of increasing adiposity. The aim of the current study was to investigate whether FAAH and MGL activities in human subcutaneous adipocytes are affected by body mass index (BMI, or other markers of adiposity and metabolism. Methods Subcutaneous abdominal mature adipocytes, fasting blood samples and anthropometric measurements were obtained from 28 metabolically healthy subjects representing a range of BMIs. FAAH and MGL activities were assayed in mature adipocytes using radiolabelled substrates. Serum glucose, insulin and adipokines were determined using ELISAs. Results MGL activity showed no relationship with BMI or other adiposity indices, metabolic markers (fasting serum insulin or glucose or serum adipokine levels (adiponectin, leptin or resistin. In contrast, FAAH activity in subcutaneous adipocytes correlated positively with BMI and waist circumference, but not with skinfold thickness, metabolic markers or serum adipokine levels. Conclusions In this study, novel evidence is provided that FAAH activity in subcutaneous mature adipocytes increases with BMI, whereas MGL activity does not. These findings support the hypothesis that some components of the ECS are upregulated with increasing adiposity in humans, and that AEA and 2-AG may be regulated differently.

  15. Simple and robust determination of the activity signature of key carbohydrate metabolism enzymes for physiological phenotyping in model and crop plants.

    Science.gov (United States)

    Jammer, Alexandra; Gasperl, Anna; Luschin-Ebengreuth, Nora; Heyneke, Elmien; Chu, Hyosub; Cantero-Navarro, Elena; Großkinsky, Dominik K; Albacete, Alfonso A; Stabentheiner, Edith; Franzaring, Jürgen; Fangmeier, Andreas; van der Graaff, Eric; Roitsch, Thomas

    2015-09-01

    The analysis of physiological parameters is important to understand the link between plant phenotypes and their genetic bases, and therefore is needed as an important element in the analysis of model and crop plants. The activities of enzymes involved in primary carbohydrate metabolism have been shown to be strongly associated with growth performance, crop yield, and quality, as well as stress responses. A simple, fast, and cost-effective method to determine activities for 13 key enzymes involved in carbohydrate metabolism has been established, mainly based on coupled spectrophotometric kinetic assays. The comparison of extraction buffers and requirement for dialysis of crude protein extracts resulted in a universal protein extraction protocol, suitable for the preparation of protein extracts from different organs of various species. Individual published kinetic activity assays were optimized and adapted for a semi-high-throughput 96-well assay format. These assays proved to be robust and are thus suitable for physiological phenotyping, enabling the characterization and diagnosis of the physiological state. The potential of the determination of distinct enzyme activity signatures as part of a physiological fingerprint was shown for various organs and tissues from three monocot and five dicot model and crop species, including two case studies with external stimuli. Differential and specific enzyme activity signatures are apparent during inflorescence development and upon in vitro cold treatment of young inflorescences in the monocot ryegrass, related to conditions for doubled haploid formation. Likewise, treatment of dicot spring oilseed rape with elevated CO2 concentration resulted in distinct patterns of enzyme activity responses in leaves.

  16. Non-metabolic functions of glycolytic enzymes in tumorigenesis.

    Science.gov (United States)

    Yu, X; Li, S

    2017-05-11

    Cancer cells reprogram their metabolism to meet the requirement for survival and rapid growth. One hallmark of cancer metabolism is elevated aerobic glycolysis and reduced oxidative phosphorylation. Emerging evidence showed that most glycolytic enzymes are deregulated in cancer cells and play important roles in tumorigenesis. Recent studies revealed that all essential glycolytic enzymes can be translocated into nucleus where they participate in tumor progression independent of their canonical metabolic roles. These noncanonical functions include anti-apoptosis, regulation of epigenetic modifications, modulation of transcription factors and co-factors, extracellular cytokine, protein kinase activity and mTORC1 signaling pathway, suggesting that these multifaceted glycolytic enzymes not only function in canonical metabolism but also directly link metabolism to epigenetic and transcription programs implicated in tumorigenesis. These findings underscore our understanding about how tumor cells adapt to nutrient and fuel availability in the environment and most importantly, provide insights into development of cancer therapy.

  17. Dietary back-calculation using stable isotopes: can activities of enzymes involved in amino acid metabolism be used to improve estimates of trophic shifts in fish?

    Science.gov (United States)

    Gaye-Siessegger, Julia; Focken, Ulfert; Abel, Hansjörg; Becker, Klaus

    2007-06-01

    The aim of this study was (1) to assess the effects of dietary protein content and feeding level on trophic shifts of C and N isotopes (Delta delta(13)C(tissue-diet) and Delta delta(15)N(tissue-diet)) and (2) to test whether the measurement of the activities of two enzymes involved in the metabolism of amino acids could improve the accuracy of estimation of the trophic shifts of C and N isotopes. For this, 36 Nile tilapia (Oreochromis niloticus) were kept under controlled conditions for 8 weeks and fed at three different levels (2, 4 and 8 g kg(-0.8) d(-1)) with three diets differing in their protein content only (20, 29 and 39 %). For each fish, food to fish body trophic shifts of C and N isotopes were measured as well as the hepatic activities of aspartate aminotransferase (ASAT) and glutamate dehydrogenase (GDH). The feeding level affected the activities of ASAT and GDH as well as the trophic shifts of C and N isotopes significantly but the dietary protein content had no significant effect except on the specific activity of ASAT. Fish fed at the lowest level had significantly higher trophic shifts of C and N isotopes than fish fed at higher levels. The trophic shifts were significantly lower in fish with a high protein utilisation. Values of the 'goodness-of-fit' for linear regressions between enzyme activities and trophic shifts were low. Thus, activities of ASAT and GDH are not suitable for predicting estimates of trophic shifts in situations where the amount of food consumed or the dietary protein content is not known. In further studies, activities of enzymes involved in the metabolism of amino acids combined with measurements of the activities of other enzymes should be used to try and improve the accuracy of estimates of trophic shifts.

  18. Metabolic rates, enzyme activities and chemical compositions of some deep-sea pelagic worms, particularly Nectonemertes mirabilis (Nemertea; Hoplonemertinea) and Poeobius meseres (Annelida; Polychaeta)

    Science.gov (United States)

    Thuesen, Erik V.; Childress, James J.

    1993-05-01

    Investigations of metabolic rate, enzyme activity and chemical composition were undertaken on two abundant deep-sea pelagic worms: Nectonemertes mirabilis (Nemertea; Hoplonemertinea) and Poeobius meseres (Annelida; Polychaeta). Six other species of worms ( Pelagonemertes brinkmanni (Nemertea) and the following polychaetes: Pelagobia species A, Tomopteris nisseni, Tomopteris pacifica, Tomopteris species A, and Traviopsis lobifera) were captured in smaller numbers and used for comparison in the physiological and biochemical measurements. Polychaete worms had the highest oxygen consumption rates and, along with N. mirabilis, displayed significant size effects on metabolic rate. Poeobius meseres had the lowest rates of oxygen consumption and displayed no significant relationship of oxygen consumption rate to wet weight. No significant effect of size on the activities of citrate synthase, lactate dehydrogenase or pyruvate kinase was observed in P. meseres or N. mirabilis. Lipid content was higher than protein content for all the worms in this study. Carbohydrate was of little significance in these worms and was usually <0.01% of the total weight. Citrate synthase activities of pelagic worms showed excellent correlation with metabolic rates. It appears that polychaete worms as a group have higher metabolic rates than bathypelagic shrimps, copepods and fishes, and may be the animals with the highest metabolic rates in the bathypelagic regions of the world's oceans.

  19. The Effect of Yokukansan, a Traditional Herbal Preparation Used for the Behavioral and Psychological Symptoms of Dementia, on the Drug-Metabolizing Enzyme Activities in Healthy Male Volunteers.

    Science.gov (United States)

    Soraoka, Hiromi; Oniki, Kentaro; Matsuda, Kazuki; Ono, Tatsumasa; Taharazako, Kosuke; Uchiyashiki, Yoshihiro; Kamihashi, Ryoko; Kita, Ayana; Takashima, Ayaka; Nakagawa, Kazuko; Yasui-Furukori, Norio; Kadowaki, Daisuke; Miyata, Keishi; Saruwatari, Junji

    2016-01-01

    The concomitant use of herb and prescription medications is increasing globally. Herb-drug interactions are therefore a clinically important problem. Yokukansan (YKS), a Japanese traditional herbal medicine, is one of the most frequently used herbal medicines. It is effective for treating the behavioral and psychological symptoms of dementia. We investigated the potential effects of YKS on drug-metabolizing enzyme activities in humans. An open-label repeat-dose study was conducted in 26 healthy Japanese male volunteers (age: 22.7±2.3 years) with no history of smoking. An 8-h urine sample was collected after a 150-mg dose of caffeine and a 30-mg dose of dextromethorphan before and after the administration of YKS (2.5 g, twice a day for 1 week). The activities of cytochrome P450 (CYP) 1A2, CYP2D6, CYP3A, xanthine oxidase (XO) and N-acetyltransferase 2 (NAT2) were assessed based on the urinary metabolic indices of caffeine and dextromethorphan, and the urinary excretion ratio of 6β-hydroxycortisol to cortisol. There were no statistically significant differences in the activities of the examined enzymes before or after the 7-d administration of YKS. Although further studies assessing the influence of YKS on the pharmacokinetics and pharmacodynamics of the substrates of the drug-metabolizing enzymes are needed to verify the present results, YKS is unlikely that a pharmacokinetic interaction will occur with concomitantly administered medications that are predominantly metabolized by the CYP1A2, CYP2D6, CYP3A, XO and NAT2.

  20. Monocrotophos induces the expression and activity of xenobiotic metabolizing enzymes in pre-sensitized cultured human brain cells.

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    Vinay K Tripathi

    Full Text Available The expression and metabolic profile of cytochrome P450s (CYPs is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y and glial (U373-MG cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC, cyclophosphamide (CPA, ethanol and known neurotoxicant- monocrotophos (MCP, a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against

  1. Monocrotophos induces the expression and activity of xenobiotic metabolizing enzymes in pre-sensitized cultured human brain cells.

    Science.gov (United States)

    Tripathi, Vinay K; Kumar, Vivek; Singh, Abhishek K; Kashyap, Mahendra P; Jahan, Sadaf; Pandey, Ankita; Alam, Sarfaraz; Khan, Feroz; Khanna, Vinay K; Yadav, Sanjay; Lohani, Mohtshim; Pant, Aditya B

    2014-01-01

    The expression and metabolic profile of cytochrome P450s (CYPs) is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y) and glial (U373-MG) cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC), cyclophosphamide (CPA), ethanol and known neurotoxicant- monocrotophos (MCP), a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h) of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against xenobiotics.

  2. Predicting tumor responses to mitomycin C on the basis of DT-diaphorase activity or drug metabolism by tumor homogenates: implications for enzyme-directed bioreductive drug development.

    Science.gov (United States)

    Phillips, R M; Burger, A M; Loadman, P M; Jarrett, C M; Swaine, D J; Fiebig, H H

    2000-11-15

    Mitomycin C (MMC) is a clinically used anticancer drug that is reduced to cytotoxic metabolites by cellular reductases via a process known as bioreductive drug activation. The identification of key enzymes responsible for drug activation has been investigated extensively with the ultimate aim of tailoring drug administration to patients whose tumors possess the biochemical machinery required for drug activation. In the case of MMC, considerable interest has been centered upon the enzyme DT-diaphorase (DTD) although conflicting reports of good and poor correlations between enzyme activity and response in vitro and in vivo have been published. The principle aim of this study was to provide a definitive answer to the question of whether tumor response to MMC could be predicted on the basis of DTD activity in a large panel of human tumor xenografts. DTD levels were measured in 45 human tumor xenografts that had been characterized previously in terms of their sensitivity to MMC in vitro and in vivo (the in vivo response profile to MMC was taken from work published previously). A poor correlation between DTD activity and antitumor activity in vitro as well as in vivo was obtained. This study also assessed the predictive value of an alternative approach based upon the ability of tumor homogenates to metabolize MMC. This approach is based on the premise that the overall rate of MMC metabolism may provide a better indicator of response than single enzyme measurements. MMC metabolism was evaluated in tumor homogenates (clarified by centrifugation at 1000 x g for 1 min) by measuring the disappearance of the parent compound by HPLC. In responsive [T/C 50%) tumors, the mean half life of MMC was 75+/-48.3 and 280+/-129.6 min, respectively. The difference between the two groups was statistically significant (P < 0.005). In conclusion, these results unequivocally demonstrate that response to MMC in vivo cannot be predicted on the basis of DTD activity. Measurement of MMC

  3. Metabolic enzymes activity and histomorphology in the liver of whitefish (Coregonus lavaretus L.) and pike (Esox lucius L.) inhabiting a mineral contaminated lake.

    Science.gov (United States)

    Churova, Maria V; Murzina, Svetlana A; Meshcheryakova, Olga V; Nemova, Nina N

    2014-12-01

    The effects of wastewater from a mining and ore-dressing mill on fish in Lake Kostomukshskoe, which is used as a cesspool of circulating water and for storage of industrial wastes produced by the Kostomuksha mining and ore-dressing mill in northwest Russia, were studied. The lake is characterized by heavy mineralization, high pH, elevated levels of K(+), Li(+), SO4 (2-), NO(2-), Cl(-), Li, Mn, and Ni, and the presence of a fine-dispersed mechanical suspension. To assess the impact of contamination on fish and determine the mechanisms of their adaptation, we investigated the biochemical indices and histology of the liver of whitefish (Coregonus lavaretus L.) and pike (Esox lucius L.) inhabiting Lake Kostomukshskoe, downstream Lake Koyvas (64° 47' 30° 59'), and Lake Kamennoe, which is located in a nature preserve and has not been affected by anthropogenic activity (64° 28' 30° 13'). Changes were detected in the activity of metabolic enzymes (cytochrome c oxidase (COX), lactate dehydrogenase, and glucose-6-phosphate dehydrogenase) in the liver. Specifically, the COX activity in the liver of both fish species from the contaminated lake decreased, indicating a low level of aerobic metabolism. Lipid infiltration was the most visible and widespread change observed in the liver of both fish species; therefore, it can be considered a marker of such long-term contamination. Lesions in pike liver demonstrated a wider range of severity than in those of whitefish. In summary, metabolic enzyme activity and histomorphology of the liver of whitefish and pike differed among lakes in a species-specific manner. The changes in enzyme activity and histomorphological alterations in fish that were observed can be applied for evaluation of freshwater systems that may be subjected to mineral pollution.

  4. Identifying metabolic enzymes with multiple types of association evidence

    Directory of Open Access Journals (Sweden)

    Vitkup Dennis

    2006-03-01

    Full Text Available Abstract Background Existing large-scale metabolic models of sequenced organisms commonly include enzymatic functions which can not be attributed to any gene in that organism. Existing computational strategies for identifying such missing genes rely primarily on sequence homology to known enzyme-encoding genes. Results We present a novel method for identifying genes encoding for a specific metabolic function based on a local structure of metabolic network and multiple types of functional association evidence, including clustering of genes on the chromosome, similarity of phylogenetic profiles, gene expression, protein fusion events and others. Using E. coli and S. cerevisiae metabolic networks, we illustrate predictive ability of each individual type of association evidence and show that significantly better predictions can be obtained based on the combination of all data. In this way our method is able to predict 60% of enzyme-encoding genes of E. coli metabolism within the top 10 (out of 3551 candidates for their enzymatic function, and as a top candidate within 43% of the cases. Conclusion We illustrate that a combination of genome context and other functional association evidence is effective in predicting genes encoding metabolic enzymes. Our approach does not rely on direct sequence homology to known enzyme-encoding genes, and can be used in conjunction with traditional homology-based metabolic reconstruction methods. The method can also be used to target orphan metabolic activities.

  5. Differential regulation of grain sucrose accumulation and metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta) revealed through gene expression and enzyme activity analysis.

    Science.gov (United States)

    Privat, Isabelle; Foucrier, Séverine; Prins, Anneke; Epalle, Thibaut; Eychenne, Magali; Kandalaft, Laurianne; Caillet, Victoria; Lin, Chenwei; Tanksley, Steve; Foyer, Christine; McCarthy, James

    2008-01-01

    * Coffea arabica (Arabica) and Coffea canephora (Robusta) are the two main cultivated species used for coffee bean production. Arabica genotypes generally produce a higher coffee quality than Robusta genotypes. Understanding the genetic basis for sucrose accumulation during coffee grain maturation is an important goal because sucrose is an important coffee flavor precursor. * Nine new Coffea genes encoding sucrose metabolism enzymes have been identified: sucrose phosphate synthase (CcSPS1, CcSPS2), sucrose phosphate phosphatase (CcSP1), cytoplasmic (CaInv3) and cell wall (CcInv4) invertases and four invertase inhibitors (CcInvI1, 2, 3, 4). * Activities and mRNA abundance of the sucrose metabolism enzymes were compared at different developmental stages in Arabica and Robusta grains, characterized by different sucrose contents in mature grain. * It is concluded that Robusta accumulates less sucrose than Arabica for two reasons: Robusta has higher sucrose synthase and acid invertase activities early in grain development - the expression of CcSS1 and CcInv2 appears to be crucial at this stage and Robusta has a lower SPS activity and low CcSPS1 expression at the final stages of grain development and hence has less capacity for sucrose re-synthesis. Regulation of vacuolar invertase CcInv2 activity by invertase inhibitors CcInvI2 and/or CcInvI3 during Arabica grain development is considered.

  6. Renal fructose-metabolizing enzymes: significance in hereditary fructose intolerance.

    Science.gov (United States)

    Kranhold, J F; Loh, D; Morris, R C

    1969-07-25

    In patients with hereditary fructose intolerance, which is characterized by deficient aldolase activity toward fructose-1-phosphate, fructose induces a renal tubular dysfunction that implicates only the proximal convoluted tubule. Because normal metabolism of fructose by way of fructose-1-phosphate requires fructokinase, aldolase "B," and triokinase, the exclusively cortical location of these enzymes indicates that the medulla is not involved in the metabolic abnormality presumably causal of the renal dysfunction.

  7. Chemoprotective potentials of homoisoflavonoids and chalcones of Dracaena cinnabari: modulations of drug-metabolizing enzymes and antioxidant activity.

    Science.gov (United States)

    Machala, M; Kubínová, R; Horavová, P; Suchý, V

    2001-03-01

    A series of homoisoflavonoids and chalcones, isolated from the endemic tropical plant Dracaena cinnabari Balf. (Agavaceae), were tested for their potential to inhibit cytochrome P4501A (CYP1A) enzymes and Fe-enhanced in vitro peroxidation of microsomal lipids in C57B1/6 mouse liver. The effects of the polyphenolic compounds were compared with those of prototypal flavonoid modulators of CYP1A and the well-known antioxidant, butylated hydroxytoluene. 2-Hydroxychalcone and partly 4,6-dihydroxychalcone were found to be strong inhibitors of CYP1A-dependent 7-ethoxyresorufin O-deethylase (EROD) activity in vitro comparable to the effects of quercetin and chrysin. The first screening of flavonoids and chalcones of Dracaena cinnabari for antioxidant activity was done in an in vitro microsomal peroxidation assay. While chalcones were shown to be poor antioxidants, 7,8-methylenedioxy-3(4-hydroxybenzyl) chromane, as one of the tested homoisoflavonoids, exhibited a strong antioxidant activity comparable to that of the strongest flavonol antioxidant, quercetin.

  8. Effects of Variation in Activities of Starch-Sugar Metabolic Enzymes on Reducing Sugar Accumulation and Processing Quality of Potato Tubers

    Institute of Scientific and Technical Information of China (English)

    CHENG Shan-han; SU Zhen-hong; XIE Cong-hua; LIU Jun

    2004-01-01

    The experiment was designed,via storing potato tubers of cv.E-Potatol and E-Potato3 in different temperatures,to explore the variation patterns of reducing sugar(RS)and total sugar(TS)contents and enzyme activities that are involved in the pathway of starch-sugar metabolism aiming at identifying the main factors that influence the chip color.The results showed that low temperature in storage was a main factor that accelerated the accumulation of RS of the stored tubers and a very significant linear relationship existed between RS content and chip color index(CCI)of the tubers.Further analysis elucidated that when tubers stored at 4℃,the activities of ADP glucose pyrophosphorylase(AGPase),UDP glucose pyrophosphorylase(UGPase)and sucrose synthase(SuSy)were negatively exponential to the RS content significantly while that of acid invertase and alkaline invertase was significantly linear to RS content.It suggested that these enzymes could play main roles in the cold sweetening of potato tubers through regulating starch-sugar metabolism.

  9. Expression of Enzymes that Metabolize Medications

    Science.gov (United States)

    Wotring, Virginia E.; Peters, C. P.

    2012-01-01

    Most pharmaceuticals are metabolized by the liver. Clinically-used medication doses are given with normal liver function in mind. A drug overdose can result if the liver is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism we want to understand the effects of spaceflight on the enzymes of the liver.

  10. Optical isomers of dihydropyridine calcium channel blockers display enantiospecific effects on the expression and enzyme activities of human xenobiotics-metabolizing cytochromes P450.

    Science.gov (United States)

    Štěpánková, Martina; Krasulová, Kristýna; Dořičáková, Aneta; Kurka, Ondřej; Anzenbacher, Pavel; Dvořák, Zdeněk

    2016-11-16

    Dihydropyridine calcium channel blockers (CCBs) are used as anti-hypertensives and in the treatment of angina pectoris. Structurally, CCBs have at least one chiral center in the molecule, thereby existing in two or more different enantiomers. In the current paper we examined effects of benidipine, felodipine and isradipine enantiomers on the expression and enzyme activities of human xenobiotics-metabolizing cytochromes P450. All CCBs dose-dependently activated aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), as revealed by gene reporter assays. Activation of AhR, but not PXR, was enantiospecific. Consistently, CCBs induced CYP1A1 and CYP1A2 mRNAs, but not protein, in human hepatocytes and HepG2 cells, with following pattern: benidipine (-)>(+), isradipine (-)>(+) and felodipine (+)>(-). All CCBs induced CYP2A6, CYP2B6 and CYP3A4 mRNA and protein in human hepatocytes, and there were not differences between the enantiomers. All CCBs transformed AhR in its DNA-binding form, as revealed by electromobility shift assay. Tested CCBs inhibited enzyme activities of CYP3A4 (benidipine (+)>(-); felodipine (-)>(+); isradipine (-)-(+)) and CYP2C9 (benidipine (-)>(+); felodipine (+)>(-); isradipine (-)>(+)). The data presented here might be of toxicological and clinical importance.

  11. Enzymes and Metabolites in Carbohydrate Metabolism of Desiccation Tolerant Plants

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    Qingwei Zhang

    2016-12-01

    Full Text Available Resurrection plants can tolerate extreme water loss. Substantial sugar accumulation is a phenomenon in resurrection plants during dehydration. Sugars have been identified as one important factor contributing to desiccation tolerance. Phylogenetic diversity of resurrection plants reflects the diversity of sugar metabolism in response to dehydration. Sugars, which accumulate during dehydration, have been shown to protect macromolecules and membranes and to scavenge reactive oxygen species. This review focuses on the performance of enzymes participating in sugar metabolism during dehydration stress. The relation between sugar metabolism and other biochemical activities is discussed and open questions as well as potential experimental approaches are proposed.

  12. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid...

  13. [Effects of nitrogen application period on the nitrogen metabolism key enzymes activities and antioxidant characteristics of high-yielding summer maize].

    Science.gov (United States)

    Lü, Peng; Zhang, Ji-Wang; Liu, Wei; Yang, Jin-Sheng; Dong, Shu-Ting; Liu, Peng; Li, Deng-Hai

    2012-06-01

    Taking the high-yielding summer maize cultivars Denghai 661 (DH661) and Zhengdan 958 (ZD958) as test materials, a field experiment was conducted to study their grain yield, nitrogen use efficiency, key enzymes activities of nitrogen metabolism, and antioxidant enzymes activities under effects of different nitrogen application periods. One-dose nitrogen application at jointing stage was not beneficial to the increase of grain yield and the nitrogen accumulation in plant and grain, while split application in combination with application after anthesis increased the nitrogen accumulation in plant and grain significantly and increased the grain yield. When the nitrogen was applied at a ratio of 2:4:4 at jointing stage, 10-leaf stage, and 10 days after anthesis, the grain yield of DH661 was up to 14123.0 kg x hm(-2); when the nitrogen was applied at a ratio of 1:2:5:2 as the basal and at jointing stage, 10-leaf stage, and 10 days after anthesis, the grain yield of ZD958 was up to 14517.1 kg x hm(-2). These two nitrogen application modes increased the grain yield of DH661 and ZD958 by 14.5% and 17.5%, respectively, as compared with one-dose nitrogen application at jointing stage. Split nitrogen application before anthesis increased plant nitrate reductase activity significantly. In the 0-42 days after anthesis under split nitrogen application, the glutamine synthetase, glutamate synthase, and glutamate dehydrogenase activities of DH661 and ZD958 were averagely increased by 32.6%, 47.1% and 50.4%, and 14.5%, 61.8% and 25.6%, and the superoxide dismutase and catalase activities were increased by 22. 0% and 36.6%, and 13.4% and 62.0%, respectively, and the malondialdehyde content was decreased significantly, as compared with one-off nitrogen application. It was suggested that for the high-yielding of summer maize, split application of nitrogen and appropriately increasing the nitrogen application ratio after anthesis could enhance the plant key nitrogen metabolism enzymes

  14. Exploration of the spontaneous fluctuating activity of single enzyme molecules

    NARCIS (Netherlands)

    Schwabe, A.; Maarleveld, T.R.; Bruggeman, F.J.

    2013-01-01

    Single enzyme molecules display inevitable, stochastic fluctuations in their catalytic activity. In metabolism, for instance, the stochastic activity of individual enzymes is averaged out due to their high copy numbers per single cell. However, many processes inside cells rely on single enzyme activ

  15. Methadone induces the expression of hepatic drug-metabolizing enzymes through the activation of pregnane X receptor and constitutive androstane receptor.

    Science.gov (United States)

    Tolson, Antonia H; Li, Haishan; Eddington, Natalie D; Wang, Hongbing

    2009-09-01

    Methadone (MD) is the most established substance abuse pharmacotherapy of choice for the management of heroin dependence. To date, drug-drug interactions involving MD have been characterized asymmetrically among existing reports, which describe how other drugs affect the metabolic or pharmacokinetic profiles of MD; however, limited information is available regarding the potential for MD to influence similar fates of coadministered drugs. Moreover, little to no mechanistic evidence has been explored. Here, we show that MD induces hepatic drug-metabolizing enzymes (DMEs) through the activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Real-time polymerase chain reaction analysis of human hepatocyte cultures revealed that MD induces the mRNA expression of CYP2B6, CYP3A4, UGT1A1, and multidrug resistance 1 in a concentration-related manner, with the maximal induction of CYP2B6 challenging that of the induction by rifampicin. Furthermore, MD-mediated induction of CYP2B6 and CYP3A4 proteins was observed in Western blot analysis. In cell-based reporter assays, MD significantly increased human (h) PXR-mediated CYP2B6 reporter activities but exhibited minimal effect on hCAR activation as a result of the constitutive activity of hCAR in HepG2 cells. Further studies revealed that treatment with MD resulted in significant nuclear accumulation of adenovirus/enhanced yellow fluorescent protein tagged-hCAR in human hepatocytes, which has been regarded as the initial step of CAR activation. Additional analysis of the two enantiomers of MD, R-(-)-MD (active) and S-(+)-MD (inactive), indicates the lack of stereoselectivity pertaining to MD-mediated DME induction. Overall, our results show that MD induces the hepatic expression of multiple DMEs by activating PXR- and CAR-mediated pathways.

  16. Identifying quantitative operation principles in metabolic pathways: a systematic method for searching feasible enzyme activity patterns leading to cellular adaptive responses

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    Sorribas Albert

    2009-11-01

    Full Text Available Abstract Background Optimization methods allow designing changes in a system so that specific goals are attained. These techniques are fundamental for metabolic engineering. However, they are not directly applicable for investigating the evolution of metabolic adaptation to environmental changes. Although biological systems have evolved by natural selection and result in well-adapted systems, we can hardly expect that actual metabolic processes are at the theoretical optimum that could result from an optimization analysis. More likely, natural systems are to be found in a feasible region compatible with global physiological requirements. Results We first present a new method for globally optimizing nonlinear models of metabolic pathways that are based on the Generalized Mass Action (GMA representation. The optimization task is posed as a nonconvex nonlinear programming (NLP problem that is solved by an outer-approximation algorithm. This method relies on solving iteratively reduced NLP slave subproblems and mixed-integer linear programming (MILP master problems that provide valid upper and lower bounds, respectively, on the global solution to the original NLP. The capabilities of this method are illustrated through its application to the anaerobic fermentation pathway in Saccharomyces cerevisiae. We next introduce a method to identify the feasibility parametric regions that allow a system to meet a set of physiological constraints that can be represented in mathematical terms through algebraic equations. This technique is based on applying the outer-approximation based algorithm iteratively over a reduced search space in order to identify regions that contain feasible solutions to the problem and discard others in which no feasible solution exists. As an example, we characterize the feasible enzyme activity changes that are compatible with an appropriate adaptive response of yeast Saccharomyces cerevisiae to heat shock Conclusion Our results

  17. The Native Fruit Geoffroea decorticans from Arid Northern Chile: Phenolic Composition, Antioxidant Activities and In Vitro Inhibition of Pro-Inflammatory and Metabolic Syndrome-Associated Enzymes

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    Felipe Jiménez-Aspee

    2017-09-01

    Full Text Available The native tree Geoffroea decorticans (chañar grows in the arid lands of northern Chile. It has been used as a food plant since prehistoric times. Phenolic-enriched extracts (PEEs of Chilean chañar fruits were assessed for their chemical composition, antioxidant properties and inhibition of pro-inflammatory and metabolic syndrome-associated enzymes. Phenolic profiles were determined by HPLC-DAD-MS/MS. The PEEs of G. decorticans showed a strong effect towards the enzymes COX-1/COX-2, with inhibition percentages ranging from inactive to 92.1% and inactive to 76.0% at 50 µg PEE/mL, respectively. The IC50 values of the PEEs towards lipoxygenase and phospholipase A2 inhibitory activity were between 43.6–96.8 and 98.9–156.0 μg PEE/mL, respectively. Samples inhibited α-glucosidase (IC50 0.8–7.3 μg PEE/mL and lipase (9.9 to >100 μg PEE/mL. However, samples did not inhibit α-amylase. The HPLC-DAD-MS analysis of the PEEs allowed the tentative identification of 53 compounds, mainly flavonol glycosides and procyanidins. The procyanidin content of the Chilean G. decorticans pulp was positively correlated with the antioxidant activity and the inhibition of the enzyme α-glucosidase. These results indicate that the Chilean chañar fruit contains bioactive polyphenols with functional properties.

  18. Effect of Ethanol Accumulation on Porcine Interferon-α Production by Pichia pastoris and Activities of Key Enzymes in Carbon Metabolism.

    Science.gov (United States)

    Ding, Jian; Gao, Minjie; Hou, Guoli; Liang, Kexue

    2015-08-01

    In production of porcine interferon α (pIFN-α) by Pichia pastoris, improper glycerol feeding strategy leads to ethanol accumulation in the last stage of growth phase. In the present study, taking two runs with low ethanol accumulation under 2 g/L as control group, effects of long-term (>4 h) and instantaneous high ethanol concentration (>10 g/L) on pIFN-α production, and activities of key enzymes in carbon metabolism were discussed. As a result, compared with control group, pIFN-α expression level was decreased about 4~12 folds under long-term high ethanol concentration, from the level above 3 g/L to the level under 1 g/L; pIFN-α expression level was decreased about 8 folds under instantaneous high ethanol concentration, reaching to the low level of 0.42 g/L. The low production of pIFN-α was caused by the severe inhibitory effect of ethanol on these enzymes.

  19. Effects of different dwarfing interstocks on key enzyme activities and the expression of genes related to malic acid metabolism in Red Fuji apples.

    Science.gov (United States)

    Shi, J; Li, F F; Ma, H; Li, Z Y; Xu, J Z

    2015-12-22

    In this experiment, the test materials were 'Red Fuji' apple trees grafted onto three interstocks (No. 53, No. 111, and No. 236), which were chosen from SH40 seeding interstocks. The content of malic acid, the enzyme activities, and the expression of genes related to malic acid metabolism were determined during fruit development.The results showed that malic acid content in the ripe fruit on interstock No. 53 was higher than that in the interstock No. 111 fruit. The malate dehydrogenase (NAD-MDH) activity in apples on interstock No. 53 was highest on Day 30, Day 100, and Day 160 after bloom, and the malic enzyme (NADP-ME) activity in apples on interstock No. 111 was higher than in the interstock No. 53 fruit from Day 70 to Day 100 after bloom. The relative expression of NAD-MDH genes in interstock No. 53 fruit was higher than in No. 236 fruit on Day 100 after bloom, but the relative expression of NADP-ME in No. 236 interstock fruit was lower than in No. 53 fruit. The relative expression of NAD-MDH genes in No. 53 interstock fruit was highest on Day 160 after bloom. This might have been the main reason for the difference in the accumulation of malic acid in the ripe apples.There was a positive correlation between the relative expression of phosphoenolpyruvate carboxylase (PEPC) and the malic acid content of the fruit, and the content of malic acid in the apples was affected by the PEPC activity during the early developmental stage.

  20. Carboxymefloquine, the major metabolite of the antimalarial drug mefloquine, induces drug-metabolizing enzyme and transporter expression by activation of pregnane X receptor.

    Science.gov (United States)

    Piedade, Rita; Traub, Stefanie; Bitter, Andreas; Nüssler, Andreas K; Gil, José P; Schwab, Matthias; Burk, Oliver

    2015-01-01

    Malaria patients are frequently coinfected with HIV and mycobacteria causing tuberculosis, which increases the use of coadministered drugs and thereby enhances the risk of pharmacokinetic drug-drug interactions. Activation of the pregnane X receptor (PXR) by xenobiotics, which include many drugs, induces drug metabolism and transport, thereby resulting in possible attenuation or loss of the therapeutic responses to the drugs being coadministered. While several artemisinin-type antimalarial drugs have been shown to activate PXR, data on nonartemisinin-type antimalarials are still missing. Therefore, this study aimed to elucidate the potential of nonartemisinin antimalarial drugs and drug metabolites to activate PXR. We screened 16 clinically used antimalarial drugs and six major drug metabolites for binding to PXR using the two-hybrid PXR ligand binding domain assembly assay; this identified carboxymefloquine, the major and pharmacologically inactive metabolite of the antimalarial drug mefloquine, as a potential PXR ligand. Two-hybrid PXR-coactivator and -corepressor interaction assays and PXR-dependent promoter reporter gene assays confirmed carboxymefloquine to be a novel PXR agonist which specifically activated the human receptor. In the PXR-expressing intestinal LS174T cells and in primary human hepatocytes, carboxymefloquine induced the expression of drug-metabolizing enzymes and transporters on the mRNA and protein levels. The crucial role of PXR for the carboxymefloquine-dependent induction of gene expression was confirmed by small interfering RNA (siRNA)-mediated knockdown of the receptor. Thus, the clinical use of mefloquine may result in pharmacokinetic drug-drug interactions by means of its metabolite carboxymefloquine. Whether these in vitro findings are of in vivo relevance has to be addressed in future clinical drug-drug interaction studies.

  1. 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.

  2. A core metabolic enzyme mediates resistance to phosphine gas.

    Science.gov (United States)

    Schlipalius, David I; Valmas, Nicholas; Tuck, Andrew G; Jagadeesan, Rajeswaran; Ma, Li; Kaur, Ramandeep; Goldinger, Anita; Anderson, Cameron; Kuang, Jujiao; Zuryn, Steven; Mau, Yosep S; Cheng, Qiang; Collins, Patrick J; Nayak, Manoj K; Schirra, Horst Joachim; Hilliard, Massimo A; Ebert, Paul R

    2012-11-09

    Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

  3. An enzyme with rhamnogalacturonase activity.

    NARCIS (Netherlands)

    Kovod, L.V.; Dalboge, H.; Andersen, L.N.; Kauppinen, M.; Christgan, S.; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A.G.J.; Schols, H.A.

    1994-01-01

    An enzyme exhibiting rhamnogalacturonase activity, which enzyme: a) is encoded by the DNA sequence shown in SEQ ID No. 1 or a sequence homologous thereto encoding a polypeptide with RGase activity, b) has the amino acid sequence shown in SEQ ID No. 2 or an analogue thereof, c) is reactive with an

  4. 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

  5. Female mice lacking active nadph-oxidase enzymes are protected against “western diet”--induced obesity and metabolic syndrome

    Science.gov (United States)

    NADPH oxidase (Nox) enzymes have been implicated in regulation of adipocyte differentiation and inflammation in a variety of tissues. We examined the effects of feeding AIN-93G or a “Western diet” (WD) (45% fat, 0.5% cholesterol) on development of obesity and “metabolic syndrome” in wild type (WT) m...

  6. Aspartate aminotransferase – key enzyme in the human systemic metabolism

    Directory of Open Access Journals (Sweden)

    Dagmara Otto-Ślusarczyk

    2016-03-01

    Full Text Available Aspartate aminotransferase is an organ - nonspecific enzyme located in many tissues of the human body where it catalyzes reversible reaction of transamination. There are two aspartate aminotransferase isoforms - cytoplasmic (AST1 and mitochondrial (AST2, that usually occur together and interact with each other metabolically. Both isoforms are homodimers containing highly conservative regions responsible for catalytic properties of enzyme. The common feature of all aspartate aminotransfeses is Lys – 259 residue covalent binding with prosthetic group - pyridoxal phosphate. The differences in the primary structure of AST isoforms determine their physico-chemical, kinetic and immunological properties. Because of the low concentration of L-aspartate (L-Asp in the blood, AST is the only enzyme, which supply of this amino acid as a substrate for many metabolic processes, such as urea cycle or purine and pyrimidine nucleotides in the liver, synthesis of L-arginine in the kidney and purine nucleotide cycle in the brain and the skeletal muscle. AST is also involved in D-aspartate production that regulates the metabolic activity at the auto-, para- and endocrine level. Aspartate aminotransferase is a part of the malate-aspartate shuttle in the myocardium, is involved in gluconeogenesis in the liver and kidney, glyceroneogenesis in the adipose tissue, and synthesis of neurotransmitters and neuro-glial pathway in the brain. Recently, the significant role of AST in glutaminolysis - normal metabolic pathway in tumor cells, was demonstrated. The article is devoted the role of AST, known primarily as a diagnostic liver enzyme, in metabolism of various human tissues and organs.

  7. A new nuclear function of the Entamoeba histolytica glycolytic enzyme enolase: the metabolic regulation of cytosine-5 methyltransferase 2 (Dnmt2 activity.

    Directory of Open Access Journals (Sweden)

    Ayala Tovy

    2010-02-01

    Full Text Available Cytosine-5 methyltransferases of the Dnmt2 family function as DNA and tRNA methyltransferases. Insight into the role and biological significance of Dnmt2 is greatly hampered by a lack of knowledge about its protein interactions. In this report, we address the subject of protein interaction by identifying enolase through a yeast two-hybrid screen as a Dnmt2-binding protein. Enolase, which is known to catalyze the conversion of 2-phosphoglycerate (2-PG to phosphoenolpyruvate (PEP, was shown to have both a cytoplasmatic and a nuclear localization in the parasite Entamoeba histolytica. We discovered that enolase acts as a Dnmt2 inhibitor. This unexpected inhibitory activity was antagonized by 2-PG, which suggests that glucose metabolism controls the non-glycolytic function of enolase. Interestingly, glucose starvation drives enolase to accumulate within the nucleus, which in turn leads to the formation of additional enolase-E.histolytica DNMT2 homolog (Ehmeth complex, and to a significant reduction of the tRNA(Asp methylation in the parasite. The crucial role of enolase as a Dnmt2 inhibitor was also demonstrated in E.histolytica expressing a nuclear localization signal (NLS-fused-enolase. These results establish enolase as the first Dnmt2 interacting protein, and highlight an unexpected role of a glycolytic enzyme in the modulation of Dnmt2 activity.

  8. Selenium concentrations and enzyme activities of glutathione metabolism in wild long-tailed ducks and common eiders

    Science.gov (United States)

    Franson, J. Christian; Hoffman, David J.; Flint, Paul L.

    2011-01-01

    The relationships of selenium (Se) concentrations in whole blood with plasma activities of total glutathione peroxidase, Se-dependent glutathione peroxidase, and glutathione reductase were studied in long-tailed ducks (Clangula hyemalis) and common eiders (Somateria mollissima) sampled along the Beaufort Sea coast of Alaska, USA. Blood Se concentrations were >8 μg/g wet weight in both species. Linear regression revealed that the activities of total and Se-dependent glutathione peroxidase were significantly related to Se concentrations only in long-tailed ducks, raising the possibility that these birds were experiencing early oxidative stress.

  9. The effect of dietary bagasse on the activities of some key enzymes of carbohydrate and lipid metabolism in mouse liver.

    Science.gov (United States)

    Stanley, J C; Newsholme, E A

    1985-09-01

    The effects of a 100 g/kg diet substitution of bagasse on the body-weight gain, food consumption and faecal dry weight of mice given a high-sucrose diet and on the activities of hepatic glucose-6-phosphate dehydrogenase (EC I.I.I.49), 6-phosphogluconate dehydrogenase (EC I.I.I.44), malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) (EC I.I.I.40), ATP-citrate (pro-3S) lyase (EC 4.I.3.8), 6-phosphofructokinase EC 2.7.I.II), pyruvate kinase (EC 2.7.I.40) and fructose-1,6-bisphosphatase (EC 3.I.3.II) were studied. Bagasse had no effect on body-weight gain, food consumption or faecal dry weight. Bagasse decreased the activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphofructokinase expressed on a wet weight basis and on a protein basis. Bagasse decreased the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase expressed on a body-weight basis. These results suggest that bagasse decreases the flux through some pathways of hepatic lipogenesis when mice are given high-sucrose diets.

  10. Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism

    Directory of Open Access Journals (Sweden)

    Yang M

    2014-08-01

    Full Text Available Ming Yang,1 Huizhong Su,1 Tomoyoshi Soga,2 Kamil R Kranc,3 Patrick J Pollard1 1Cancer Biology and Metabolism Group, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; 2Institute for Advanced Biosciences, Keio University, Mizukami, Tsuruoka, Yamagata, Japan; 3MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Abstract: The hypoxia-inducible factor (HIF prolyl hydroxylase domain enzymes (PHDs regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2 availability enables its association with the von Hippel-Lindau (VHL tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFβ and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism. Keywords: prolyl hydroxylase domain (PHD

  11. STUDY ON THE SUGAR-ACID RATIO AND RELEVANT METABOLIZING ENZYME ACTIVITIES IN NAVEL ORANGE FRUITS FROM DIFFERENT ECO-REGIONS

    Directory of Open Access Journals (Sweden)

    GONG RONGGAO

    2015-12-01

    Full Text Available ABSTRACT The flavor quality of citrus fruits is largely determined by the sugar-acid ratio, but it remains uncertain how sugar- and/or acid-metabolizing enzymes regulate the sugar-acid ratio of navel oranges and further affect the fruit quality. In the present study, Robertson navel oranges (Citrus sinesis Osb. were collected from six representative habitats in three eco-regions of Sichuan, China. The changes in the sugar-acid ratio and the activities of sucrose phosphate synthase (SPS, sucrose synthase (SS, cytosolic cio-aconitase (ACO, and isocitrate dehydrogenase (IDH were examined in navel oranges during fruit development. The results indicated that the sugar-acid ratio of fruits in different eco-regions changed significantly from 150 days after full bloom. The SPS and cytosolic ACO fruit activities had minor changes among different ecoregions throughout the experimental periods, whereas the activities of SS and IDH changed significantly in fruits among three eco-regions. Furthermore, the sugar-acid ratio and the activities of SS in the synthetic direction and IDH were the highest in south subtropics and the lowest in north mid-subtropics, probably due to the effects of climate conditions and/or other relevant eco-factors. It demonstrated that SS in the synthetic direction and IDH were of greater importance in regulating the sugar-acid ratio of navel oranges in different eco-regions, which provided new insights into the factors that determine the flavor quality of navel oranges and valuable data for guiding relevant agricultural practices.

  12. Effects of Cyclamen trochopteranthum on hepatic drug-metabolizing enzymes

    Directory of Open Access Journals (Sweden)

    Arslan Sevki

    2011-01-01

    Full Text Available The modulatory effects of the Cyclamen trochopterantum tuber extract on hepatic drug-metabolizing enzymes, including aniline 4-hydroxylase (A4H; CYP2E1, ethoxyresorufin O-deethylase (EROD; CYP1A, methoxyresorufin O-demethylase (MROD; CYP1A, caffeine N-demethylase (C3ND; CYP1A2 aminopyrene N-demethylase (APND; CYP2C6, and erythromycin N-demethylase (ERND; CYP3A1, were examined in vivo in rats. The activities of all of these enzymes were induced by the cyclamen extract. In addition, Western-blot and RT-PCR results clearly showed that CYP2E1, CYP1A1/CYP1A2 and CYP2C6 protein and mRNA levels were substantially increased by four different doses of cyclamen. Although, the CYP3A1 protein level was increased significantly, the mRNA level was not changed. These results indicate that cyclamen tuber extract might have a potential not only to inhibit and/or induce the metabolism of certain co-administered drugs but also influence the development of toxicity and carcinogenesis due to the induction of the cytochrome P450-dependent drug-metabolizing enzymes.

  13. Metabolic enzymes link morphine withdrawal with metabolic disorder

    Institute of Scientific and Technical Information of China (English)

    Xi Jiang; Jing Li; Lan Ma

    2007-01-01

    @@ Energy metabolism is a fundamental biological process that is vital for the survival of all species. Disorders in the metabolic system result in deficiency or redundancy of certain nutrients, including carbohydrates, lipids, amino acids, etc. Abnormality of the energy metabolism system leads to a number of metabolic diseases, such as the metabolic syndrome. Broadly speaking, the term "metabolic diseases" now tends to be widened to the category that refers to all diseases with metabolism disorder.

  14. Evolution of enzymes in metabolism: a network perspective.

    Science.gov (United States)

    Alves, Rui; Chaleil, Raphael A G; Sternberg, Michael J E

    2002-07-19

    Several models have been proposed to explain the origin and evolution of enzymes in metabolic pathways. Initially, the retro-evolution model proposed that, as enzymes at the end of pathways depleted their substrates in the primordial soup, there was a pressure for earlier enzymes in pathways to be created, using the later ones as initial template, in order to replenish the pools of depleted metabolites. Later, the recruitment model proposed that initial templates from other pathways could be used as long as those enzymes were similar in chemistry or substrate specificity. These two models have dominated recent studies of enzyme evolution. These studies are constrained by either the small scale of the study or the artificial restrictions imposed by pathway definitions. Here, a network approach is used to study enzyme evolution in fully sequenced genomes, thus removing both constraints. We find that homologous pairs of enzymes are roughly twice as likely to have evolved from enzymes that are less than three steps away from each other in the reaction network than pairs of non-homologous enzymes. These results, together with the conservation of the type of chemical reaction catalyzed by evolutionarily related enzymes, suggest that functional blocks of similar chemistry have evolved within metabolic networks. One possible explanation for these observations is that this local evolution phenomenon is likely to cause less global physiological disruptions in metabolism than evolution of enzymes from other enzymes that are distant from them in the metabolic network.

  15. Increase in sphingolipid catabolic enzyme activity during aging

    Institute of Scientific and Technical Information of China (English)

    Santosh J SACKET; Hae-young CHUNG; Fumikazu OKAJIMA; Dong-soon IM

    2009-01-01

    Aim:To understand the contribution of sphingolipid metabolism and its metabolites to development and aging.Methods: A systemic analysis on the changes in activity of sphingolipid metabolic enzymes in kidney, liver and brain tissues during development and aging was conducted. The study was conducted using tissues from 1-day-old to 720-day-old rats.Results: Catabolic enzyme activities as well as the level of sphingomyelinase (SMase) and ceramidase (CDase) were higher than that of anabolic enzyme activities, sphingomyelin synthase and ceramide synthase. This suggested an accumulation of ceramide and sphingosine during development and aging. The liver showed the highest neutral-SMase activity among the tested enzymes while the kidney and brain exhibited higher neutral-SMase and ceramidase activities, indicating a high production of ceramide in liver and ceramide/sphingosine in the kidney and brain. The activities of sphingolipid metabolic enzymes were significantly elevated in all tested tissues during development and aging, although the onset of significant increase in activity varied on the tissue and enzyme type. During aging, 18 out of 21 enzyme activities were further increased on day 720 compared to day 180.Conclusion: Differential increases in sphingolipid metabolic enzyme activities suggest that sphingolipids including ceramide and sphingosine might play important and dynamic roles in proliferation, differentiation and apoptosis during development and aging.

  16. Exploration of the spontaneous fluctuating activity of single enzyme molecules

    NARCIS (Netherlands)

    A. Schwabe (Anne); T.R. Maarleveld (Timo); F.J. Bruggeman (Frank)

    2013-01-01

    htmlabstractSingle enzyme molecules display inevitable, stochastic fluctuations in their catalytic activity. In metabolism, for instance, the stochastic activity of individual enzymes is averaged out due to their high copy numbers per single cell. However, many processes inside cells rely on single

  17. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Directory of Open Access Journals (Sweden)

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  18. Interplay of drug metabolizing enzymes with cellular transporters.

    Science.gov (United States)

    Böhmdorfer, Michaela; Maier-Salamon, Alexandra; Riha, Juliane; Brenner, Stefan; Höferl, Martina; Jäger, Walter

    2014-11-01

    Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.

  19. Alginate Immobilization of Metabolic Enzymes (AIME) for High ...

    Science.gov (United States)

    Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput screening (HTS) assays to assess chemical perturbations of molecular and cellular endpoints. A key criticism of using HTS assays for toxicity assessment is the lack of xenobiotic metabolism (XM) which precludes both metabolic detoxification as well as bioactivation of chemicals tested in vitro thereby mischaracterizing the potential risk posed by these chemicals. To address this deficiency, we have developed an extracellular platform to retrofit existing HTS assays with XM activity. This platform utilizes the S9 fraction of liver homogenate encapsulated in an alginate gel network which reduces the cytotoxicity caused by direct addition of S9 to cells in culture. Alginate microspheres containing encapsulated human liver S9 were cross-linked to solid supports extending from a 96-well plate lid and were assayed using a pro-luciferin substrate specific for CYP3A4 (IPA). We demonstrate that S9 was successfully encapsulated and remained enzymatically active post-encapsulation with 5-10X the CYP3A4 activity as compared to 1 µg solubilized human liver S9. Ketoconazole, a known inhibitor of human CYP3A4, inhibited CYP3A4 activity in a concentration-dependent manner (IC50: 0.27 µM) and inhibiti

  20. Effects of glucose, insulin and triiodothyroxine on leptin and leptin receptor expression and the effects of leptin on activities of enzymes related to glucose metabolism in grass carp (Ctenopharyngodon idella) hepatocytes.

    Science.gov (United States)

    Lu, Rong-Hua; Zhou, Yi; Yuan, Xiao-Chen; Liang, Xu-Fang; Fang, Liu; Bai, Xiao-Li; Wang, Min; Zhao, Yu-Hua

    2015-08-01

    Leptin is an important regulator of appetite and energy expenditure in mammals, but its role in fish metabolism control is poorly understood. Our previous studies demonstrated that leptin has an effect on the regulation of food intake and energy expenditure as well as lipid metabolism (stimulation of lipolysis and inhibition of adipogenesis) in the grass carp Ctenopharyngodon idella. To further investigate the role of leptin in fish, the effects of glucose, insulin and triiodothyroxine (T3) on the expression levels of leptin and leptin receptor (Lepr) and the effects of leptin on the activities of critical glucose metabolism enzymes in grass carp hepatocytes were evaluated in the present study. Our data indicated that leptin gene expression was induced by glucose in a dose-dependent manner, while Lepr gene expression exhibited a biphasic change. A high dose of insulin (100 ng/mL) significantly up-regulated the expression of leptin and Lepr. Leptin expression was markedly up-regulated by a low concentration of T3 but inhibited by a high concentration of T3. T3 up-regulated Lepr expression in a dose-dependent manner. Together, these data suggest that leptin had a close relationship with three factors (glucose, insulin and T3) and might participate in the regulation of glucose metabolism in grass carp. In addition, we also found that leptin affected the activities of key enzymes that are involved in glucose metabolism, which might be mediated by insulin receptor substrate-phosphoinositol 3-kinase signaling.

  1. Comparison of metabolism of sesamin and episesamin by drug-metabolizing enzymes in human liver.

    Science.gov (United States)

    Yasuda, Kaori; Ikushiro, Shinichi; Wakayama, Shuto; Itoh, Toshimasa; Yamamoto, Keiko; Kamakura, Masaki; Munetsuna, Eiji; Ohta, Miho; Sakaki, Toshiyuki

    2012-10-01

    Sesamin and episesamin are two epimeric lignans that are found in refined sesame oil. Commercially available sesamin supplements contain both sesamin and episesamin at an approximate 1:1 ratio. Our previous study clarified the sequential metabolism of sesamin by cytochrome P450 (P450) and UDP-glucuronosyltransferase in human liver. In addition, we revealed that sesamin caused a mechanism-based inhibition (MBI) of CYP2C9, the P450 enzyme responsible for sesamin monocatecholization. In the present study, we compared the metabolism and the MBI of episesamin with those of sesamin. Episesamin was first metabolized to the two epimers of monocatechol, S- and R-monocatechols in human liver microsomes. The P450 enzymes responsible for S- and R-monocatechol formation were CYP2C9 and CYP1A2, respectively. The contribution of CYP2C9 was much larger than that of CYP1A2 in sesamin metabolism, whereas the contribution of CYP2C9 was almost equal to that of CYP1A2 in episesamin metabolism. Docking of episesamin to the active site of CYP1A2 explained the stereoselectivity in CYP1A2-dependent episesamin monocatecholization. Similar to sesamin, the episesamin S- and R-monocatechols were further metabolized to dicatechol, glucuronide, and methylate metabolites in human liver; however, the contribution of each reaction was significantly different between sesamin and episesamin. The liver microsomes from CYP2C19 ultra-rapid metabolizers showed a significant amount of episesamin dicatechol. In this study, we have revealed significantly different metabolism by P450, UDP-glucuronosyltransferase, and catechol-O-methyltransferase for sesamin and episesamin, resulting in different biological effects.

  2. Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

    Directory of Open Access Journals (Sweden)

    Norris Vic

    2013-02-01

    Full Text Available Abstract Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined.

  3. Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

    Science.gov (United States)

    Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2014-02-01

    Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects.

  4. 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.

  5. Tools and strategies for discovering novel enzymes and metabolic pathways

    Directory of Open Access Journals (Sweden)

    John A. Gerlt

    2016-12-01

    Full Text Available The number of entries in the sequence databases continues to increase exponentially – the UniProt database is increasing with a doubling time of ∼4 years (2% increase/month. Approximately 50% of the entries have uncertain, unknown, or incorrect function annotations because these are made by automated methods based on sequence homology. If the potential in complete genome sequences is to be realized, strategies and tools must be developed to facilitate experimental assignment of functions to uncharacterized proteins discovered in genome projects. The Enzyme Function Initiative (EFI; previously supported by U54GM093342 from the National Institutes of Health, now supported by P01GM118303 developed web tools for visualizing and analyzing (1 sequence and function space in protein families (EFI-EST and (2 genome neighbourhoods in microbial and fungal genomes (EFI-GNT to assist the design of experimental strategies for discovering the in vitro activities and in vivo metabolic functions of uncharacterized enzymes. The EFI developed an experimental platform for large-scale production of the solute binding proteins (SBPs for ABC, TRAP, and TCT transport systems and their screening with a physical ligand library to identify the identities of the ligands for these transport systems. Because the genes that encode transport systems are often co-located with the genes that encode the catabolic pathways for the transported solutes, the identity of the SBP ligand together with the EFI-EST and EFI-GNT web tools can be used to discover new enzyme functions and new metabolic pathways. This approach is demonstrated with the characterization of a novel pathway for ethanolamine catabolism.

  6. [Noradrenaline and glycogen content and the activity of several enzymes of carbohydrate metabolism in normal, embryonic, and partly denervated livers and in hepatomas of the rat].

    Science.gov (United States)

    Iljin, S V; Shanigina, K I; Sydow, G; Parfhenova, N S

    1977-01-01

    The noradrenaline and glycogen contents as well as hexokinase, glucokinase and glucose-6-phosphatase activities were determined in normal, embryonic and partially denervated (bilateral dissection of the Nervus splanchnicus or Nervus vagus) rat liver and in two transplantable hepatomas. In embryonic liver and hepatomas a strong decrease or complete loss of noradrenaline and glycogen levels and glucokinase and glucose-6-phosphatase activities is demonstrable as compared to the livers of adult animals, while the hexokinase activity is enhanced. Following bilateral splanchnicotomy the glycogen content and hexokinase activity are enhanced; the glucose-6-phosphatase activity is reduced, and the liver does not contain any noradrenaline. Bilateral vagotomy causes decrease of the glycogen content, of the hexokinase and glucokinase activities and an enhancement of glucose-6-phosphatase activity. The results lend support to the idea of antagonistic action of the sympathetic and parasympathetic nervous systems upon several partial reactions of carbohydrate metabolism of liver. In addition, it can be assumed that the alterations of the carbohydrate metabolism demonstrable in hepatomas as compared to normal liver are not solely attributable to disturbance or breakdown of the nervous regulation.

  7. [Enzyme kinetics of ligustilide metabolism in rat liver microsomes].

    Science.gov (United States)

    Qian, Min; Shi, Li-fu; Hu, Jin-hong

    2009-04-01

    To study the enzyme kinetics of ligustilide metabolism and the effects of selective CYP450 inhibitors on the metabolism of ligustilide in liver microsomes of rat, a LC-MS method was established for quantitative analysis of ligustilide in liver microsomes incubation system with nitrendipine as internal standard. The determination m/z for ligustilide was 173, and for nitrendipine, 315. An optimum incubation system was found and various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of ligustilide. The results showed that enzyme kinetics of ligustilide could be significantly inhibited by ketoconazole, trimethoprim and a-naphthoflavon but scarcely inhibited by omeprazole, 4-methylpyrazole and quinidine. Therefore, CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme participated in in vitro metabolism of ligustilide.

  8. Diced electrophoresis gel assay for screening enzymes with specified activities.

    Science.gov (United States)

    Komatsu, Toru; Hanaoka, Kenjiro; Adibekian, Alexander; Yoshioka, Kentaro; Terai, Takuya; Ueno, Tasuku; Kawaguchi, Mitsuyasu; Cravatt, Benjamin F; Nagano, Tetsuo

    2013-04-24

    We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.

  9. Disruption of androgen metabolism, regulation and effects : involvement of steroidogenic enzymes

    OpenAIRE

    Fürstenberger, Cornelia

    2014-01-01

    Communication between organs and tissues is predominately controlled by hormones. Hormones regulate a vast variety of physiological and behavioural activities, including metabolism, growth and development, reproduction, sleep and mood. Steroid hormones are characterized by their sterane backbone and are regulated by distinct enzymes which control the balance between their active and their inactive forms. The present studies of this thesis focus on the enzymes which selectively...

  10. Effects of surfactants on the contents of metallothionein, heme and hemoproteins and on the activities of heme oxygenase and drug-metabolizing enzymes in rats pretreated with phenobarbital or. beta. -naphthoflavone

    Energy Technology Data Exchange (ETDEWEB)

    Ariyoshi, Toshihiko; Hasegawa, Hiroyuki; Matsumoto, Hideki; Arizono, Koji (Nagasaki Univ. (Japan))

    1991-01-01

    Synthetic surfactants as major constituent of detergent products are widely used in consumer and industrial fields, and hence environmental and toxicological investigations of surfactants are numerous. In the previous study, the authors observed that intraperitoneal administration of surfactants such as sodium dodecyl sulfate (SDS), sodium n-dodecylbenzenesulfonate (LAS) and polyoxyethyleneglycol nonylphenyl ether (Emulgen 913) to rats depressed the content of microsomal cytochrome P-450, while they enhanced markedly the activity of heme oxygenase, the first and rate-limiting enzyme in heme degradation. In addition, they noted an increase of metallothionein content in the liver of rats treated with LAS. In this study, the authors investigated the effects of surfactants on metallothionein, heme, hemoproteins, heme oxygenase and drug-metabolizing enzymes in the liver of rats pretreated with phenobarbital or {beta}-naphthoflavone.

  11. Enzyme kinetics in drug metabolism: fundamentals and applications.

    Science.gov (United States)

    Nagar, Swati; Argikar, Upendra A; Tweedie, Donald J

    2014-01-01

    Enzymes are protein catalysts that lower the energy barrier for a reaction and speed the rate of a chemical change. The kinetics of reactions catalyzed by enzymes, as well as several mechanisms underlying the kinetics, have been comprehensively studied and written in textbooks (1, 2). The importance of quantitative evaluation of enzymatic processes has been recognized in many fields of study, including biochemistry, molecular biology, and pharmaceutical sciences to name a few. In pharmaceutical sciences, the applications of enzyme kinetics range from hit finding efforts for new chemical entities on a pharmacological target to concentration effect relationships to large-scale biosynthesis. The study of the science of drug metabolism has two principal concepts-rate and extent. While understanding disposition pathways and identification of metabolites provides an insight into the extent of metabolism, kinetics of depletion of substrates (endogenous or exogenous) and formation of metabolites deals with the rate of metabolism. The current textbook specifically focuses on kinetics of drug-metabolizing enzymes, detailing specific enzyme classes, and discusses kinetics as they apply to drug transporters. This textbook also outlines additional factors that contribute to the kinetics of reactions catalyzed by these proteins such as variability in isoforms (pharmacogenomics) and experimental factors including key concepts such as alterations of substrate concentrations due to binding. Applications of these approaches in predicting kinetic parameters and alternative approaches for enzymes (systems biology) and transporters are also discussed. The final section focuses on real-life examples (case studies) to try and exemplify the applications of enzyme kinetic principles. This chapter provides a brief overview outlining some key concepts within each of the sections and the chapters within this textbook.

  12. Doubling the CO{sub 2} concentration enhanced the activity of carbohydrate-metabolism enzymes, source carbohydrate production, photoassimilate transport, and sink strength for Opuntia ficus-indica

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ning; Nobel, P.S. [Univ. of California, Los Angeles, CA (United States)

    1996-03-01

    After exposure to a doubled CO{sub 2} concentration of 750 {mu}mol mol{sup -1} air for about 3 months, glucose and starch in the chlorenchyma of basal cladodes of Opuntia ficus-indica increased 175 and 57%, respectively, compared with the current CO{sub 2} concentration of 370 {mu}mol mol{sup -1}, but sucrose content was virtually unaffected. Doubling the CO{sub 2} concentration increased the noncturnal malate production in basal cladodes by 75%, inorganic phosphate (Pi) by 32% soluble starch synthase activity by 30%, and sucrose-Pi synthase activity by 146%, but did not affect the activity of hexokinase. Doubling CO{sub 2} accelerated phloem transport of sucrose out of the basal cladodes, resulting in a 73% higher dry weight for the daughter cladodes. Doubling CO{sub 2} increased the glucose content in 14-d-old daughter cladodes by 167%, increased nocturnal malate production by 22%, decreased total amino acid content by 61%, and increased soluble starch synthase activity by 30% and sucrose synthase activity by 62%. No downward acclimation of photosynthesis during long-term exposure to elevated CO{sub 2} concentrations occurs for O. ficus-indica, consistent with its higher source capacity and sink strength than under current CO{sub 2}. These changes apparently do not result in Pi limitation of photosynthesis or suppression of genes governing photosynthesis for this perennial Crassulacean acid metabolism species, as occur for some annual crops.

  13. Effects of frying oil and Houttuynia cordata thunb on xenobiotic-metabolizing enzyme system of rodents

    Institute of Scientific and Technical Information of China (English)

    Ya-Yen Chen; Chiao-Ming Chen; Pi-Yu Chao; Tsan-Ju Chang; Jen-Fang Liu

    2005-01-01

    AIM: To evaluate the effects of frying oil and Houttuynia cordata Thunb (H. cordata), a vegetable traditionally consumed in Taiwan, on the xenobiotic-metabolizing enzyme system of rodents.METHODS: Forty-eight Sprague-Dawley rats were fed with a diet containing 0%, 2% or 5% H. cordata powder and 15% fresh soybean oil or 24-h oxidized frying oil (OFO)for 28 d respectively. The level of microsomal protein, total cytochrome 450 content (CYP450) and enzyme activities including NADPH reductase, ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-dealkylase (PROD), aniline hydroxylase (ANH), aminopyrine demethylase (AMD), and quinone reductase (QR) were determined. QR represented phase Ⅱ enzymes, the rest of the enzymes tested represented phase Ⅰ enzymes.RESULTS: The oxidized frying oil feeding produced a significant increase in phase Ⅰ and Ⅱ enzyme systems,including the content of CYP450 and microsomal protein,and the activities of NADPH reductase, EROD, PROD, ANH,AMD and QR in rats (P<0.05). In addition, the activities of EROD, ANH and AMD decreased and QR increased after feeding with H. cordata in OFO-fed group (P<0.05). The feeding with 2% H. cordata diet showed the most significant effect.CONCLUSION: The OFO diet induces phases Ⅰ and Ⅱ enzyme activity, and the 2% H. cordata diet resulted in a better regulation of the xenobiotic-metabolizing enzyme system.

  14. Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets--"Sand Out and Gold Stays".

    Science.gov (United States)

    Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T; Wang, Hong; Yang, Xiao-feng

    2016-02-01

    To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: (1) Histone enzymes are differentially expressed in cardiovascular, immune, and other tissues; (2) our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, and histone methylation/demethylation are in the highest varieties; and (3) histone enzymes are more downregulated than upregulated in metabolic diseases and regulatory T cell (Treg) polarization/ differentiation, but not in tumors. These results have demonstrated a new working model of "Sand out and Gold stays," where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity.

  15. Key Metabolic Enzymes Underlying Astrocytic Upregulation of GABAergic Plasticity

    Directory of Open Access Journals (Sweden)

    Przemysław T. Kaczor

    2017-05-01

    Full Text Available GABAergic plasticity is recognized as a key mechanism of shaping the activity of the neuronal networks. However, its description is challenging because of numerous neuron-specific mechanisms. In particular, while essential role of glial cells in the excitatory plasticity is well established, their involvement in GABAergic plasticity only starts to emerge. To address this problem, we used two models: neuronal cell culture (NC and astrocyte-neuronal co-culture (ANCC, where we chemically induced long-term potentiation at inhibitory synapses (iLTP. iLTP could be induced both in NC and ANCC but in ANCC its extent was larger. Importantly, this functional iLTP manifestation was accompanied by an increase in gephyrin puncta size. Furthermore, blocking astrocyte Krebs cycle with fluoroacetate (FA in ANCC prevented enhancement of both mIPSC amplitude and gephyrin puncta size but this effect was not observed in NC, indicating a key role in neuron-astrocyte cross-talk. Blockade of monocarboxylate transport with α-Cyano-4-hydroxycinnamic acid (4CIN abolished iLTP both in NC and ANCC and in the latter model prevented also enlargement of gephyrin puncta. Similarly, blockade of glycogen phosphorylase with BAYU6751 prevented enlargement of gephyrin puncta upon iLTP induction. Finally, block of glutamine synthetase with methionine sulfoxide (MSO nearly abolished mIPSC increase in both NMDA stimulated cell groups but did not prevent enlargement of gephyrin puncta. In conclusion, we provide further evidence that GABAergic plasticity is strongly regulated by astrocytes and the underlying mechanisms involve key metabolic enzymes. Considering the strategic role of GABAergic interneurons, the plasticity described here indicates possible mechanism whereby metabolism regulates the network activity.

  16. Common catabolic enzyme patterns in a microplankton community of the Humboldt Current System off northern and central-south Chile: Malate dehydrogenase activity as an index of water-column metabolism in an oxygen minimum zone

    Science.gov (United States)

    González, R. R.; Quiñones, R. A.

    2009-07-01

    An extensive subsurface oxygen minimum zone off northern and central-south Chile, associated with the Peru-Chile undercurrent, has important effects on the metabolism of the organisms inhabiting therein. Planktonic species deal with the hypoxic and anoxic environments by relying on biochemical as well as physiological processes related to their anaerobic metabolisms. Here we characterize, for the first time, the potential enzymatic activities involved in the aerobic and anaerobic energy production pathways of microplanktonic organisms (catabolic pathways in the oxygen minimum zone. Malate dehydrogenase had the highest oxidizing activity of nicotinamide adenine dinucleotide (reduced form) in the batch of catabolic enzymatic activities assayed, including potential pyruvate oxidoreductases activity, the electron transport system, and dissimilatory nitrate reductase. Malate dehydrogenase correlated significantly with almost all the enzymes analyzed within and above the oxygen minimum zone, and also with the oxygen concentration and microplankton biomass in the water column of the Humboldt Current System, especially in the oxygen minimum zone off Iquique. These results suggest a possible specific pattern for the catabolic activity of the microplanktonic realm associated with the oxygen minimum zone spread along the Humboldt Current System off Chile. We hypothesize that malate dehydrogenase activity could be an appropriate indicator of microplankton catabolism in the oxygen minimum zone and adjacent areas.

  17. 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.

  18. Long-term fasting in the anadromous Arctic charr is associated with downregulation of metabolic enzyme activity and upregulation of leptin A1 and SOCS expression in the liver.

    Science.gov (United States)

    Jørgensen, Even Hjalmar; Martinsen, Mads; Strøm, Vidar; Hansen, Kristin Elisa Ruud; Ravuri, Chandra Sekhar; Gong, Ningping; Jobling, Malcolm

    2013-09-01

    The life strategy of the anadromous Arctic charr (Salvelinus alpinus) includes several months of voluntary fasting during overwintering in freshwater, leading to emaciation prior to seawater migration in spring. In this study we compared changes in condition, substrate utilization and liver metabolism between captive anadromous charr subjected to food deprivation during late winter and spring, and conspecifics fed in excess. In March, nine out of the 10 sampled fed fish had not eaten, indicating that they were in a voluntary anorexic state. In June, the fed fish were eating and all had higher body mass, condition factor and adiposity than in March. In fasted fish there were only small decreases in body mass, condition factor and adiposity between March and May, but all these parameters decreased markedly from May to June. The fasted fish were depleted in fat and glycogen in June, had suppressed activity of hepatic enzymes involved in lipid metabolism (G6PDH and HOAD) and seemed to rely on protein-derived glucose as a major energy source. This was associated with upregulated liver gene expression of leptin A1, leptin A2, SOCS1, SOCS2 and SOCS3, and reduced IGF-I expression. In an in vitro study with liver slices it was shown that recombinant rainbow trout leptin stimulated SOCS1 and SOCS3 expression, but not SOCS2, IGF-I or genes of enzymes involved in lipid (G6PDH) and amino acid (AspAT) metabolism. It is concluded that liver leptin interacts with SOCS in a paracrine fashion to suppress lipolytic pathways and depress metabolism when fat stores are depleted.

  19. Doubling the CO2 Concentration Enhanced the Activity of Carbohydrate-Metabolism Enzymes, Source Carbohydrate Production, Photoassimilate Transport, and Sink Strength for Opuntia ficus-indica.

    Science.gov (United States)

    Wang, N.; Nobel, P. S.

    1996-01-01

    After exposure to a doubled CO2 concentration of 750 [mu]mol mol-1 air for about 3 months glucose and starch in the chlorenchyma of basal cladodes of Opuntia ficus-indica increased 175 and 57%, respectively, compared with the current CO2 concentration of 370 [mu]mol mol-1, but sucrose content was virtually unaffected. Doubling the CO2 concentration increased the nocturnal malate production in basal cladodes by 75%, inorganic phosphate (Pi) by 32%, soluble starch synthase activity by 30%, and sucrose-Pi synthase activity by 146%, but did not affect the activity of hexokinase. Doubling CO2 accelerated phloem transport of sucrose out of the basal cladodes, resulting in a 73% higher dry weight for the daughter cladodes. Doubling CO2 increased the glucose content in 14-d-old daughter cladodes by 167%, increased nocturnal malate production by 22%, decreased total amino acid content by 61%, and increased soluble starch synthase activity by 30% and sucrose synthase activity by 62%. No downward acclimation of photosynthesis during long-term exposure to elevated CO2 concentrations occurs for O. ficus-indica (M. Cui, P.M. Miller, P.S. Nobel [1993] Plant Physiol 103: 519-524; P.S. Nobel, A.A. Israel [1994] J Exp Bot 45: 295-303), consistent with its higher source capacity and sink strength than under current CO2. These changes apparently do not result in Pi limitation of photosynthesis or suppression of genes governing photosynthesis for this perennial Crassulacean acid metabolism species, as occur for some annual crops. PMID:12226228

  20. Doubling the CO2 Concentration Enhanced the Activity of Carbohydrate-Metabolism Enzymes, Source Carbohydrate Production, Photoassimilate Transport, and Sink Strength for Opuntia ficus-indica.

    Science.gov (United States)

    Wang, N.; Nobel, P. S.

    1996-03-01

    After exposure to a doubled CO2 concentration of 750 [mu]mol mol-1 air for about 3 months glucose and starch in the chlorenchyma of basal cladodes of Opuntia ficus-indica increased 175 and 57%, respectively, compared with the current CO2 concentration of 370 [mu]mol mol-1, but sucrose content was virtually unaffected. Doubling the CO2 concentration increased the nocturnal malate production in basal cladodes by 75%, inorganic phosphate (Pi) by 32%, soluble starch synthase activity by 30%, and sucrose-Pi synthase activity by 146%, but did not affect the activity of hexokinase. Doubling CO2 accelerated phloem transport of sucrose out of the basal cladodes, resulting in a 73% higher dry weight for the daughter cladodes. Doubling CO2 increased the glucose content in 14-d-old daughter cladodes by 167%, increased nocturnal malate production by 22%, decreased total amino acid content by 61%, and increased soluble starch synthase activity by 30% and sucrose synthase activity by 62%. No downward acclimation of photosynthesis during long-term exposure to elevated CO2 concentrations occurs for O. ficus-indica (M. Cui, P.M. Miller, P.S. Nobel [1993] Plant Physiol 103: 519-524; P.S. Nobel, A.A. Israel [1994] J Exp Bot 45: 295-303), consistent with its higher source capacity and sink strength than under current CO2. These changes apparently do not result in Pi limitation of photosynthesis or suppression of genes governing photosynthesis for this perennial Crassulacean acid metabolism species, as occur for some annual crops.

  1. Recuperative effect of Semecarpus anacardium linn. nut milk extract on carbohydrate metabolizing enzymes in experimental mammary carcinoma-bearing rats.

    Science.gov (United States)

    Sujatha, Venugopal; Sachdanandam, Panchanatham

    2002-03-01

    Semecarpus anacardium Linn. of the family Anacardiaceae has many applications in the Ayurvedic and Siddha systems of medicine. We have tested the antitumour activity of Semecarpus anacardium nut extract against experimental mammary carcinoma in animals. As there is a direct relationship between the proliferation of tumour cells and the activities of the glycolytic and gluconeogenic enzymes, we studied changes in the activities of enzymes involved in this metabolic pathway in the liver and kidney. The enzymes investigated were glycolytic enzymes, namely hexokinase, phosphoglucoisomerase, aldolase and the gluconeogenic enzymes, namely glucose-6-phosphatase and fructose-1,6-biphosphatase in experimental rats. A significant rise in glycolytic enzyme activities and a simultaneous fall in gluconeogenic enzyme activities were found in mammary carcinoma bearing rats. Drug administration returned these enzyme activities to their respective control activities. Copyright 2002 John Wiley & Sons, Ltd.

  2. Regional variation in muscle metabolic enzymes in individual American shad (Alosa sapidissima)

    Science.gov (United States)

    Leonard, J.B.K.

    1999-01-01

    Evaluation of the activity of metabolic enzymes is often used to asses metabolic capacity at the tissue level, but the amount of regional variability within a tissue in an individual fish of a given species is frequently unknown. The activities of four enzymes (citrate synthase (CS), phosphofructokinase, lactate dehydrogenase (LDH), and ??-hydroxyacyl coenzyme A dehydrogenase (HOAD) were assayed in red and white muscle at 10 sites along the body of adult American shad (Alosa sapidissima). Red and white muscle HOAD and white muscle CS and LDH varied significantly, generally increasing posteriorly. Maximal variation occurs in red muscle HOAD (~450%) and white muscle LDH (~60%) activity. Differences between the sexes also vary with sampling location. This study suggests that the variability in enzyme activity may be linked to functional differences in the muscle at different locations, and also provides guidelines for sample collection in this species.

  3. Cell organelles from crassulacean acid metabolism (CAM) plants : II. Compartmentation of enzymes of the crassulacean acid metabolism.

    Science.gov (United States)

    Schnarrenberger, C; Groß, D; Burkhard, C; Herbert, M

    1980-02-01

    The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH4)2SO4 gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed.

  4. 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)…

  5. 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)…

  6. Patterns of extracellular enzyme activities and microbial metabolism in an Arctic fjord of Svalbard and in the northern Gulf of Mexico: contrasts in carbon processing by pelagic microbial communities

    Directory of Open Access Journals (Sweden)

    Carol eArnosti

    2013-10-01

    Full Text Available The microbial community composition of polar and temperate ocean waters differs substantially, but the potential functional consequences of these differences are largely unexplored. We measured bacterial production, glucose metabolism, and the abilities of microbial communities to hydrolyze a range of polysaccharides in an Arctic fjord of Svalbard (Smeerenburgfjord, and thus to initiate remineralization of high-molecular weight organic matter. We compared these data with similar measurements previously carried out in the northern Gulf of Mexico in order to investigate whether differences in the spectrum of enzyme activities measurable in Arctic and temperate environments are reflected in ‘downstream’ aspects of microbial metabolism (metabolism of monomers and biomass production. Only 4 of 6 polysaccharide substrates were hydrolyzed in Smeerenburgfjord; all were hydrolyzed in the upper water column of the Gulf. These patterns are consistent on an interannual basis. Bacterial protein production was comparable at both locations, but the pathways of glucose utilization differed. Glucose incorporation rate constants were comparatively higher in Svalbard, but glucose respiration rate constants were higher in surface waters of the Gulf. As a result, at the time of sampling ca. 75% of the glucose was incorporated into biomass in Svalbard, but in the northern Gulf of Mexico most of the glucose was respired to CO2. A limited range of enzyme activities is therefore not a sign of a dormant community or one unable to further process substrates resulting from extracellular enzymatic hydrolysis. The ultimate fate of carbohydrates in marine waters, however, is strongly dependent upon the specific capabilities of heterotrophic microbial communities in these disparate environments.

  7. Patterns of extracellular enzyme activities and microbial metabolism in an Arctic fjord of Svalbard and in the northern Gulf of Mexico: contrasts in carbon processing by pelagic microbial communities.

    Science.gov (United States)

    Arnosti, Carol; Steen, Andrew D

    2013-01-01

    The microbial community composition of polar and temperate ocean waters differs substantially, but the potential functional consequences of these differences are largely unexplored. We measured bacterial production, glucose metabolism, and the abilities of microbial communities to hydrolyze a range of polysaccharides in an Arctic fjord of Svalbard (Smeerenburg Fjord), and thus to initiate remineralization of high-molecular weight organic matter. We compared these data with similar measurements previously carried out in the northern Gulf of Mexico in order to investigate whether differences in the spectrum of enzyme activities measurable in Arctic and temperate environments are reflected in "downstream" aspects of microbial metabolism (metabolism of monomers and biomass production). Only four of six polysaccharide substrates were hydrolyzed in Smeerenburg Fjord; all were hydrolyzed in the upper water column of the Gulf. These patterns are consistent on an interannual basis. Bacterial protein production was comparable at both locations, but the pathways of glucose utilization differed. Glucose incorporation rate constants were comparatively higher in Svalbard, but glucose respiration rate constants were higher in surface waters of the Gulf. As a result, at the time of sampling ca. 75% of the glucose was incorporated into biomass in Svalbard, but in the northern Gulf of Mexico most of the glucose was respired to CO2. A limited range of enzyme activities is therefore not a sign of a dormant community or one unable to further process substrates resulting from extracellular enzymatic hydrolysis. The ultimate fate of carbohydrates in marine waters, however, is strongly dependent upon the specific capabilities of heterotrophic microbial communities in these disparate environments.

  8. AMPK Activation Affects Glutamate Metabolism in Astrocytes

    DEFF Research Database (Denmark)

    Voss, Caroline Marie; Pajęcka, Kamilla; Stridh, Malin H

    2015-01-01

    on glutamate metabolism in astrocytes was studied using primary cultures of these cells from mouse cerebral cortex during incubation in media containing 2.5 mM glucose and 100 µM [U-(13)C]glutamate. The metabolism of glutamate including a detailed analysis of its metabolic pathways involving the tricarboxylic...... acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon...... affected by a reduction of the flux of glutamate derived carbon through the malic enzyme and pyruvate carboxylase catalyzed reactions. Finally, it was found that in the presence of glutamate as an additional substrate, glucose metabolism monitored by the use of tritiated deoxyglucose was unaffected by AMPK...

  9. Radiation Exposure Alters Expression of Metabolic Enzyme Genes in Mice

    Science.gov (United States)

    Wotring, V. E.; Mangala, L. S.; Zhang, Y.; Wu, H.

    2011-01-01

    Most administered pharmaceuticals are metabolized by the liver. The health of the liver, especially the rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand the effects of spaceflight on the enzymes of the liver and exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments. Additionally, it has been previous noted that pre-exposure to small radiation doses seems to confer protection against later and larger radiation doses. This protective power of pre-exposure has been called a priming effect or radioadaptation. This study is an effort to examine the drug metabolizing effects of radioadaptation mechanisms that may be triggered by early exposure to low radiation doses.

  10. 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.

  11. Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Nielsen, John E; Jørgensen, Anne

    2010-01-01

    , since it is not solely dependent on VDR expression, but also on cellular uptake of circulating VD and presence and activity of VD metabolizing enzymes. Expression of VD metabolizing enzymes has not previously been investigated in human testis and male reproductive tract. Therefore, we performed......The vitamin D receptor (VDR) is expressed in human testis, and vitamin D (VD) has been suggested to affect survival and function of mature spermatozoa. Indeed, VDR knockout mice and VD deficient rats show decreased sperm counts and low fertility. However, the cellular response to VD is complex...

  12. Polycyclic aromatic hydrocarbon (PAH) metabolizing enzyme activities in human lung, and their inducibility by exposure to naphthalene, phenanthrene, pyrene, chrysene, and benzo(a)pyrene as shown in the rat lung and liver

    Energy Technology Data Exchange (ETDEWEB)

    Elovaara, Eivor; Mikkola, Jouni; Stockmann-Juvala, Helene; Vainio, Harri [Finnish Institute of Occupational Health, Helsinki (Finland); Luukkanen, Leena [Finnish Institute of Occupational Health, Helsinki (Finland); University of Helsinki, Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Helsinki (Finland); Keski-Hynnilae, Helena; Kostiainen, Risto [University of Helsinki, Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Helsinki (Finland); Pasanen, Markku [University of Oulu, Department of Pharmacology and Toxicology, Oulu (Finland); University of Kuopio, Department of Pharmacology and Toxicology, Kuopio (Finland); Pelkonen, Olavi [University of Oulu, Department of Pharmacology and Toxicology, Oulu (Finland)

    2007-03-15

    In order to survey changes and activities in the polycyclic aromatic hydrocarbon (PAH)-metabolizing enzymes implicated in lung cancer susceptibility studies, we investigated enzyme induction by 2-5-ring-sized 'biomarker' PAHs in rat liver and lung, and the activities in five human lung specimens. Naphthalene, phenanthrene, pyrene, chrysene, and benzo[a]pyrene (BaP) were administered to rats for 3 days (25-128 mg/kg/day) and the responses compared with those of model inducers. PAH treatment increased the CYP1A-catalyzed activity of pyrene 1-hydroxylation and 7-ethoxyresorufin O-deethylation in rat liver by up to 28- and 279-fold, and in rat lung by up to 22- and 51-fold, respectively. 1-Naphthol (hUGT1A6), 1-hydroxypyrene (hUGT1A6/1A9), and entacapone (hUGT1A9) are markers of PAH-glucuronidating human uridine diphosphate-glucuronosyltransferases (UGT). These activities increased up to 6.4-fold in rat liver and up to 1.9-fold in rat lung. NADPH:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase activities increased up to 5.3- and 1.6-fold (liver), and up to 4.4- and 1.4-fold (lung), respectively. CYP1A showed the best liver-to-lung relationship (R {sup 2} = 0.90). The inducing efficiency by PAHs differed extensively: control {<=} naphthalene < phenanthrene, pyrene << chrysene < BaP. In human lung (non-smokers), the marker activities of CYP1A1, UGT1A6/1A9, and NQO1 were lower than those in rat lung. Epoxide hydrolase activity was 1,000-fold higher than the pulmonary CYP1A1 activities. Human UGT and NQO1 displayed large variations (>60-fold), many times greater than the experimental (inducible/constitutive) variation in the rat. Kinetics of 1-hydroxypyrene glucuronidation showed two low-K{sub m} forms both in rat and human lung. Since the 2-4-ring PAHs (major constituents) were poor enzyme inducers, it appears that the PAH-metabolizing pathways are mainly induced by BaP-type minor constituents. Gene-environmental interactions which magnify

  13. Enhancing flora balance in the gastrointestinal tract of mice by lactic acid bacteria from Chinese sourdough and enzyme activities indicative of metabolism of protein, fat, and carbohydrate by the flora.

    Science.gov (United States)

    Yang, Dong; Yu, Xiaomin; Wu, Yaoping; Chen, Xingxing; Wei, Hua; Shah, Nagendra P; Xu, Feng

    2016-10-01

    In this study, we investigated the effect of administration of 5 strains of lactic acid bacteria (LAB) isolated from traditional Chinese sourdough on the flora balance of gastrointestinal tract of mice. We specifically measured Enterococcus, Enterobacter, Bacteroides, and Lactobacillus by plate count and real-time PCR methods, and α-glucosidase, lactate dehydrogenase, esterase, and aminopeptidase activities as indicative of metabolism of sugar, fat, and protein from LAB isolated from feces of mice in vitro. The results showed that administration of Lactobacillus acidophilus LAC0201 and Lactobacillus fermentum LFE0302 lowered the uricacid index of serum. Lactobacillus acidophilus LAC0201, L. fermentum LFE0302, as well as Lactobacillus curvatus LCU0401 administration resulted in a reduction in the opportunistic pathogens (i.e., Enterococcus and Enterobacter), meanwhile, administration of L. fermentum LFE0302 and Lactobacillus sp. ULA0104 resulted in an increase in the counts of Lactobacillus. Lactobacillus fermentum LFE0302 administration increased starch digestion of intestinal flora after 4wk of feeding and also resulted in increased α-glucosidase activity in the intestinal flora after 3wk of feeding. We found a similar trend in esterase activity after administration of L. acidophilus LAC0201 for 3wk. Hence, our study suggested that LAB from Chinese sourdough might be used as potential probiotics to strengthen the flora balance in gastrointestinal tract and positively change the metabolism of nutrients through bacterial enzyme activities. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Human Metabolic Enzymes Deficiency: A Genetic Mutation Based Approach

    Science.gov (United States)

    Chaturvedi, Swati; Singh, Ashok K.; Maity, Siddhartha; Sarkar, Srimanta

    2016-01-01

    One of the extreme challenges in biology is to ameliorate the understanding of the mechanisms which emphasize metabolic enzyme deficiency (MED) and how these pretend to have influence on human health. However, it has been manifested that MED could be either inherited as inborn error of metabolism (IEM) or acquired, which carries a high risk of interrupted biochemical reactions. Enzyme deficiency results in accumulation of toxic compounds that may disrupt normal organ functions and cause failure in producing crucial biological compounds and other intermediates. The MED related disorders cover widespread clinical presentations and can involve almost any organ system. To sum up the causal factors of almost all the MED-associated disorders, we decided to embark on a less traveled but nonetheless relevant direction, by focusing our attention on associated gene family products, regulation of their expression, genetic mutation, and mutation types. In addition, the review also outlines the clinical presentations as well as diagnostic and therapeutic approaches. PMID:27051561

  15. Low salinity acclimation and thyroid hormone metabolizing enzymes in gilthead seabream (Sparus auratus)

    NARCIS (Netherlands)

    Klaren, P.H.M.; Guzman, J.M.; Reutelingsperger, S.J.; Mancera, J.M.; Flik, G.

    2007-01-01

    We investigated the effect of acclimation to low salinity water of gilthead seabream (Sparus auratus), a euryhaline seawater teleost, on the activities of thyroid hormone-metabolizing enzymes in gills, kidney, and liver. Following acclimation to low salinity water, the plasma free thyroxine (T(4))

  16. Low salinity acclimation and thyroid hormone metabolizing enzymes in gilthead seabream (Sparus auratus)

    NARCIS (Netherlands)

    Klaren, P.H.M.; Guzman, J.M.; Reutelingsperger, S.J.; Mancera, J.M.; Flik, G.

    2007-01-01

    We investigated the effect of acclimation to low salinity water of gilthead seabream (Sparus auratus), a euryhaline seawater teleost, on the activities of thyroid hormone-metabolizing enzymes in gills, kidney, and liver. Following acclimation to low salinity water, the plasma free thyroxine (T(4)) c

  17. [Glutathione redox system, immune status, antioxidant enzymes and metabolism of purine nucleotides in hypothyroidism].

    Science.gov (United States)

    Tapbergenov, S O; Sovetov, B S; Bekbosynova, R B; Bolysbekova, S M

    2015-01-01

    The immune status, components of the glutathione redox system, the activity of antioxidant enzymes and metabolism of purine nucleotides have been investigated in animals with experimental hypothyroidism. On day 8 after an increase in the number of leukocytes, lymphocytes, T-helpers and T-suppressors as well as increased number of B-lymphocytes was found in blood of thyroidectomized rats. This was accompanied by decreased activity of adenosine deaminase (AD), AMP-deaminase (AMPD), and 5'-nucleotidase (5'N) in blood, but the ratio of enzyme activity AD/AMPD increased. These changes in the activity of enzymes, involved in purine catabolism can be regarded as increased functional relationships between T and B lymphocytes in hypothyroidism. The functional changes of immune system cells were accompanied by increased activity of glutathione peroxidase (GPx), a decrease in the activity of superoxide dismutase (SOD), glutathione reductase (GR) and the ratio GH/GPx. Thyroidectomized rats had increased amounts of total, oxidized (GSSG) and reduced glutathione (GSH), but the ratio GSH/GSSG decerased as compared with control animals. In the liver, hypothyroidism resulted in activation of SOD, GPx, decreased activity of GR and decreased ratio GR/GPx. At the same time, the levels of total, oxidized, and reduced glutathione increased, but the ratio GSH/GSSG as well as activities of enzymes involved in purine nucleotide metabolism ratio (and their ratio 5'N/AD + AMPD) decreased. All these data suggest a functional relationship of the glutathione redox system not only with antioxidant enzymes, but also activity of enzymes involved purine nucleotide metabolism and immune status.

  18. Enzymes of energy metabolism in hatchlings of amazonian freshwater turtles (Testudines, Podocnemididae).

    Science.gov (United States)

    Duncan, W P; Marcon, J L

    2009-05-01

    The metabolic profiles of selected tissues were analyzed in hatchlings of the Amazonian freshwater turtles Podocnemis expansa, P. unifilis and P. sextuberculata. Metabolic design in these species was judged based on the key enzymes of energy metabolism, with special emphasis on carbohydrate, lipid, amino acid and ketone body metabolism. All species showed a high glycolytic potential in all sampled tissues. Based on low levels of hexokinase, glycogen may be an important fuel for these species. The high lactate dehydrogenase activity in the liver may play a significant role in carbohydrate catabolism, possibly during diving. Oxidative metabolism in P. sextuberculata appears to be designed for the use of lipids, amino acids and ketone bodies. The maximal activities of 3-hydroxyacyl-CoA dehydrogenase, malate dehydrogenase, glutamine dehydrogenase, alanine aminotransferase and succinyl-CoA keto transferase display high aerobic potential, especially in muscle and liver tissues of this species. Although amino acids and ketone bodies may be important fuels for oxidative metabolism, carbohydrates and lipids are the major fuels used by P. expansa and P. unifilis. Our results are consistent with the food habits and lifestyle of Amazonian freshwater turtles. The metabolic design, based on enzyme activities, suggests that hatchlings of P. unifilis and P. expansa are predominately herbivorous, whereas P. sextuberculata rely on a mixed diet of animal matter and vegetation.

  19. Enzymes of energy metabolism in hatchlings of amazonian freshwater turtles (Testudines, Podocnemididae

    Directory of Open Access Journals (Sweden)

    WP. Duncan

    Full Text Available The metabolic profiles of selected tissues were analyzed in hatchlings of the Amazonian freshwater turtles Podocnemis expansa, P. unifilis and P. sextuberculata. Metabolic design in these species was judged based on the key enzymes of energy metabolism, with special emphasis on carbohydrate, lipid, amino acid and ketone body metabolism. All species showed a high glycolytic potential in all sampled tissues. Based on low levels of hexokinase, glycogen may be an important fuel for these species. The high lactate dehydrogenase activity in the liver may play a significant role in carbohydrate catabolism, possibly during diving. Oxidative metabolism in P. sextuberculata appears to be designed for the use of lipids, amino acids and ketone bodies. The maximal activities of 3-hydroxyacyl-CoA dehydrogenase, malate dehydrogenase, glutamine dehydrogenase, alanine aminotransferase and succinyl-CoA keto transferase display high aerobic potential, especially in muscle and liver tissues of this species. Although amino acids and ketone bodies may be important fuels for oxidative metabolism, carbohydrates and lipids are the major fuels used by P. expansa and P. unifilis. Our results are consistent with the food habits and lifestyle of Amazonian freshwater turtles. The metabolic design, based on enzyme activities, suggests that hatchlings of P. unifilis and P. expansa are predominately herbivorous, whereas P. sextuberculata rely on a mixed diet of animal matter and vegetation.

  20. Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

    Science.gov (United States)

    Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

    2015-01-01

    In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively.

  1. Enzyme Activity Experiments Using a Simple Spectrophotometer

    Science.gov (United States)

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  2. Enzyme Activity Experiments Using a Simple Spectrophotometer

    Science.gov (United States)

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  3. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    Directory of Open Access Journals (Sweden)

    Fiona Karen Harlan

    Full Text Available Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research

  4. Co-administration of creatine plus pyruvate prevents the effects of phenylalanine administration to female rats during pregnancy and lactation on enzymes activity of energy metabolism in cerebral cortex and hippocampus of the offspring.

    Science.gov (United States)

    Bortoluzzi, Vanessa Trindade; de Franceschi, Itiane Diehl; Rieger, Elenara; Wannmacher, Clóvis Milton Duval

    2014-08-01

    Phenylketonuria (PKU) is the most frequent inborn error of metabolism. It is caused by deficiency in the activity of phenylalanine hydroxylase, leading to accumulation of phenylalanine and its metabolites. Untreated maternal PKU or hyperphenylalaninemia may result in nonphenylketonuric offspring with low birth weight and neonatal sequelae, especially microcephaly and intellectual disability. The mechanisms underlying the neuropathology of brain injury in maternal PKU syndrome are poorly understood. In the present study, we evaluated the possible preventive effect of the co-administration of creatine plus pyruvate on the effects elicited by phenylalanine administration to female Wistar rats during pregnancy and lactation on some enzymes involved in the phosphoryltransfer network in the brain cortex and hippocampus of the offspring at 21 days of age. Phenylalanine administration provoked diminution of body, brain cortex an hippocampus weight and decrease of adenylate kinase, mitochondrial and cytosolic creatine kinase activities. Co-administration of creatine plus pyruvate was effective in the prevention of those alterations provoked by phenylalanine, suggesting that altered energy metabolism may be important in the pathophysiology of maternal PKU. If these alterations also occur in maternal PKU, it is possible that pyruvate and creatine supplementation to the phenylalanine-restricted diet might be beneficial to phenylketonuric mothers.

  5. Establishing population distribution of drug-metabolizing enzyme activities for the use of salivary caffeine as a dynamic liver function marker in a Singaporean Chinese population.

    Science.gov (United States)

    Chia, Hazel Yiting; Yau, Wai-Ping; Ho, Han Kiat

    2016-04-01

    The salivary paraxanthine/caffeine molar ratio has been proposed as a novel dynamic liver function test to guide dose adjustments of drugs hepatically cleared by CYP1A2. Its usability requires an established population norm as well as the factors influencing the ratio and actual concentrations. To address this knowledge gap, salivary caffeine and paraxanthine concentrations were measured at 4 h post caffeine dose in healthy Chinese individuals who had undergone 24 h of caffeine abstinence. The metabolic ratio was calculated and statistical analysis was performed. From the 52 participants (26 males; 30 regular caffeine consumers) recruited, the salivary paraxanthine/caffeine molar ratio was normally distributed with a mean and SD of 0.5 ± 0.2. No statistically significant factors (BMI, body weight, gender and regularity of caffeine intake) affecting the metabolic ratio were found. The caffeine concentration and total caffeine plus paraxanthine concentrations were lower in males than in females, and lower in regular caffeine consumers than in non-regular caffeine consumers. The 4 h salivary metabolic ratio (mean: 0.5) was generally not significantly different from the literature reported salivary, serum and plasma ratios measured at 4-9 h in healthy individuals (mean range 0.4-0.7) but was significantly higher than the literature reported 6 h plasma ratio and salivary ratios measured at 1-6 h in patients with liver disease or mild abnormal liver function tests (mean range 0.03-0.2). Overall, the population norm of the salivary metabolic ratio in a Singaporean Chinese population established in this study is distinct from individuals with liver disease or mild abnormal liver function tests and provides the benchmark for dosage adjustments of drugs metabolized by CYP1A2. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Regulation of eNOS enzyme activity by posttranslational modification.

    Science.gov (United States)

    Heiss, Elke H; Dirsch, Verena M

    2014-01-01

    The regulation of endothelial NO synthase (eNOS) employs multiple different cellular control mechanisms impinging on level and activity of the enzyme. This review aims at summarizing the current knowledge on the posttranslational modifications of eNOS, including acylation, nitrosylation, phosphorylation, acetylation, glycosylation and glutathionylation. Sites, mediators and impact on enzyme localization and activity of the single modifications will be discussed. Moreover, interdependence, cooperativity and competition between the different posttranslational modifications will be elaborated with special emphasis on the susceptibility of eNOS to metabolic cues.

  7. Functions for diverse metabolic activities in heterochromatin.

    Science.gov (United States)

    Su, Xue Bessie; Pillus, Lorraine

    2016-03-15

    Growing evidence demonstrates that metabolism and chromatin dynamics are not separate processes but that they functionally intersect in many ways. For example, the lysine biosynthetic enzyme homocitrate synthase was recently shown to have unexpected functions in DNA damage repair, raising the question of whether other amino acid metabolic enzymes participate in chromatin regulation. Using an in silico screen combined with reporter assays, we discovered that a diverse range of metabolic enzymes function in heterochromatin regulation. Extended analysis of the glutamate dehydrogenase 1 (Gdh1) revealed that it regulates silent information regulator complex recruitment to telomeres and ribosomal DNA. Enhanced N-terminal histone H3 proteolysis is observed in GDH1 mutants, consistent with telomeric silencing defects. A conserved catalytic Asp residue is required for Gdh1's functions in telomeric silencing and H3 clipping. Genetic modulation of α-ketoglutarate levels demonstrates a key regulatory role for this metabolite in telomeric silencing. The metabolic activity of glutamate dehydrogenase thus has important and previously unsuspected roles in regulating chromatin-related processes.

  8. Concentration of specific amino acids at the catalytic/active centers of highly-conserved "housekeeping" enzymes of central metabolism in archaea, bacteria and Eukaryota: is there a widely conserved chemical signal of prebiotic assembly?

    Science.gov (United States)

    Pollack, J Dennis; Pan, Xueliang; Pearl, Dennis K

    2010-06-01

    In alignments of 1969 protein sequences the amino acid glycine and others were found concentrated at most-conserved sites within approximately 15 A of catalytic/active centers (C/AC) of highly conserved kinases, dehydrogenases or lyases of Archaea, Bacteria and Eukaryota. Lysine and glutamic acid were concentrated at least-conserved sites furthest from their C/ACs. Logistic-regression analyses corroborated the "movement" of glycine towards and lysine away from their C/ACs: the odds of a glycine occupying a site were decreased by 19%, while the odds for a lysine were increased by 53%, for every 10 A moving away from the C/AC. Average conservation of MSA consensus sites was highest surrounding the C/AC and directly decreased in transition toward model's peripheries. Findings held with statistical confidence using sequences restricted to individual Domains or enzyme classes or to both. Our data describe variability in the rate of mutation and likelihoods for phylogenetic trees based on protein sequence data and endorse the extension of substitution models by incorporating data on conservation and distance to C/ACs rather than only using cumulative levels. The data support the view that in the most-conserved environment immediately surrounding the C/AC of taxonomically distant and highly conserved essential enzymes of central metabolism there are amino acids whose identity and degree of occupancy is similar to a proposed amino acid set and frequency associated with prebiotic evolution.

  9. The combined effects of reactant kinetics and enzyme stability explain the temperature dependence of metabolic rates.

    Science.gov (United States)

    DeLong, J P; Gibert, J P; Luhring, T M; Bachman, G; Reed, B; Neyer, A; Montooth, K L

    2017-06-01

    A mechanistic understanding of the response of metabolic rate to temperature is essential for understanding thermal ecology and metabolic adaptation. Although the Arrhenius equation has been used to describe the effects of temperature on reaction rates and metabolic traits, it does not adequately describe two aspects of the thermal performance curve (TPC) for metabolic rate-that metabolic rate is a unimodal function of temperature often with maximal values in the biologically relevant temperature range and that activation energies are temperature dependent. We show that the temperature dependence of metabolic rate in ectotherms is well described by an enzyme-assisted Arrhenius (EAAR) model that accounts for the temperature-dependent contribution of enzymes to decreasing the activation energy required for reactions to occur. The model is mechanistically derived using the thermodynamic rules that govern protein stability. We contrast our model with other unimodal functions that also can be used to describe the temperature dependence of metabolic rate to show how the EAAR model provides an important advance over previous work. We fit the EAAR model to metabolic rate data for a variety of taxa to demonstrate the model's utility in describing metabolic rate TPCs while revealing significant differences in thermodynamic properties across species and acclimation temperatures. Our model advances our ability to understand the metabolic and ecological consequences of increases in the mean and variance of temperature associated with global climate change. In addition, the model suggests avenues by which organisms can acclimate and adapt to changing thermal environments. Furthermore, the parameters in the EAAR model generate links between organismal level performance and underlying molecular processes that can be tested for in future work.

  10. Activation of interfacial enzymes at membrane surfaces

    DEFF Research Database (Denmark)

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi;

    2006-01-01

    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s......PLA2), are only activated at the interface between water and membrane surfaces, where they lead to a break-down of the lipid molecules into lysolipids and free fatty acids. The activation is critically dependent on the physical properties of the lipid-membrane substrate. A topical review is given...

  11. Effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism.

    Science.gov (United States)

    Klaassen, C D; Hood, A M

    2001-01-01

    The effects of microsomal enzyme inducers on thyroid hormone homeostasis and the thyroid gland are of concern. We have investigated the effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism in rats. We have shown that small increases in serum TSH can result in large increases in thyroid follicular cell proliferation. Furthermore, only those microsomal enzyme inducers that increase serum TSH--that is, phenobarbital (PB) and pregnenolone-16alpha-carbonitrile (PCN)-increase thyroid follicular cell proliferation, whereas those microsomal enzyme inducers that do not increase serum TSH--that is, 3-methylcholanthrene (3MC) and Aroclor 1254 (PCB)-do not increase thyroid follicular cell proliferation. Deiodination does not appear to be the reason why serum T3 concentrations are maintained in microsomal enzyme inducer-treated rats. We have also shown that those microsomal enzyme inducers that increase serum TSH increase T3 UDP-glucuronosyltransferase (UGT) activity, whereas those microsomal enzyme inducers that do not increase serum TSH do not increase T3 UGT activity. This finding suggests that induction of T3 glucuronidation, rather than T4 glucuronidation, mediates increases in serum TSH of microsomal enzyme inducer treated rats.

  12. A survey of orphan enzyme activities

    Directory of Open Access Journals (Sweden)

    Pouliot Yannick

    2007-07-01

    Full Text Available Abstract Background Using computational database searches, we have demonstrated previously that no gene sequences could be found for at least 36% of enzyme activities that have been assigned an Enzyme Commission number. Here we present a follow-up literature-based survey involving a statistically significant sample of such "orphan" activities. The survey was intended to determine whether sequences for these enzyme activities are truly unknown, or whether these sequences are absent from the public sequence databases but can be found in the literature. Results We demonstrate that for ~80% of sampled orphans, the absence of sequence data is bona fide. Our analyses further substantiate the notion that many of these enzyme activities play biologically important roles. Conclusion This survey points toward significant scientific cost of having such a large fraction of characterized enzyme activities disconnected from sequence data. It also suggests that a larger effort, beginning with a comprehensive survey of all putative orphan activities, would resolve nearly 300 artifactual orphans and reconnect a wealth of enzyme research with modern genomics. For these reasons, we propose that a systematic effort to identify the cognate genes of orphan enzymes be undertaken.

  13. Enzymes of yeast polyphosphate metabolism: structure, enzymology and biological roles.

    Science.gov (United States)

    Gerasimaitė, Rūta; Mayer, Andreas

    2016-02-01

    Inorganic polyphosphate (polyP) is found in all living organisms. The known polyP functions in eukaryotes range from osmoregulation and virulence in parasitic protozoa to modulating blood coagulation, inflammation, bone mineralization and cellular signalling in mammals. However mechanisms of regulation and even the identity of involved proteins in many cases remain obscure. Most of the insights obtained so far stem from studies in the yeast Saccharomyces cerevisiae. Here, we provide a short overview of the properties and functions of known yeast polyP metabolism enzymes and discuss future directions for polyP research.

  14. Metaproteogenomic analysis of a sulfate-reducing enrichment culture reveals genomic organization of key enzymes in the m-xylene degradation pathway and metabolic activity of proteobacteria.

    Science.gov (United States)

    Bozinovski, Dragana; Taubert, Martin; Kleinsteuber, Sabine; Richnow, Hans-Hermann; von Bergen, Martin; Vogt, Carsten; Seifert, Jana

    2014-10-01

    This study aimed to ascertain the functional and phylogenetic relationships within an m-xylene degrading sulfate-reducing enrichment culture, which had been maintained for several years in the laboratory with m-xylene as the sole source of carbon and energy. Previous studies indicated that a phylotype affiliated to the Desulfobacteraceae was the main m-xylene assimilating organism. In the present study, genes and gene products were identified by a metaproteogenomic approach using LC-MS/MS analysis of the microbial community, and 2426 peptides were identified from 576 proteins. In the metagenome of the community, gene clusters encoding enzymes involved in fumarate addition to a methyl moiety of m-xylene (nms, bss), as well as gene clusters coding for enzymes involved in modified beta-oxidation to (3-methyl)benzoyl-CoA (bns), were identified in two separate contigs. Additionally, gene clusters containing homologues to bam genes encoding benzoyl-CoA reductase (Bcr) class II, catalyzing the dearomatization of (3-methyl)benzoyl-CoA, were identified. Time-resolved protein stable isotope probing (protein-SIP) experiments using (13)C-labeled m-xylene showed that the respective gene products were highly (13)C-labeled. The present data suggested the identification of gene products that were similar to those involved in methylnaphthalene degradation even though the consortium was not capable of growing in the presence of naphthalene, methylnaphthalene or toluene as substrates. Thus, a novel branch of enzymes was found that was probably specific for anaerobic m-xylene degradation.

  15. The RNA world and the origin of metabolic enzymes.

    Science.gov (United States)

    Ralser, Markus

    2014-08-01

    An RNA world has been placed centre stage for explaining the origin of life. Indeed, RNA is the most plausible molecule able to form both a (self)-replicator and to inherit information, necessities for initiating genetics. However, in parallel with self-replication, the proto-organism had to obtain the ability to catalyse supply of its chemical constituents, including the ribonucleotide metabolites required to replicate RNA. Although the possibility of an RNA-catalysed metabolic network has been considered, it is to be questioned whether RNA molecules, at least on their own, possess the required catalytic capacities. An alternative scenario for the origin of metabolism involves chemical reactions that are based on environmental catalysts. Recently, we described a non-enzymatic glycolysis and pentose phosphate pathway-like reactions catalysed by metal ions [mainly Fe(II)] and phosphate, simple inorganic molecules abundantly found in Archaean sediments. While the RNA world can serve to explain the origin of genetics, the origin of the metabolic network might thus date back to constraints of environmental chemistry. Interestingly, considering a metal-catalysed origin of metabolism gives rise to an attractive hypothesis about how the first enzymes could have formed: simple RNA or (poly)peptide molecules could have bound the metal ions, and thus increased their solubility, concentration and accessibility. In a second step, this would have allowed substrate specificity to evolve.

  16. 不同酸化条件对菜豆幼苗碳氮代谢酶系活性的影响%Effects of Acid on Enzyme Activity of Carbon-Nitrogen Metabolic in Kidney Bean Seedling

    Institute of Scientific and Technical Information of China (English)

    孙海燕; 郭伟

    2013-01-01

    Four different pH (pH 7, 6, 5, 4) nutrient solutions were applied to simulate the environment of germination and growth for kidney bean (Phaseolus vulgari L.). Seed germination, seedling growth characteristics, antioxidase enzyme activities of root and leaf and carbon-nitrogen metabolism enzyme activities and product levels of leaf were studied with decreasing pH in order to explain the response mechanism of carbon-nitrogen metabolism to acidification stress for kidney bean. The results showed that germinating potential and germination rate decreased, seedling and root dry weight reduced, and glutamine synthetase (GS) activity decreased and sucrose phosphate synthase (SPS) activity increased which increased the contents of sucrose, fructose and total soluble sugar, decreased the contents of superoxide anion free radical (O-·2) and malondialdehyde (MDA) of root, and increased the contents of O-·2 and MDA of leaf with decreasing pH. However, the change trend of antioxidase in root was the same as that in leaf, namely, the activity of superoxide dismutase (SOD) and catalase (CAT) increased, peroxidase (POD) activity decreased. Carbon metabolism was increased, however, nitrogen metabolism was inhibited under acidification stress, increased O-·2 content and reduced POD activity of leaf resulted in the increase of membrane lipid peroxidation.%采用4个不同pH值(pH 7,6,5,4)培养液模拟菜豆萌发和幼苗生长环境,研究了酸化条件下菜豆种子萌发和幼苗生长特性、幼苗根系和幼叶的抗氧化酶系活性以及叶片的碳氮代谢的主要酶系活性和产物水平,以期明确菜豆的碳氮代谢对酸化胁迫的响应机制.结果表明:随着pH的降低,菜豆发芽势和发芽率降低,幼苗和根系干物质量减少;谷氨酰胺合成酶活性降低,蔗糖磷酸合成酶活性增强,从而增加了蔗糖、果糖和总可溶性糖含量;根系的超氧阴离子自由基(O2-)和丙二醛(MDA)含量降低,叶片的O2-和MDA含

  17. The Impact of Non-Enzymatic Reactions and Enzyme Promiscuity on Cellular Metabolism during (Oxidative) Stress Conditions.

    Science.gov (United States)

    Piedrafita, Gabriel; Keller, Markus A; Ralser, Markus

    2015-09-10

    Cellular metabolism assembles in a structurally highly conserved, but functionally dynamic system, known as the metabolic network. This network involves highly active, enzyme-catalyzed metabolic pathways that provide the building blocks for cell growth. In parallel, however, chemical reactivity of metabolites and unspecific enzyme function give rise to a number of side products that are not part of canonical metabolic pathways. It is increasingly acknowledged that these molecules are important for the evolution of metabolism, affect metabolic efficiency, and that they play a potential role in human disease-age-related disorders and cancer in particular. In this review we discuss the impact of oxidative and other cellular stressors on the formation of metabolic side products, which originate as a consequence of: (i) chemical reactivity or modification of regular metabolites; (ii) through modifications in substrate specificity of damaged enzymes; and (iii) through altered metabolic flux that protects cells in stress conditions. In particular, oxidative and heat stress conditions are causative of metabolite and enzymatic damage and thus promote the non-canonical metabolic activity of the cells through an increased repertoire of side products. On the basis of selected examples, we discuss the consequences of non-canonical metabolic reactivity on evolution, function and repair of the metabolic network.

  18. Comparative analysis on the key enzymes of the glycerol cycle metabolic pathway in Dunaliella salina under osmotic stresses.

    Science.gov (United States)

    Chen, Hui; Lu, Yan; Jiang, Jian-Guo

    2012-01-01

    The glycerol metabolic pathway is a special cycle way; glycerol-3-phosphate dehydrogenase (G3pdh), glycerol-3-phosphate phosphatase (G3pp), dihydroxyacetone reductase (Dhar), and dihydroxyacetone kinase (Dhak) are the key enzymes around the pathway. Glycerol is an important osmolyte for Dunaliella salina to resist osmotic stress. In this study, comparative activities of the four enzymes in D. salina and their activity changes under various salt stresses were investigated, from which glycerol metabolic flow direction in the glycerol metabolic pathway was estimated. Results showed that the salinity changes had different effects on the enzymes activities. NaCl could stimulate the activities of all the four enzymes in various degrees when D. salina was grown under continuous salt stress. When treated by hyperosmotic or hypoosmotic shock, only the activity of G3pdh in D. salina was significantly stimulated. It was speculated that, under osmotic stresses, the emergency response of the cycle pathway in D. salina was driven by G3pdh via its response to the osmotic stress. Subsequently, with the changes of salinity, other three enzymes started to respond to osmotic stress. Dhar played a role of balancing the cycle metabolic pathway by its forward and backward reactions. Through synergy, the four enzymes worked together for the effective flow of the cycle metabolic pathways to maintain the glycerol requirements of cells in order to adapt to osmotic stress environments.

  19. Visualization of enzyme activities inside earthworm pores

    Science.gov (United States)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  20. 红火蚁幼虫的杀虫剂敏感性及代谢酶活性研究%Insecticide Sensitivity and Metabolic Enzyme Activity of the Larvae of the Red Imported Fire Ant(Solenopsis invicta Buren)

    Institute of Scientific and Technical Information of China (English)

    鄢勤; 曾鑫年; 苗建忠

    2011-01-01

    The insecticide sensitivity of larvae of the red imported fire ant {Solenopsis invieta Buren) was evaluated by the analysis of the contact toxicity of 17 insecticides of various types against the fourth instars reproductive larvae. It was found that the toxicities of the tested insecticides were relatively low with LC50 from 639.28 μg/mL to 3124.55 μg/mL, amongst the LC50 of 88.24% insecticides were higher than 1000 μg/mL. It was indicated that the larvae were insensitive to insecticides. In comparison of the activities of carboxyl esterases, phosphorases, and glutathione-S-transferases of the reproductive larval ants with those of worker ants, it was found that the specific activities of the measured metabolic enzymes in larvae were significantly higher than those in worker ants, especially the activity of phosphorases at folds of above 100, suggesting that strong activity of metabolic enzymes was an important factor for the insensitivity of larval ants to insecticides.%为确定红火蚁幼蚁的杀虫剂敏感性,笔者利用点滴法测定了17种不同类型杀虫剂对红火蚁4龄幼蚁的毒力.毒力测定结果表明,供试杀虫剂对红火蚁4龄幼蚁的毒力均相当低,其LC50在639.28~3124.55μg/mL范围内,其中88.24%的药剂的LC50大于1000 μg/mL,显示红火蚁幼蚁对杀虫剂的敏感性低.通过比较测定红火蚁4龄幼蚁和工蚁体内的羧酸酯酶(CarE)、磷酸酯酶、及谷胱甘肽-S-转移酶(GSTs)的活性,发现幼蚁的代谢酶比活力显著高于工蚁,特别是磷酸酯酶的比活力是工蚁的100倍以上.因此,认为幼蚁体内代谢酶活性高是其杀虫剂敏感性低的重要因素.

  1. How thiamine diphosphate is activated in enzymes.

    Science.gov (United States)

    Kern, D; Kern, G; Neef, H; Tittmann, K; Killenberg-Jabs, M; Wikner, C; Schneider, G; Hübner, G

    1997-01-03

    The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

  2. Effect of Chicory on Uric Acid and Uricopoiesis Metabolic Enzymes Activities of Hyperuricemia Quail%菊苣干预高尿酸血症鹌鹑尿酸及相关代谢酶活性研究

    Institute of Scientific and Technical Information of China (English)

    黄胜男; 林志健; 张冰; 赓迪; 牛红娟; 朱春胜; 王雪洁; 孙博喻

    2015-01-01

    Objective To investigate the effect of chicory on uric acid and uricopoiesis metabolic enzymes activities in hyperuricemia quail and to explore the possible mechanism of chicory in preventing and treating hyperuricemia. Methods Sixty quails were evenly randomized into 5 groups according to the body weight, namely normal group, model group, benzbromarone(20 mg·kg-1·d-1) group, high- and low-dosage chicory groups(10, 5 g·kg-1·d-1 respectively). Except for the normal group, the quails in other groups were given high purine diet(ordinary forage mixed with 15 g·kg-1·d-1 of yeast powder) to induce hyperuricemia(HUA)model. And then we observed the changes of serum uric acid level and activities of uricopoiesis metabolic enzymes dynamically during the chicory treatment for 28 days. Results On the chicory administration day 7, 14, 21 and 28, serum uric acid level of quail was decreased obviously, and the activities of uricopoiesis metabolic enzymes of 5'-nucleotidase(5'-NT), adenosine deaminase (ADA),purine nucleoside phosphorylase(PNP),guanine deaminase(GD),xanthine oxidase(XO)were inhibited to various degrees in chicory groups(P<0.05, P<0.01 compared with the model group). Conclusion Chicory has the effect on lowering serum uric acid level in quail hyperuricemia model, which may be associated with reducing the activities of 5'-NT,ADA,PNP,GD,and XO.%目的:探讨菊苣对高尿酸血症(HUA)鹌鹑尿酸及相关代谢酶活性的影响,阐释菊苣防治HUA的可能机制。方法将60只鹌鹑,按体质量随机分为5组,即正常组,模型组,苯溴马隆组(20 mg·kg-1·d-1),菊苣高、低剂量组(10,5 g·kg-1·d-1),每组12只。除正常组喂饲鹌鹑普通饲料外,其余各组均给予高嘌呤饲料(普通饲料拌入酵母干粉15 g·kg-1·d-1)复制HUA模型。以菊苣治疗28 d,动态观察鹌鹑尿酸(uric acid,UA)水平及UA生成代谢酶活性的变化。结果给药7,14,21,28 d,菊苣高、低

  3. Soil Enzyme Activities with Greenhouse Subsurface Irrigation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-Long; WANG Yao-Sheng

    2006-01-01

    Various environmental conditions determine soil enzyme activities, which are important indicators for changes of soil microbial activity, soil fertility, and land quality. The effect of subsurface irrigation scheduling on activities of three soil enzymes (phosphatase, urease, and catalase) was studied at five depths (0-10, 10-20, 20-30, 30-40, and 40-60 cm) of a tomato greenhouse soil. Irrigation was scheduled when soil water condition reached the maximum allowable depletion(MAD) designed for different treatments (-10, -16, -25, -40, and -63 kPa). Results showed that soil enzyme activities had significant responses to the irrigation scheduling during the period of subsurface irrigation. The neutral phosphatase activity and the catalase activity were found to generally increase with more frequent irrigation (MAD of -10 and -16kPa). This suggested that a higher level of water content favored an increase in activity of these two enzymes. In contrast,the urease activity decreased under irrigation, with less effect for MAD of -40 and -63 kPa. This implied that relatively wet soil conditions were conducive to retention of urea N, but relatively dry soil conditions could result in increasing loss of urea N. Further, this study revealed that soil enzyme activities could be alternative natural bio-sensors for the effect of irrigation on soil biochemical reactions and could help optimize irrigation management of greenhouse crop production.

  4. Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, Edith E., E-mail: ed.mueller@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Mayr, Johannes A., E-mail: h.mayr@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Zimmermann, Franz A., E-mail: f.zimmermann@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Feichtinger, Rene G., E-mail: r.feichtinger@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Stanger, Olaf, E-mail: o.stanger@rbht.nhs.uk [Department of Cardiac Surgery, Paracelsus Medical University, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Sperl, Wolfgang, E-mail: w.sperl@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Kofler, Barbara, E-mail: b.kofler@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.

  5. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

    Science.gov (United States)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

    2016-02-10

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  6. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    Science.gov (United States)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  7. Enzyme activity in dialkyl phosphate ionic liquids

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, M.F.; Dunn, J.; Li, L.-L.; Handley-Pendleton, J. M.; van der lelie, D.; Wishart, J. F.

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  8. Enzyme activity in dialkyl phosphate ionic liquids.

    Science.gov (United States)

    Thomas, Marie F; Li, Luen-Luen; Handley-Pendleton, Jocelyn M; van der Lelie, Daniel; Dunn, John J; Wishart, James F

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  9. Pivotal Enzyme in Glutamate Metabolism of Poly-γ-Glutamate-Producing Microbes

    OpenAIRE

    Tohru Kamei; Takashi Yamamoto; Makoto Ashiuchi

    2013-01-01

    The extremely halophilic archaeon Natrialba aegyptiaca secretes the L-homo type of poly-g-glutamate (PGA) as an extremolyte. We examined the enzymes involved in glutamate metabolism and verified the presence of L-glutamate dehydrogenases, L-aspartate aminotransferase, and L-glutamate synthase. However, neither glutamate racemase nor D-amino acid aminotransferase activity was detected, suggesting the absence of sources of D-glutamate. In contrast, D-glutamate-rich PGA producers mostly possess ...

  10. Extract from Eugenia punicifolia is an antioxidant and inhibits enzymes related to metabolic syndrome.

    Science.gov (United States)

    Lopes Galeno, Denise Morais; Carvalho, Rosany Piccolotto; Boleti, Ana Paula de Araújo; Lima, Arleilson Sousa; Oliveira de Almeida, Patricia Danielle; Pacheco, Carolina Carvalho; Pereira de Souza, Tatiane; Lima, Emerson Silva

    2014-01-01

    The present study aimed to investigate in vitro biological activities of extract of Eugenia punicifolia leaves (EEP), emphasizing the inhibitory activity of enzymes related to metabolic syndrome and its antioxidant effects. The antioxidant activity was analyzed by free radicals scavengers in vitro assays: DPPH·, ABTS(·+), O2(·−), and NO· and a cell-based assay. EEP were tested in inhibitory colorimetric assays using α-amylase, α-glucosidase, xanthine oxidase, and pancreatic lipase enzymes. The EEP exhibited activity in ABTS(·+), DPPH·, and O2(·−) scavenger (IC50 = 10.5 ± 1.2, 28.84 ± 0.54, and 38.12 ± 2.6 μg/mL), respectively. EEP did not show cytotoxic effects, and it showed antioxidant activity in cells in a concentration-dependent manner. EEP exhibited inhibition of α-amylase, α-glucosidase, and xanthine oxidase activities in vitro assays (IC50 = 122.8 ± 6.3; 2.9 ± 0.1; 23.5 ± 2.6), respectively; however, EEP did not inhibit the lipase activity. The findings supported that extract of E. punicifolia leaves is a natural antioxidant and inhibitor of enzymes, such as α-amylase, α-glucosidase, and xanthine oxidase, which can result in a reduction in the carbohydrate absorption rate and decrease of risks factors of cardiovascular disease, thereby providing a novel dietary opportunity for the prevention of metabolic syndrome.

  11. From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes.

    Science.gov (United States)

    Corcoran, Callan C; Grady, Cameron R; Pisitkun, Trairak; Parulekar, Jaya; Knepper, Mark A

    2017-03-01

    The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database (https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database (Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct.

  12. 温度对刺参繁殖期消化酶和代谢酶活力的影响%Effects of temperature on digestive and metabolic enzymes activities of sea cucumber (Apostichopus japonicus) brood-stock

    Institute of Scientific and Technical Information of China (English)

    茹小尚; 高天翔; 刘石林; 宋盛亮; 杨红生

    2015-01-01

    为探究刺参(Apostichopus japonicus)繁殖期消化、代谢生理特点,作者以刺参亲参(240 g±15 g)为实验材料,设置了5个温度水平(6、10、14、18、21℃),研究了温度对刺参繁殖期内肠道消化酶和体壁肌肉代谢酶活力的影响。结果发现:温度对亲参繁殖期消化酶、代谢酶活力受培育水温影响显著(P<0.05)。其中,胰蛋白酶与淀粉酶活力随水温升高呈先升高后降低的趋势,在10℃时活力最高且达到峰值;脂肪酶活力随着养殖水温升高呈下降趋势,在6℃时活力最高。己糖激酶、丙酮酸激酶、乳酸脱氢酶、苹果酸脱氢酶活力随水温升高出现先增高后降低的趋势,在10℃时达到峰值;而琥珀酸脱氢酶活力随温度升高呈下降趋势。研究表明,10~14℃温度下,亲参消化酶与代谢酶活力均处于较高水平,是理想的亲参室内促熟温度参数。%To investigate the digestive and metabolic performance of adult sea cucumber (Apostichopus japonicus) during breeding season, the activities of digestive and metabolic enzymes including trypsin, lipase, amylase, hexokinase, pyruvate kinase, lactate dehydrogenase, succinate dehydrogenase and malate dehydrogenase of sea cucumber broodstock cultured at different water temperatures (6, 10, 14, 18 and 21℃) were detected. The results indicated that digestive and metabolic enzymes activities were affected by water temperature significantly (P<0.05). The activities of trypsin, amylase, hexokinase, pyruvate kinase, lactate dehydrogenase and malate dehydrogenase increased when the water temperature increased from 6 to 10℃, then decreased when the water temperature in-creased from 14 to 20℃, and the highest activities were observed at 10℃. While the activities of lipase and succi-nate dehydrogenase decreased as water temperature rose. Based on results of this study, the optimal temperature range for gonad development of sea cucumber is from 10 to 14℃.

  13. Rhodanese functions as sulfur supplier for key enzymes in sulfur energy metabolism.

    Science.gov (United States)

    Aussignargues, Clément; Giuliani, Marie-Cécile; Infossi, Pascale; Lojou, Elisabeth; Guiral, Marianne; Giudici-Orticoni, Marie-Thérèse; Ilbert, Marianne

    2012-06-08

    How microorganisms obtain energy is a challenging topic, and there have been numerous studies on the mechanisms involved. Here, we focus on the energy substrate traffic in the hyperthermophilic bacterium Aquifex aeolicus. This bacterium can use insoluble sulfur as an energy substrate and has an intricate sulfur energy metabolism involving several sulfur-reducing and -oxidizing supercomplexes and enzymes. We demonstrate that the cytoplasmic rhodanese SbdP participates in this sulfur energy metabolism. Rhodaneses are a widespread family of proteins known to transfer sulfur atoms. We show that SbdP has also some unusual characteristics compared with other rhodaneses; it can load a long sulfur chain, and it can interact with more than one partner. Its partners (sulfur reductase and sulfur oxygenase reductase) are key enzymes of the sulfur energy metabolism of A. aeolicus and share the capacity to use long sulfur chains as substrate. We demonstrate a positive effect of SbdP, once loaded with sulfur chains, on sulfur reductase activity, most likely by optimizing substrate uptake. Taken together, these results lead us to propose a physiological role for SbdP as a carrier and sulfur chain donor to these key enzymes, therefore enabling channeling of sulfur substrate in the cell as well as greater efficiency of the sulfur energy metabolism of A. aeolicus.

  14. The Effects of Pharmaceutical Excipients on Gastrointestinal Tract Metabolic Enzymes and Transporters-an Update.

    Science.gov (United States)

    Zhang, Wenpeng; Li, Yanyan; Zou, Peng; Wu, Man; Zhang, Zhenqing; Zhang, Tao

    2016-07-01

    Accumulating evidence from the last decade has shown that many pharmaceutical excipients are not pharmacologically inert but instead have effects on metabolic enzymes and/or drug transporters. Hence, the absorption, distribution, metabolism, and elimination (ADME) of active pharmaceutical ingredients (APIs) may be altered due to the modulation of their metabolism and transport by excipients. The impact of excipients is a potential concern for Biopharmaceutics Classification System (BCS)-based biowaivers, particularly as the BCS-based biowaivers have been extended to class 3 drugs in certain dosage forms. The presence of different excipients or varying amounts of excipients between formulations may result in bio-inequivalence. The excipient impact may lead to significant variations in clinical outcomes as well. The aim of this paper is to review the recent findings of excipient effects on gastrointestinal (GI) absorption, focusing on their interactions with the metabolic enzymes and transporters in the GI tract. A wide range of commonly used excipients such as binders, diluents, fillers, solvents, and surfactants are discussed here. We summarized the reported effects of those excipients on GI tract phase I and phase II enzymes, uptake and efflux transporters, and relevant clinical significance. This information can enhance our understanding of excipient influence on drug absorption and is useful in designing pharmacokinetic studies and evaluating the resultant data.

  15. PCB126 enhanced the genotoxicity of BaP in HepG2 cells by modulating metabolic enzyme and DNA repair activities.

    Science.gov (United States)

    Wei, Wei; Zhang, Chi; Liu, Ai-Lin; Xie, Shao-Hua; Chen, Xue-Min; Lu, Wen-Qing

    2009-09-10

    Both of benzo(a)pyrene (BaP) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) are ubiquitous and persistent environmental pollutants. These two chemicals coexist in various environmental media and human samples and thus may have combined effects on human health. However, the toxic effects and related mechanism of co-exposure to BaP and PCB126 remain unknown. In a series of experiments using the HepG2 cells exposed to BaP (50microM) or/and PCB126 (0.01, 0.1, 1 and 10nM), we measured the rate of micronucleus (MN) formation, CYP1A1 activity and expression of nucleotide excision repair (NER) proteins (XPA and XPC). We found that the exposure to BaP or PCB126 alone could effectively increase the CYP1A1 activity and the XPA expression. BaP alone had a profound enhancement of MN formation. Compared with BaP alone, co-exposure to both BaP and PCB126 significantly enhanced the CYP1A1 activity and the formation of MN but reduced the expression of both XPA and XPC. The synergistic effect of PCB126 on BaP-induced MN formation was inhibited by alpha-naphthoflavone (ANF), an inhibitor of CYP1A1. Our findings suggest that PCB126 may enhance BaP-induced DNA damage and genotoxicity by increasing cytochrome P450 1A activity and decreasing the NER capacity.

  16. Enzyme activities by indicator of quality in organic soil

    Science.gov (United States)

    Raigon Jiménez, Mo; Fita, Ana Delores; Rodriguez Burruezo, Adrián

    2016-04-01

    The analytical determination of biochemical parameters, as soil enzyme activities and those related to the microbial biomass is growing importance by biological indicator in soil science studies. The metabolic activity in soil is responsible of important processes such as mineralization and humification of organic matter. These biological reactions will affect other key processes involved with elements like carbon, nitrogen and phosphorus , and all transformations related in soil microbial biomass. The determination of biochemical parameters is useful in studies carried out on organic soil where microbial processes that are key to their conservation can be analyzed through parameters of the metabolic activity of these soils. The main objective of this work is to apply analytical methodologies of enzyme activities in soil collections of different physicochemical characteristics. There have been selective sampling of natural soils, organic farming soils, conventional farming soils and urban soils. The soils have been properly identified conserved at 4 ° C until analysis. The enzyme activities determinations have been: catalase, urease, cellulase, dehydrogenase and alkaline phosphatase, which bring together a representative group of biological transformations that occur in the soil environment. The results indicate that for natural and agronomic soil collections, the values of the enzymatic activities are within the ranges established for forestry and agricultural soils. Organic soils are generally higher level of enzymatic, regardless activity of the enzyme involved. Soil near an urban area, levels of activities have been significantly reduced. The vegetation cover applied to organic soils, results in greater enzymatic activity. So the quality of these soils, defined as the ability to maintain their biological productivity is increased with the use of cover crops, whether or spontaneous species. The practice of cover based on legumes could be used as an ideal choice

  17. Activity assessment of microbial fibrinolytic enzymes.

    Science.gov (United States)

    Kotb, Essam

    2013-08-01

    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  18. Something Old, Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism.

    Science.gov (United States)

    Moghe, Gaurav D; Last, Robert L

    2015-11-01

    Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks.

  19. Something Old, Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism1

    Science.gov (United States)

    Moghe, Gaurav D.; Last, Robert L.

    2015-01-01

    Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks. PMID:26276843

  20. Allosteric ACTion: the varied ACT domains regulating enzymes of amino-acid metabolism.

    Science.gov (United States)

    Lang, Eric J M; Cross, Penelope J; Mittelstädt, Gerd; Jameson, Geoffrey B; Parker, Emily J

    2014-12-01

    Allosteric regulation of enzyme activity plays important metabolic roles. Here we review the allostery of enzymes of amino-acid metabolism conferred by a discrete domain known as the ACT domain. This domain of 60-70 residues has a βαββαβ topology leading to a four-stranded β4β1β3β2 antiparallel sheet with two antiparallel helices on one face. Extensive sequence variation requires a combined sequence/structure/function analysis for identification of the ACT domain. Common features include highly varied modes of self-association of ACT domains, ligand binding at domain interfaces, and transmittal of allosteric signals through conformational changes and/or the manipulation of quaternary equilibria. A recent example illustrates the relatively facile adoption of this versatile module of allostery by gene fusion.

  1. Quality assessment of oosperm of turbot (Scophthalmus maximus) based on energy metabolism-related relative enzyme activities%基于能量代谢酶相对活力评价大菱鲆卵质

    Institute of Scientific and Technical Information of China (English)

    肖永双; 马道远; 肖志忠; 刘清华; 徐世宏; 李军

    2012-01-01

    We assessed the oosperm quality of turbot (Scophthalmus maximus) based on the morphological comparison of oosperm diameter and energy metabolism-related relative enzyme activities. The results show that the average oosperm diameter for turbot is (1. 053 ±0. 020) mm and no significant difference is found among different batches. Bivariate correlation statistics reveal that the correlation is insignificant among oosperm diameter and suspensibility as well as fertilization rate and hatching rate. The statistics of energy metabolism-related relative enzyme activities indicate that significant correlation is found between hatching rate and aspartate amino transferase (AST) [EAST≥(15.159 ±3.300) U·mg-1, Hhatching >20% , Spearman coefficient R= 0.675, P = 0.003 ]; the correlation among suspensibility, fertilization rate and AST is significant No significant correlation is found among pyruvate kinase ( PK) , suspensibility, fertilization rate and hatching rate. Thus, the relative activity of AST can be used as a physiological and biochemical parameter to assess the oosperm quality of turbot, and protein is an important energetic source for the energy metabolism of turbot's oosperm.%采用卵径形态比较和能量代谢关键酶生理生化分析方法,对大菱鲆(Scophthalmus maximus卵质进行了评价.结果显示,大菱鲆受精卵卵径平均值为(1.053±0.020) mm,不同批次的受精卵卵径间无显著差异.双变量相关统计分析结果显示,大菱鲆卵径与悬浮率、受精率和孵化率间相关性皆不显著.基于能量代谢关键酶统计结果显示,大菱鲆受精卵孵化率与参与蛋白代谢关键酶谷草转氨酶(AST)相对活力存在显著相关性[EAST≥( 15.159±3.300) U·mg-1,HHatching rate>20%,Spearman相关系数为R=0.675,P=0.003],悬浮率和受精率与AST显著相关;大菱鲆受精卵悬浮率、受精率和孵化率与参与糖代谢关键酶丙酮酸激酶(PK)间相关性均不显著.该研

  2. Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

    Energy Technology Data Exchange (ETDEWEB)

    Lee, T. (Oak Ridge Associated Univ., TN); Malone, B.; Wasserman, S.I.; Fitzgerald, V.; Snyder, F.

    1982-04-29

    Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (l-alkyl-2-acetyl-sn-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the O-alkyl linkage in lipids), l-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-sn-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-sn-glycero-3-phospho-choline synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.

  3. Ammonium Metabolism Enzymes Aid Helicobacter pylori Acid Resistance

    OpenAIRE

    2014-01-01

    The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH3 that is immediately protonated to form NH4+. This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) th...

  4. Determination of Intestinal Enzyme Activities During Infancy Period

    Directory of Open Access Journals (Sweden)

    Emel Örün

    2016-08-01

    Full Text Available Introduction: Intestinal enzyme activities are indirect indicators that reflect the existence and metabolic activity of bacteria living in the intestinal flora. The purpose of the study was to measure fecal beta (β-glucuronidase, β-glucosidase and urease enzyme activities and to determine the factors that affect levels in 6 week old and 8 month old babies. Materials and Methods: The study comprised 100 healthy infants at 6 weeks of age. Feces samples were collected from all infants. However, 17 of the feces samples were not included due to the lack of particles in the feces. The same samples were also taken from 35 infants at 8 months of age. Twenty-five of the infants had given feces samples at both 6 weeks and 8 months of age. Urease, β-glucuronidase and β-glucosidase enzyme activities (nmol/min-1/mg-protein-1 were measured. Results: In repeated measures, the levels of β-glucuronidase and urease declined over time and β-glucosidase levels increased. At 8 months of age, higher β-glucuronidase levels were obtained in premature infants. At 6 weeks of age, lower levels of urease were measured in babies who were started breastfeeding at the first hour of life and were bottle-fed. Exclusive breastfeeding had no influence on the intestinal enzyme activities. Conclusions: In early infancy period when microflora is structured, intestinal enzyme activities are important that show indirectly functionality of the microflora. However, it is difficult to highlight what affects the levels of intestinal enzymes because activities vary according to the age.

  5. Topography and function of androgen-metabolizing enzymes in the central nervous system.

    Science.gov (United States)

    Tsuruo, Yoshihiro

    2005-03-01

    The present review describes concisely the topography and function of the three androgen-metabolizing enzymes, namely aromatase, 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase, in the central nervous system (CNS). Aromatase, estrogen synthetase, is the key enzyme for converting androgens to estrogens. Aromatase is indispensable for the sexual differentiation of the brain and the enzyme activity and expression of aromatase are high during the critical period of neural development, which extends from the late embryonal to the early neonatal period in rodents. Aromatase is expressed in neurons within specific hypothalamic and limbic regions. The locations of aromatase-immunoreactive neurons are divided into three groups according to the period of enzyme expression. Steroid 5alpha-reductase converts a number of steroids with a C3 ketone group and a C4-C5 double bond (delta4; androgens, progestins and glucocorticoids) to their 5alpha-reduced metabolites. Two isoforms of 5alpha-reductase are found and type 1 is predominant in neural tissues. The enzyme activity of 5alpha-reductase is found widely in the CNS and is high in white matter regions. The enzyme expression of 5alpha-reductase peaks during the late embryonic period. 3alpha-Hydroxysteroid dehydrogenase is the oxidoreductase that interconverts 3-ketosteroids to 3alpha-hydroxysteroids. Four isozymes have been found in humans and only one type has been found in rats. The enzyme converts 5alpha-reduced steroids (e.g. 5alpha-dihydroprogesterone) to tetrahydrosteroids (e.g. 3alpha,5alpha-tetrahydroprogesterone). The latter steroid is a potent stimulator of the GABA(A) receptor. The activity of 3alpha-hydroxysteroid dehydrogenase is high during the first 1-2 postnatal weeks, decreases with development and this enzyme is highly expressed in astrocytes.

  6. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

    Directory of Open Access Journals (Sweden)

    Nursen Keser

    2014-04-01

    Full Text Available Folate is a vital B vitamin which is easily water-soluble. It is a natural source which is found in the herbal and animal foods. Folate has important duties in the human metabolism, one of them is the adjustment of the level of plasma homocysteine. Reduction in MTHFR (methylenetetrahydrofolate reductase,which is in charge of the metabolism of homocysteine activity affects the level of homocysteine. Therefore MTHFR is an important enzyme in folate metabolism. Some of the mutations occurring in the MTHFR gene is a risk factor for various diseases and may be caused the hyperhomocysteinemia or the homocystinuria, and they also may lead to metabolic problems. MTHFR is effective in the important pathways such as DNA synthesis, methylation reactions and synthesis of RNA. C677T and A1298C are the most commonly occurring polymorphisms in the gene of MTHFR. The frequency of these polymorphisms show differences in the populations. MTHFR, folate distribution, metabolism of homocysteine and S-adenosylmethionine, by the MTHFR methylation the genetic defects have the potential of affecting the risk of disease in the negative or positive way.

  7. Effects of Buyang Huanwu Decoction on antioxidant and drug-metabolizing enzymes in rat liver.

    Science.gov (United States)

    Fan, Xing-Hua; Shi, Wei-Zhou; Cheng, Yun-Xiang; Yang, Xiu-Fen

    2014-06-01

    To study the effect of Buyang Huanwu Decoction (BYHWD) on the antioxidant enzymes and drug-metabolizing enzymes in rat liver. Following treatment of rats with BYHWD at 6.42, 12.83, or 25.66 g·kg(-1) per day for 15 days, microsomes and cytosols isolated from the liver tissues were prepared by differential centrifugation according to standard procedures. The activities of the antioxidant enzymes and cytochrome b5, NADPH-cytochrome P450 reductase, CYP3A, CYP2E1, UGT, and GST of the rat livers were determined by UV-Vis spectrophotometer. The activities of ALT, AST, antioxidant enzymes, and the Hepatosomatic Index in serum were not significantly affected. In cytosols, the activity of CAT was significantly increased at the dosage of 12.83 g·kg(-1), and all the other antioxidant activities and MDA levels were not affected by this treatment. BYHWD had no effect on cytochrome b5, NADPH-cytochrome P450 reductase, CYP3A, and UGT. At the highest dose (25.66 g·kg(-1)), the activity of CYP2E1 was significantly inhibited, and the activities of GST and the level of GSH were increased. BYHWD is safe for the liver, and has the functions of detoxification and antioxidant. Patients should be cautioned about the herb-drug interaction of BYHWD and CYP2E1 substrates. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  8. Modulation of Ethanol-Metabolizing Enzymes by Developmental Lead Exposure: Effects in Voluntary Ethanol Consumption

    Directory of Open Access Journals (Sweden)

    Miriam B. Virgolini

    2017-05-01

    Full Text Available This review article provides evidence of the impact of the environmental contaminant lead (Pb on the pattern of the motivational effects of ethanol (EtOH. To find a mechanism that explains this interaction, the focus of this review article is on central EtOH metabolism and the participating enzymes, as key factors in the modulation of brain acetaldehyde (ACD accumulation and resulting effect on EtOH intake. Catalase (CAT seems a good candidate for the shared mechanism between Pb and EtOH due to both its antioxidant and its brain EtOH-metabolizing properties. CAT overactivation was reported to increase EtOH consumption, while CAT blockade reduced it, and both scenarios were modified by Pb exposure, probably as the result of elevated brain and blood CAT activity. Likewise, the motivational effects of EtOH were enhanced when brain ACD metabolism was prevented by ALDH2 inhibition, even in the Pb animals that evidenced reduced brain ALDH2 activity after chronic EtOH intake. Overall, these results suggest that brain EtOH metabolizing enzymes are modulated by Pb exposure with resultant central ACD accumulation and a prevalence of the reinforcing effects of the metabolite in brain against the aversive peripheral ACD accumulation. They also support the idea that early exposure to an environmental contaminant, even at low doses, predisposes at a later age to differential reactivity to challenging events, increasing, in this case, vulnerability to acquiring addictive behaviors, including excessive EtOH intake.

  9. 温度对采后香蕉果实糖代谢及其酶活性的影响%Effect of Temperature on Sugar Metabolism and Enzyme Activities in Postharvest Ripening Banana Fruit

    Institute of Scientific and Technical Information of China (English)

    袁扬静; 胡玉林; 谢江辉

    2011-01-01

    以香蕉为试材,研究采后不同温度(20、4℃)贮藏对果实的呼吸作用、糖代谢及其相关酶活性变化的影响.结果表明:20℃贮藏早期(0~8 d),果实呼吸作用平缓上升,SPS,NI和AI酶活性较低,蔗糖、果糖和葡萄糖含量较少;随着果实进入快速软化阶段(8~13 d),3种酶活性与蔗糖含量分别在第13天和第12天达到峰值,然后急剧下降,变化趋势类似于CO2释放量.而果糖和葡萄糖含量在20℃贮藏过程中持续上升,SS活性无明显变化.相比之下,4℃贮藏可明显抑制果实的呼吸作用,CO2含量先降低后缓慢增加,SPS、NI和AI酶活性和搪含量上升缓慢,SS活性下降.由此,低温贮藏可明显抑制香蕉体内碳素代谢,降低代谢酶活性和糖分积累速度,有利于提高果实品质,延缓贮藏时间.%The effect of different temperatures (20 and 4 ℃ )on respiration characteristic, sugar content and sugarmetabolizing enzyme activities from banana fruits was studied during storage. In the early 0~8 d, fruit respiration gradually increased. Moreover, there were all low in the activities of invertase (Al), neutral invertase (NI), sucrose synthase(SS), sucrose phosphate synthase (SPS) and the contents of sucrose, fructose and glucose. The peak values of the three enzymes and the sucrose content were in the 13th and 12th during softening stage 8~13 d, respectively, and then rapidly decreased. This tendency was consistent with the change of CO2. However, the fructose and glucose increased slowly, and SS activity did not change significantly during the total process of postharvest. In 4 ℃ storage, CO2 respiration was inhibited, first decreased and then increased slowly. The activities of SPS, NI, and Al and the content of sugar gradually went up. But SS activity declined during the fruit storage. So low temperature storage in vitro can significantly inhibit the metabolism of carbohydrate the banana, reduce the activity of the enzymes and the

  10. Reactive phosphate esters as affinity labels for enzymes of carbohydrate metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, F. C.; Norton, I. L.; Stringer, C. D.; Schloss, J. V.

    1978-01-01

    Many enzymes of carbohydrate metabolism have an affinity for small organic anions. An attempt was made to exploit the common binding specificities of enzymes, whose substrates are phosphate esters, in the design and use of affinity labels. The basic premise is that small phosphate esters that contain chemically reactive substituents will be active-site-specific probes for some enzymes with affinities for phosphate esters. This paper describes three recent endeavors: (1) an attempt to design an affinity label for the catalytic group of triosephosphate isomerase that polarizes the substrate carbonyl, (2) the use of N-bromoacetylethanolamine phosphate (BrAcNHEtOP) as an affinity label for rabbit and chicken phosphoglycerate mutase, and (3) the use of BrAcNHEtOP as an affinity label for D-ribulose 1,5-bisphosphate carboxylase/oxygenase.

  11. Effects of Grafting on Tobacco Key Enzyme Activities of Nitrogen Metabolism and the Contents of Chlorophyll%嫁接对烤烟氮代谢关键酶活性及叶绿素含量的影响

    Institute of Scientific and Technical Information of China (English)

    梅芳; 杨铁钊; 刘剑君; 张小全; 张广普; 武云杰; 刘中威; 李洪臣

    2012-01-01

    Using Jin Xing 6007 as rootstock and Nong Da 202 as scion, grafted plants were made to compare the differences in dry leaf weight, some key enzyme activities of nitrogen metabolism (nitrate reductase,glutamine synthetase)and the contents of chlorophyll between grafted and non-grafted tobacco seedlings grown in pot with the different amount of nitrogen. The results showed that nitrate reductase activities, glutamine synthetase activities and the contents of chlorophyll in leaves of grafted seedlings were significantly higher than those of non-grafted seedlings under two different amount of nitrogen. Nitrate reductase activities, glutamine synthetase activities and the contents of chlorophyll of grafted and non-grafted plants were decreasing with the decreasing nitrogen level, they were also decreasing when tobacco leaves were turning to mature. The speed of decreasing in grafted seedlings was slower than that in non-grafted seedlings. These results indicated that higher nitrate reductase activities, glutamine synthetase activities and the contents of chlorophyll might be resulted from the stronger nitrogen efficiency in grafted tobacco seedlings.%以金星6007为砧木,农大202为接穗,比较盆栽试验下不同施氮水平对烤烟嫁接苗和接穗自根苗的烟叶干质量、氮代谢关键酶(硝酸还原酶和谷氨酰胺合成酶)活性和叶绿素含量的影响.结果表明,烟叶干质量、硝酸还原酶活性、谷氨酰胺合成酶活性和叶绿素含量在两种氮水平下均为嫁接苗大于自根苗,且差异极显著.随着施氮量的降低和生育期的推进,嫁接苗和自根苗氮代谢关键酶活性和叶绿素含量都下降,但降幅不同,嫁接苗的降幅较低.以上结果说明,低氮水平下较高的硝酸还原酶活性、谷氨酰胺合成酶活性和叶绿素含量与烤烟嫁接苗的氮效率增强有关.

  12. 本草消渴丹对实验性糖尿病大鼠糖代谢酶活性的影响%Effect of Bencao Xiaoke Pill on Carbohydrate Metabolism Enzyme Activity in Type 2 Diabetic Rats

    Institute of Scientific and Technical Information of China (English)

    杨宏莉; 张宏馨; 李兰会; 王燕; 卢淑兰; 孙淑敏; 张伟伟

    2009-01-01

    Objective To study the effect of Chinese Herbal Compound-Bencao Xiaoke pill on the activity of key enzymes - hexokinase and malate dehydrogenase in the carbohydrate metabolism of type 2 diabetic rats and provide scientific basis for clinical application.Methods Type 2 diabetic rat model was built by small dose injection of streptozotocin (STZ) (25 mg/kg,intraperitoneal injection) and intake of high lipid die.After the model was established,the rats with diabetes were treated with Compound-Bencao Xiaoke pill and metformin.Blood glucose was detected every weekend by cutting rats' tails.After four weeks treatment the blood glucose,activities of muscle hexokinase(HK)and liver malate dehydrogenase(MDH) were tested.Results The blood glucose was significantly decreased as compared with model groups,and there had significant differences in the activities of HK of the muscle tissue and MDH of the liver.Conclusion One of the hypoglycemic mechanism for materia medica Xiaoke pills is to increese directly or indirectly the activity of carbohydrate metabolic enzymes.The Compound-Bencao xiaoke pill has certain effect on the weight loss of diabetic body.%目的 探讨中药复方-本草消渴丹对2型糖尿病大鼠糖代谢的关键酶己糖激酶、苹果酸脱氢酶活性的影响,为临床用药提供科学依据.方法 选择高脂、高糖饲料加小剂量链脲佐菌素(STZ,25 mg/kg,腹腔注射)的方法建立大鼠2型糖尿病模型,分别以本草消渴丹和西药二甲双胍进行干预,每周末断尾取血检测空腹血糖值.治疗4周后检测大鼠空腹血糖值、肌肉己糖激酶(HK)和肝脏苹果酸脱氢酶活性(MDH).结果 本草消渴丹治疗可以显著降低糖尿病大鼠血糖,其肌肉组织HK、肝脏组织MDH活性与模型组对比有显著性差异.结论 中药复方-本草消渴丹降低血糖的作用机制之一可能是直接或间接提高了葡萄糖氧化分解的酶活性;本草消渴丹对对抗糖尿病体重降低有一定作用.

  13. An NMR Study of Enzyme Activity.

    Science.gov (United States)

    Peterman, Keith E.; And Others

    1989-01-01

    A laboratory experiment designed as a model for studying enzyme activity with a basic spectrometer is presented. Included are background information, experimental procedures, and a discussion of probable results. Stressed is the value of the use of Nuclear Magnetic Resonance in biochemistry. (CW)

  14. An NMR Study of Enzyme Activity.

    Science.gov (United States)

    Peterman, Keith E.; And Others

    1989-01-01

    A laboratory experiment designed as a model for studying enzyme activity with a basic spectrometer is presented. Included are background information, experimental procedures, and a discussion of probable results. Stressed is the value of the use of Nuclear Magnetic Resonance in biochemistry. (CW)

  15. Androgenic/estrogenic balance in the male rat cerebral circulation: metabolic enzymes and sex steroid receptors.

    Science.gov (United States)

    Gonzales, Rayna J; Ansar, Saema; Duckles, Sue P; Krause, Diana N

    2007-11-01

    Tissues from males can be regulated by a balance of androgenic and estrogenic effects because of local metabolism of testosterone and expression of relevant steroid hormone receptors. As a critical first step to understanding sex hormone influences in the cerebral circulation of males, we investigated the presence of enzymes that metabolize testosterone to active products and their respective receptors. We found that cerebral blood vessels from male rats express 5alpha-reductase type 2 and aromatase, enzymes responsible for conversion of testosterone into dihydrotestosterone (DHT) and 17beta-estradiol, respectively. Protein levels of these enzymes, however, were not modulated by long-term in vivo hormone treatment. We also showed the presence of receptors for both androgens (AR) and estrogens (ER) from male cerebral vessels. Western blot analysis showed bands corresponding to the full-length AR (110 kDa) and ERalpha (66 kDa). Long-term in vivo treatment of orchiectomized rats with testosterone or DHT, but not estrogen, increased AR levels in cerebral vessels. In contrast, ERalpha protein levels were increased after in vivo treatment with estrogen but not testosterone. Fluorescent immunostaining revealed ERalpha, AR, and 5alpha-reductase type 2 in both the endothelial and smooth muscle layers of cerebral arteries, whereas aromatase staining was solely localized to the endothelium. Thus, cerebral vessels from males are target tissues for both androgens and estrogen. Furthermore, local metabolism of testosterone might balance opposing androgenic and estrogenic influences on cerebrovascular as well as brain function in males.

  16. Effects of the agrochemicals butachlor, pretilachlor and isoprothiolane on rat liver xenobiotic-metabolizing enzymes.

    Science.gov (United States)

    Ishizuka, M; Iwata, H; Kazusaka, A; Hatakeyama, S; Fujita, S

    1998-11-01

    1. The herbicides butachlor (2-chloro-2',6',diethyl-N-[buthoxymethyl] acetanilide) and pretilachlor (2-chloro-2',6'-diethyl-N-[2-propoxyethyl] acetanilide) are widely used in Asia, South America, Europe and Africa. Isoprothiolane (diisopropyl-1,3-dithiolan-2-ylidenemalonate) is used as a fungicide and an insecticide in rice paddies. We administered these agrochemicals to the male rat and examined their effects on cytochrome P450 (P450), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UDPGT), and NAD(P)H-quinone oxidoreductase 1 (NQO1)-related metabolism in the liver. 2. Administration of isoprothiolane, butachlor or pretilachlor to rat induced hepatic P4502B subfamily-dependent enzyme activities (pentoxyresorufin O-depentylation and testosterone 16 beta-hydroxylation) up to 271-413% of control, which coincided with the increase in expression levels of the P4502B apoprotein. 3. Activities of GST toward 1-chloro-2,4-nitrobenzene and 3,4-dichloronitrobenzene were slightly induced (127-133% of control) in the liver of the rat treated with these pesticides. On the other hand, marked elevations of UDPGT activities toward p-nitrophenol (164-281% of control) were observed. NQO1-related metabolism (menadione reductase activity) was also induced (123-176% of control) in the liver of rat treated with these agrochemicals. 4. These results indicate that some of the agrochemicals currently in use are capable of inducing phase I and II xenobiotic-metabolizing enzyme activities in an isozyme selective manner. The induction of these activities may disrupt normal physiologic functions related to these enzymes in exposed animals.

  17. Predicting Drug Extraction in the Human Gut Wall: Assessing Contributions from Drug Metabolizing Enzymes and Transporter Proteins using Preclinical Models.

    Science.gov (United States)

    Peters, Sheila Annie; Jones, Christopher R; Ungell, Anna-Lena; Hatley, Oliver J D

    2016-06-01

    Intestinal metabolism can limit oral bioavailability of drugs and increase the risk of drug interactions. It is therefore important to be able to predict and quantify it in drug discovery and early development. In recent years, a plethora of models-in vivo, in situ and in vitro-have been discussed in the literature. The primary objective of this review is to summarize the current knowledge in the quantitative prediction of gut-wall metabolism. As well as discussing the successes of current models for intestinal metabolism, the challenges in the establishment of good preclinical models are highlighted, including species differences in the isoforms; regional abundances and activities of drug metabolizing enzymes; the interplay of enzyme-transporter proteins; and lack of knowledge on enzyme abundances and availability of empirical scaling factors. Due to its broad specificity and high abundance in the intestine, CYP3A is the enzyme that is frequently implicated in human gut metabolism and is therefore the major focus of this review. A strategy to assess the impact of gut wall metabolism on oral bioavailability during drug discovery and early development phases is presented. Current gaps in the mechanistic understanding and the prediction of gut metabolism are highlighted, with suggestions on how they can be overcome in the future.

  18. The effects of space flight on some rat liver enzymes regulating carbohydrate and lipid metabolism

    Science.gov (United States)

    Abraham, S.; Lin, C. Y.; Klein, H. P.; Volkmann, C.

    1981-01-01

    The effects of space flight conditions on the activities of certain enzymes regulating carbohydrate and lipid metabolism in rat liver are investigated in an attempt to account for the losses in body weight observed during space flight despite preflight caloric consumption. Liver samples were analyzed for the activities of 32 cytosolic and microsomal enzymes as well as hepatic glycogen and individual fatty acid levels for ground control rats and rats flown on board the Cosmos 936 biosatellite under normal space flight conditions and in centrifuges which were sacrificed upon recovery or 25 days after recovery. Significant decreases in the activities of glycogen phosphorylase, alpha-glycerol phosphate acyl transferase, diglyceride acyl transferase, aconitase and 6-phosphogluconate dehydrogenase and an increase in palmitoyl CoA desaturase are found in the flight stationary relative to the flight contrifuged rats upon recovery, with all enzymes showing alterations returning to normal values 25 days postflight. The flight stationary group is also observed to be characterized by more than twice the amount of liver glycogen of the flight centrifuged group as well as a significant increase in the ratio of palmitic to palmitoleic acid. Results thus indicate metabolic changes which may be involved in the mechanism of weight loss during weightlessness, and demonstrate the equivalence of centrifugation during space flight to terrestrial gravity.

  19. Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense).

    Science.gov (United States)

    Tamura, Ken-ichi; Sanada, Yasuharu; Tase, Kazuhiro; Yoshida, Midori

    2014-07-01

    Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0°C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to -3°C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering.

  20. Pharmacokinetic interactions between herbal medicines and prescribed drugs: focus on drug metabolic enzymes and transporters.

    Science.gov (United States)

    Meng, Qiang; Liu, Kexin

    2014-01-01

    Herbal medicines have been widely used for thousands of years, and now are gaining continued popularity worldwide as a complementary or alternative treatment for a variety of diseases, rehabilitation and health care. Since herbal medicines contain more than one pharmacologically active ingredient and are commonly used with many prescribed drugs, there are potential herb-drug interactions. A variety of reported herb-drug interactions are of pharmacokinetic origin, arising from the effects of herbal medicines on metabolic enzymes and/or transporters. Such an alteration in metabolism or transport can result in changes in absorption, distribution, metabolism, and excretion (e.g., induction or inhibition of metabolic enzymes, and modulation of uptake and efflux transporters), leading to changed pharmacokinetics of the concomitantly prescribed drugs. Pharmacokinetic herb-drug interactions have more clinical significance as pharmacokinetic parameters such as the area under the plasma concentration-time curve (AUC), the maximum plasma concentration (Cmax) or the elimination half-life (t1/2) of the concomitant drug alter. This review summarizes the mechanism underlying herb-drug interactions and the approaches to identify the interactions, and discusses pharmacokinetic interactions of eight widely used herbal medicines (Ginkgo biloba, ginseng, garlic, black cohosh, Echinacea, milk thistle, kava, and St. John's wort) with conventional drugs, using various in vitro, animal in vivo, and clinical studies. The increasing understanding of pharmacokinetic herb-drug interactions will make health care professionals and patients pay more attention to the potential interactions.

  1. Gene expression analysis of a critical enzyme in intermediary metabolism in oyster pathogen Perkinsus marinus .

    Science.gov (United States)

    Noell, K.

    2016-02-01

    A key regulatory component in the Krebs cycle pathway is the mitochondrial aconitase enzyme which has been posited to balance energy needs and oxidative growth total storage via citrate utilization. The presence of a cytosolic aconitase (cAcon) activity which serves as a competitor for citrate substrate has been recognized for years. cAcon is a dual function protein with mutually exclusive roles as a post transcriptional regulator of animal cell iron metabolism or as the cytosolic isoform of the iron sulfur enzyme aconitase. We are interested in establishing the role of this orthologue in Perkinsus marnius metabolism through demonstrating its function as aconitase, by looking at gene expression under certain environmental conditions. P. marinus is a close evolutionary relative of the dinoflagellates and is the causative agent of Dermo disease, which has significantly impacted oyster populations along the eastern seaboard. An understanding of intermediary metabolism will yield important insights into how c-aconitase may be involved in stress response systems such as oxidative tension and metabolite deficiency, which could be used to help aquaculturists alleviate the severe impact of "dermo" on the on the oyster population. This study will present data regarding our preliminary analysis of the gene aconitase and its role in intermediary metabolism.

  2. Shemamruthaa, herbal formulation modulates xenobiotic metabolizing enzymes and energy metabolism in 7,12-Dimethylbenz[a]anthracene-induced breast cancer in rats

    Directory of Open Access Journals (Sweden)

    Purushothaman Ayyakkannu

    2014-12-01

    Full Text Available There is an increasing interest in identifying naturally occurring potent preventive and therapeutic agents for cancer. Shemamruthaa, a phytochemical formulation was evaluated for the first time with a view to potentiate more intense anticancer property. Adult female Sprague-Dawley rats (8-week-old were used for the study and were divided into 4 Groups. Group I control animals received standard pellet diet and water ad libitum. Group II animals were induced for mammary carcinoma with a single oral dose of 25 mg of DMBA, whereas another set of DMBA-induced rats were treated with SM (400 mg/kg body weight/day orally by gastric intubation for 14 days after 3 months of induction period (Group III. Group IV animals served as SM-control. The status xenobiotic metabolizing enzymes, glycolytic and gluconeogenic enzymes were analysed in control and experimental rats. Our findings revealed that the SM formulation has potential to induce Phase-II enzyme activities, associated mainly with carcinogen detoxification and inhibit the Phase I enzyme activities. The activities of glycolytic and gluconeogenic enzymes were significantly brought back to near normal levels in SM treated animals. The results demonstrated unequivocally the effect of SM on inhibition of tumor progression by altering xenobiotic metabolizing enzymes and restoring energy metabolism

  3. Curcumin and resveratrol in combination modulate drug-metabolizing enzymes as well as antioxidant indices during lung carcinogenesis in mice.

    Science.gov (United States)

    Liu, Y; Wu, Y-M; Yu, Y; Cao, C-S; Zhang, J-H; Li, K; Zhang, P-Y

    2015-06-01

    This study investigated combined chemopreventive potential of curcumin and resveratrol during benzo(a)pyrene (BP)-induced lung carcinogenesis in mice. The mice were segregated into five groups that included normal control, BP-treated, BP + curcumin-treated, BP + resveratrol-treated, and BP + curcumin + resveratrol-treated groups. A statistically significant increase in the levels of lipid peroxidation (LPO) was observed in the lungs of mice after 22 weeks of single dose of benzo(a)pyrene. Further, BP treatment also resulted in a significant increase in the enzyme activities of aryl hydrocarbon hydroxylase as well as drug-metabolizing enzymes, namely cytocrome P450 and cytochrome b5. On the other hand, reduced glutathione (GSH) levels, the activities of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione-S-transferase (GST) were found to be significantly decreased following BP treatment. Supplementation with curcumin and resveratrol to BP-treated mice significantly decreased the LPO levels, GSH levels, and enzyme activities of drug-metabolizing enzymes. Further, treatment of curcumin and resveratrol to BP-treated mice significantly elevated the activities of SOD, GR, and GST. Histoarchitectural studies showed well-differentiated signs of lung carcinogenesis following BP administration to mice. However, combined treatment with curcumin and resveratrol resulted in a noticeable improvement in the lung histoarchitecture. This study, therefore, concludes that curcumin and resveratrol when supplemented in combination regulate drug-metabolizing enzymes as well as antioxidant enzymes during lung carcinogenesis in mice. © The Author(s) 2014.

  4. Phosphorylation of lipid metabolic enzymes by yeast protein kinase C requires phosphatidylserine and diacylglycerol.

    Science.gov (United States)

    Dey, Prabuddha; Su, Wen-Min; Han, Gil-Soo; Carman, George M

    2017-04-01

    Protein kinase C in Saccharomyces cerevisiae, i.e., Pkc1, is an enzyme that plays an important role in signal transduction and the regulation of lipid metabolic enzymes. Pkc1 is structurally similar to its counterparts in higher eukaryotes, but its requirement of phosphatidylserine (PS) and diacylglycerol (DAG) for catalytic activity has been unclear. In this work, we examined the role of these lipids in Pkc1 activity with protein and peptide substrates. In agreement with previous findings, yeast Pkc1 did not require PS and DAG for its activity on the peptide substrates derived from lipid metabolic proteins such as Pah1 [phosphatidate (PA) phosphatase], Nem1 (PA phosphatase phosphatase), and Spo7 (protein phosphatase regulatory subunit). However, the lipids were required for Pkc1 activity on the protein substrates Pah1, Nem1, and Spo7. Compared with DAG, PS had a greater effect on Pkc1 activity, and its dose-dependent interaction with the protein kinase was shown by the liposome binding assay. The Pkc1-mediated degradation of Pah1 was attenuated in the cho1Δ mutant, which is deficient in PS synthase, supporting the notion that the phospholipid regulates Pkc1 activity in vivo. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  5. Effects of cow ghee (clarified butter oil) & soybean oil on carcinogen-metabolizing enzymes in rats.

    Science.gov (United States)

    Rani, Rita; Kansal, Vinod K

    2012-09-01

    Our previous study showed that cow ghee relative to soybean oil had a protective effect against carcinogen induced mammary cancer in rats. The objective of this study was to elucidate its biochemical mechanism. Two groups of 21 day old rats (20 each) were fed for 44 wk diet containing cow ghee or soybean oil (10%). Five animals from each group were sacrificed at 0 day and at 5, 21 and 44 wk for analysis of phase I and phase II pathways enzymes of carcinogen metabolism. Dietary cow ghee relative to soybean oil decreased the activities of cytochrome P450 (CYP) enzymes, CYP1A1, CYP1A2, CYP1B1 and CYP2B1, responsible for activation of carcinogen in liver. Carcinogen detoxification activities of uridinediphospho-glucuronosyl transferase (UDPGT) and quinone reductase (QR) in liver, and γ-glutamyltranspeptidase (GGTP) and QR in mammary tissue were significantly higher in cow ghee fed rats than in soybean oil fed rats. The hepatic GGTP activity decreased on soybean oil diet; while in cow ghee group it remained unaffected. Our findings show that dietary cow ghee compared to soybean oil downregulates the enzyme activities responsible for carcinogen activation in liver and upregulates carcinogen detoxification activities in liver and mammary tissues.

  6. Approaches to biosynthesis of salidroside and its key metabolic enzymes

    Institute of Scientific and Technical Information of China (English)

    Shi Ling-ling; Wang Li; Zhang Yan-xia; Liu Yu-jun

    2007-01-01

    As a main component of efficiency in Rhodiola plants. salidroside is a promising environmental acclamation medicine and possesses specific medical properties against symptoms of fatigue,old age,microwave radiation, viral infections and tumors. Salidroside plays important roles,especially in military, aerospace,sport and healthcare medicine and has,therefore,recently, drawn more and closer attention. This article probes mainly into theprobable biosynthetic pathway of salidroside following a brief introduction of the exploitation and utilization values of Rhodiola plants and the current condition of its natural resources. We have come to the conclusion that tyrosol,the aglycon of salidroside,is biosynthesized through the well-characterized shikimic acid pathway. A molecule of glucose is transferred by the UDP-glucosyltransferase (or possibly by the β-D-glucosidase too) to the tyrosol to form salidroside. On the other hand, salidroside may be degraded into tyrosol and glucose by β-D-glucosidase. Progress in research of these two key-enzymes. involved in the metabolism of salidroside,is finally elaborated.

  7. Activity Changes of Carbohydrate Metabolism Related Enzymes of S. cerevisiae after A Satellite Flight%卫星搭载啤酒酵母糖代谢相关酶类活性变化

    Institute of Scientific and Technical Information of China (English)

    易宗春; 薛明; 韩庆玲; 吴小荣; 孙艳

    2009-01-01

    The viable conditions and activities of carbohydrate metabolism related enzymes (CMRE) of Saccharomy-ces cerevisiae took a flight in Practice 8 recoverable satellite for 15 days (space yeast) were analyzed. The yeast was cultured in liquid YPD medium overnight, then diluted with fresh YPD to 10~(-6) times before taking the satellite flight and filled another one to keep on the ground for 15 days as a control. After recovery, the cell viability was determines by colony counting on fiat plate, and the activities of CMRE including hexokinase, suceinate dehydrogenase, malate dehydrogenase, and the content of glycogen in whole cells of both yeasts (satellite and ground) were tested and deter-mined with zymolysis combined with spectrophotometry. The results showed that the colony forming amount of space yeast samples was obviously higher than that of the ground control samples, as high as 3.1 times. However, the activ-ities of hexokinase and snccinate dehydrogenase of space yeast were obviously lower than the ground control one; but the activity of malate dehydrogenase of space yeast was higher than the ground control one. The content of glycogen of space yeast was lower than the ground control one. These indicated that under the space flight could increase the via-bility of the yeast, and at the same time it accompanied with the changes of the activities of related enzymes and the level of glycogen that conduced to the viability of the yeast in space flight conditions.%分析了搭载于实践八号育种卫星的啤酒酵母的存活情况和糖代谢相关酶类活性.啤酒酵母于YPD液体培养基培养,培养过夜后用新鲜YPD稀释10~(-6)倍,分装后分剐置于地面和卫星搭栽两种条件下15 d.返回地面后收集样品,利用稀释平板计数法检测啤酒酵母活力,采用酶解结合分光光度法检测酵母糖原水平,分光光度法检测己糖激酶、琥珀酸脱氢酶和苹果酸脱氢酶的活性.结果发现,卫星搭载样品的菌

  8. The effects of the continuous administration of N,N-dimethyl-4-phenylazoaniline (DAB) on the activities and the inducibilities of some drug-metabolizing enzymes in rat liver

    DEFF Research Database (Denmark)

    1975-01-01

    of dye feeding on some of the enzyme activities in the two major liver lobes and differences were found. (3) The effect of phenobarbital (PB) pretreatment on the DAB-fed rats was studied at 4-week intervals. The activities of DAB-azoreductase and of nitroreductase increased throughout the whole period...

  9. Enzyme Activities in Waste Water and Activated Sludge

    DEFF Research Database (Denmark)

    Nybroe, Ole; Jørgensen, Per Elberg; Henze, Mogens

    1992-01-01

    The purpose of the present study was to evaluate the potential of selected enzyme activity assays to determine microbial abundance and heterotrophic activity in waste water and activated sludge. In waste water, esterase and dehydrogenase activities were found to correlate with microbial abundance...... measured as colony forming units of heterotrophic bacteria. A panel of four enzyme activity assays, α-glucosidase, alanine-aminopeptidase, esterase and dehydrogenase were used to characterize activated sludge and anaerobic hydrolysis sludge from a pilot scale plant. The enzymatic activity profiles were...... distinctly different, suggesting that microbial populations were different, or had different physiological properties, in the two types of sludge. Enzyme activity profiles in activated sludge from four full-scale plants seemed to be highly influenced by the composition of the inlet. Addition of hydrolysed...

  10. Effect of music sound wave on respiratory metabolism and energy metabolism enzyme activities of Litopenaeus vannamei%音乐声波对凡纳滨对虾呼吸代谢和能量代谢酶活力的影响

    Institute of Scientific and Technical Information of China (English)

    朱玉杰; 王芳

    2012-01-01

    An experiment was carried out to investigate the effects of music sound wave on oxygen consumption rates, ammonia excretion rates and energy metabolism enzyme activities of Litopenaeus vannamei. There were control group and music group two groups. After a 60-day feeding trial, the results were as follows: under conditions of music, no significant differences were found in oxygen consumption rates and ammonia excretion rates of shrimps (P > 0.05), no significant difference was found in the muscle pyruvate kinase activity (P>0.05), but the muscle succinate actitivity was significantly higher(P <0.05), and the muscle lactate dehydrogenase activity was significantly lower (P <0.01). The results showed that music sound wave can increase the level of aerobic metabolism and decrease the level of anaerobic metabolism of Litopenaeus vannamei.%实验室条件下研究了音乐声波对凡纳滨对虾呼吸代谢和能量代谢酶活力的影响.实验设对照组和音乐组两个处理组,凡纳滨对虾在两种条件下的驯化时间为60 d.实验结果表明:播放音乐条件下,凡纳滨对虾的耗氧率和排氨率无明显变化(P>0.05),对虾肌肉丙酮酸激酶活力无明显变化(P>0.05),但琥珀酸脱氢酶活力显著提高(P<0.05),乳酸脱氢酶活力显著降低(P<0.01).结果表明,音乐声波能够提高凡纳滨对虾肌肉的有氧代谢水平,降低其无氧代谢水平.

  11. Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: Phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase.

    Science.gov (United States)

    Rivero, Luz Amira; Concepción, Juan Luis; Quintero-Troconis, Ender; Quiñones, Wilfredo; Michels, Paul A M; Acosta, Héctor

    2016-06-01

    Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite.

  12. Hydrolytic enzyme activity enhanced by Barium supplementation

    Directory of Open Access Journals (Sweden)

    Camilo Muñoz

    2016-10-01

    Full Text Available Hydrolysis of polymers is a first and often limiting step during the degradation of plant residues. Plant biomass is generally a major component of waste residues and a major renewable resource to obtain a variety of secondary products including biofuels. Improving the performance of enzymatic hydrolysis of plant material with minimum costs and limiting the use of additional microbial biomass or hydrolytic enzymes directly influences competitiveness of these green biotechnological processes. In this study, we cloned and expressed a cellulase and two esterases recovered from environmental thermophilic soil bacterial communities and characterize their optimum activity conditions including the effect of several metal ions. Results showed that supplementing these hydrolytic reactions with Barium increases the activity of these extracellular hydrolytic enzymes. This observation represents a simple but major improvement to enhance the efficiency and competitiveness of this process within an increasingly important biotechnological sector.

  13. Engineered N-acetylhexosamine-active enzymes in glycoscience.

    Science.gov (United States)

    Slámová, Kristýna; Bojarová, Pavla

    2017-08-01

    In recent years, enzymes modifying N-acetylhexosamine substrates have emerged in numerous theoretical studies as well as practical applications from biology, biomedicine, and biotechnology. Advanced enzyme engineering techniques converted them into potent synthetic instruments affording a variety of valuable glycosides. This review presents the diversity of engineered enzymes active with N-acetylhexosamine carbohydrates: from popular glycoside hydrolases and glycosyltransferases to less known oxidases, epimerases, kinases, sulfotransferases, and acetylases. Though hydrolases in natura, engineered chitinases, β-N-acetylhexosaminidases, and endo-β-N-acetylglucosaminidases were successfully employed in the synthesis of defined natural and derivatized chitooligomers and in the remodeling of N-glycosylation patterns of therapeutic antibodies. The genes of various N-acetylhexosaminyltransferases were cloned into metabolically engineered microorganisms for producing human milk oligosaccharides, Lewis X structures, and human-like glycoproteins. Moreover, mutant N-acetylhexosamine-active glycosyltransferases were applied, e.g., in the construction of glycomimetics and complex glycostructures, industrial production of low-lactose milk, and metabolic labeling of glycans. In the synthesis of biotechnologically important compounds, several innovative glycoengineered systems are presented for an efficient bioproduction of GlcNAc, UDP-GlcNAc, N-acetylneuraminic acid, and of defined glycosaminoglycans. The above examples demonstrate that engineering of N-acetylhexosamine-active enzymes was able to solve complex issues such as synthesis of tailored human-like glycoproteins or industrial-scale production of desired oligosaccharides. Due to the specific catalytic mechanism, mutagenesis of these catalysts was often realized through rational solutions. Specific N-acetylhexosamine glycosylation is crucial in biological, biomedical and biotechnological applications and a good

  14. Multifaceted roles of metabolic enzymes of the Paracoccidioides species complex

    Directory of Open Access Journals (Sweden)

    Caroline Maria Marcos

    2014-12-01

    Full Text Available Paracoccidioides species are dimorphic fungi, and are the etiologic agents of paracoccidioidomycosis (PCM, a serious disease of multiple organs. The large number of tissues colonized by this fungus suggests the presence of a variety of surface molecules involved in adhesion. A surprising finding is that the majority of enzymes in the glycolytic pathway, tricarboxylic acid (TCA cycle and glyoxylate cycle in Paracoccidioides spp. has adhesive properties that aid in the interaction with the host extracellular matrix, and so act as ‘moonlighting’ proteins. Moonlighting proteins have multiple functions and add another dimension to cellular complexity, while benefiting cells in several ways. This phenomenon occurs in both eukaryotes and prokaryotes. For example, moonlighting proteins from the glycolytic pathway or TCA cycle can play roles in bacterial pathogens, either by acting as proteins secreted in a conventional pathway or not and/or as cell surface component that facilitate adhesion or adherence . This review outlines the multifuncionality exposed by a variety of Paracoccidioides spp. enzymes including aconitase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate lyase, malate synthase, triose phosphate isomerase, fumarase and enolase. The roles that moonlighting activities play in the virulence characteristics of this fungus and several other human pathogens during their interactions with the host are discussed.

  15. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

    Science.gov (United States)

    Lietzan, Adam D; St Maurice, Martin

    2014-02-15

    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  16. Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat

    DEFF Research Database (Denmark)

    Breinholt, V.; Lauridsen, S. T.; Daneshvar, B.

    2000-01-01

    to be affected by prior. lycopene exposure. The level of PhIP-DNA adducts in the liver or colon was likewise not affected by lycopene at any dose. Overall, the present study provides evidence that lycopene administered in the diet of young female rats exerts minor modifying effects toward antioxidant and drug-metabolizing...... enzymes involved in the protection against oxidative stress and cancer. The fact that these enzymatic activities are induced at all of these very low plasma levels, could be taken to suggest that modulation of antioxidant and drug-metabolizing enzymes map indeed be relevant to humans, which in general......The administration of lycopene to female rats at doses ranging from 0.001 to 0.1 g/kg b.w, per day for 2 weeks was found to alter the drug-metabolizing capacity and antioxidant status of the exposed animals. An investigation of four cytochrome P450-dependent enzymes revealed that benzyloxyresorufin...

  17. Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways.

    Science.gov (United States)

    Hutson, Susan M; Islam, Mohammad Mainul; Zaganas, Ioannis

    2011-09-01

    Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5'-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5'-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.

  18. The Activity Reaction Core and Plasticity of Metabolic Networks.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available Understanding the system-level adaptive changes taking place in an organism in response to variations in the environment is a key issue of contemporary biology. Current modeling approaches, such as constraint-based flux-balance analysis, have proved highly successful in analyzing the capabilities of cellular metabolism, including its capacity to predict deletion phenotypes, the ability to calculate the relative flux values of metabolic reactions, and the capability to identify properties of optimal growth states. Here, we use flux-balance analysis to thoroughly assess the activity of Escherichia coli, Helicobacter pylori, and Saccharomyces cerevisiae metabolism in 30,000 diverse simulated environments. We identify a set of metabolic reactions forming a connected metabolic core that carry non-zero fluxes under all growth conditions, and whose flux variations are highly correlated. Furthermore, we find that the enzymes catalyzing the core reactions display a considerably higher fraction of phenotypic essentiality and evolutionary conservation than those catalyzing noncore reactions. Cellular metabolism is characterized by a large number of species-specific conditionally active reactions organized around an evolutionary conserved, but always active, metabolic core. Finally, we find that most current antibiotics interfering with bacterial metabolism target the core enzymes, indicating that our findings may have important implications for antimicrobial drug-target discovery.

  19. Effect of Chromium(VI Toxicity on Enzymes of Nitrogen Metabolism in Clusterbean (Cyamopsis tetragonoloba L.

    Directory of Open Access Journals (Sweden)

    Punesh Sangwan

    2014-01-01

    Full Text Available Heavy metals are the intrinsic component of the environment with both essential and nonessential types. Their excessive levels pose a threat to plant growth and yield. Also, some heavy metals are toxic to plants even at very low concentrations. The present investigation (a pot experiment was conducted to determine the affects of varying chromium(VI levels (0.0, 0.5, 1.0, 2.0, and 4.0 mg chromium(VI kg−1 soil in the form of potassium dichromate on the key enzymes of nitrogen metabolism in clusterbean. Chromium treatment adversely affect nitrogenase, nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate dehydrogenase in various plant organs at different growth stages as specific enzyme activity of these enzymes decreased with an increase in chromium(VI levels from 0 to 2.0 mg chromium(VI kg−1 soil and 4.0 mg chromium(VI kg−1 soil was found to be lethal to clusterbean plants. In general, the enzyme activity increased with advancement of growth to reach maximum at flowering stage and thereafter decreased at grain filling stage.

  20. Polymorphisms in genes encoding acetylsalicylic acid metabolizing enzymes are unrelated to upper gastrointestinal health in cardiovascular patients on acetylsalicylic acid.

    NARCIS (Netherlands)

    Oijen, M.G.H. van; Huybers, S.; Peters, W.H.M.; Drenth, J.P.H.; Laheij, R.J.F.; Verheugt, F.W.A.; Jansen, J.B.M.J.

    2005-01-01

    BACKGROUND: As acetylsalicylic acid is metabolized by UDP-glucuronosyltransferase 1A6 (UGT1A6) and cytochrome P450 2C9 (CYP2C9), interindividual differences in activity of these enzymes may modulate the effects and side-effects of acetylsalicylic acid. The objective of this study was to assess wheth

  1. Assessment of Mercaptopurine (6MP) Metabolites and 6MP Metabolic Key-Enzymes in Childhood Acute Lymphoblastic Leukemia

    NARCIS (Netherlands)

    Wojtuszkiewicz, A.; Barcelos, A.; Dubbelman, B.; Abreu, R.A. de; Brouwer, C.; Bökkerink, J.P.M.; Haas, V. de; Groot-Kruseman, H. de; Jansen, Gert; Kaspers, G.L.; Cloos, J.; Peters, G.J.

    2014-01-01

    Pediatric acute lymphoblastic leukemia (ALL) is treated with combination chemotherapy including mercaptopurine (6MP) as an important component. Upon its uptake, 6MP undergoes a complex metabolism involving many enzymes and active products. The prognostic value of all the factors engaged in this path

  2. Following Enzyme Activity with Infrared Spectroscopy

    Directory of Open Access Journals (Sweden)

    Saroj Kumar

    2010-03-01

    Full Text Available Fourier transform infrared (FTIR spectroscopy provides a direct, "on-line" monitor of enzymatic reactions. Measurement of enzymatic activity is based on the fact that the infrared spectra of reactants and products of an enzymatic reaction are usually different. Several examples are given using the enzymes pyruvate kinase, fumarase and alcohol dehydrogenase. The main advantage of the infrared method is that it observes the reaction of interest directly, i.e.,no activity assay is required to convert the progress of the reaction into an observable quantity.

  3. Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia

    Directory of Open Access Journals (Sweden)

    Mahnaz Darvish-Damavandi

    2016-09-01

    Full Text Available Propionic acidemia (PA is a life-threatening disease caused by the deficiency of a mitochondrial biotin-dependent enzyme known as propionyl coenzyme-A carboxylase (PCC. This enzyme is responsible for degrading the metabolic intermediate, propionyl coenzyme-A (PP-CoA, derived from multiple metabolic pathways. Currently, except for drastic surgical and dietary intervention that can only provide partial symptomatic relief, no other form of therapeutic option is available for this genetic disorder. Here, we examine a novel approach in protein delivery by specifically targeting and localizing our protein candidate of interest into the mitochondrial matrix of the cells. In order to test this concept of delivery, we have utilized cell penetrating peptides (CPPs and mitochondria targeting sequences (MTS to form specific fusion PCC protein, capable of translocating and localizing across cell membranes. In vitro delivery of our candidate fusion proteins, evaluated by confocal images and enzymatic activity assay, indicated effectiveness of this strategy. Therefore, it holds immense potential in creating a new paradigm in site-specific protein delivery and enzyme replacement therapeutic for PA.

  4. Changes in metabolome and in enzyme activities during germination of Trichoderma atroviride conidia.

    Science.gov (United States)

    Kaliňák, Michal; Simkovič, Martin; Zemla, Peter; Matata, Matej; Molnár, Tomáš; Liptaj, Tibor; Varečka, L'udovít; Hudecová, Daniela

    2014-08-01

    The aim of this work was to study the metabolic changes during germination of Trichoderma atroviride conidia along with selected marker enzyme activities. The increase in proteinogenic amino acid concentrations together with the increase in glutamate dehydrogenase activity suggests a requirement for nitrogen metabolism. Even though the activities of tricarboxylic acid cycle enzymes also increased, detected organic acid pools did not change, which predisposes this pathway to energy production and supply of intermediates for further metabolism. The concentrations of many metabolites, including the main osmolytes mannitol and betaine, also increased during the formation of germ tubes. The activities of H(+)-ATPase and GDPase, the only marker enzymes that did not have detectable activity in non-germinated conidia, were shown with germ tubes.

  5. Immunohistochemical localization of key arachidonic acid metabolism enzymes during fracture healing in mice.

    Directory of Open Access Journals (Sweden)

    Hsuan-Ni Lin

    Full Text Available This study investigated the localization of critical enzymes involved in arachidonic acid metabolism during the initial and regenerative phases of mouse femur fracture healing. Previous studies found that loss of cyclooxygenase-2 activity impairs fracture healing while loss of 5-lipoxygenase activity accelerates healing. These diametric results show that arachidonic acid metabolism has an essential function during fracture healing. To better understand the function of arachidonic acid metabolism during fracture healing, expression of cyclooxygenase-1 (COX-1, cyclooxygenase -2 (COX-2, 5-lipoxygenase (5-LO, and leukotriene A4 hydrolase (LTA4H was localized by immunohistochemistry in time-staged fracture callus specimens. All four enzymes were detected in leukocytes present in the bone marrow and attending inflammatory response that accompanied the fracture. In the tissues surrounding the fracture site, the proportion of leukocytes expressing COX-1, COX-2, or LTA4H decreased while those expressing 5-LO remained high at 4 and 7 days after fracture. This may indicate an inflammation resolution function for 5-LO during fracture healing. Only COX-1 was consistently detected in fracture callus osteoblasts during the later stages of healing (day 14 after fracture. In contrast, callus chondrocytes expressed all four enzymes, though 5-LO appeared to be preferentially expressed in newly differentiated chondrocytes. Most interestingly, osteoclasts consistently and strongly expressed COX-2. In addition to bone surfaces and the growth plate, COX-2 expressing osteoclasts were localized at the chondro-osseous junction of the fracture callus. These observations suggest that arachidonic acid mediated signaling from callus chondrocytes or from callus osteoclasts at the chondro-osseous junction regulate fracture healing.

  6. Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole.

    Science.gov (United States)

    Kobayashi, Yasuna; Suzuki, Michiya; Ohshiro, Naomi; Sunagawa, Takashi; Sasaki, Tadanori; Oguro, Takiko; Tokuyama, Shogo; Yamamoto, Toshinori; Yoshida, Takemi

    2002-01-01

    To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long-Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5'-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver.

  7. Enzymes of creatine biosynthesis, arginine and methionine metabolism in normal and malignant cells.

    Science.gov (United States)

    Bera, Soumen; Wallimann, Theo; Ray, Subhankar; Ray, Manju

    2008-12-01

    The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

  8. Effect of insulin and glucose on adenosine metabolizing enzymes in human B lymphocytes.

    Science.gov (United States)

    Kocbuch, Katarzyna; Sakowicz-Burkiewicz, Monika; Grden, Marzena; Szutowicz, Andrzej; Pawelczyk, Tadeusz

    2009-01-01

    In diabetes several aspects of immunity are altered, including the immunomodulatory action of adenosine. Our study was undertaken to investigate the effect of different glucose and insulin concentrations on activities of adenosine metabolizing enzymes in human B lymphocytes line SKW 6.4. The activity of adenosine deaminase in the cytosolic fraction was very low and was not affected by different glucose concentration, but in the membrane fraction of cells cultured with 25 mM glucose it was decreased by about 35% comparing to the activity in cells maintained in 5 mM glucose, irrespective of insulin concentration. The activities of 5'-nucleotidase (5'-NT) and ecto-5'-NT in SKW 6.4 cells depended on insulin concentration, but not on glucose. Cells cultured with 10(-8) M insulin displayed an about 60% lower activity of cytosolic 5'-NT comparing to cells maintained at 10(-11) M insulin. The activity of ecto-5'-NT was decreased by about 70% in cells cultured with 10(-8) M insulin comparing to cells grown in 10(-11) M insulin. Neither insulin nor glucose had an effect on adenosine kinase (AK) activity in SKW 6.4 cells or in human B cells isolated from peripheral blood. The extracellular level of adenosine and inosine during accelerated catabolism of cellular ATP depended on glucose, but not on insulin concentration. Concluding, our study demonstrates that glucose and insulin differentially affect the activities of adenosine metabolizing enzymes in human B lymphocytes, but changes in those activities do not correlate with the adenosine level in cell media during accelerated ATP catabolism, implying that nucleoside transport is the primary factor determining the extracellular level of adenosine.

  9. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

    Science.gov (United States)

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5-50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans.

  10. Testing biochemistry revisited: how in vivo metabolism can be understood from in vitro enzyme kinetics.

    Directory of Open Access Journals (Sweden)

    Karen van Eunen

    Full Text Available A decade ago, a team of biochemists including two of us, modeled yeast glycolysis and showed that one of the most studied biochemical pathways could not be quite understood in terms of the kinetic properties of the constituent enzymes as measured in cell extract. Moreover, when the same model was later applied to different experimental steady-state conditions, it often exhibited unrestrained metabolite accumulation.Here we resolve this issue by showing that the results of such ab initio modeling are improved substantially by (i including appropriate allosteric regulation and (ii measuring the enzyme kinetic parameters under conditions that resemble the intracellular environment. The following modifications proved crucial: (i implementation of allosteric regulation of hexokinase and pyruvate kinase, (ii implementation of V(max values measured under conditions that resembled the yeast cytosol, and (iii redetermination of the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase under physiological conditions.Model predictions and experiments were compared under five different conditions of yeast growth and starvation. When either the original model was used (which lacked important allosteric regulation, or the enzyme parameters were measured under conditions that were, as usual, optimal for high enzyme activity, fructose 1,6-bisphosphate and some other glycolytic intermediates tended to accumulate to unrealistically high concentrations. Combining all adjustments yielded an accurate correspondence between model and experiments for all five steady-state and dynamic conditions. This enhances our understanding of in vivo metabolism in terms of in vitro biochemistry.

  11. The interactive effects of mercury and selenium on metabolic profiles, gene expression and antioxidant enzymes in halophyte Suaeda salsa.

    Science.gov (United States)

    Liu, Xiaoli; Lai, Yongkai; Sun, Hushan; Wang, Yiyan; Zou, Ning

    2016-04-01

    Suaeda salsa is the pioneer halophyte in the Yellow River Delta and was consumed as a popular vegetable. Mercury has become a highly risky contaminant in the sediment of intertidal zones of the Yellow River Delta. In this work, we investigated the interactive effects of mercury and selenium in S. salsa on the basis of metabolic profiling, antioxidant enzyme activities and gene expression quantification. Our results showed that mercury exposure (20 μg L(-1)) inhibited plant growth of S. salsa and induced significant metabolic responses and altered expression levels of INPS, CMO, and MDH in S. salsa samples, together with the increased activities of antioxidant enzymes including SOD and POD. Overall, these results indicated osmotic and oxidative stresses, disturbed protein degradation and energy metabolism change in S. salsa after mercury exposures. Additionally, the addition of selenium could induce both antagonistic and synergistic effects including alleviating protein degradation and aggravating osmotic stress caused by mercury.

  12. Epigenetics of dominance for enzyme activity

    Indian Academy of Sciences (India)

    Kuldip S Trehan; Kulbir S Gill

    2002-03-01

    We have isolated and purified two parental homodimers and a unique heterodimer of acid phosphatase [coded by Acph-11.05() and Acph-10.95()] from isogenic homozygotes and heterozygotes of Drosophila malerkotliana. and produce qualitatively different allozymes and the two alleles are expressed equally within and across all three genotypes and and play an equal role in the epigenetics of dominance. Subunit interaction in the heterodimer over a wide range of H+ concentrations accounts for the epigenetics of dominance for enzyme activity.

  13. Protein stability and enzyme activity at extreme biological temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Feller, Georges, E-mail: gfeller@ulg.ac.b [Laboratory of Biochemistry, Centre for Protein Engineering, Institute of Chemistry B6a, University of Liege, B-4000 Liege (Belgium)

    2010-08-18

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 {sup 0}C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

  14. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  15. Neuron-astrocyte interaction enhance GABAergic synaptic transmission in a manner dependent on key metabolic enzymes.

    Directory of Open Access Journals (Sweden)

    Przemysław eKaczor

    2015-04-01

    Full Text Available GABA is the major inhibitory neurotransmitter in the adult brain and mechanisms of GABAergic inhibition have been intensely investigated in the past decades. Recent studies provided evidence for an important role of astrocytes in shaping GABAergic currents. One of the most obvious, but yet poorly understood, mechanisms of the cross-talk between GABAergic currents and astrocytes is metabolism including neurotransmitter homeostasis. In particular, how modulation of GABAergic currents by astrocytes depends on key enzymes involved in cellular metabolism remains largely unknown. To address this issue, we have considered two simple models of neuronal cultures: nominally astrocyte-free neuronal culture (NC and neuronal-astrocytic co-cultures (ANCC and miniature Inhibitory Postsynaptic Currents (mIPSCs were recorded in control conditions and in the presence of respective enzyme blockers. We report that enrichment of neuronal culture with astrocytes results in a marked increase in mIPSC frequency. This enhancement of GABAergic activity was accompanied by increased number of GAD65 and vGAT puncta, indicating that at least a part of the frequency enhancement was due to increased number of synaptic contacts. Inhibition of glutamine synthetase (with MSO strongly reduced mIPSC frequency in ANCC but had no effect in NC. Moreover, treatment of ANCC with inhibitor of glycogen phosphorylase (BAYU6751 or with selective inhibitor of astrocytic Krebs cycle,fluoroacetate, resulted in a marked reduction of mIPSC frequency in ANCC having no effect in NC. We conclude that GABAergic synaptic transmission strongly depends on neuron-astrocyte interaction in a manner dependent on key metabolic enzymes as well as on the Krebs cycle.

  16. Comparisons of different muscle metabolic enzymes and muscle fiber types in Jinhua and Landrace pigs.

    Science.gov (United States)

    Guo, J; Shan, T; Wu, T; Zhu, L N; Ren, Y; An, S; Wang, Y

    2011-01-01

    Western and indigenous Chinese pig breeds show obvious differences in muscle growth and meat quality, however, the underlying molecular mechanism remains unclear. The main objective of this study was to evaluate the breed-specific mechanisms controlling meat quality and postmortem muscle metabolism. The specific purpose was to investigate the variations in meat quality, muscle fiber type, and enzyme activity between local Jinhua and exotic Landrace pigs at the same age (180 d of age), as well as the same BW of 64 kg, respectively. We compared differentially expressed muscle fiber types such as types I and IIa (oxidative), type IIb (glycolytic), as well as type IIx (intermediate) fibers in LM and soleus muscles of Jinhua and Landrace pigs using real-time reverse-transcription PCR. Furthermore, the metabolic enzyme activities of lactate dehydrogenase, as well as succinic dehydrogenase and malate dehydrogenase, were used as markers of glycolytic and oxidative capacities, respectively. Results showed that Jinhua pigs exhibited greater intramuscular fat content and less drip loss compared with the Landrace (P Landrace (P Landrace (P Landrace pigs. These results may provide valuable information for understanding the molecular mechanism responsible for breed specific differences in growth performance and meat quality.

  17. In vitro metabolism of fenofibric acid by carbonyl reducing enzymes.

    Science.gov (United States)

    Malátková, Petra; Kanavi, Matthildi; Nobilis, Milan; Wsól, Vladimír

    2016-10-25

    Fenofibric acid is a hypolipidemic drug that is used as an active ingredient per se or is administered in the form of fenofibrate that releases fenofibric acid after absorption. The metabolism of fenofibric acid is mediated primarily by glucuronidation. However, the other part of fenofibric acid is excreted as reduced fenofibric acid. Enzymes responsible for the formation of reduced fenofibric acid as well as their subcellular localization have remained unknown until now. We have found that the predominant site of fenofibric acid reduction is the human liver cytosol, whereas liver microsomes reduced fenofibric acid to a lower extent and exhibited a lower affinity for this drug (Km > 1000 μM). Of nine carbonyl-reducing enzymes (CREs) tested, CBR1 exhibited the greatest activity for fenofibric acid reduction (CLint = 85.975 μl/mg protein/min). CBR1 predominantly formed (-)-enantiomers of reduced fenofibric acid similar to liver cytosol and in accordance with the in vivo data. AKR1C1, AKR1C2, AKR1C3 and AKR1B1 were also identified as reductases of fenofibric acid but are expected to play only a minor role in fenofibric acid metabolism. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

    Science.gov (United States)

    Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

    2016-01-01

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. PMID:26365239

  19. Trace elements as an activator of antioxidant enzymes

    Directory of Open Access Journals (Sweden)

    Marta Wołonciej

    2016-12-01

    Full Text Available Oxidative stress is a state of impaired balance between the formation of free radicals and antioxidant capacity of the body. It causes many defects of the body, e.g. lipid peroxidation, DNA and protein damage. In order to prevent the effects of oxidative stress, the organism has developed defence mechanisms. These mechanisms capture and inhibit the formation of free radicals and also chelate ion metals that catalyse free radical reactions. Trace elements are components of antioxidant enzymes involved in antioxidant mechanisms. Selenium, as a selenocysteine, is a component of the active site of glutathione peroxidase (GPx. The main function of GPx is neutralization of hydrogen peroxide (H2O2 and organic peroxide (LOOH. Furthermore, selenium is a structural part of a large group of selenoproteins that are necessary for proper functioning of the body. Manganese, copper and zinc are a part of the group of superoxide dismutase enzymes (MnSOD, Cu/ZnSOD, which catalyse the superoxide anion dismutation into hydrogen peroxide and oxygen. Formed hydrogen peroxide is decomposed into water and oxygen by catalase or glutathione peroxidase. An integral component of catalase (CAT is iron ions. The concentration of these trace elements has a significant influence on the activity of antioxidant enzymes, and thus on defence against oxidative stress. Even a small change in the level of trace elements in the tissue causes a disturbance in their metabolism, leading to the occurrence of many diseases.

  20. Metabolism of Monoterpenes: Acetylation of (-)-Menthol by a Soluble Enzyme Preparation from Peppermint (Mentha piperita) Leaves.

    Science.gov (United States)

    Croteau, R; Hooper, C L

    1978-05-01

    The essential oil from mature leaves of flowering peppermint (Mentha piperita L.) contains up to 15% (-)-menthyl acetate, and leaf discs converted exogenous (-)-[G-(3)H]menthol into this ester in approximately 15% yield of the incorporated precursor. Leaf extracts catalyzed the acetyl coenzyme A-dependent acetylation of (-)-[G-(3)H]menthol and the product of this transacetylase reaction was identified by radiochromatographic techniques. Transacetylase activity was located mainly in the 100,000g supernatant fraction, and the preparation was partially purified by combination of Sephadex G-100 gel filtration and chromatography on O-diethylaminoethyl-cellulose. The transacetylase had a molecular weight of about 37,000 as judged by Sephadex G-150 gel filtration, and a pH optimum near 9. The apparent K(m) and velocity for (-)-menthol were 0.3 mm and 16 nmol/hr. mg of protein, respectively. The saturation curve for acetyl coenzyme A was sigmoidal, showing apparent saturation near 0.1 mm. Dithioerythritol was required for maximum activity and stability of the enzyme, and the enzyme was inhibited by thiol directed reagents such as p-hydroxymercuribenzoate. Diisopropylfluorophosphate also inhibited transacylation suggesting the involvement of a serine residue in catalysis. The transacylase was highly specific for acetyl coenzyme A; propionyl coenzyme A and butyryl coenzyme A were not nearly as efficient as acyl donors (11% and 2%, respectively). However, the enzyme was much less selective with regard to the alcohol substrate, suggesting that the nature of the acetate ester synthesized in mint is more dependent on the type of alcohol available than on the specificity of the transacetylase. This is the first report on an enzyme involved in monoterpenol acetylation in plants. A very similar enzyme, catalyzing this key reaction in the metabolism of menthol, was also isolated from the flowers of peppermint.

  1. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  2. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.

    Science.gov (United States)

    Shinohara, Naoki; Uyama, Toru; Jin, Xing-Hua; Tsuboi, Kazuhito; Tonai, Takeharu; Houchi, Hitoshi; Ueda, Natsuo

    2011-11-01

    A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

  3. A modern mode of activation for nucleic acid enzymes.

    Directory of Open Access Journals (Sweden)

    Dominique Lévesque

    Full Text Available Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes, a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process.

  4. Enzyme activities of demersal fishes from the shelf to the abyssal plain

    Science.gov (United States)

    Drazen, Jeffrey C.; Friedman, Jason R.; Condon, Nicole E.; Aus, Erica J.; Gerringer, Mackenzie E.; Keller, Aimee A.; Elizabeth Clarke, M.

    2015-06-01

    The present study examined metabolic enzyme activities of 61 species of demersal fishes (331 individuals) trawled from a 3000 m depth range. Citrate synthase, lactate dehydrogenase, malate dehydrogenase, and pyruvate kinase activities were measured as proxies for aerobic and anaerobic activity and metabolic rate. Fishes were classified according to locomotory mode, either benthic or benthopelagic. Fishes with these two locomotory modes were found to exhibit differences in metabolic enzyme activity. This was particularly clear in the overall activity of citrate synthase, which had higher activity in benthopelagic fishes. Confirming earlier, less comprehensive studies, enzyme activities declined with depth in benthopelagic fishes. For the first time, patterns in benthic species could be explored and these fishes also exhibited depth-related declines in enzyme activity, contrary to expectations of the visual interactions hypothesis. Trends were significant when using depth parameters taken from the literature as well as from the present trawl information, suggesting a robust pattern regardless of the depth metric used. Potential explanations for the depth trends are discussed, but clearly metabolic rate does not vary simply as a function of mass and habitat temperature in fishes as shown by the substantial depth-related changes in enzymatic activities.

  5. Estrogen metabolizing enzymes : biomarkers of exposure, effect and susceptibility for carcinogenesis

    NARCIS (Netherlands)

    Duursen, Majorie Beatrix Maria van

    2005-01-01

    In the etiology of breast cancer, estrogens and its metabolites play a key role as tumor initiators and promoters. Co-expression of estrogen synthesizing enzymes (aromatase and steroid sulfatase) and estrogen metabolizing enzymes (CYP1A1 and CYP1B1) in breast tissue makes it plausible that locally f

  6. An Extended Polyanion Activation Surface in Insulin Degrading Enzyme.

    Directory of Open Access Journals (Sweden)

    Eun Suk Song

    Full Text Available Insulin degrading enzyme (IDE is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ, glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use a computational approach to define three potential ATP binding sites and mutagenesis and kinetic studies to confirm the relevance of these sites. Mutations were made at four positively charged residues (Arg 429, Arg 431, Arg 847, Lys 898 within the polyanion-binding region, converting them to polar or hydrophobic residues. We find that mutations in all three ATP binding sites strongly decrease the degree of activation by ATP and can lower basal activity and cooperativity. Computational analysis suggests conformational changes that result from polyanion binding as well as from mutating residues involved in polyanion binding. These findings indicate the presence of multiple polyanion binding modes and suggest the anion-binding surface plays an important conformational role in controlling IDE activity.

  7. Discovery of new enzymes and metabolic pathways by using structure and genome context.

    Science.gov (United States)

    Zhao, Suwen; Kumar, Ritesh; Sakai, Ayano; Vetting, Matthew W; Wood, B McKay; Brown, Shoshana; Bonanno, Jeffery B; Hillerich, Brandan S; Seidel, Ronald D; Babbitt, Patricia C; Almo, Steven C; Sweedler, Jonathan V; Gerlt, John A; Cronan, John E; Jacobson, Matthew P

    2013-10-31

    Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns. We and others are developing computation-guided strategies for functional discovery with 'metabolite docking' to experimentally derived or homology-based three-dimensional structures. Bacterial metabolic pathways often are encoded by 'genome neighbourhoods' (gene clusters and/or operons), which can provide important clues for functional assignment. We recently demonstrated the synergy of docking and pathway context by 'predicting' the intermediates in the glycolytic pathway in Escherichia coli. Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB 2PMQ), 2-epimerization of trans-4-hydroxy-L-proline betaine (tHyp-B) and cis-4-hydroxy-D-proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.

  8. 杜仲叶绿原酸提取物对脂代谢关键酶活性的影响%Effects of Chlorogenic Acid Extract from Leaves of Eucommia ulmoides on Key Enzyme Activities in Lipid Metabolism

    Institute of Scientific and Technical Information of China (English)

    李文娜; 韩宇东; 刘银花; 陈阳; 肖苑

    2012-01-01

    目的 研究杜仲叶绿原酸提取物对脂代谢关键酶的影响,探讨其降血脂作用机理.方法 测定杜仲叶绿原酸提取物对离体胆固醇微胶粒形成,猪肝HMG-CoA还原酶活性以及胰脂肪酶活性的影响.结果 杜仲叶绿原酸提取物抑制离体胆固醇微胶粒形成作用(IC5o=64.8μg· mL-1强于绿原酸(IC50=82.2 μg· mL-1);抑制HMG-CoA还原酶效价强于辛伐他汀;抑制胰脂肪酶活性(IC5o =2.6 μg·mL-1强于绿原酸(IC5o=4.0 μg·mL-1)但略低于奥利司他(IC50 =1.7 μg·mL-1.结论 杜仲叶绿原酸提取物降血脂机理可能与抑制脂质的吸收转化、抑制肠道胆固醇的吸收和减少肝脏胆固醇的合成有关.%Objective To observe the effects of chlorogenic acid extract from leaves of Eucommia ulmoides on key enzyme activities in lipid metabolism and to explore its anti-hyperlipidemia mechanism. Methods The lipid-lowering effect and mechanism of chlorogenic acid extract in leaves of Eucommia ulmoides were studied by observing the influ ence on the formation of cholesterol micelles and on the inhibition of 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase from normal pig liver as well as pancreatic lipase in vitro. Results The chlorogenic acid extract from leaves of Eucommia ulmoides had stronger inhibitory effects on cholesterol micelles formation (IC50=64.8 μg·mL-1)than chloro- genie acid(IC50=82.2 μg·mL-1), and had stronger inhibitory potency on HMG-CoA reductase than simvastatin. The chlorogenic acid extract also had stronger inhibition on the activity of pancreatic lipase (IC50 =2.6 μg·mL-1) than chlorogenic acid (IC50=4.0 μg·mL-1) , but the inhibition of chlorogenic acid extract was inferior to orlistat (IC50 = 1.7 μg·mL-1). Conclusion The mechanism of chlorogenic acid extract from leaves of Eucommia ulmoides in reducing blood lipids perhaps was related with the inhibition of absorption and transformation of lipids and with the inhibition of intestinal absorption

  9. Discovery of a sesamin-metabolizing microorganism and a new enzyme.

    Science.gov (United States)

    Kumano, Takuto; Fujiki, Etsuko; Hashimoto, Yoshiteru; Kobayashi, Michihiko

    2016-08-09

    Sesamin is one of the major lignans found in sesame oil. Although some microbial metabolites of sesamin have been identified, sesamin-metabolic pathways remain uncharacterized at both the enzyme and gene levels. Here, we isolated microorganisms growing on sesamin as a sole-carbon source. One microorganism showing significant sesamin-degrading activity was identified as Sinomonas sp. no. 22. A sesamin-metabolizing enzyme named SesA was purified from this strain and characterized. SesA catalyzed methylene group transfer from sesamin or sesamin monocatechol to tetrahydrofolate (THF) with ring cleavage, yielding sesamin mono- or di-catechol and 5,10-methylenetetrahydrofolate. The kinetic parameters of SesA were determined to be as follows: Km for sesamin = 0.032 ± 0.005 mM, Vmax = 9.3 ± 0.4 (μmol⋅min(-1)⋅mg(-1)), and kcat = 7.9 ± 0.3 s(-1) Next, we investigated the substrate specificity. SesA also showed enzymatic activity toward (+)-episesamin, (-)-asarinin, sesaminol, (+)-sesamolin, and piperine. Growth studies with strain no. 22, and Western blot analysis revealed that SesA formation is inducible by sesamin. The deduced amino acid sequence of sesA exhibited weak overall sequence similarity to that of the protein family of glycine cleavage T-proteins (GcvTs), which catalyze glycine degradation in most bacteria, archaea, and all eukaryotes. Only SesA catalyzes C1 transfer to THF with ring cleavage reaction among GcvT family proteins. Moreover, SesA homolog genes are found in both Gram-positive and Gram-negative bacteria. Our findings provide new insights into microbial sesamin metabolism and the function of GcvT family proteins.

  10. Reconstruction of phylogenetic relationships from metabolic pathways based on the enzyme hierarchy and the gene ontology.

    Science.gov (United States)

    Clemente, José C; Satou, Kenji; Valiente, Gabriel

    2005-01-01

    There has been much interest in the structural comparison and alignment of metabolic pathways. Several techniques have been conceived to assess the similarity of metabolic pathways of different organisms. In this paper, we show that the combination of a new heuristic algorithm for the comparison of metabolic pathways together with any of three enzyme similarity measures (hierarchical, information content, and gene ontology) can be used to derive a metabolic pathway similarity measure that is suitable for reconstructing phylogenetic relationships from metabolic pathways. Experimental results on the Glycolysis pathway of 73 organisms representing the three domains of life show that our method outperforms previous techniques.

  11. [Progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes].

    Science.gov (United States)

    Wang, Huanhuan; Lu, Yayao; Peng, Bo; Qian, Xiaohong; Zhang, Yangjun

    2015-06-01

    Cytochrome P450 (CYP) enzymes and uridine 5-diphospho-glucuronosyltransferase (UGT) enzymes are critical enzymes for drug metabolism. Both chemical drugs and traditional Chinese medicines are converted to more readily excreted compounds by drug metabolizing enzymes in human livers. Because of the disparate expression of CYP and UGT enzymes among different individuals, accurate quantification of these enzymes is essential for drug pharmacology, drug-drug interactions and drug clinical applications. The research progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes in the recent decade is reviewed.

  12. Update on the Genetic Polymorphisms of Drug-Metabolizing Enzymes in Antiepileptic Drug Therapy

    Directory of Open Access Journals (Sweden)

    Junji Saruwatari

    2010-08-01

    Full Text Available Genetic polymorphisms in the genes that encode drug-metabolizing enzymes are implicated in the inter-individual variability in the pharmacokinetics and pharmaco-dynamics of antiepileptic drugs (AEDs. However, the clinical impact of these polymorphisms on AED therapy still remains controversial. The defective alleles of cytochrome P450 (CYP 2C9 and/or CYP2C19 could affect not only the pharmacokinetics, but also the pharmacodynamics of phenytoin therapy. CYP2C19 deficient genotypes were associated with the higher serum concentration of an active metabolite of clobazam, N-desmethylclobazam, and with the higher clinical efficacy of clobazam therapy than the other CYP2C19 genotypes. The defective alleles of CYP2C9 and/or CYP2C19 were also found to have clinically significant effects on the inter-individual variabilities in the population pharmacokinetics of phenobarbital, valproic acid and zonisamide. EPHX1 polymorphisms may be associated with the pharmacokinetics of carbamazepine and the risk of phenytoin-induced congenital malformations. Similarly, the UDP-glucuronosyltransferase 2B7 genotype may affect the pharmacokinetics of lamotrigine. Gluthatione S-transferase null genotypes are implicated in an increased risk of hepatotoxicity caused by carbamazepine and valproic acid. This article summarizes the state of research on the effects of mutations of drug-metabolizing enzymes on the pharmacokinetics and pharmacodynamics of AED therapies. Future directions for the dose-adjustment of AED are discussed.

  13. The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes

    Science.gov (United States)

    McMillan, Duncan G. G.; Marritt, Sophie J.; Kemp, Gemma L.; Gordon-Brown, Piers; Butt, Julea N.; Jeuken, Lars J. C.

    2014-01-01

    It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes. PMID:24634538

  14. The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes.

    Science.gov (United States)

    McMillan, Duncan G G; Marritt, Sophie J; Kemp, Gemma L; Gordon-Brown, Piers; Butt, Julea N; Jeuken, Lars J C

    2013-11-01

    It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes.

  15. [Detection of enzyme activity in decontaminated spices in industrial use].

    Science.gov (United States)

    Müller, R; Theobald, R

    1995-03-01

    A range of decontaminated species of industrial use have been examined for their enzymes (catalase, peroxidase, amylase, lipase activity). The genuine enzymes remain fully active in irradiated spices, whereas the microbial load is clearly reduced. In contrast steam treated spices no longer demonstrate enzyme activities. Steam treatment offers e.g. black pepper without lipase activity, which can no longer cause fat deterioration. Low microbial load in combination with clearly detectable enzyme activity in spices is an indication for irradiation, whereas, reduced microbial contamination combined with enzyme inactivation indicate steam treatment of raw material.

  16. Discovery of a pyruvylated peptide-metabolizing enzyme using a fluorescent substrate-based protein discovery technique.

    Science.gov (United States)

    Yoshioka, Kentaro; Komatsu, Toru; Hanaoka, Kenjiro; Ueno, Tasuku; Terai, Takuya; Nagano, Tetsuo; Urano, Yasuteru

    2016-03-21

    We employed a fluorescent substrate-based target discovery approach to screen the enzymome for metabolic activity towards pyruvyl-amidated peptides, and identified an acylamino acid-releasing enzyme (APEH). Cells overexpressing APEH exhibited higher metabolic activity towards the probe, N-pyruvyl-leucyl-7-amido-4-methylcoumarin (Pyr-Leu-AMC), while the selective APEH inhibitor AA74-1 blocked the reaction. Metabolism of various pyruvylated peptides in liver lysate was almost completely blocked by AA74-1. Pyruvyl peptides are synthesized in response to oxidative stress, but their biological role is poorly understood; identification of a key contributor to their metabolism should stimulate research on pathways leading from oxidative stress to protein modification and biological output.

  17. Modeling the role of covalent enzyme modification in Escherichia coli nitrogen metabolism

    Science.gov (United States)

    Kidd, Philip B.; Wingreen, Ned S.

    2010-03-01

    In the bacterium Escherichia coli, the enzyme glutamine synthetase (GS) converts ammonium into the amino acid glutamine. GS is principally active when the cell is experiencing nitrogen limitation, and its activity is regulated by a bicyclic covalent modification cascade. The advantages of this bicyclic-cascade architecture are poorly understood. We analyze a simple model of the GS cascade in comparison to other regulatory schemes and conclude that the bicyclic cascade is suboptimal for maintaining metabolic homeostasis of the free glutamine pool. Instead, we argue that the lag inherent in the covalent modification of GS slows the response to an ammonium shock and thereby allows GS to transiently detoxify the cell, while maintaining homeostasis over longer times.

  18. Malate metabolism in Bacillus subtilis: distinct roles for three classes of malate-oxidizing enzymes.

    Science.gov (United States)

    Meyer, Frederik M; Stülke, Jörg

    2013-02-01

    The Gram-positive soil bacterium Bacillus subtilis uses glucose and malate as the preferred carbon sources. In the presence of either glucose or malate, the expression of genes and operons for the utilization of secondary carbon sources is subject to carbon catabolite repression. While glucose is a preferred substrate in many organisms from bacteria to man, the factors that contribute to the preference for malate have so far remained elusive. In this work, we have studied the contribution of the different malate-metabolizing enzymes in B. subtilis, and we have elucidated their distinct functions. The malate dehydrogenase and the phosphoenolpyruvate carboxykinase are both essential for malate utilization; they introduce malate into gluconeogenesis. The NADPH-generating malic enzyme YtsJ is important to establish the cellular pools of NADPH for anabolic reactions. Finally, the NADH-generating malic enzymes MaeA, MalS, and MleA are involved in keeping the ATP levels high. Together, this unique array of distinct activities makes malate a preferred carbon source for B. subtilis.

  19. Maternal and embryonic control of uterine sphingolipid-metabolizing enzymes during murine embryo implantation.

    Science.gov (United States)

    Kaneko-Tarui, Tomoko; Zhang, Ling; Austin, Kathleen J; Henkes, Luiz E; Johnson, Joshua; Hansen, Thomas R; Pru, James K

    2007-10-01

    During early gestation in invasively implanting species, the uterine stromal compartment undergoes dramatic remodeling, defined by the differentiation of stromal fibroblast cells into decidual cells. Lipid signaling molecules from a number of pathways are well-established functional components of this decidualization reaction. Because of a correlation in the events that transpire in the uterus during early implantation with known functions of bioactive sphingolipid metabolites established from studies in other organ systems, we hypothesized that uterine sphingolipid metabolism would change during implantation. By a combination of Northern blot, Western blot, and immunohistochemical analyses, we establish that enzymes at each of the major catalytic steps in the sphingolipid cascade become transcriptionally up-regulated in the uterus during decidualization. Each of the enzymes analyzed was up-regulated from Days of Pregnancy (DOP) 4.5-7.5. When comparing embryo-induced decidualization (decidual) with mechanically induced decidualization (deciduomal), sphingomyelin phosphodiesterase 1 (Smpd1) mRNA and sphingosine kinase 1 (SPHK1) protein were shown to be dually regulated in the endometrium by both maternal and embryonic factors. As measured by the diacyl glycerol kinase assay, ceramide levels rose in parallel with Smpd1 gene expression, suggesting that elevated transcription of sphingolipid enzymes results in heightened catalytic activity of the pathway. Altogether, these findings place sphingolipids on a growing list of lipid signaling molecules that become increasingly present at the maternal-embryonic interface.

  20. Effect of a PCB-based transformer oil on testicular steroidogenesis and xenobiotic-metabolizing enzymes.

    Science.gov (United States)

    Andric, Nebojsa L; Kostic, Tatjana S; Zoric, Sonja N; Stanic, Bojana D; Andric, Silvana A; Kovacevic, Radmila Z

    2006-07-01

    Pyralene is a PCB-based transformer oil with a unique PCB congener profile when compared to other mixtures. We studied the influence of Pyralene on testicular steroidogenesis and the status of xenobiotic-metabolizing enzymes in the testis and liver of rats during oral exposure (10 and 50 mg/kg body weight, p.o. daily for 1 week) and a 3-week post-treatment recovery period. As expected, Pyralene induced a rapid and sustained increase in mRNA transcripts for CYP1A1 and CYP2B1 in hepatocytes that was associated with a dramatic increase in ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD) activities. Testicular androgenesis and the conversion of progesterone to testosterone in testicular microsomes were bidirectionally affected. An increase in these parameters was observed 24h after the initial administration of Pyralene, followed by inhibition that lasted until the fourth post-treatment day. Expression PCR analysis revealed a significant decrease in 17beta-hydroxysteroid dehydrogenase (17betaHSD) transcript abundance at 48 h after Pyralene administration. In contrast, transcripts for several other steroidogenic enzymes and for testicular CYP1A1, CYP1B1, and CYP2B1 were unaffected under the same conditions. These results in the rat indicate that a sub-chronic exposure to Pyralene disrupted testicular steroidogenesis and suggest the mechanism may involve direct action on the regulation of specific steroidogenic enzymes such as 17betaHSD.

  1. Predicting metabolic pathways of small molecules and enzymes based on interaction information of chemicals and proteins.

    Science.gov (United States)

    Gao, Yu-Fei; Chen, Lei; Cai, Yu-Dong; Feng, Kai-Yan; Huang, Tao; Jiang, Yang

    2012-01-01

    Metabolic pathway analysis, one of the most important fields in biochemistry, is pivotal to understanding the maintenance and modulation of the functions of an organism. Good comprehension of metabolic pathways is critical to understanding the mechanisms of some fundamental biological processes. Given a small molecule or an enzyme, how may one identify the metabolic pathways in which it may participate? Answering such a question is a first important step in understanding a metabolic pathway system. By utilizing the information provided by chemical-chemical interactions, chemical-protein interactions, and protein-protein interactions, a novel method was proposed by which to allocate small molecules and enzymes to 11 major classes of metabolic pathways. A benchmark dataset consisting of 3,348 small molecules and 654 enzymes of yeast was constructed to test the method. It was observed that the first order prediction accuracy evaluated by the jackknife test was 79.56% in identifying the small molecules and enzymes in a benchmark dataset. Our method may become a useful vehicle in predicting the metabolic pathways of small molecules and enzymes, providing a basis for some further analysis of the pathway systems.

  2. Sedentary activity associated with metabolic syndrome independent of physical activity

    DEFF Research Database (Denmark)

    Bankoski, Andrea; Harris, Tamara B; McClain, James J

    2011-01-01

    This study examined the association between objectively measured sedentary activity and metabolic syndrome among older adults.......This study examined the association between objectively measured sedentary activity and metabolic syndrome among older adults....

  3. Screening the ToxCast Phase I Chemical Library for inhibition of Deiodinase Type I enzyme activity

    Science.gov (United States)

    Thyroid hormone (TH) signaling in vertebrates is dependent upon coordination of multiple key events including iodide uptake, hormone synthesis, metabolism and elimination, to maintain proper homeostasis of the hormones. Deiodinase enzymes interconvert THs between less active and...

  4. Changes in activities of tissues enzymes in rats administered Ficus ...

    African Journals Online (AJOL)

    enzymes in the serum, liver, kidney and heart of albino rats. ... significant alteration in enzyme activities in the serum and tissues as well as relative organ ... powder using an electrical blender. ... (average weight 160 g) at the Central Animal.

  5. Gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes.

    Science.gov (United States)

    Ow, Yin-Yin; Stupans, Ieva

    2003-06-01

    Gallic acid and its structurally related compounds are found widely distributed in fruits and plants. Gallic acid, and its catechin derivatives are also present as one of the main phenolic components of both black and green tea. Esters of gallic acid have a diverse range of industrial uses, as antioxidants in food, in cosmetics and in the pharmaceutical industry. In addition, gallic acid is employed as a source material for inks, paints and colour developers. Studies utilising these compounds have found them to possess many potential therapeutic properties including anti-cancer and antimicrobial properties. In this review, studies of the effects of gallic acid, its esters, and gallic acid catechin derivatives on Phase I and Phase II enzymes are examined. Many published reports of the effects of the in vitro effects of gallic acid and its derivatives on drug metabolising enzymes concern effects directly on substrate (generally drug or mutagen) metabolism or indirectly through observed effects in Ames tests. In the case of the Ames test an antimutagenic effect may be observed through inhibition of CYP activation of indirectly acting mutagens and/or by scavenging of metabolically generated mutagenic electrophiles. There has been considerable interest in the in vivo effects of the gallate esters because of their incorporation into foodstuffs as antioxidants and in the catechin gallates with their potential role as chemoprotective agents. Principally an induction of Phase II enzymes has been observed however more recent studies using HepG2 cells and primary cultures of human hepatocytes provide evidence for the overall complexity of actions of individual components versus complex mixtures, such as those in food. Further systematic studies of mechanisms of induction and inhibition of drug metabolising enzymes by this group of compounds are warranted in the light of their distribution and consequent ingestion, current uses and suggested therapeutic potential. However, it

  6. Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

    Science.gov (United States)

    Shisler, Krista A; Broderick, Joan B

    2014-03-15

    The glycyl radical enzyme activating enzymes (GRE-AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe-4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE-AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE-AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE-AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes. Copyright © 2014. Published by Elsevier Inc.

  7. Correlating structure and function of drug-metabolizing enzymes: progress and ongoing challenges.

    Science.gov (United States)

    Johnson, Eric F; Connick, J Patrick; Reed, James R; Backes, Wayne L; Desai, Manoj C; Xu, Lianhong; Estrada, D Fernando; Laurence, Jennifer S; Scott, Emily E

    2014-01-01

    This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology held April 20-24 in Boston, MA. Presentations discussed the status of cytochrome P450 (P450) knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drug-metabolizing P450 enzymes is known. However, the flexibility of these active sites can limit the predictive value of one structure for other ligands. A second limitation is our coarse-grain understanding of P450 interactions with membranes, other P450 enzymes, NADPH-cytochrome P450 reductase, and cytochrome b5. Recent work has examined differential P450 interactions with reductase in mixed P450 systems and P450:P450 complexes in reconstituted systems and cells, suggesting another level of functional control. In addition, protein nuclear magnetic resonance is a new approach to probe these protein/protein interactions, identifying interacting b5 and P450 surfaces, showing that b5 and reductase binding are mutually exclusive, and demonstrating ligand modulation of CYP17A1/b5 interactions. One desired outcome is the application of such information to control drug metabolism and/or design selective P450 inhibitors. A final presentation highlighted development of a CYP3A4 inhibitor that slows clearance of human immunodeficiency virus drugs otherwise rapidly metabolized by CYP3A4. Although understanding P450 structure/function relationships is an ongoing challenge, translational advances will benefit from continued integration of existing and new biophysical approaches.

  8. Homology modeling of mosquito cytochrome P450 enzymes involved in pyrethroid metabolism: insights into differences in substrate selectivity

    Directory of Open Access Journals (Sweden)

    Rongnoparut Pornpimol

    2011-09-01

    Full Text Available Abstract Background Cytochrome P450 enzymes (P450s have been implicated in insecticide resistance. Anopheles minumus mosquito P450 isoforms CYP6AA3 and CYP6P7 are capable of metabolizing pyrethroid insecticides, however CYP6P8 lacks activity against this class of compounds. Findings Homology models of the three An. minimus P450 enzymes were constructed using the multiple template alignment method. The predicted enzyme model structures were compared and used for molecular docking with insecticides and compared with results of in vitro enzymatic assays. The three model structures comprise common P450 folds but differences in geometry of their active-site cavities and substrate access channels are prominent. The CYP6AA3 model has a large active site allowing it to accommodate multiple conformations of pyrethroids. The predicted CYP6P7 active site is more constrained and less accessible to binding of pyrethroids. Moreover the predicted hydrophobic interface in the active-site cavities of CYP6AA3 and CYP6P7 may contribute to their substrate selectivity. The absence of CYP6P8 activity toward pyrethroids appears to be due to its small substrate access channel and the presence of R114 and R216 that may prevent access of pyrethroids to the enzyme heme center. Conclusions Differences in active site topologies among CYPAA3, CYP6P7, and CYP6P8 enzymes may impact substrate binding and selectivity. Information obtained using homology models has the potential to enhance the understanding of pyrethroid metabolism and detoxification mediated by P450 enzymes.

  9. 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.

  10. Mitigating role of baicalein on lysosomal enzymes and xenobiotic metabolizing enzyme status during lung carcinogenesis of Swiss albino mice induced by benzo(a)pyrene.

    Science.gov (United States)

    Naveenkumar, Chandrashekar; Raghunandakumar, Subramanian; Asokkumar, Selvamani; Binuclara, John; Rajan, Balan; Premkumar, Thandavamoorthy; Devaki, Thiruvengadam

    2014-06-01

    The lungs mainly serve as a primary site for xenobiotic metabolism and constitute an important defense mechanism against inhalation of carcinogens. Our current study aimed to evaluate the chemotherapeutic efficacy of baicalein (BE) in Swiss albino mice exposed to tobacco-specific carcinogen benzo(a)pyrene [B(a)P] for its ability to mitigate pulmonary carcinogenesis. Here, we report that altered activities/levels of lysosomal enzymes (cathepsin-D, cathepsin-B, acid phosphatase, β-D-galactosidase, β-D-glucuronidase, and β-D-N-acetyl glucosaminidase), phase I biotransformation enzymes (cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, and NADH-cytochrome b5 reductase), and phase II enzymes (glutathione S-transferase, UDP-glucuronyl transferase, and DT-diaphorase) were observed in the B(a)P-induced mice. Treatment with BE significantly restored back the activities/levels of lysosomal enzymes, phase I and phase II biotransformation enzymes. Moreover, assessment of lysosomal abnormalities by transmission electron microscopic examination revealed that BE treatment effectively counteract B(a)P-induced oxidative damages. Protein expression levels studied by immunohistochemistry, immunofluorescence, and immunoblot analysis of CYP1A1 revealed that BE treatment effectively negate B(a)P-induced upregulated expression of CYP1A1. Further analysis of scanning electron microscopic studies in lung was carried out to substantiate the anticarcinogenic effect of BE. The overall data suggest that BE treatment significantly inhibits lysosomal and microsomal dysfunction, thus revealing its potent anticarcinogenic effect.

  11. Effect of Dietary Energy Levels on Activity of Lipids Metabolic Enzymes in Wujin Pigs%日粮能量水平对乌金猪脂肪代谢相关酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    潘洪彬; 陈强; 黄英; 张永云; 赵素梅; 高士争

    2012-01-01

    The aim of the study was to investigate the effects of dietary energy levels on lipids metabolic enzymes of liver and adipose tissue in Wujin pigs. Fifty-four Wujin pigs were randomly allotted into three groups fed diet containing different energy levels, there were eighteen pigs in each group with three replicates , and six pigs were used in per replicates. The dietary energy levels were high energy (HE, 14. 22 MJ/kg), medium energy (ME, 12. 89 MJ/kg) and low energy (LE, 11. 74 MJ/kg) respectively. At the body weight of 30, 60 and 100 kg, Wujin pigs were slaughtered and liver and back subcutaneous fatty tissue were taken for analyzing the activity of lipase ( DPS) , glucose-6 phosphate dehydrogenase (G-6-PDH) and malate dehydrogenase (MDH). The results showed that the activity of G-6-PDH and MDH in liver and back subcutaneous fatty tissue increased as Wujin pigs fed dietary of energy level increased, but, contents of UPS in liver and back subcutaneous fatty tissue decreased as Wujin pigs fed dietary of energy level increased, At 30kg body weight, the activity of LPS, G-6-PDH and MDH in liver and back subcutaneous fatty tissue of Wujin pigs were affected not significantly by the dietary energy level ( P > 0. 05 ). At 60 and 100 kg body weight, the LPS and G-6-PDH activity in liver and back subcutaneous fatty tissue of Wujin pigs fed on the high energy level dietary were significantly lower than the low energy level dietary (P <0. 05) , the MDH activity in liver and back subcutaneous fatty tissue of Wujin pigs fed on the high energy level dietary were significantly higher than the low energy level dietary (P < 0. 05 ). The results indicated that higher energy level of dietary could decrease endogenous energy release, increase exogenous energy availability and promote fat deposition in Wujin pigs at growth midanaphase.%为研究日粮不同能量水平对乌金猪脂肪代谢相关酶活性的影响,选取体重约15 kg的乌金猪54头,随机分为3组,下设3

  12. Undercover: Gene control by metabolites and metabolic enzymes

    NARCIS (Netherlands)

    J.A. van der Knaap (Jan); C.P. Verrijzer (Peter)

    2016-01-01

    textabstractTo make the appropriate developmental decisions or maintain homeostasis, cells and organisms must coordinate the expression of their genome and metabolic state. However, the molecular mechanisms that relay environmental cues such as nutrient availability to the appropriate gene expressio

  13. Ketone Body Metabolic Enzyme OXCT1 Regulates Prostate Cancer Chemoresistance

    Science.gov (United States)

    2015-12-01

    cellular respiration , we measured oxygen consumption in prostate cancer cells after docetaxel treatment. The results showed that upon docetaxel...and cellular energy homeostasis. Analysis of patient data indicated that higher OXCT1 levels are associated with docetaxel chemotherapy resistance...Objectives To determine the role of OXCT1-mediated ketone body utilization in regulating prostate cancer cell response to docetaxel, cellular metabolism

  14. Characterisation of a major enzyme of bovine nitrogen metabolism

    CSIR Research Space (South Africa)

    Mathomu, LM

    2010-09-01

    Full Text Available of cellular protein metabolism (Curthoys & Watford, 1995; Meister, 1974). Glutamine functions as a major inter-organ transport form of nitrogen, carbon and serves as a source of energy between tissues such as brain, liver, kidney and even muscles...

  15. Effect of sprint cycle training on activities of antioxidant enzymes in human skeletal muscle

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Apple, F. S.; Sjödin, B.

    1996-01-01

    The effect of intermittent sprint cycle training on the level of muscle antioxidant enzyme protection was investigated. Resting muscle biopsies, obtained before and after 6 wk of training and 3, 24, and 72 h after the final session of an additional 1 wk of more frequent training, were analyzed...... for activities of the antioxidant enzymes glutathione peroxidase (GPX), glutathione reductase (GR), and superoxide dismutase (SOD). Activities of several muscle metabolic enzymes were determined to assess the effectiveness of the training. After the first 6-wk training period, no change in GPX, GR, or SOD...... the level of antioxidant protection in the muscle....

  16. Disturbance of Antioxidant Enzymes and Purine Metabolism in the Ejaculate of Men Living in Disadvantaged Areas of Kyzylorda Region

    Directory of Open Access Journals (Sweden)

    Valentihna N. Kislitskaya

    2015-07-01

    CONCLUSIONS: According to the results of study, it was put the influence  of negative factors of the Aral Sea region in men’s sperm of reproductive  age gives to disability free-radical processes, that proves changing of ferments of ant oxidative protection Catalase and adenosine deaminase (ADA.  This disturbance in men’s sperm of reproductive age leading to increased level of oxidative stress and impaired activity of  antioxidant enzymes and purine metabolism, responsible for the abnormal transmembrane and intracellular processes, reflecting the degree of imbalance of enzymes.

  17. Therapeutic effect of tamoxifen and energy-modulating vitamins on carbohydrate-metabolizing enzymes in breast cancer.

    Science.gov (United States)

    Perumal, Selvanathan Saravana; Shanthi, Palanivelu; Sachdanandam, Panchanadham

    2005-07-01

    increases in gluconeogenic enzymes. The activities of the mitochondrial Krebs cycle enzymes isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, and respiratory chain enzymes NADH dehydrogenase and cytochrome c oxidase were significantly reduced in both tumour and surrounding tissue of the mammary carcinoma-bearing rats. These biochemical disturbances were effectively counteracted by supplementation with EMV, which restored the activities of all these enzyme to their respective control levels. Combination therapy of TAM with EMV not only alters carbohydrate metabolism but can also prevent body weight loss by enhancing the host energy metabolism.

  18. VISCOSITY DICTATES METABOLIC ACTIVITY of Vibrio ruber

    Directory of Open Access Journals (Sweden)

    Maja eBoric

    2012-07-01

    Full Text Available Little is known about metabolic activity of bacteria, when viscosity of their environment changes. In this work, bacterial metabolic activity in media with viscosity ranging from 0.8 to 29.4 mPas was studied. Viscosities up to 2.4 mPas did not affect metabolic activity of Vibrio ruber. On the other hand, at 29.4 mPas respiration rate and total dehydrogenase activity increased 8 and 4-fold, respectively. The activity of glucose-6-phosphate dehydrogenase increased up to 13-fold at higher viscosities. However, intensified metabolic activity did not result in faster growth rate. Increased viscosity delayed the onset as well as the duration of biosynthesis of prodigiosin. As an adaptation to viscous environment V. ruber increased metabolic flux through the pentose phosphate pathway and reduced synthesis of a secondary metabolite. In addition, V. ruber was able to modify the viscosity of its environment.

  19. In vitro metabolic stability and intestinal transport of P57AS3 (P57) from Hoodia gordonii and its interaction with drug metabolizing enzymes.

    Science.gov (United States)

    Madgula, Vamsi L M; Avula, Bharathi; Pawar, Rahul S; Shukla, Yatin J; Khan, Ikhlas A; Walker, Larry A; Khan, Shabana I

    2008-08-01

    Hoodia gordonii, a succulent cactus-like plant growing in South Africa, has been used in traditional medicine for its appetite suppressant properties. Its use as a dietary supplement to promote weight loss has recently gained popularity. An oxypregnane steroidal glycoside P57AS3 (P57) is reported to be the active constituent of the sap extract responsible for anorexigenic activity. No information is available about its metabolic stability, intestinal transport and interaction with drug metabolizing enzymes. In the present investigation, the metabolic stability of P57 in human liver microsomes and its interaction with drug metabolizing enzymes (CYP1A2, 2C9, 3A4 and 2D6) were determined. Intestinal transport of P57 was studied in the Caco-2 cell model of intestinal transport and absorption. P57 was metabolically stable in the presence of human liver microsomes. The compound inhibited CYP3A4 activity with an IC50 value of 45 microM, whereas the activity of CYP 1A2, 2C9 and 2D6 was not inhibited. In the Caco-2 model, P57 exhibited a higher transport in the secretory direction than in the absorptive direction with efflux ratios of 3.1 and 3.8 at 100 and 200 microM, respectively. The efflux was inhibited by selective inhibitors of multidrug resistance associated proteins MRP1/MRP2 (MK-571) and P-gp (verapamil). In conclusion, intestinal transport of P57 was mediated by P-gp and MRP transporters. The compound was metabolically stable and showed weak inhibition of CYP 3A4.

  20. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes

    Directory of Open Access Journals (Sweden)

    Cho YY

    2014-11-01

    Full Text Available Yong-Yeon Cho,1 Hyeon-Uk Jeong,1 Jeong-Han Kim,2 Hye Suk Lee1 1College of Pharmacy, The Catholic University of Korea, Bucheon, Korea; 2Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea Abstract: Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP, UDP-glucuronosyltransferase (UGT, and sulfotransferase 2A1 (SULT2A1, were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 µM increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 µM did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19 or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1 in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. Keywords: honokiol, human hepatocytes, drug interactions, cytochrome P450, UDP-glucuronosyltransferases

  1. Genome-wide identification of gibberellins metabolic enzyme genes and expression profiling analysis during seed germination in maize.

    Science.gov (United States)

    Song, Jian; Guo, Baojian; Song, Fangwei; Peng, Huiru; Yao, Yingyin; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2011-08-15

    Gibberellin (GA) is an essential phytohormone that controls many aspects of plant development. To enhance our understanding of GA metabolism in maize, we intensively screened and identified 27 candidate genes encoding the seven GA metabolic enzymes including ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox), using all available public maize databases. The results indicate that maize genome contains three CPS, four KS, two KO and one KAO genes, and most of them are arranged separately on the maize genome, which differs from that in rice. In addition, the enzymes catalyzing the later steps (ZmGA20ox, ZmGA3ox and ZmGA2ox) are also encoded by gene families in maize, but GA3ox enzyme is likely to be encoded by single gene. Expression profiling analysis exhibited that transcripts of 15 GA metabolic genes could be detected during maize seed germination, which provides further evidence for the notion that increased synthesis of active GA in the embryo is required for triggering germination events. Moreover, a variety of temporal genes expression patterns of GA metabolic genes were detected, which revealed the complexity of underlying mechanism for GA regulated seed germination.

  2. Detection of Extracellular Enzyme Activities in Ganoderma neo-japonicum

    OpenAIRE

    Jo, Woo-Sik; Park, Ha-Na; Cho, Doo-Hyun; Yoo, Young-Bok; Park, Seung-Chun

    2011-01-01

    The ability of Ganoderma to produce extracellular enzymes, including β-glucosidase, cellulase, avicelase, pectinase, xylanase, protease, amylase, and ligninase was tested in chromogenic media. β-glucosidase showed the highest activity, among the eight tested enzymes. In particular, Ganoderma neo-japonicum showed significantly stronger activity for β-glucosidase than that of the other enzymes. Two Ganoderma lucidum isolates showed moderate activity for avicelase; however, Ganoderma neo-japonic...

  3. Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes.

    Science.gov (United States)

    Weng, Zuquan; Greenhaw, James; Salminen, William F; Shi, Qiang

    2012-11-15

    Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1-100μM for 30min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1μM EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1μM EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults.

  4. Immobilized formaldehyde-metabolizing enzymes from Hansenula polymorpha for removal and control of airborne formaldehyde.

    Science.gov (United States)

    Sigawi, Sasi; Smutok, Oleh; Demkiv, Olha; Zakalska, Oksana; Gayda, Galina; Nitzan, Yeshayahu; Nisnevitch, Marina; Gonchar, Mykhaylo

    2011-05-20

    Formaldehyde (FA)-containing indoor air has a negative effect on human health and should be removed by intensive ventilation or by catalytic conversion to non-toxic products. FA can be oxidized by alcohol oxidase (AOX) taking part in methanol metabolism of methylotrophic yeasts. In the present work, AOX isolated from a Hansenula polymorpha C-105 mutant (gcr1 catX) overproducing this enzyme in glucose medium, was tested for its ability to oxidize airborne FA. A continuous fluidized bed bioreactor (FBBR) was designed to enable an effective bioconversion of airborne FA by AOX or by permeabilized mutant H. polymorpha C-105 cells immobilized in calcium alginate beads. The immobilized AOX having a specific activity of 6-8 U mg⁻¹ protein was shown to preserve 85-90% of the initial activity. The catalytic parameters of the immobilized enzyme were practically the same as for the free enzyme (k(cat)/K(m) was 2.35×10³ M⁻¹ s⁻¹ vs 2.89×10³ M⁻¹ s⁻¹, respectively). The results showed that upon bubbling of air containing from 0.3 up to 18.5 ppm FA through immobilized AOX in the range of 1.3-26.6 U g⁻¹ of the gel resulted in essential decrease of FA concentration in the outlet gas phase (less than 0.02-0.03 ppm, i.e. 10-fold less than the threshold limit value). It was also demonstrated that a FBBR with immobilized permeabilized C-105 cells provided more than 90% elimination of airborne FA. The process was monitored by a specially constructed enzymatic amperometric biosensor based on FA oxidation by NAD+ and glutathione-dependent formaldehyde dehydrogenase from the recombinant H. polymorpha Tf 11-6 strain.

  5. Drug Metabolism in Human Brain: High Levels of Cytochrome P4503A43 in Brain and Metabolism of Anti-Anxiety Drug Alprazolam to Its Active Metabolite

    OpenAIRE

    Varsha Agarwal; Reddy P. Kommaddi; Khader Valli; Daniel Ryder; Hyde, Thomas M.; Kleinman, Joel E; Strobel, Henry W.; Vijayalakshmi Ravindranath

    2008-01-01

    Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabo...

  6. Crystallographic B factor of critical residues at enzyme active site

    Institute of Scientific and Technical Information of China (English)

    张海龙; 宋时英; 林政炯

    1999-01-01

    Thirty-seven sets of crystallographic enzyme data were selected from Protein Data Bank (PDB, 1995). The average temperature factors (B) of the critical residues at the active site and the whole molecule of those enzymes were calculated respectively. The statistical results showed that the critical residues at the active site of most of the enzymes had lower B factors than did the whole molecules, indicating that in the crystalline state the critical residues at the active site of the natural enzymes possess more stable conformation than do the whole molecules. The flexibility of the active site during the unfolding by denaturing was also discussed.

  7. An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

    DEFF Research Database (Denmark)

    Rasmussen, Randi Engelberth; Erstad, Simon Matthé; Ramos Martinez, Erick Miguel

    2016-01-01

    microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls...... and subsequent activity assays were successfully adapted to the 96-well plate system. CONCLUSIONS: An easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using...... radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner....

  8. Pesticide exposure and genetic variation in xenobiotic-metabolizing enzymes interact to induce biochemical liver damage.

    Science.gov (United States)

    Hernández, Antonio F; Gil, Fernando; Lacasaña, Marina; Rodríguez-Barranco, Miguel; Tsatsakis, Aristidis M; Requena, Mar; Parrón, Tesifón; Alarcón, Raquel

    2013-11-01

    Metabolic activation of pesticides in the liver may result in highly reactive intermediates capable of impairing various cellular functions. Nevertheless, the knowledge about the effect of pesticide exposure on liver function is still limited. This study assessed whether exposure to pesticides elicits early biochemical changes in biomarkers of liver function and looked for potential gene-environmental interactions between pesticide exposure and polymorphisms of pesticide-metabolizing genes. A longitudinal study was conducted in farm-workers from Andalusia (South Spain), during two periods of the same crop season with different degree of pesticide exposure. Blood samples were taken for the measurement of serum and erythrocyte cholinesterase activities as well as for determining clinical chemistry parameters as biomarkers of liver function. Serum lipid levels were also measured as they may help to monitor the progress of toxic liver damage. A reduction in serum cholinesterase was associated with decreased levels of all clinical chemistry parameters studied except HDL-cholesterol. Conversely, a decreased erythrocyte cholinesterase (indicating long-term pesticide exposure) was associated with increased levels of aspartate aminotransferase and alkaline phosphatase and increased levels of triglycerides, total cholesterol and LDL-cholesterol, but reduced levels of HDL-cholesterol. Changes in liver biomarkers were particularly associated with the PON155M/192R haplotype. The obtained results therefore support the hypothesis that pesticide exposure results in subtle biochemical liver toxicity and highlight the role of genetic polymorphisms in pesticide-metabolizing enzymes as biomarkers of susceptibility for developing adverse health effects. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.

    Science.gov (United States)

    Paiardini, Alessandro; Tramonti, Angela; Schirch, Doug; Guiducci, Giulia; di Salvo, Martino Luigi; Fiascarelli, Alessio; Giorgi, Alessandra; Maras, Bruno; Cutruzzolà, Francesca; Contestabile, Roberto

    2016-11-01

    The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP.

  10. Emerging roles for brain drug-metabolizing cytochrome P450 enzymes in neuropsychiatric conditions and responses to drugs.

    Science.gov (United States)

    Toselli, Francesca; Dodd, Peter R; Gillam, Elizabeth M J

    2016-08-01

    P450s in the human brain were originally considered unlikely to contribute significantly to the clearance of drugs and other xenobiotic chemicals, since their overall expression was a small fraction of that found in the liver. However, it is now recognized that P450s play substantial roles in the metabolism of both exogenous and endogenous chemicals in the brain, but in a highly cell type- and region-specific manner, in line with the greater functional heterogeneity of the brain compared to the liver. Studies of brain P450 expression and the characterization of the catalytic activity of specific forms expressed as recombinant enzymes have suggested possible roles for xenobiotic-metabolizing P450s in the brain. It is now possible to confirm these roles through the use of intracerebroventricular administration of selective P450 inhibitors in animal models, coupled with brain sampling techniques to measure drug concentrations in vivo, and modern neuroimaging techniques. The purpose of this review is to discuss the evidence behind the functional importance of P450s from the "xenobiotic-metabolizing" families, CYP1, CYP2 and CYP3 in the brain. Approaches used to define the quantitative and qualitative significance of these P450s in determining tissue-specific levels of xenobiotics in brain will be considered. Finally, the possible roles of these enzymes in brain biochemistry will be examined in light of the demonstrated activity of these enzymes in vitro and the association of particular P450 forms with disease states.

  11. Effects of Dietary Carbohydrate Sources on Growth Performance, Digestive Enzyme and Carbohydrate Metabolic Key Enzyme Activities of Large Yellow Croaker ( Larmichthys crocea Richardson)%饲料中糖源对大黄鱼生长性能及消化酶、糖代谢关键酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    李弋; 周飘苹; 邱红; 候迎梅; 申屠基康; 周歧存

    2015-01-01

    为评估饲料中糖源对大黄鱼生长性能及消化酶、糖代谢关键酶活性的影响,进行为期8周的养殖试验. 以鱼粉、小麦蛋白粉和豆粕为蛋白质源,鱼油、豆油和大豆卵磷脂为脂肪源,葡萄糖、蔗糖、糊精、土豆淀粉、玉米淀粉和小麦淀粉分别为糖源,配制成6 种等氮等能的试验饲料. 每种试验饲料设3个重复,每个重复放养初始平均体重为(7.06±0.48) g的大黄鱼幼鱼50尾,以重复为单位养殖于浮伐式网箱(1.5 m×1.5 m×2.0 m)中,每天饲喂2次. 结果表明:小麦淀粉和玉米淀粉组大黄鱼的增重率、特定生长率、饲料效率和蛋白质效率均显著高于蔗糖和葡萄糖组( P0.05),但小麦淀粉、土豆淀粉和玉米淀粉组的全鱼粗脂肪含量显著高于糊精、蔗糖和葡萄糖组( P0.05). 由结果得出,大黄鱼对小麦淀粉和玉米淀粉等结构复杂多糖的利用能力要高于蔗糖和葡萄糖.%An 8-week feeding trial was conducted to evaluate the effects of dietary carbohydrate sources on growth performance, digestive enzyme and carbohydrate metabolic key enzyme activities of large yellow croak-er ( Larmichthys crocea Richardson) . Fish meal, wheat gluten and soybean meal were used as protein sources, fish oil, soybean oil and soybean lecithin were used as lipid sources, and wheat starch, corn starch, potato starch, dextrin, saccharose and glucose were used as carbohydrate sources, respectively, six isonitrogenous and isoenergetic diets were prepared. Each diet was randomly assigned to 3 replicates, and each replicate had 50 juvenile large yellow croaker with the initial average body weight of (7.06±0.48) g which were stocked in floating net cages (1.5 m×1.5 m×2.0 m), and were fed twice daily. The results showed as follows: fish fed the diets containing wheat starch or corn starch had significantly higher weight gain ratio ( WGR ) , specific growth rate ( SGR) , feed efficiency ( FE) and protein efficiency ratio

  12. EFFECTS OF DIETARY CARBOHYDRATE SOURCES AND LEVELS ON THE ACTIVITIES OF CARBOHYDRATE METABOLIC ENZYMES IN TURBOT%不同糖源及糖水平对大菱鲆糖代谢酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    聂琴; 苗惠君; 苗淑彦; 陈尘悦; 李静; 张文兵; 麦康森

    2013-01-01

    采用3×4双因素实验设计,以初始质量为(8.06士0.08)g的大菱鲆幼鱼(Scophthalmus maximus L.)为对象,研究在饲料中添加3种糖源(葡萄糖、蔗糖和糊精)及4个水平(0、5%、15%、28%)对大菱鲆肝脏糖酵解关键酶己糖激酶(HK)、葡萄糖激酶(GK)、磷酸果糖激酶(PFK)、丙酮酸激酶(PK)和糖异生关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)、l,6-二磷酸果糖酶(FBPase)活性的影响.结果表明:饲料糖添加量从0升高到15%时,大菱鲆的糖酵解酶GK和PK活性随饲料葡萄糖或糊精含量的增加而增加;当饲料中葡萄糖或糊精含量为28%时,GK和PK活性有下降的趋势.3种糖源的4个添加水平对HK和PFK活性均无显著影响(P>0.05).添加不同水平的葡萄糖对大菱鲆糖异生途径的PEPCK活性无显著影响(P>0.05),但在饲料中葡萄糖添加量为5%时显著促进了FBPase活性(P<0.05),当葡萄糖添加量升高为15%或28%时,FBPase活性与对照组无显著差异(P>0.05).糊精作为饲料糖源时抑制了大菱鲆肝脏FBPase和PEPCK的活性,而添加不同水平的蔗糖对FBPase和PEPCK活性的影响均不显著(P>0.05).总的来说,从大菱鲆幼鱼肝脏糖代谢角度而言,在饲料中添加15%的葡萄糖或糊精时,可以有效促进大菱鲆肝脏糖酵解能力;较添加葡萄糖,糊精在促进大菱鲆肝脏糖酵解的同时对糖异生存在一定程度的抑制.蔗糖作为饲料糖源时,仅在添加量为28%时显著促进糖酵解酶GK活性,糖酵解其他酶活性以及糖异生酶活性均不受蔗糖水平的显著影响.%A 3×4 two-factorial experiment was conducted to investigate the effects of dietary carbohydrate sources (glucose,sucrose and dextrin) and levels (0,5%,15% and 28%) on the activities of glycolytic and gluconeogenic enzymes in turbot (Scophthalmus maximus L.).The initial weight of the turbot was (8.06±0.08) g.Each diet was fed to triplicate groups of turbot in a flow

  13. Increments and duplication events of enzymes and transcription factors influence metabolic and regulatory diversity in prokaryotes.

    Directory of Open Access Journals (Sweden)

    Mario Alberto Martínez-Núñez

    Full Text Available In this work, the content of enzymes and DNA-binding transcription factors (TFs in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM and structural domains (Superfamily. For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22% than in the bacterial ones (27% was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected.

  14. Increments and duplication events of enzymes and transcription factors influence metabolic and regulatory diversity in prokaryotes.

    Science.gov (United States)

    Martínez-Núñez, Mario Alberto; Poot-Hernandez, Augusto Cesar; Rodríguez-Vázquez, Katya; Perez-Rueda, Ernesto

    2013-01-01

    In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected.

  15. Increments and Duplication Events of Enzymes and Transcription Factors Influence Metabolic and Regulatory Diversity in Prokaryotes

    Science.gov (United States)

    Martínez-Núñez, Mario Alberto; Poot-Hernandez, Augusto Cesar; Rodríguez-Vázquez, Katya; Perez-Rueda, Ernesto

    2013-01-01

    In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected. PMID:23922780

  16. Spatial distribution of enzyme activities in the rhizosphere

    Science.gov (United States)

    Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    The rhizosphere, the tiny zone of soil surrounding roots, certainly represents one of the most dynamic habitat and interfaces on Earth. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. That is why there is an urgent need in spatially explicit methods for the determination of the rhizosphere extension and enzyme distribution. Recently, zymography as a new technique based on diffusion of enzymes through the 1 mm gel plate for analysis has been introduced (Spohn & Kuzyakov, 2013). We developed the zymography technique to visualize the enzyme activities with a higher spatial resolution. For the first time, we aimed at quantitative imaging of enzyme activities as a function of distance from the root tip and the root surface in the soil. We visualized the two dimensional distribution of the activity of three enzymes: β-glucosidase, phosphatase and leucine amino peptidase in the rhizosphere of maize using fluorogenically labelled substrates. Spatial-resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography visualized heterogeneity of enzyme activities along the roots. The activity of all enzymes was the highest at the apical parts of individual roots. Across the roots, the enzyme activities were higher at immediate vicinity of the roots (1.5 mm) and gradually decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify spatial distribution of enzyme activities in the rhizosphere hotspots. References Spohn, M., Kuzyakov, Y., 2013. Phosphorus mineralization can be driven by microbial need for carbon. Soil Biology & Biochemistry 61: 69-75

  17. A systems biology framework for modeling metabolic enzyme inhibition of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Reifman Jaques

    2009-09-01

    Full Text Available Abstract Background Because metabolism is fundamental in sustaining microbial life, drugs that target pathogen-specific metabolic enzymes and pathways can be very effective. In particular, the metabolic challenges faced by intracellular pathogens, such as Mycobacterium tuberculosis, residing in the infected host provide novel opportunities for therapeutic intervention. Results We developed a mathematical framework to simulate the effects on the growth of a pathogen when enzymes in its metabolic pathways are inhibited. Combining detailed models of enzyme kinetics, a complete metabolic network description as modeled by flux balance analysis, and a dynamic cell population growth model, we quantitatively modeled and predicted the dose-response of the 3-nitropropionate inhibitor on the growth of M. tuberculosis in a medium whose carbon source was restricted to fatty acids, and that of the 5'-O-(N-salicylsulfamoyl adenosine inhibitor in a medium with low-iron concentration. Conclusion The predicted results quantitatively reproduced the experimentally measured dose-response curves, ranging over three orders of magnitude in inhibitor concentration. Thus, by allowing for detailed specifications of the underlying enzymatic kinetics, metabolic reactions/constraints, and growth media, our model captured the essential chemical and biological factors that determine the effects of drug inhibition on in vitro growth of M. tuberculosis cells.

  18. Type IV collagen-degrading enzyme activity in human serum.

    OpenAIRE

    Hashimoto, Noriaki; Kobayashi, Michio; Watanabe,Akiharu; Higashi, Toshiro; Tsuji, Takao

    1988-01-01

    Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful...

  19. Type IV collagen-degrading enzyme activity in human serum.

    Directory of Open Access Journals (Sweden)

    Hashimoto,Noriaki

    1988-02-01

    Full Text Available Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful for estimating the degradation of type IV collagen.

  20. Thin film voltammetry of metabolic enzymes in rat liver microsomes

    Science.gov (United States)

    Krishnan, Sadagopan; Rusling, James F.

    2007-01-01

    We report herein thin film voltammetry and kinetics of electron transfer for redox proteins in rat liver microsomes for the first time. Films were made layer-by-layer from liver microsomes and polycations on pyrolytic graphite electrodes. Cyclic voltammograms were chemically reversible with a midpoint potential of −0.48 V vs SCE at 0.1 V s−1 in pH 7.0 phosphate buffer. Reduction peak potentials shifted negative at higher scan rates, and oxidation-reduction peak current ratios were ∼1 consistent with non-ideal quasireversible thin film voltammetry. Analysis of oxidation-reduction peak separations gave an average apparent surface electron transfer rate constant of 30 s−1. Absence of significant electrocatalytic reduction of O2 or H2O2 and lack of shift in midpoint potential when CO is added that indicates lack of an iron heme cofactor suggest that peaks can be attributed to oxidoreductases present in the microsomes rather than cytochrome P450 enzymes. PMID:18037986

  1. Kinetics of soil enzyme activities under different ecosystems: An index of soil quality

    Directory of Open Access Journals (Sweden)

    Monty Kujur

    2014-03-01

    Full Text Available Soil microbial activity plays an important role in regulating biotransformation, nutrient cycling and hence the microbiological processes are at the center of many ecological functions. The kinetic parameters (Vmax and KmMichaelis constant of different enzymes (amylase, invertase, protease, urease, and dehydrogenase were determined in order to assess the metabolic response of soil. The maximum reaction velocity (Vmax represents a maximum rate of activity when all enzymes are saturated, which markedly increased in forest soil as compared to fresh mine spoil due to the gradual accumulation of soil organic matter. Smaller Km value was estimated in forest soil (FS as compared to fresh mine spoil (FMS, suggesting the greater affinity of soil enzymes for substrate in FS. The catalytic efficiency (Vmax /Km reflects an impression on microbial community composition with a change in soil enzymes. These enzyme characters (activities and kinetic parameters have greater significance as early and sensitive indicators of the changes in soil properties induced by different management systems. These parameters (Vmax and Km can be useful markers to assess changes in microbial activity of soil, since they represent quantity and affinity of enzymes respectively. The metabolic index (dehydrogenase activity/organic carbon (OC was found to be correlated with Vmax of dehydrogenase (r = 0.953; p < 0.01 and OC (r = 0.880; p < 0.01. Principal component analysis was able to discriminate seven different soil samples into seven independent clusters based on their enzyme activities and kinetic parameters. Indeed, the study revealed the importance of kinetics study of soil enzymes, which can be considered valid parameters to monitor the evolution of microbiological activity in soil, and hence an index of soil quality.

  2. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    Science.gov (United States)

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2014-01-01

    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  3. Upgrading HepG2 cells with adenoviral vectors that encode drug-metabolizing enzymes: application for drug hepatotoxicity testing.

    Science.gov (United States)

    Gómez-Lechón, M José; Tolosa, Laia; Donato, M Teresa

    2017-02-01

    Drug attrition rates due to hepatotoxicity are an important safety issue considered in drug development. The HepG2 hepatoma cell line is currently being used for drug-induced hepatotoxicity evaluations, but its expression of drug-metabolizing enzymes is poor compared with hepatocytes. Different approaches have been proposed to upgrade HepG2 cells for more reliable drug-induced liver injury predictions. Areas covered: We describe the advantages and limitations of HepG2 cells transduced with adenoviral vectors that encode drug-metabolizing enzymes for safety risk assessments of bioactivable compounds. Adenoviral transduction facilitates efficient and controlled delivery of multiple drug-metabolizing activities to HepG2 cells at comparable levels to primary human hepatocytes by generating an 'artificial hepatocyte'. Furthermore, adenoviral transduction enables the design of tailored cells expressing particular metabolic capacities. Expert opinion: Upgraded HepG2 cells that recreate known inter-individual variations in hepatic CYP and conjugating activities due to both genetic (e.g., polymorphisms) or environmental (e.g., induction, inhibition) factors seems a suitable model to identify bioactivable drug and conduct hepatotoxicity risk assessments. This strategy should enable the generation of customized cells by reproducing human pheno- and genotypic CYP variability to represent a valuable human hepatic cell model to develop new safer drugs and to improve existing predictive toxicity assays.

  4. Metabolism of (-)-cis- and (-)-trans-rose oxide by cytochrome P450 enzymes in human liver microsomes.

    Science.gov (United States)

    Nakahashi, Hiroshi; Yamamura, Yuuki; Usami, Atsushi; Rangsunvigit, Pramoch; Malakul, Pomthong; Miyazawa, Mitsuo

    2015-12-01

    The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes.

  5. Effects of Lanthanum on Hydrolytic Enzyme Activities in Red Soil

    Institute of Scientific and Technical Information of China (English)

    褚海燕; 朱建国; 谢祖彬; 李振高; 曹志洪; 曾青; 林先贵

    2002-01-01

    The effects of La on some hydrolytic enzyme activities in red soil were studied in incubation and pot culture experiments. In the incubation experiment, La slightly stimulates the activities of urease and acidic phosphatase in soil and strongly stimulates sucrase activity in soil. In the pot culture experiment, La stimulates the activities of urease, acidic phosphatase and sucrase to different degrees. The stimulative effects of rare earth elements (REE) on hydrolytic enzyme activities in soil may result in increasing yield of crops.

  6. Enzymes of heme metabolism in the kidney: regulation by trace metals which do not form heme complexes.

    Science.gov (United States)

    Maines, M D; Kappas, A

    1977-11-01

    The in vivo regulation by metal ions of the enzymes of heme metabolism in kidney-particularly of ALAS, the rate-limiting enzyme in heine formation- was investigated. Ni(2+) and Pt(4+), metals which do not enzymatically form metalloporphyrins, were found to regulate ALAS in kidney as they do in liver. The pattern of this regulation was generally similar to that observed with heme and metal ions in liver, i.e., a late increase in enzyme activity after an early period in which ALAS activity was unaltered or inhibited. The metals did not interact with the enzyme in vitro to alter its activity. In this study no direct reciprocal relationship between ALAS activity and total cellular heine content was demonstrated. The metal ions, particularly Pt(4+), also altered the activity of other enzymes of heme biosynthesis in kidney. Pt(4+) severely inhibited the activity of ALAD and UROS. Ni(2+) and Pt(4+) were potent inducers of heme oxygenase, the initial and rate-limiting enzyme in heine degradation. It is proposed that the physiological regulation of ALAS is mediated through the action of metal ions, rather than by the cellular content of heine, and that the regulation of ALAS by heine reflects the action of the central metal ion of heme rather than that of the entire metalloporphyrin complex. In this proposed mechanism for metal ion regulation of ALAS, the tetrapyrrole moiety of heine is considered to function principally as an efficient carrier of metal to the regulatory site for ALAS production, inasmuch as the tetrapyrrole ring itself has been shown in earlier studies not to have any effect on ALAS activity. The production of heine oxygenase is believed to be similarly regulated.

  7. Proteinaceous inhibitors of carbohydrate-active enzymes in cereals: implication in agriculture, cereal processing and nutrition

    DEFF Research Database (Denmark)

    Juge, N.; Svensson, Birte

    2006-01-01

    Enzymes that degrade, modify, or create glycosidic bonds are involved in carbohydrate biosynthesis and remodelling. Microbial carbohydrate-active enzymes form the basis of current green technology in the food, feed, starch, paper and pulp industries and the revolution in genomics may offer long......-term gains on the quality and quantity of the raw materials. Proteinaceous inhibitors of carbohydrate-active enzymes (alpha-amylase, limit-dextrinase, polygalacturonase, pectin lyase, pectin methylesterase, invertase and xyloglucan endoglucanase) naturally occur in plants where they are involved in various...... roles from plant defence to metabolism. Xylanase inhibitors represent the latest addition to this growing family. In this review, we will focus on the inhibitors of carbohydrate-active enzymes present in cereals, mostly represented by et-amylase and xylanase inhibitors, and summarise the existing...

  8. Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

    Science.gov (United States)

    Allison, S. D.; Jastrow, J. D.

    2004-12-01

    Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

  9. Changes in the inorganic status and enzyme activities in senescent leaves of chickpea, Cicer arietinum L.

    Directory of Open Access Journals (Sweden)

    Chandrashekkhar V. Murumkar

    2014-01-01

    Full Text Available The changes in the level of some inorganic constituents and the activities of some important enzyme systems in senescent leaves of chickpea (Cicer arietinum L. have been studied. In senescent leaves, a marked decline in the potassium and phosphorus contents was evident which was accompanied by the accumulation of calcium, silicon, chloride and manganese. Leaf senescence was accompanied by a great increase in hydrolytic processes, as revealed by the increase in the activities of acid phosphatase, alkaline phosphatase, ATPase, inorganic pyrophosphatase and 3-phosphoglycerate phosphatase. The activities of nitrogen metabolism enzymes, namely nitrate reductase, nitrite reductase, glutamine synthetase and alanine aminotransferase, and of photorespiratory enzymes -- phosphoglycolate phosphatase, glycolate oxidase and catalase, were lower in senescent leaves. Leaf senescence was further associated with an increase in the activities of peroxidase and polyphenol oxidase, a considerable depression in pyruvate kinase activity, and a slight elevation in aldolase activity.

  10. Differentiated effect of ageing on the enzymes of Krebs' cycle, electron transfer complexes and glutamate metabolism of non-synaptic and intra-synaptic mitochondria from cerebral cortex.

    Science.gov (United States)

    Villa, R F; Gorini, A; Hoyer, S

    2006-11-01

    The effect of ageing on the activity of enzymes linked to Krebs' cycle, electron transfer chain and glutamate metabolism was studied in three different types of mitochondria of cerebral cortex of 1-year old and 2-year old male Wistar rats. We assessed the maximum rate (V(max)) of the mitochondrial enzyme activities in non-synaptic perikaryal mitochondria, and in two populations of intra-synaptic mitochondria. The results indicated that: (i) in normal, steady-state cerebral cortex the values of the catalytic activities of the enzymes markedly differed in the various populations of mitochondria; (ii) in intra-synaptic mitochondria, ageing affected the catalytic properties of the enzymes linked to Krebs' cycle, electron transfer chain and glutamate metabolism; (iii) these changes were more evident in intra-synaptic "heavy" than "light" mitochondria. These results indicate a different age-related vulnerability of subpopulations of mitochondria in vivo located into synapses than non-synaptic ones.

  11. Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. II. Metabolic characteristics of the enzyme

    Science.gov (United States)

    Leznicki, A. J.; Bandurski, R. S.

    1988-01-01

    The synthesis of indole-3-acetyl-1-O-beta-D-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as rho-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed.

  12. A Simple and Accurate Method for Measuring Enzyme Activity.

    Science.gov (United States)

    Yip, Din-Yan

    1997-01-01

    Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

  13. A Simple and Accurate Method for Measuring Enzyme Activity.

    Science.gov (United States)

    Yip, Din-Yan

    1997-01-01

    Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

  14. Arachidonic acid-metabolizing cytochrome P450 enzymes are targets of {omega}-3 fatty acids.

    Science.gov (United States)

    Arnold, Cosima; Markovic, Marija; Blossey, Katrin; Wallukat, Gerd; Fischer, Robert; Dechend, Ralf; Konkel, Anne; von Schacky, Clemens; Luft, Friedrich C; Muller, Dominik N; Rothe, Michael; Schunck, Wolf-Hagen

    2010-10-22

    Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ω-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these ω-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca(2+)-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms ω-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantly ω- and ω minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary ω-3 fatty acids.

  15. Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism

    OpenAIRE

    Lee, Su Hyeon; Kim, Eung Ju; Lee, Dong-Hyoung; Lee, Won-Yong; Chung, Bong Chul; Seo, Hong Seog; Choi, Man Ho

    2016-01-01

    Background Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. Methods Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatograp...

  16. Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

    Science.gov (United States)

    Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

    2013-12-01

    Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed.

  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. Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

    Science.gov (United States)

    Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

  19. Genetic polymorphism of metabolic enzymes modifies the risk of chronic solvent-induced encephalopathy

    NARCIS (Netherlands)

    S. Kezic; F. Calkoen; M.A.M. Wenker; J.J.L. Jacobs; M.M. Verberk

    2006-01-01

    In the present study, we investigate whether genetic polymorphism in enzymes involved in the metabolism of organic solvents influences susceptibility to chronic solvent encephalopathy (CSE), which is one of the major effects of long-term exposure to organic solvents. Polymorphisms in the genes encod

  20. Phenotype prediction of nonsynonymous single nucleotide polymorphisms in human phase II drug/xenobiotic metabolizing enzymes: perspectives on molecular evolution

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Nonsynonymous single nucleotide polymorphisms (nsSNPs) in coding regions can lead to amino acid changes that might alter the protein’s function and account for susceptibility to disease and altered drug/xenobiotic response. Many nsSNPs have been found in genes encoding human phase II metabolizing enzymes; however, there is little known about the relationship between the genotype and phenotype of nsSNPs in these enzymes. We have identified 923 validated nsSNPs in 104 human phase II enzyme genes from the Ensembl genome database and the NCBI SNP database. Using PolyPhen, Panther, and SNAP algorithms, 44%?59% of nsSNPs in phase II enzyme genes were predicted to have functional impacts on protein function. Predictions largely agree with the available experimental annotations. 68% of deleterious nsSNPs were correctly predicted as damaging. This study also identified many amino acids that are likely to be functionally critical, but have not yet been studied experimentally. There was significant concordance between the predicted results of Panther and PolyPhen, and between SNAP non-neutral predictions and PolyPhen scores. Evolutionarily non-neutral (destabilizing) amino acid substitutions are thought to be the pathogenetic basis for the alteration of phase II enzyme activity and to be associated with disease susceptibility and drug/xenobiotic toxicity. Furthermore, the molecular evolutionary patterns of phase II enzymes were characterized with regards to the predicted deleterious nsSNPs.

  1. Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism

    Science.gov (United States)

    Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

    2017-01-01

    Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis. PMID:28241048

  2. The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown.

    Science.gov (United States)

    Seppälä, Susanna; Wilken, St Elmo; Knop, Doriv; Solomon, Kevin V; O'Malley, Michelle A

    2017-09-21

    A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that are currently in biotechnological use, and explore more basal, non-conventional fungi and their underexploited biomass-degrading mechanisms as promising agents in the transition towards a bio-based society. Of special interest are anaerobic fungi like the Neocallimastigomycota, which were recently found to harbor the largest diversity of biomass-degrading enzymes among the fungal kingdom. Enzymes sourced from these basal fungi have been used to metabolically engineer substrate utilization in yeast, and may offer new paths to lignin breakdown and tunneled biocatalysis. We also contrast classic enzymology approaches with emerging 'omics'-based tools to decipher function within novel fungal isolates and identify new promising enzymes. Recent developments in genome editing are expected to accelerate discovery and metabolic engineering within these systems, yet are still limited by a lack of high-resolution genomes, gene regulatory regions, and even appropriate culture conditions. Finally, we present new opportunities to harness the biomass-degrading potential of undercharacterized fungi via heterologous expression and engineered microbial consortia. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  3. Functional diversity of carbohydrate-active enzymes enabling a bacterium to ferment plant biomass.

    Science.gov (United States)

    Boutard, Magali; Cerisy, Tristan; Nogue, Pierre-Yves; Alberti, Adriana; Weissenbach, Jean; Salanoubat, Marcel; Tolonen, Andrew C

    2014-11-01

    Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

  4. Functional diversity of carbohydrate-active enzymes enabling a bacterium to ferment plant biomass.

    Directory of Open Access Journals (Sweden)

    Magali Boutard

    2014-11-01

    Full Text Available Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme assays, RNA sequencing (RNA-seq, and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

  5. Human liver enzymes responsible for metabolic elimination of tyramine; a vasopressor agent from daily food.

    Science.gov (United States)

    Niwa, Toshiro; Murayama, Norie; Umeyama, Hiromi; Shimizu, Makiko; Yamazaki, Hiroshi

    2011-08-01

    Dietary tyramine is associated with hypertensive crises because of its ability to induce the release of catecholamines. The roles of monoamine oxidase (MAO); flavin-containing monooxygenase (FMO); and cytochrome P450 2D6 (CYP2D6) were studied in terms of the enzymatic elimination of tyramine in vitro at a substrate concentration of 1.0 µM; which is relevant to in vivo serum concentrations. Tyramine elimination by human liver supernatant fractions was decreased by ˜70% in the absence of NADPH. Pargyline; an MAO inhibitor; decreased tyramine elimination rates by ˜30%. Among recombinant P450 and FMO enzymes; CYP2D6 had a high activity in terms of tyramine elimination. Tyramine elimination rates were inhibited by quinidine and significantly correlated with bufuralol 1'-hydroxylation activities (a CYP2D6 marker). Liver microsomes genotyped for CYP2D6*10/*10 and CYP2D6*4/*4 showed low and undetectable activities; respectively; compared with the wild-type CYP2D6*1/*1. The present results suggest that tyramine is eliminated mainly by polymorphic CYP2D6. Tyramine toxicity resulting from differences in individual metabolic elimination is thus genetically determined.

  6. Genetic polymorphisms in alcohol-metabolizing enzymes and chronic pancreatitis.

    NARCIS (Netherlands)

    Verlaan, M.; Morsche, R.H.M. te; Roelofs, H.M.J.; Laheij, R.J.F.; Jansen, J.B.M.J.; Peters, W.H.M.; Drenth, J.P.H.

    2004-01-01

    AIMS: Alcohol misuse is now regarded as an important risk factor for development of chronic pancreatitis (CP). However, not every alcohol misuser develops CP and it therefore might be suggested that susceptibility could be further influenced by inter-individual variations in the activities of alcoho

  7. A Bayesian method for identifying missing enzymes in predicted metabolic pathway databases

    Directory of Open Access Journals (Sweden)

    Karp Peter D

    2004-06-01

    Full Text Available Abstract Background The PathoLogic program constructs Pathway/Genome databases by using a genome's annotation to predict the set of metabolic pathways present in an organism. PathoLogic determines the set of reactions composing those pathways from the enzymes annotated in the organism's genome. Most annotation efforts fail to assign function to 40–60% of sequences. In addition, large numbers of sequences may have non-specific annotations (e.g., thiolase family protein. Pathway holes occur when a genome appears to lack the enzymes needed to catalyze reactions in a pathway. If a protein has not been assigned a specific function during the annotation process, any reaction catalyzed by that protein will appear as a missing enzyme or pathway hole in a Pathway/Genome database. Results We have developed a method that efficiently combines homology and pathway-based evidence to identify candidates for filling pathway holes in Pathway/Genome databases. Our program not only identifies potential candidate sequences for pathway holes, but combines data from multiple, heterogeneous sources to assess the likelihood that a candidate has the required function. Our algorithm emulates the manual sequence annotation process, considering not only evidence from homology searches, but also considering evidence from genomic context (i.e., is the gene part of an operon? and functional context (e.g., are there functionally-related genes nearby in the genome? to determine the posterior belief that a candidate has the required function. The method can be applied across an entire metabolic pathway network and is generally applicable to any pathway database. The program uses a set of sequences encoding the required activity in other genomes to identify candidate proteins in the genome of interest, and then evaluates each candidate by using a simple Bayes classifier to determine the probability that the candidate has the desired function. We achieved 71% precision at a

  8. Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum: presence of a mitochondrial NADPH dehydrogenase.

    Science.gov (United States)

    Harris, Diana M; Diderich, Jasper A; van der Krogt, Zita A; Luttik, Marijke A H; Raamsdonk, Léonie M; Bovenberg, Roel A L; van Gulik, Walter M; van Dijken, Johannes P; Pronk, Jack T

    2006-03-01

    Based on assumed reaction network structures, NADPH availability has been proposed to be a key constraint in beta-lactam production by Penicillium chrysogenum. In this study, NADPH metabolism was investigated in glucose-limited chemostat cultures of an industrial P. chrysogenum strain. Enzyme assays confirmed the NADP(+)-specificity of the dehydrogenases of the pentose-phosphate pathway and the presence of NADP(+)-dependent isocitrate dehydrogenase. Pyruvate decarboxylase/NADP(+)-linked acetaldehyde dehydrogenase and NADP(+)-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. Although the NADPH requirement of penicillin-G-producing chemostat cultures was calculated to be 1.4-1.6-fold higher than that of non-producing cultures, in vitro measured activities of the major NADPH-providing enzymes were the same. Isolated mitochondria showed high rates of antimycin A-sensitive respiration of NADPH, thus indicating the presence of a mitochondrial NADPH dehydrogenase that oxidises cytosolic NADPH. The presence of this enzyme in P. chrysogenum might have important implications for stoichiometric modelling of central carbon metabolism and beta-lactam production and may provide an interesting target for metabolic engineering.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ogunwole, J.O.A.

    1984-01-01

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

  10. Operating Conditions Effects Onenzyme Activity: Case Enzyme Protease

    Directory of Open Access Journals (Sweden)

    Adel Oueslati

    2014-09-01

    Full Text Available The Proteases an enzyme added to detergents to degrade the protein spots origin.Their action is manifested through its activity the middle of washing clothes. This activity depends on the operating conditions. In this article, the effects of temperature and pH of the reaction and the substrate concentration and time of washing medium on the enzyme activity were studied. There action mechanism has been shown. The activity measurements were made by absorption spectrometry

  11. Industry as a metabolic activity.

    OpenAIRE

    Smart, B.(SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom)

    1992-01-01

    The concept of "industrial economic metabolism" can provide a bridge to better understanding between environmentalists and industry. In nature each individual or species reacts to natural stimuli, competing with others for resources, extending its domain until it loses comparative advantage and comes to equilibrium with an adjacent competitor. Those species that succeed over time flourish; those that do not, diminish or disappear. Nature's rule book has no moral or ethical ingredient beyond s...

  12. Diffusional correlations among multiple active sites in a single enzyme.

    Science.gov (United States)

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  13. Effect of commercially available green and black tea beverages on drug-metabolizing enzymes and oxidative stress in Wistar rats.

    Science.gov (United States)

    Yao, Hsien-Tsung; Hsu, Ya-Ru; Lii, Chong-Kuei; Lin, Ai-Hsuan; Chang, Keng-Hao; Yang, Hui-Ting

    2014-08-01

    The effect of commercially available green tea (GT) and black tea (BT) drinks on drug metabolizing enzymes (DME) and oxidative stress in rats was investigated. Male Wistar rats were fed a laboratory chow diet and GT or BT drink for 5 weeks. Control rats received de-ionized water instead of the tea drinks. Rats received the GT and BT drinks treatment for 5 weeks showed a significant increase in hepatic microsomal cytochrome P450 (CYP) 1A1 and CYP1A2, and a significant decrease in CYP2C, CYP2E1 and CYP3A enzyme activities. Results of immunoblot analyses of enzyme protein contents showed the same trend with enzyme activity. Significant increase in UDP-glucuronosyltransferase activity and reduced glutathione content in liver and lungs were observed in rats treated with both tea drinks. A lower lipid peroxide level in lungs was observed in rats treated with GT drink. Electrophoretic mobility shift assay revealed that both tea drinks decreased pregnane X receptor binding to DNA and increased nuclear factor-erythroid 2 p45-related factor 2 binding to DNA. These results suggest that feeding of both tea drinks to rats modulated DME activities and reduced oxidative stress in liver and lungs. GT drink is more effective on reducing oxidative stress than BT drink.

  14. 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 ...

  15. Lysine acetylation is a common post-translational modification of key metabolic pathway enzymes of the anaerobe Porphyromonas gingivalis.

    Science.gov (United States)

    Butler, Catherine A; Veith, Paul D; Nieto, Matthew F; Dashper, Stuart G; Reynolds, Eric C

    2015-10-14

    Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a keystone pathogen in the development of the bacterial-associated inflammatory oral disease chronic periodontitis. Although post-translational modifications (PTMs) of proteins are commonly found to modify protein function in eukaryotes and prokaryotes, PTMs such as lysine acetylation have not been examined in P. gingivalis. Lysine acetylation is the addition of an acetyl group to a lysine which removes this amino acid's positive charge and can induce changes in a protein's secondary structure and reactivity. A proteomics based approach combining immune-affinity enrichment with high sensitivity Orbitrap mass spectrometry identified 130 lysine acetylated peptides from 92 P. gingivalis proteins. The majority of these peptides (71) were attributed to 45 proteins with predicted metabolic activity; these proteins could be mapped to several P. gingivalis metabolic pathways where enzymes catalysing sequential reactions within the same pathway were often found acetylated. In particular, the catabolic pathways of complex anaerobic fermentation of amino acids to produce energy had 12 enzymes lysine acetylated. The results suggest that lysine acetylation may be an important mechanism in metabolic regulation in P. gingivalis, which is vital for P. gingivalis survival and adaptation of its metabolism throughout infection. Statement of significance. Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The ability of the pathogen to induce dysbiosis and disease is related to an array of specific virulence factors and metabolic regulation that enables the bacterium to proliferate in an inflamed periodontal pocket. The mechanisms P. gingivalis uses to adapt to a changing and hostile environment are poorly understood and here we show, for the first time, that enzymes of critical metabolic pathways for energy

  16. Microbial antimony biogeochemistry: Enzymes, regulation, and related metabolic pathways

    Science.gov (United States)

    Li, Jingxin; Qian Wang,; Oremland, Ronald S.; Kulp, Thomas R.; Rensing, Christopher; Wang, Gejiao

    2016-01-01

    Antimony (Sb) is a toxic metalloid that occurs widely at trace concentrations in soil, aquatic systems, and the atmosphere. Nowadays, with the development of its new industrial applications and the corresponding expansion of antimony mining activities, the phenomenon of antimony pollution has become an increasingly serious concern. In recent years, research interest in Sb has been growing and reflects a fundamental scientific concern regarding Sb in the environment. In this review, we summarize the recent research on bacterial antimony transformations, especially those regarding antimony uptake, efflux, antimonite oxidation, and antimonate reduction. We conclude that our current understanding of antimony biochemistry and biogeochemistry is roughly equivalent to where that of arsenic was some 20 years ago. This portends the possibility of future discoveries with regard to the ability of microorganisms to conserve energy for their growth from antimony redox reactions and the isolation of new species of “antimonotrophs.”

  17. Incorporation of enzyme concentrations into FBA and identification of optimal metabolic pathways

    Directory of Open Access Journals (Sweden)

    Mukhopadhyay Subhasis

    2008-07-01

    Full Text Available Abstract Background In the present article, we propose a method for determining optimal metabolic pathways in terms of the level of concentration of the enzymes catalyzing various reactions in the entire metabolic network. The method, first of all, generates data on reaction fluxes in a pathway based on steady state condition. A set of constraints is formulated incorporating weighting coefficients corresponding to concentration of enzymes catalyzing reactions in the pathway. Finally, the rate of yield of the target metabolite, starting with a given substrate, is maximized in order to identify an optimal pathway through these weighting coefficients. Results The effectiveness of the present method is demonstrated on two synthetic systems existing in the literature, two pentose phosphate, two glycolytic pathways, core carbon metabolism and a large network of carotenoid biosynthesis pathway of various organisms belonging to different phylogeny. A comparative study with the existing extreme pathway analysis also forms a part of this investigation. Biological relevance and validation of the results are provided. Finally, the impact of the method on metabolic engineering is explained with a few examples. Conclusions The method may be viewed as determining an optimal set of enzymes that is required to get an optimal metabolic pathway. Although it is a simple one, it has been able to identify a carotenoid biosynthesis pathway and the optimal pathway of core carbon metabolic network that is closer to some earlier investigations than that obtained by the extreme pathway analysis. Moreover, the present method has identified correctly optimal pathways for pentose phosphate and glycolytic pathways. It has been mentioned using some examples how the method can suitably be used in the context of metabolic engineering.

  18. Unraveling the toxicity mechanisms of the herbicide diclofop-methyl in rice: modulation of the activity of key enzymes involved in citrate metabolism and induction of cell membrane anion channels.

    Science.gov (United States)

    Ding, Haiyan; Lu, Haiping; Lavoie, Michel; Xie, Jun; Li, Yali; Lv, Xiaolu; Fu, Zhengwei; Qian, Haifeng

    2014-11-01

    Residual soil concentrations of the herbicide diclofop-methyl (DM) can be toxic to other nontarget plant species, but the toxicity mechanisms at play are not fully understood. In the present study, we analyzed the toxic effect of DM on root growth and metabolism in the rice species Oryza sativa. The results show that a 48-h exposure to a trace level (5 μg/L) of DM inhibits rice root growth by almost 70%. A 48-h exposure to 5 μg/L DM also leads to an ≈2.5-fold increase in citrate synthase (CS) activity (and CS gene transcription) and an ≈2-fold decrease in the citrate lyase gene transcripts, which lead to an increase in the intracellular concentration of citrate and in citrate exudation rate. Addition of a specific inhibitor of cell membrane anion channel, anthracene-9-carboxylic acid, decreased citrate release in the culture, suggesting that DM-induced citrate loss from the cells is mediated by a specific membrane-bound channel protein. This study brings new insights into the key biochemical mechanisms leading to DM toxicity in rice.

  19. Tumour-selective targeting of drug metabolizing enzymes to treat metastatic cancer.

    Science.gov (United States)

    Wierdl, Monika; Tsurkan, Lyudmila; Hatfield, M Jason; Potter, Philip M

    2016-10-01

    Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of ester-containing xenobiotics. This hydrolysis reaction results in the formation of the corresponding carboxylic acid and alcohol. Due to their highly plastic active site, CEs can hydrolyze structurally very distinct and complex molecules. Because ester groups significantly increase the water solubility of compounds, they are frequently used in the pharmaceutical industry to make relatively insoluble compounds more bioavailable. By default, this results in CEs playing a major role in the distribution and metabolism of these esterified drugs. However, this can be exploited to selectively improve compound hydrolysis, and using specific in vivo targeting techniques can be employed to generate enhanced drug activity. Here, we seek to detail the human CEs involved in esterified molecule hydrolysis, compare and contrast these with CEs present in small mammals and describe novel methods to improve drug therapy by specific delivery of CEs to cells in vivo. Finally, we will discuss the development of such approaches for their potential application towards malignant disease.

  20. The Angiotensin Converting Enzyme Insertion/Deletion Polymorphism Modifies Exercise-Induced Muscle Metabolism.

    Directory of Open Access Journals (Sweden)

    David Vaughan

    Full Text Available A silencer region (I-allele within intron 16 of the gene for the regulator of vascular perfusion, angiotensin-converting enzyme (ACE, is implicated in phenotypic variation of aerobic fitness and the development of type II diabetes. We hypothesised that the reportedly lower aerobic performance in non-carriers compared to carriers of the ACE I-allele, i.e. ACE-DD vs. ACE-ID/ACE-II genotype, is associated with alterations in activity-induced glucose metabolism and capillarisation in exercise muscle.Fifty-three, not-specifically trained Caucasian men carried out a one-legged bout of cycling exercise to exhaustion and/or participated in a marathon, the aim being to identify and validate genotype effects on exercise metabolism. Respiratory exchange ratio (RER, serum glucose and lipid concentration, glycogen, and metabolite content in vastus lateralis muscle based on ultra-performance lipid chromatography-mass spectrometry (UPLC-MS, were assessed before and after the cycling exercise in thirty-three participants. Serum metabolites were measured in forty subjects that completed the marathon. Genotype effects were assessed post-hoc.Cycling exercise reduced muscle glycogen concentration and this tended to be affected by the ACE I-allele (p = 0.09. The ACE-DD genotype showed a lower maximal RER and a selective increase in serum glucose concentration after exercise compared to ACE-ID and ACE-II genotypes (+24% vs. +2% and -3%, respectively. Major metabolites of mitochondrial metabolism (i.e. phosphoenol pyruvate, nicotinamide adenine dinucleotide phosphate, L-Aspartic acid, glutathione were selectively affected in vastus lateralis muscle by exercise in the ACE-DD genotype. Capillary-to-fibre ratio was 24%-lower in the ACE-DD genotype. Individuals with the ACE-DD genotype demonstrated an abnormal increase in serum glucose to 7.7 mM after the marathon.The observations imply a genetically modulated role for ACE in control of glucose import and oxidation in

  1. Highlighting the Need for Systems-Level Experimental Characterization of Plant Metabolic Enzymes.

    Science.gov (United States)

    Engqvist, Martin K M

    2016-01-01

    The biology of living organisms is determined by the action and interaction of a large number of individual gene products, each with specific functions. Discovering and annotating the function of gene products is key to our understanding of these organisms. Controlled experiments and bioinformatic predictions both contribute to functional gene annotation. For most species it is difficult to gain an overview of what portion of gene annotations are based on experiments and what portion represent predictions. Here, I survey the current state of experimental knowledge of enzymes and metabolism in Arabidopsis thaliana as well as eleven economically important crops and forestry trees - with a particular focus on reactions involving organic acids in central metabolism. I illustrate the limited availability of experimental data for functional annotation of enzymes in most of these species. Many enzymes involved in metabolism of citrate, malate, fumarate, lactate, and glycolate in crops and forestry trees have not been characterized. Furthermore, enzymes involved in key biosynthetic pathways which shape important traits in crops and forestry trees have not been characterized. I argue for the development of novel high-throughput platforms with which limited functional characterization of gene products can be performed quickly and relatively cheaply. I refer to this approach as systems-level experimental characterization. The data collected from such platforms would form a layer intermediate between bioinformatic gene function predictions and in-depth experimental studies of these functions. Such a data layer would greatly aid in the pursuit of understanding a multiplicity of biological processes in living organisms.

  2. Why do crown ethers activate enzymes in organic solvents?

    NARCIS (Netherlands)

    Unen, van Dirk-Jan; Engbersen, Johan F.J.; Reinhoudt, David N.

    2002-01-01

    One of the major drawbacks of enzymes in nonaqueous solvents is that their activity is often dramatically low compared to that in water. This limitation can be largely overcome by crown ether treatment of enzymes. In this paper, we describe a number of carefully designed new experiments that have im

  3. Stimulatory effects of chlordiazepoxide, diazepam and oxazepam on the drug-metabolizing enzymes in microsomes.

    Science.gov (United States)

    Jablońska, J K; Knobloch, K; Majka, J; Wiśniewska-Knypl, J M

    1975-09-01

    5 days' exposure of rats to daily doses of 400 mg/kg body wt. of chlordiazepoxide, diazepam and oxazepam stimulated the microsomal metabolism in the liver, as evidenced by acceleration of both p-hydroxylation of aniline and hydroxylation of benzene. The effect was accompanied by an increased concentration of liver microsomal protein and by the development of tolerance to the drugs. Similar effects were found after exposure of rats to lower doses of the drugs. The metabolism of aniline in vivo in rats treated with chlordiazepoxide was accelerated; this was correlated with development of tolerance to these drugs. It is suggested that both the stimulation of microsomal metabolism and the development of tolerance are associated with the induction of microsomal drug-metabolizing enzymes.

  4. Ecological effects of atmospheric nitrogen deposition on soil enzyme activity

    Institute of Scientific and Technical Information of China (English)

    WANG Cong-yan; Lv Yan-na; LIU Xue-yan Liu; WANG Lei

    2013-01-01

    The continuing increase in human activities is causing global changes such as increased deposition of atmospheric nitrogen.There is considerable interest in understanding the effects of increasing atmospheric nitrogen deposition on soil enzyme activities,specifically in terms of global nitrogen cycling and its potential future contribution to global climate change.This paper summarizes the ecological effects of atmospheric nitrogen deposition on soil enzyme activities,including size-effects,stage-effects,site-effects,and the effects of different levels and forms of atmospheric nitrogen deposition.We discuss needs for further research on the relationship between atmospheric nitrogen deposition and soil enzymes.

  5. Involvement of anti-oxidative enzymes, photosynthetic pigments and flavonoid metabolism in the adaptation of Reaumuria soongorica to salt stress

    Institute of Scientific and Technical Information of China (English)

    YuBing Liu; Bo Cao; MeiLing Liu

    2013-01-01

    Reaumuria soongorica is a short woody shrub widely found in semi-arid areas of China. It can survive severe environ-mental stress including high salinity in its natural habitat. Thus, we investigated the involvement of anti-oxidative enzymes, photosynthetic pigments and flavonoid metabolism in the adaptation of R. soongorica to saline environments. R. soon-gorica was treated with 0, 100, 200 and 400 mM NaCl solutions for 14 days. Soil salt content increased significantly by watering with high content of NaCl solution, and no variation between 8 and 14 days during treatment. The levels of pe-roxidation of lipid membranes (measured by malondialdehyde content) and the activities of three antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX)) increased under salt stress. Chlorophyll and carotenoid content decreased with increasing salt content. The ratio of Chl a/Chl b and carotenoid/Chl exhibited sig-nificant increase under 400 mM NaCl. However, total flavonoid and anthocyanin contents and key enzyme activities in the flavonoid pathway including phenylalanine ammonialyase (PAL) and Chalcone isomerase (CHI) decreased under salt stress. These findings possibly suggest that R. soongorica has an adaptation protection mechanism against salt-induced oxidative damage by inducing the activity of antioxidant enzymes and maintaining a steady level of carotenoid/Chl.

  6. Liver enzymes and metabolic syndrome: a large-scale case-control study.

    Science.gov (United States)

    Zhang, Lu; Ma, Xiangyu; Jiang, Zhi; Zhang, Kejun; Zhang, Mengxuan; Li, Yafei; Zhao, Xiaolan; Xiong, Hongyan

    2015-09-29

    Previous studies suggested that elevated liver enzymes could be used as potential novel biomarkers of Metabolic syndrome (MetS) and its clinical outcomes, although the results were inconsistent and the conclusions were underpowered. A case-control study with 6,268 MetS subjects and 6,330 frequency-matched healthy controls was conducted to systematically evaluated levels of four liver enzymes (ALT, AST, GGT and ALP), both in overall populations and in subjects with normal liver enzymes, with MetS risk using both quartiles and continuous unit of liver enzymes. We found significant associations were detected for all above analyses. Compared with quartile 1 (Q1), other quartiles have significant higher MetS risk, with ORs ranging from 1.15 to 18.15. The highest effected was detected for GGT, for which the OR value for the highest versus lowest quartile was 18.15 (95% CI: 15.7-20.9). Mutual adjustment proved the independence of the relations for all four liver enzymes. Sensitivity analyses didn't materially changed the trend. To the best of our knowledge, this study should be the largest, which aimed at evaluating the association between liver enzymes measures and MetS risk. The results can better support that liver enzyme levels could be used as clinical predictors of MetS.

  7. Stereochemical course, isotope effects, and enzyme inhibitor studies of glaucine metabolism in fungi

    Energy Technology Data Exchange (ETDEWEB)

    Kerr, K.M.

    1986-01-01

    The microbial transformation of the aporphine alkaloid glaucine by the fungi Fusarium solani (ATCC 12823) and Aspergillus flavipes (ATCC 1030) proceeds with complete substrate stereoselectivity. The fungus F. solani metabolizes only S-(+)-glaucine (1) to dehydroglaucine (3), and A. flavipes metabolizes only R-(-)-glaucine (2) to dehydroglaucine. This facile microbiological reaction is useful in the destructive resolution of racemic mixtures of glaucine, and may provide a model for producing pure enantiomers (either R or S) of other aporphines from racemic mixtures. In order to extend the reaction to other aporphines and related alkaloids, the overall stereochemical course and enzyme(s) involved in the reaction, and the substrate requirements of the enzyme were investigated. The overall stereochemical course of the transformation was examined using C-7 methyl-blocked analogs of glaucine, cis- and trans-7-methylglaucine, as substrates for the fungi. Isolation and examination of residual substrates from semi-preparative scale incubations by MS, PMR, PMR with a chiral shift reagent, OR and ORD indicated that the transformation was enantioselective in the case of A. flavipes. However, only a 10% enrichment of 6aR,7R-cis-7-methylglaucine was observed in F. solani cultures. The oxidation of glaucine can be envisioned as proceeding through one of several mechanisms, each involving a different enzyme system. Deuterium isotope, induction and enzyme inhibitor experiments helped to distinguish between the three mechanisms.

  8. A comparison of maximal bioenergetic enzyme activities obtained with commonly used homogenization techniques.

    Science.gov (United States)

    Grace, M; Fletcher, L; Powers, S K; Hughes, M; Coombes, J

    1996-12-01

    Homogenization of tissue for analysis of bioenergetic enzyme activities is a common practice in studies examining metabolic properties of skeletal muscle adaptation to disease, aging, inactivity or exercise. While numerous homogenization techniques are in use today, limited information exists concerning the efficacy of specific homogenization protocols. Therefore, the purpose of this study was to compare the efficacy of four commonly used approaches to homogenizing skeletal muscle for analysis of bioenergetic enzyme activity. The maximal enzyme activity (Vmax) of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured from homogenous muscle samples (N = 48 per homogenization technique) and used as indicators to determine which protocol had the highest efficacy. The homogenization techniques were: (1) glass-on-glass pestle; (2) a combination of a mechanical blender and a teflon pestle (Potter-Elvehjem); (3) a combination of the mechanical blender and a biological detergent; and (4) the combined use of a mechanical blender and a sonicator. The glass-on-glass pestle homogenization protocol produced significantly higher (P < 0.05) enzyme activities compared to all other protocols for both enzymes. Of the four protocols examined, the data demonstrate that the glass-on-glass pestle homogenization protocol is the technique of choice for studying bioenergetic enzyme activity in skeletal muscle.

  9. Enzyme activity in the aestivating green-striped burrowing frog (Cyclorana alboguttata).

    Science.gov (United States)

    Mantle, Beth L; Guderley, Helga; Hudson, Nicholas J; Franklin, Craig E

    2010-10-01

    Green-striped burrowing frogs (Cyclorana alboguttata) can depress their resting metabolism by more than 80% during aestivation. Previous studies have shown that this species is able to withstand long periods of immobilisation during aestivation while apparently maintaining whole muscle mass and contractile performance. The aim of this study was to determine the effect of prolonged aestivation on the levels of metabolic enzymes (CCO, LDH and CS) in functionally distinct skeletal muscles (cruralis, gastrocnemius, sartorius, iliofibularis and rectus abdominus) and liver of C. alboguttata. CS activity was significantly reduced in all tissues except for the cruralis, gastrocnemius and the liver. LDH activity was significantly reduced in the sartorius and rectus abdominus, but remained at control (active) levels in the other tissues. CCO activity was significantly reduced in the gastrocnemius and rectus abdominus, and unchanged in the remaining tissues. Muscle protein was significantly reduced in the sartorius and iliofibularis during aestivation, and unchanged in the remaining muscles. The results suggest that the energy pathways involved in the production and consumption of ATP are remodelled during prolonged aestivation but selective. Remodelling and subsequent down-regulation of metabolic activity seem to target the smaller non-jumping muscles, while the jumping muscles retain enzyme activities at control levels during aestivation. These results suggest a mechanism by which aestivating C. alboguttata are able to maintain metabolic depression while ensuring that the functional capacity of critical muscles is not compromised upon emergence from aestivation.

  10. Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response.

    Directory of Open Access Journals (Sweden)

    Ohad Yogev

    2010-03-01

    Full Text Available In eukaryotes, fumarase (FH in human is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC. A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase, thereby causing stabilization of HIF (Hypoxia-inducible factor by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor

  11. Glucoraphasatin and glucoraphenin, a redox pair of glucosinolates of brassicaceae, differently affect metabolizing enzymes in rats.

    Science.gov (United States)

    Barillari, Jessica; Iori, Renato; Broccoli, Massimiliano; Pozzetti, Laura; Canistro, Donatella; Sapone, Andrea; Bonamassa, Barbara; Biagi, Gian Luigi; Paolini, Moreno

    2007-07-11

    Brassica vegetables are an important dietary source of glucosinolates (GLs), whose breakdown products exhibit anticancer activity. The protective properties of Brassicaceae are believed to be due to the inhibition of Phase-I or induction of Phase-II xenobiotic metabolizing enzymes (XMEs), thus enhancing carcinogen clearance. To study whether GLs affect XMEs and the role of their chemical structure, we focused on two alkylthio GLs differing in the oxidation degree of the side chain sulfur. Male Sprague-Dawley rats were supplemented (per oral somministration by gavage) with either glucoraphasatin (4-methylthio-3-butenyl GL; GRH) or glucoraphenin (4-methylsulfinyl-3-butenyl GL; GRE), at 24 or 120 mg/kg body weight in a single or repeated fashion (daily for four consecutive days), and hepatic microsomes were prepared for XME analyses. Both GLs were able to induce XMEs, showing different induction profiles. While the inductive effect was stronger after multiple administration of the higher GRH dosage, the single lower GRE dose was the most effective in boosting cytochrome P-450 (CYP)-associated monooxygenases and the postoxidative metabolism. CYP3A1/2 were the most affected isoforms by GRH treatment, whereas GRE induced mainly CYP1A2 supported oxidase. Glutathione S-transferase increased up to approximately 3.2-fold after a single (lower) GRE dose and UDP-glucuronosyl transferase up to approximately 2-fold after four consecutive (higher) GRH doses. In conclusion, the induction profile of these GLs we found is not in line with the chemopreventive hypothesis. Furthermore, the oxidation degree of the side chain sulfur of GLs seems to exert a crucial role on XME modulation.

  12. Effects of resveratrol on drug- and carcinogen-metabolizing enzymes, implications for cancer prevention.

    Science.gov (United States)

    Guthrie, Ariane R; Chow, H-H Sherry; Martinez, Jessica A

    2017-02-01

    Resveratrol is a polyphenol found in grape skins and peanuts that has demonstrated many health benefits including protection against aging, cardiovascular and metabolic disease, neurological decline, and cancer. The anticancer properties of resveratrol have been attributed to a variety of mechanisms, including its general inhibition of phase I metabolism and induction of phase II metabolism. The effects of resveratrol on these enzymes, however, are still unclear, as in vitro evidence often contrasts with animal studies and clinical trials. Reasons for these variances could include the low bioavailability of resveratrol and the effects of resveratrol metabolites. Due to resveratrol's interactions with drug-metabolizing enzymes and drug transporters, individuals concurrently taking pharmacological doses of resveratrol with other supplements or medications could potentially experience nutrient-drug interactions. This review summarizes the known effects of resveratrol and its main metabolites on drug metabolism in order to help characterize which populations might benefit from resveratrol for the prevention of cancer, as well as those that may need to avoid supplementation due to potential drug interactions.

  13. Enzyme activity in banana fruits rotted by Botryodiplodia theobromae Pat.

    Directory of Open Access Journals (Sweden)

    Nityananda Chakraborty

    2015-06-01

    Full Text Available Peroxidase and polyphenol oxidase activities in fruits of two cultivars of banana, 'champa' and 'kanthali' rotted by Botryodiplodia theobromae Pat. was studied. The enzymes showed much higher activities in infected than that in uninfected 'tissues. Increase in peroxidase activity was evidently inhibited by cycloheximide. Polyphenol oxidase activity was also inhibited in presence of phenylthiourea and Na-diethyldithiocarbamate more strongly by the former. Increase in activities seemed to be due to increased sytheses of the enzymes. In an in vitro culture, the fungus exhibited some peroxidase but no polyphenoloxidase activity.

  14. Therapeutic effect of Semecarpus anacardium Linn. nut milk extract on carbohydrate metabolizing and mitochondrial TCA cycle and respiratory chain enzymes in mammary carcinoma rats.

    Science.gov (United States)

    Arathi, G; Sachdanandam, P

    2003-09-01

    Semecarpus anacardium Linn. of the family Anacardiaceae has many applications in the Ayurvedic and Siddha systems of medicine. We have evaluated the effect of S. anacardium nut milk extract on carbohydrate metabolizing enzymes and mitochondrial tricarboxylic acid cycle and respiratory enzymes in liver and kidney mitochondria of dimethyl benzanthracene-induced mammary carcinoma in Sprague-Dawley rats. Mammary carcinoma-bearing rats showed a significant rise in glycolytic enzymes (hexokinase, phosphoglucoisomerase and aldolase) and a simultaneous fall in gluconeogenic enzymes (glucose-6-phosphatase and fructose 1,6-diphosphatase). The activities of mitochondrial enzymes isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH-dehydrogenase and cytochrome C oxidase were significantly lowered in mammary carcinoma-bearing rats when compared with control rats. S. anacardium nut extract administration to tumour-induced animals significantly lowered the glycolytic enzyme activities (hexokinase, phosphoglucoisomerase and aldolase) and there was a rise in gluconeogenic enzymes (glucose-6-phosphatase and fructose 1,6-diphosphatase), which indicated an antitumour and anticancer effect. Comparison of normal control rats and rats administered S. anacardium only as drug control animals showed no significant variations in enzyme activities. S. anacardium nut extract administration to dimethyl benzanthracene-tumour-induced animals significantly increased the activities of mitochondrial enzymes, thereby suggesting its role in mitochondrial energy production.

  15. activity of enzyme trypsin immobilized onto macroporous poly(epoxy ...

    African Journals Online (AJOL)

    dell

    The immobilization yield and the influence of pH and temperature conditions on the activity of the ... immobilized enzyme that might change the .... trypsin was packed in a rubber stoppered ..... elastic polyacrylamide gels prepared at subzero ...

  16. Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities

    National Research Council Canada - National Science Library

    Ismail H Al-abdullah; Karine Bagramyan; Shiela Bilbao; Meirigeng Qi; Markus Kalkum

    2017-01-01

    A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay...

  17. A moonlighting enzyme links Escherichia coli cell size with central metabolism.

    Directory of Open Access Journals (Sweden)

    Norbert S Hill

    Full Text Available Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing Escherichia coli cells are more than twice as large as their slow growing counterparts. Here we report the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. Biochemical analysis is consistent with OpgH sequestering FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no homology, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis reveals conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.

  18. Heparin and related polysaccharides: Synthesis using recombinant enzymes and metabolic engineering

    Science.gov (United States)

    Suflita, Matthew; Fu, Li; He, Wenqin; Koffas, Mattheos; Linhardt, Robert J.

    2015-01-01

    Glycosaminoglycans are linear anionic polysaccharides that exhibit a number of important biological and pharmacological activities. The two most prominent members of this class of polysaccharides are heparin/heparan sulfate and the chondroitin sulfates (including dermatan sulfate). These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of most animal cells. The chemical synthesis of these glycosaminoglycans is precluded by their structural complexity. Today, we depend on food animal tissues for their isolation and commercial production. Ton quantities of these glycosaminoglycans are used annually as pharmaceuticals and nutraceuticals. The variability of animal-sourced glycosaminoglycans, their inherent impurities, the limited availability of source tissues, the poor control of these source materials, and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans. This mini-review focuses on the use of recombinant enzymes and metabolic engineering for the production of heparin and chondroitin sulfates. PMID:26219501

  19. Inhibitory effects of kale ingestion on metabolism by cytochrome P450 enzymes in rats.

    Science.gov (United States)

    Yamasaki, Izumi; Yamada, Masayoshi; Uotsu, Nobuo; Teramoto, Sachiyuki; Takayanagi, Risa; Yamada, Yasuhiko

    2012-01-01

    Kale (Brassica oleracea L. var acephala DC) is a leafy green vegetable belonging to the cabbage family (Brassicaceae) that contains a large amount of health-promoting phytochemicals. There are any reports about the effects of kale ingestion on the chemoprevention function and mechanism, but the interactions between kale and drugs have not been researched. We investigated the effects of kale intake on cytochrome P450 (CYP) metabolism by using cocktail probe drugs, including midazolam (for CYP3A4), caffeine (for CYP1A2), dextromethorphan (for CYP2D6), tolbutamide (for CYP2C9), omeprazole (for CYP2C19), and chlorzoxazone (for CYP2E1). Cocktail drugs were administered into rats treated with kale and cabbage (2000 mg/kg) for a week. The results showed that kale intake induced a significant increase in plasma levels and the AUC of midazolam, caffeine, and dextromethorphan. In addition, the plasma concentration and AUC of omeprazole tended to increase. Additionally, no almost differences in the mRNA expression levels of CYP enzymes in the liver were observed. In conclusion, kale ingestion was considered to have an inhibitory effect on the activities of CYP3A4, 1A2, 2D6, and 2C19 for a reason competitive inhibition than inhibitory changes in the mRNA expressions.

  20. Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering.

    Science.gov (United States)

    Suflita, Matthew; Fu, Li; He, Wenqin; Koffas, Mattheos; Linhardt, Robert J

    2015-09-01

    Glycosaminoglycans are linear anionic polysaccharides that exhibit a number of important biological and pharmacological activities. The two most prominent members of this class of polysaccharides are heparin/heparan sulfate and the chondroitin sulfates (including dermatan sulfate). These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of most animal cells. The chemical synthesis of these glycosaminoglycans is precluded by their structural complexity. Today, we depend on food animal tissues for their isolation and commercial production. Ton quantities of these glycosaminoglycans are used annually as pharmaceuticals and nutraceuticals. The variability of animal-sourced glycosaminoglycans, their inherent impurities, the limited availability of source tissues, the poor control of these source materials, and their manufacturing processes suggest a need for new approaches for their production. Over the past decade, there have been major efforts in the biotechnological production of these glycosaminoglycans. This mini-review focuses on the use of recombinant enzymes and metabolic engineering for the production of heparin and chondroitin sulfates.

  1. Electrolyte & water metabolism in sports activities.

    Science.gov (United States)

    Whang, R

    1998-01-01

    Few studies in water and electrolyte metabolism during sports activities have directed attention to magnesium. Addition of magnesium to sports beverages in appropriate concentrations appears to be safe. This article considers the potential role and availability of magnesium in fluid repletion during sports activities.

  2. Impact of expression of EMP enzymes on glucose metabolism in Zymomonas mobilis.

    Science.gov (United States)

    Chen, Rachel Ruizhen; Agrawal, Manoj; Mao, Zichao

    2013-06-01

    Zymomonas mobilis is the only known microorganism that utilizes the Entner-Doudoroff (ED) pathway anaerobically. In this work, we investigated whether the overexpression of a phosphofructokinase (PFK), the only missing Embden-Meyerhof-Parnas (EMP) pathway enzyme, could establish the pathway in this organism. Introduction of a pyrophosphate-dependent PFK, along with co-expression of homologous fructose-1,6-bisphosphate aldolase and triosephosphate isomerase, did not result in an EMP flux to any appreciable level. However, the metabolism of glucose was impacted significantly. Eight percent of glucose was metabolized to form a new metabolite, dihydroxyacetone. Reducing flux through the ED pathway by as much as 40 % through antisense of a key enzyme, ED aldolase, did not result in a fully functional EMP pathway, suggesting that the ED pathway, especially the lower arm, downstream from glyceraldehyde-3-phosphate, is very rigid, possibly due to redox balance.

  3. Enzyme activity measurement via spectral evolution profiling and PARAFAC

    DEFF Research Database (Denmark)

    Baum, Andreas; Meyer, Anne S.; Garcia, Javier Lopez

    2013-01-01

    The recent advances in multi-way analysis provide new solutions to traditional enzyme activity assessment. In the present study enzyme activity has been determined by monitoring spectral changes of substrates and products in real time. The method relies on measurement of distinct spectral...... fingerprints of the reaction mixture at specific time points during the course of the whole enzyme catalyzed reaction and employs multi-way analysis to detect the spectral changes. The methodology is demonstrated by spectral evolution profiling of Fourier Transform Infrared (FTIR) spectral fingerprints using...

  4. Correlation-based network analysis of metabolite and enzyme profiles reveals a role of citrate biosynthesis in modulating N and C metabolism in zea mays

    Science.gov (United States)

    To investigate the natural variability of leaf metabolism and enzymatic activity in a maize inbred population, statistical and network analyses were employed on metabolite and enzyme profiles. The test of coefficient of variation showed that sugars and amino acids displayed opposite trends in their ...

  5. The Analysis of Metabolic Flux and Enzyme Activities of Pyruvate Branch Point of Rhizopus oryzae As3.2686%米根霉As3.2686丙酮酸分支点代谢通量分析及相关酶活性研究

    Institute of Scientific and Technical Information of China (English)

    柯玮; 郑志; 姜绍通; 罗水忠; 吴学凤; 杨培周; 李兴江

    2014-01-01

    The metabolic flux equations of Rhizopus oryzae As3.2686 were established and the metabolic fluxes of different ventilations in the steady-state in a fermentor were calculated by metabolic flux analysis (MFA) method. The changes of the enzyme activities at pyruvate branch point including PDC (pyruvate decarboxylase), PC (pyruvate carboxylase), PDH (pyruvate dehydrogenase), and LDH (lactate dehydrogenase) were analyzed by enzyme-linked immunosorbent assay (ELISA) and spectrophotometry. These results illustrated that the flux of fumarate and ethanol were relatively higher at the ventilations of 0.5 and 1.0 L⋅(L⋅min)-1. At the ventilations of 1.5 and 2.0 L⋅(L⋅min)-1, over half of flux at pyruvate branch point flowed to L-lactate. When the ventilation was 2.5 L⋅(L⋅min)-1, more metabolic flux in the steady-state flowed to TCA cycle. According to the comparison of the activities of the same enzyme at different ventilations, the enzyme activities of PDC, PC, and PDH increased at beginning then declined with increasing ventilations, and inconsistent with ethanol, oxaloacetate and acetyl coenzyme fluxes. The activities of LDH and ADH increased gradually with the raise of ventilation, but ADH enzyme activity inconsistent with ethanol flux. More flux flowed to L-lactic acid at 2.0 L⋅(L⋅min)-1 than 1.5 L⋅(L⋅min)-1, but less flux of L-lactic acid outside the cells were obtained. Conclusion: the function of single enzyme to regulate the pyruvate flux is limited, several enzymes around the pyruvate branch point corporately control the metabolic flux, there are other enzymes affecting pyruvate to transform to L-lactic acid.%利用代谢通量分析(MFA)研究建立米根霉As3.2686的代谢通量方程,计算发酵罐不同通气条件下丙酮酸分支点的稳态代谢通量,并分别使用酶联免疫吸附法(ELISA)和分光光度法测定丙酮酸分支酶 PDC(丙酮酸脱羧酶)、PC(丙酮酸羧化酶)、PDH(丙酮酸脱氢酶)和LDH(乳酸脱氢

  6. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data

    OpenAIRE

    Barker, Brandon E.; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R.; Xi, Hongwei; Locasale, Jason W.; Gu, Zhenglong

    2014-01-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with highthroughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability resulting in improved understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the cr...

  7. The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro

    OpenAIRE

    Stadelmann, Britta; Hanevik, Kurt; Andersson, Mattias; Bruserud, Øystein; Staffan G Svärd

    2013-01-01

    Background: Arginine is a conditionally essential amino acid important in growing individuals and under nonhomeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and argini...

  8. Effects of supplemental zinc source and level on antioxidant ability and fat metabolism-related enzymes of broilers.

    Science.gov (United States)

    Liu, Z H; Lu, L; Wang, R L; Lei, H L; Li, S F; Zhang, L Y; Luo, X G

    2015-11-01

    The objective of the present study was to investigate the effects of dietary supplemental Zinc (Zn) source and level on antioxidant ability and fat metabolism-related enzymes of broilers. Dietary treatments included the Zn-unsupplemented corn-soybean meal basal diet (control) and basal diets supplemented with 60, 120, or 180 mg Zn/kg as Zn sulfate, Zn amino acid chelate with a weak chelation strength of 6.5 quotient of formation (Qf) (11.93% Zn) (Zn-AA W), Zn proteinate with a moderate chelation strength of 30.7 Qf (13.27% Zn) (Zn-Pro M), or Zn proteinate with an extremely strong chelation strength of 944.0 Qf (18.61% Zn) (Zn-Pro S). The results showed that dietary supplemental Zn increased (P enzymes in the abdominal fat and liver of broilers. Dietary Zn source, and an interaction between Zn source and level, had no effects on any measurements. It is concluded that dietary Zn supplementation improved Zn status and resulted in promoting antioxidant ability and activities and gene expressions of fat metabolism-related enzymes of broilers regardless of Zn source and level, and the addition of 60 mg Zn/kg to the corn-soybean meal basal diet (a total dietary Zn of approximately 90 mg/kg) was appropriate for improving the above aspects of broilers.

  9. 套袋梨贮藏过程中糖代谢及相关酶活性变化特征%Characteristics of Sugar Metabolism and Related Enzyme Activity of Bagged Pear Fruits during Storage

    Institute of Scientific and Technical Information of China (English)

    杨青松; 李晓刚; 王中华; 常有宏; 蔺经

    2012-01-01

    Double-layer bagged and unbagged pear fruits of Pyrus pyrifolia Nakai cv. 'Cuiguan' were used in this study,to explore the variation patterns of sucrose,fructose,glucose,sorbitol and related enzyme activities. The results showed that, compared to unbagged fruit, the bagged fruits had lower fructose, glucose, sorbitol and sucrose. In comparison with unbagged fruits, the sorbitol dehydrogenase activity of bagged fruits was lower during the former 5 days storage,but higher after 10 days storage. The activity of sucrose phosphate synthase (SPS) and sucrose synthase (SS) in the fruits had the same tendency,was high for early period and low for late period during storage. The correlation analysis of the 4 kind sugars in the fruit during storage was made,we got that sorbitol and fructose had a significant negative correlation. Also, sucrose and glucose showed significant negative correlation. We concluded that sorbitol transformed to fructose,and sucrose transformed to glucose during storage.%以‘翠冠,梨为材料,研究了套双层遮光纸袋梨果实贮藏过程中蔗糖、果糖、葡萄糖、山梨醇及糖代谢中酶活性的变化规律.结果表明,贮藏套袋梨果实中果糖、葡萄糖、山梨醇和蔗糖含量都低于未套袋对照;套袋梨果实中山梨醇脱氢酶活性在贮藏的前5d都低于对照,贮藏10 d后活性均高于对照,且与山梨醇含量呈现极显著正相关;贮藏套袋梨果实中蔗糖磷酸合酶(SPS)及蔗糖合酶(SS)分解和合成方向活性都是前期低于对照,贮藏后期都高于对照,且蔗糖含量与蔗糖磷酸合酶和蔗糖合酶(分解方向)活性都呈显著正相关;贮藏的套袋梨果实和对照中的山梨醇含量与果糖含量均呈极显著负相关,蔗糖含量与葡萄糖含量呈极显著负相关,即在贮藏过程中山梨醇可能转化为果糖,而蔗糖则转化成葡萄糖.

  10. Pharmacogenomics of Drug Metabolizing Enzymes and Transporters:Relevance to Precision Medicine

    Institute of Scientific and Technical Information of China (English)

    Shabbir Ahmed; Zhan Zhou; Jie Zhou; Shu-Qing Chen

    2016-01-01

    The interindividual genetic variations in drug metabolizing enzymes and transporters influence the efficacy and toxicity of numerous drugs. As a fundamental element in precision med-icine, pharmacogenomics, the study of responses of individuals to medication based on their genomic information, enables the evaluation of some specific genetic variants responsible for an individual’s particular drug response. In this article, we review the contributions of genetic polymorphisms to major individual variations in drug pharmacotherapy, focusing specifically on the pharmacoge-nomics of phase-I drug metabolizing enzymes and transporters. Substantial frequency differences in key variants of drug metabolizing enzymes and transporters, as well as their possible functional consequences, have also been discussed across geographic regions. The current effort illustrates the common presence of variability in drug responses among individuals and across all geographic regions. This information will aid health-care professionals in prescribing the most appropriate treatment aimed at achieving the best possible beneficial outcomes while avoiding unwanted effects for a particular patient.

  11. Effect of Dietary Protein Levels on Activity of Lipids Metabolic Enzymes in Wujin Pigs%日粮蛋白质水平对乌金猪脂肪代谢相关酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    潘洪彬; 陈强; 黄英; 张永云; 赵素梅; 张曦; 葛长荣; 高士争

    2012-01-01

    为研究日粮不同蛋白质水平对乌金猪脂肪代谢相关酶活性的影响.选取体重约15 kg的乌金猪54头,随机分为3组,每组3个重复,每个重复6头,在15~30 kg体重阶段饲喂18%(高蛋白质组)、16%(中蛋白质组)和14%(低蛋白质组)蛋白质水平的日粮,30~60 kg体重阶段饲喂16%(高蛋白质组)、14%(中蛋白质组)和12%(低蛋白质组)蛋白质水平的日粮,60~100 kg体重阶段饲喂14%(高蛋白质组)、12%(中蛋白质组)和10%(低蛋白质组)蛋白质水平日粮,在体重30、60和100kg体重时分批屠宰,取肝脏及背部皮下脂肪组织测定脂肪酶(LPS)、葡萄糖-6-磷酸脱氢酶(G-6-PDH)和苹果酸脱氢酶(MDH)活性.结果表明,在30 kg体重时,日粮蛋白质水平对乌金猪肝脏和脂肪组织中脂肪代谢相关酶活性差异不显着(P>0.05),在60和100 kg体重时,高蛋白质日粮组乌金猪肝脏和脂肪组织LPS的活性明显高于低蛋白质日粮组(P<0.05),G-6-PDH和MDH活性则明显低于低蛋白质日粮组(P<0.05).因此,高蛋白质日粮增加了生长中后期乌金猪的内源性能量释放,可能影响了体脂沉积比例.%The aim of the study was to investigate the effects of dietary energy levels on lipids metabolic enzymes of liver and adipose tissue in Wujin pigs. Fifty-four Wujin pigs were randomly allotted into three groups fed diet containing different energy levels, there were eighteen pigs in each group with three replicates, six pigs were used in per replicates. Wujin pigs fed on the dietary protein levels were 18%, 16%, 14% and 16%,14%, 12% and 14%,12%, 10% during the growth stages of 15 to 30,30 to 60 and 60 to 100 kg body weight, respectively, during the growth stages of 15 to 30,30 to 60 and 60 to 100 kg body weight, the dietary trace element contents in one group were constant. At the body weight of 30, 60 and 100 kg, Wujin pigs were slaughtered and liver and back subcutaneous fatty tissue were taken for

  12. Influence of dietary macronutrients on induction of hepatic drug metabolizing enzymes by Coleus forskohlii extract in mice.

    Science.gov (United States)

    Yokotani, Kaori; Chiba, Tsuyoshi; Sato, Yoko; Nakanishi, Tomoko; Murata, Masatsune; Umegaki, Keizo

    2013-01-01

    From studies in mice, we have reported that Coleus forskohlii extract (CFE), a popular herbal weight-loss ingredient, markedly induced hepatic drug metabolizing enzymes, especially cytochrome P450 (CYP), and interacted with co-administered drugs. This study was designed to examine how the induction of drug metabolizing enzymes by CFE was influenced by different levels of macronutrients in the diet. Mice were fed a non-purified diet or semi-purified diet with and without CFE (0.3-0.5%) for 14-18 d, and changes in the ratio of liver weight to body weight, an indicator of hepatic CYP induction, and hepatic drug metabolizing enzymes were analyzed. The ratio of liver weight to body weight, content and activities of CYPs, and activity of glutathione S-transferase were higher in a semi-purified standard diet (AIN93G formula) group than in high sucrose (62.9%) and high fat (29.9%) diet groups. Different levels of protein (7%, 20%, and 33%) in the diets did not influence CFE-induced CYP induction or increase the ratio of liver weight to body weight. The effect of CFE on the ratio of liver weight to body weight was higher with a semi-purified diet than with a non-purified diet, and was similar between dietary administration and intragastric gavage when the CFE dose and the diet were the same. There was a positive correlation between CFE-induced CYP induction and the content of starch in the diets, suggesting that dietary starch potentiates CFE-induced CYP induction in mice. The mechanism of enhanced CYP induction remains unclear.

  13. Oxidative bioelectrocatalysis: From natural metabolic pathways to synthetic metabolons and minimal enzyme cascades.

    Science.gov (United States)

    Minteer, Shelley D

    2016-05-01

    Anodic bioelectrodes for biofuel cells are more complex than cathodic bioelectrodes for biofuel cells, because laccase and bilirubin oxidase can individually catalyze four electron reduction of oxygen to water, whereas most anodic enzymes only do a single two electron oxidation of a complex fuel (i.e. glucose oxidase oxidizing glucose to gluconolactone while generating 2 electrons of the total 24 electrons), so enzyme cascades are typically needed for complete oxidation of the fuel. This review article will discuss the lessons learned from natural metabolic pathways about multi-step oxidation and how those lessons have been applied to minimal or artificial enzyme cascades. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  14. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data.

    Science.gov (United States)

    Barker, Brandon E; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R; Xi, Hongwei; Locasale, Jason W; Gu, Zhenglong

    2015-12-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with high-throughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability and improve our understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present an algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation, the ability to handle large enzyme complex rules that may incorporate multiple isoforms, and either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB.

  15. Effects of cadium, zinc and lead on soil enzyme activities

    Institute of Scientific and Technical Information of China (English)

    YANG Zhi-xin; LIU Shu-qing; ZHENG Da-wei; FENG Sheng-dong

    2006-01-01

    Heavy metal (HM) is a major hazard to the soil-plant system. This study investigated the combined effects of cadium (Cd),zinc (Zn) and lead (Pb) on activities of four enzymes in soil, including calatase, urease, invertase and alkalin phosphatase. HM content in tops of canola and four enzymes activities in soil were analyzed at two months after the metal additions to the soil. Pb was not significantly inhibitory than the other heavy metals for the four enzyme activities and was shown to have a protective role on calatase activity in the combined presence of Cd, Zn and Pb; whereas Cd significantly inhibited the four enzyme activities, and Zn only inhibited urease and calatase activities. The inhibiting effect of Cd and Zn on urease and calatase activities can be intensified significantly by the additions of Zn and Cd. There was a negative synergistic inhibitory effect of Cd and Zn on the two enzymes in the presence of Cd, Zn and Pb. The urease activity was inhibited more by the HM combinations than by the metals alone and reduced approximately 20%-40% of urease activity. The intertase and alkaline phosphatase activities significantly decreased only with the increase of Cd concentration in the soil. It was shown that urease was much more sensitive to HM than the other enzymes. There was a obvious negative correlation between the ionic impulsion of HM in soil, the ionic impulsion of HM in canola plants tops and urease activity. It is concluded that the soil urease activity may be a sensitive tool for assessing additive toxic combination effect on soil biochemical parameters.

  16. Costus afer Possesses Carbohydrate Hydrolyzing Enzymes Inhibitory Activity and Antioxidant Capacity In Vitro

    Directory of Open Access Journals (Sweden)

    Armelle D. Tchamgoue

    2015-01-01

    Full Text Available Diabetes mellitus is a metabolic disorder of glucose metabolism which correlates with postprandial hyperglycemia and oxidative stress. Control of blood glucose level is imperative in the management of diabetes. The present study tested the hypothesis that Costus afer, an antihyperglycemic medicinal plant, possesses inhibitory activity against carbohydrate hydrolyzing enzymes. Hexane, ethyl acetate, methanol, and water extracts were prepared from the leaf, stem, and rhizome of C. afer and subjected to phytochemical screening, assayed for α-amylase and α-glucosidase inhibitory activities and antioxidant capacity (determined by total phenolic and total flavonoids contents, ferric reducing antioxidant power (FRAP, and DPPH radical scavenging activity. All extracts inhibited α-amylase and α-glucosidase activities. Ethyl acetate rhizome and methanol leaf extracts exhibited the best inhibitory activity against α-amylase and α-glucosidase (IC50: 0.10 and 5.99 mg/mL, respectively. Kinetic analysis revealed two modes of enzyme inhibition (competitive and mixed. All extracts showed antioxidant capacity, with hexane extracts exhibiting the best activity. DPPH assay revealed that methanol leaf, rhizome, and ethyl acetate stem extracts (IC50 < 5 mg/mL were the best antioxidants. The presence of bioactive compounds such as flavonoids, alkaloids, phenols, and tannins may account for the antioxidant capacity and carbohydrate hydrolyzing enzyme inhibitory activity of C. afer.

  17. Costus afer Possesses Carbohydrate Hydrolyzing Enzymes Inhibitory Activity and Antioxidant Capacity In Vitro

    Science.gov (United States)

    Tchamgoue, Armelle D.; Tchokouaha, Lauve R. Y.; Tarkang, Protus A.; Kuiate, Jules-Roger; Agbor, Gabriel A.

    2015-01-01

    Diabetes mellitus is a metabolic disorder of glucose metabolism which correlates with postprandial hyperglycemia and oxidative stress. Control of blood glucose level is imperative in the management of diabetes. The present study tested the hypothesis that Costus afer, an antihyperglycemic medicinal plant, possesses inhibitory activity against carbohydrate hydrolyzing enzymes. Hexane, ethyl acetate, methanol, and water extracts were prepared from the leaf, stem, and rhizome of C. afer and subjected to phytochemical screening, assayed for α-amylase and α-glucosidase inhibitory activities and antioxidant capacity (determined by total phenolic and total flavonoids contents, ferric reducing antioxidant power (FRAP), and DPPH radical scavenging activity). All extracts inhibited α-amylase and α-glucosidase activities. Ethyl acetate rhizome and methanol leaf extracts exhibited the best inhibitory activity against α-amylase and α-glucosidase (IC50: 0.10 and 5.99 mg/mL), respectively. Kinetic analysis revealed two modes of enzyme inhibition (competitive and mixed). All extracts showed antioxidant capacity, with hexane extracts exhibiting the best activity. DPPH assay revealed that methanol leaf, rhizome, and ethyl acetate stem extracts (IC50 < 5 mg/mL) were the best antioxidants. The presence of bioactive compounds such as flavonoids, alkaloids, phenols, and tannins may account for the antioxidant capacity and carbohydrate hydrolyzing enzyme inhibitory activity of C. afer. PMID:26246844

  18. Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells.

    Science.gov (United States)

    Choi, S; Sainz, B; Corcoran, P; Uprichard, S; Jeong, H

    2009-03-01

    The objective of this study was to characterize Huh7 cells' baseline capacity to metabolize drugs and to investigate whether the drug metabolism was enhanced upon treatment with dimethyl sulfoxide (DMSO). The messenger RNA (mRNA) levels of major Phase I and Phase II enzymes were determined by quantitative real-time-polymerase chain reaction (RT-PCR), and activities of major drug-metabolizing enzymes were examined using probe drugs by analysing relevant metabolite production rates. The expression levels of drug-metabolizing enzymes in control Huh7 cells were generally very low, but DMSO treatment dramatically increased the mRNA levels of most drug-metabolizing enzymes as well as other liver-specific proteins. Importantly, functionality assays confirmed concomitant increases in drug-metabolizing enzyme activity. Additionally, treatment of the Huh7 cells with 3-methylcholanthrene induced cytochrome P450 (CYP) 1A1 expression. The results indicate that DMSO treatment of Huh7 cells profoundly enhances their differentiation state, thus improving the usefulness of this common cell line as an in vitro hepatocyte model.

  19. Metabolic benefits of physical activity

    Directory of Open Access Journals (Sweden)

    Špela Volčanšek

    2014-10-01

    Full Text Available Physical activity is the most beneficial intervention in prevention and treatment of chronic diseases. Life style, which has become mostly sedentary, leads to growing incidence in obesity, what could cause the first so far reduction in life expectancy in developed countries.Physical activity reduces the chronic low-grade inflammation, which plays an important role in the pathogenesis of type 2 diabetes, cardiovascular disease and certain types of cancer. Regular physical activity exerts two anti-inflammatory effects: reduction of visceral fat, which produces the majority of pro-inflammatory cytokines, and production of myokines. It has been proposed that cytokines and other peptides that are produced by muscle fibers should be classified as myokines that exert autocrine, paracrine and endocrine effects. Myokines induce muscle hypertrophy and myogenesis, stimulate fat oxidation, improve insulin sensitivity and have an anti-inflammatory effect.  Therefore, skeletal muscle has been identified as a secretory organ and this provides the basis for understanding how muscles communicate with other organs, such as adipose tissue, liver, pancreas, gut, bones and brain. Physical inactivity leads to an altered myokine profile, associating sedentary life style with some chronic diseases.Physical activity is recommended as a tool for weight management and prevention of weight gain, for weight loss and for prevention of weight regain. High quality studies have confirmed the important impact of exercise on improving blood glucose control in diabetic patients, and on preventing or delaying the onset of type 2 diabetes in predisposed populations. Prescribing specific exercise tailored to individual's needs is an intervention strategy for health improvement. Physical fitness counteracts the detrimental effects of obesity reducing morbidity and mortality.

  20. Inhibition of existing denitrification enzyme activity by chloramphenicol

    Science.gov (United States)

    Brooks, M.H.; Smith, R.L.; Macalady, D.L.

    1992-01-01

    Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

  1. Dynamic relationships between microbial biomass, respiration, inorganic nutrients and enzyme activities: informing enzyme based decomposition models

    Directory of Open Access Journals (Sweden)

    Daryl L Moorhead

    2013-08-01

    Full Text Available We re-examined data from a recent litter decay study to determine if additional insights could be gained to inform decomposition modeling. Rinkes et al. (2013 conducted 14-day laboratory incubations of sugar maple (Acer saccharum or white oak (Quercus alba leaves, mixed with sand (0.4% organic C content or loam (4.1% organic C. They measured microbial biomass C, carbon dioxide efflux, soil ammonium, nitrate, and phosphate concentrations, and β-glucosidase (BG, β-N-acetyl-glucosaminidase (NAG, and acid phosphatase (AP activities on days 1, 3, and 14. Analyses of relationships among variables yielded different insights than original analyses of individual variables. For example, although respiration rates per g soil were higher for loam than sand, rates per g soil C were actually higher for sand than loam, and rates per g microbial C showed little difference between treatments. Microbial biomass C peaked on day 3 when biomass-specific activities of enzymes were lowest, suggesting uptake of litter C without extracellular hydrolysis. This result refuted a common model assumption that all enzyme production is constitutive and thus proportional to biomass, and/or indicated that part of litter decay is independent of enzyme activity. The length and angle of vectors defined by ratios of enzyme activities (BG/NAG versus BG/AP represent relative microbial investments in C (length, and N and P (angle acquiring enzymes. Shorter lengths on day 3 suggested low C limitation, whereas greater lengths on day 14 suggested an increase in C limitation with decay. The soils and litter in this study generally had stronger P limitation (angles > 45˚. Reductions in vector angles to < 45˚ for sand by day 14 suggested a shift to N limitation. These relational variables inform enzyme-based models, and are usually much less ambiguous when obtained from a single study in which measurements were made on the same samples than when extrapolated from separate studies.

  2. Chimeric enzymes with improved cellulase activities

    Science.gov (United States)

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  3. Enzyme activities in mitochondria isolated from ripening tomato fruit.

    Science.gov (United States)

    Jeffery, D; Goodenough, P W; Weitzman, P D

    1986-09-01

    Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.

  4. Insecticide Susceptibility and Activity of Metabolic Enzymes of Brown Planthopper,Nilaparvata lugens(St(a)l)(Hemiptera: Deiphacidae)Feeding on Transgenic Bt Rice%取食转Bt基因水稻褐飞虱对杀虫剂的敏感性及其代谢酶的活性

    Institute of Scientific and Technical Information of China (English)

    李兆亮; 姚洪渭; 陈洋; 田俊策; 彭于发; 叶恭银

    2011-01-01

    测定比较了褐飞虱Nilaparvata lugens在转cry1Ab基因水稻KMD1和KMD2及其对照秀水11上取食不同世代的室内种群和田间种群对5种杀虫剂的敏感水平及其体内解毒酶(酯酶和谷胱甘肽S-转移酶)、靶标酶(乙酰胆碱酯酶)和保护酶(超氧化物歧化酶、过氧化氢酶和过氧化物酶)的活性,以及体内Cry1Ab蛋白的含量.结果表明,5种杀虫剂对分别持续取食KMD1和KMD2的褐飞虱室内种群和田间种群LD50值与取食对照的无显著差异.在KMD1和KMD2上取食1代和9代的褐飞虱室内种群以及田间种群体内解毒酶、靶标酶和保护酶等活性与取食对照的差异不显著.取食KMD的褐飞虱不同种群体内的Cry1Ab蛋白含量相对稳定,其中以取食KMD2的较高.上述结果表明,转cry1Ab基因水稻KMD1和KMD2对褐飞虱的杀虫剂敏感性和代谢酶活性无显著影响.%The toxicities of five commonly-used insecticides against different populations of rice brown planthopper, Nilaparvata lugens, fed on transgenic rice with crylAb gene, KMD1 and KMD2 for different generations were measured and compared with those on non-transgenic parental rice variety, Xiushui 11.Activities of detoxification enzymes (EST and GST), target enzyme (AChE), and protective enzymes (SOD, CAT and POD) from different populations of N.lugens fed on KMD1, KMD2, and Xiushui 11 and the content of Cry protein were also detected.For all five insecticides tested,there were no significant differences in LD50 values among different populations of N.lugens fed on KMD1 KMD2, and Xiushui 11 continuously in the laboratory and collected from the rice fields.For all six metabolic enzymes tested, there was no significant difference of enzyme activities among different populations of N.lugens fed on KMD1, KMD2 and Xiushui 11 for 1 and 9 generations in the laboratory or collected from the rice fields.The content of Cry protein was relatively stable in different populations of N.lugens fed on Bt

  5. Changes of drug metabolizing enzymes in the liver of male sheep exposed to either cypermethrin or dimethoate.

    Science.gov (United States)

    Sheweita, S A; Yousef, M I; Baghdadi, H H; Elshemy, A G

    2012-03-01

    Xenobiotics such as insecticides are metabolized to more or less toxic metabolites by drug-metabolizing enzymes including cytochrome P450 (Cyp P450), cytochrome b5 (Cyp b5), NADPH-cytochrome c reductase (Cyt.c R), N-nitrosdimethylamine-N-demethylase I (NDMA-dI), glutathione (GSH), glutathione s-transferase (GST), and glutathione reductase (GR). Therefore, the present study showed the influence of oral administration of cypermethrin (6 and 12 mg/kg/day) and dimethoate (1.6 and 3.2 mg/kg/day) for 63 consecutive days on the activities of the above mentioned enzymes in the livers of male sheep. Low and high-treatments of sheep with cypermethrin significantly increased the levels of Cyp P450 by 56% and 98%, Cyp b5 by 65% and 80%, GSH by 68% and 74%, and Cyt.c R by 67% and 98%, respectively in a dose-dependent manner. However, low dose of cypermethrin increased the activities of GST and GR by 56% and 91% respectively. In addition, low and high dose-treatments with dimethoate increased the hepatic contents of Cyp P450 by 27% and 40%, GSH by 259% and 132%, whereas NDMA-dI decreased by 27 and 55% respectively, and no change in the content of Cyp b5 and the activity of Cyt.c-R at any given dose of this compound. It is concluded that exposure to cypermethrin and dimethoate significantly changed the hepatic activity of phases I & II drugmetabolizing enzymes in sheep, and these changes are mainly dependent on the administred dose, and also on the type of the tested insecticides. Also, such changes should be considered when therapeutic drugs administered to people exposed to such insecticides.

  6. Catalytically active nanomaterials: a promising candidate for artificial enzymes.

    Science.gov (United States)

    Lin, Youhui; Ren, Jinsong; Qu, Xiaogang

    2014-04-15

    Natural enzymes, exquisite biocatalysts mediating every biological process in living organisms, are able to accelerate the rate of chemical reactions up to 10(19) times for specific substrates and reactions. However, the practical application of enzymes is often hampered by their intrinsic drawbacks, such as low operational stability, sensitivity of catalytic activity to environmental conditions, and high costs in preparation and purification. Therefore, the discovery and development of artificial enzymes is highly desired. Recently, the merging of nanotechnology with biology has ignited extensive research efforts for designing functional nanomaterials that exhibit various properties intrinsic to enzymes. As a promising candidate for artificial enzymes, catalytically active nanomaterials (nanozymes) show several advantages over natural enzymes, such as controlled synthesis in low cost, tunability in catalytic activities, as well as high stability against stringent conditions. In this Account, we focus on our recent progress in exploring and constructing such nanoparticulate artificial enzymes, including graphene oxide, graphene-hemin nanocomposites, carbon nanotubes, carbon nanodots, mesoporous silica-encapsulated gold nanoparticles, gold nanoclusters, and nanoceria. According to their structural characteristics, these enzyme mimics are categorized into three classes: carbon-, metal-, and metal-oxide-based nanomaterials. We aim to highlight the important role of catalytic nanomaterials in the fields of biomimetics. First, we provide a practical introduction to the identification of these nanozymes, the source of the enzyme-like activities, and the enhancement of activities via rational design and engineering. Then we briefly describe new or enhanced applications of certain nanozymes in biomedical diagnosis, environmental monitoring, and therapeutics. For instance, we have successfully used these biomimetic catalysts as colorimetric probes for the detection of

  7. Optimization to Low Temperature Activity in Psychrophilic Enzymes

    Directory of Open Access Journals (Sweden)

    Caroline Struvay

    2012-09-01

    Full Text Available Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts.

  8. Enzyme activity in forest peat soils

    OpenAIRE

    Błońska, Ewa

    2010-01-01

    The aim of the study was to determine the activity of dehydrogenases and urease in forest peat soils of different fertility. There were selected 23 experimental plots localised in central and northern Poland. The research was conducted on forest fens, transition bogs and raised bogs. The biggest differences in soil physical and chemical properties were detected between fen and raised bog soils while raised bog soils and transition bog soils differed the least. Statistically significant dif...

  9. Enzymatic activity of Lecithin:retinol acyltransferase: a thermostable and highly active enzyme with a likely mode of interfacial activation.

    Science.gov (United States)

    Horchani, Habib; Bussières, Sylvain; Cantin, Line; Lhor, Mustapha; Laliberté-Gemme, Jean-Sébastien; Breton, Rock; Salesse, Christian

    2014-06-01

    Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in detail. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70°C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated β-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100°C. The results of this study highly improved our understanding of this enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Physical activity in obesity and metabolic syndrome

    Science.gov (United States)

    Strasser, Barbara

    2013-01-01

    Biological aging is typically associated with a progressive increase in body fat mass and a loss of lean body mass. Owing to the metabolic consequences of reduced muscle mass, it is understood that normal aging and/or decreased physical activity may lead to a higher prevalence of metabolic disorders. Lifestyle modification, specifically changes in diet, physical activity, and exercise, is considered the cornerstone of obesity management. However, for most overweight people it is difficult to lose weight permanently through diet or exercise. Thus, prevention of weight gain is thought to be more effective than weight loss in reducing obesity rates. A key question is whether physical activity can extenuate age-related weight gain and promote metabolic health in adults. Current guidelines suggest that adults should accumulate about 60 minutes of moderate-intensity physical activity daily to prevent unhealthy weight gain. Because evidence suggests that resistance training may promote a negative energy balance and may change body fat distribution, it is possible that an increase in muscle mass after resistance training may be a key mediator leading to better metabolic control. PMID:23167451

  11. Improving Activity of Salt-Lyophilized Enzymes in Organic Media

    Science.gov (United States)

    Borole, Abhijeet P.; Davison, Brian H.

    Lyophilization with salts has been identified as an important method of activating enzymes in organic media. Using salt-activated enzymes to transform molecules tethered to solid surfaces in organic phase requires solubilization of enzymes in the solvents. Methods of improving performance of salt-lyophilized enzymes, further, via chemical modification, and use of surfactants and surfactants to create fine emulsions prior to lyophilization are investigated. The reaction system used is transesterification of N-acetyl phenylalanine ethyl ester with methanol or propanol. Initial rate of formation of amino acid esters by subtilisin Carlsberg (SC) was studied and found to increase two to sevenfold by either chemical modification or addition of surfactants in certain solvents, relative to the salt (only)-lyophilized enzyme. The method to prepare highly dispersed enzymes in a salt-surfactant milieu also improved activity by two to threefold. To test the effect of chemical modification on derivatization of drug molecules, acylation of bergenin was investigated using chemically modified SC.

  12. [Effects of starvation on digestive enzyme activities of Monopterus albus].

    Science.gov (United States)

    Yang, Dai-qin; Chen, Fang; Ruan, Guo-liang; Hu, Cheng-wen; Cao, Sheng-huan

    2007-05-01

    Starvation is a major environmental stress, which has a broad effect on the physiology and ecology of aquatic animals. In this study, Monopterus albus was starved for 30 days at (20 +/- 0.5) degrees C, and the activities of protease, trypsin, amylase and lipase in its digestive organs were measured on the 0, 3rd, 5th, 10th, 15th, 20th, and 30th day of starvation. The results showed that starvation had definite effects on the activities of all test enzymes. With the prolongation of starvation, the activities of test enzymes decreased, which was most significant when the fish was starved for 5-10 days. After 10 days of starvation, the decreasing trend of the enzyme activities became less obvious.

  13. Extraction of Active Enzymes from "Hard-to-Break-Cells"

    DEFF Research Database (Denmark)

    Ottaviani, Alessio; Tesauro, Cinzia; Fjelstrup, S

    We present the utilization of a rolling circle amplification (RCA) based assay to investigate the extraction efficiency of active enzymes from a class of “hard-to-break” cells, yeast Saccaramyces cerevisiae. Current analyses of microorganisms, such as pathogenic bacteria, parasites or particular...... life stages of microorganisms (e.g. spores from bacteria or fungi) is hampered by the lack of efficient lysis protocols that preserve the activity and integrity of the cellular content. Presented herein is a flexible scheme to screen lysis protocols for active enzyme extraction. We also report a gentle...... yet effective approach for extraction of active enzymes by entrapping cells in microdroplets. Combined effort of optimized extraction protocols and effective analytical approaches is expected to generate impact in future disease diagnosis and environmental safety....

  14. Glyphosate on digestive enzymes activity in piava (Leporinus obtusidens

    Directory of Open Access Journals (Sweden)

    Joseânia Salbego

    2014-09-01

    Full Text Available The effects of glyphosate, a nonselective herbicide (1.0 or 5.0mg L-1 on digestive enzymes activity (stomach and intestine were evaluated in juveniles of piava (Leporinus obtusidens after 90 days of exposure. The activity of acid protease, trypsin, chymotrypsin and amylase increased with the increase of glyphosate concentration. These results indicate that glyphosate affects digestive enzyme activities in this species, and may be an indicator of poor nutrient availability when fish survive in herbicide-contaminated water.

  15. Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus.

    Science.gov (United States)

    Doi, Hidetaka; Tokura, Yuriko; Mori, Yukiko; Mori, Kenichi; Asakura, Yoko; Usuda, Yoshihiro; Fukuda, Hiroo; Chinen, Akito

    2017-02-01

    Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2(T) was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into L-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

  16. Lipid peroxidation and antioxidant enzymes activity in avian semen.

    Science.gov (United States)

    Partyka, Agnieszka; Lukaszewicz, Ewa; Niżański, Wojciech

    2012-10-01

    The present study compared the antioxidant system and lipid peroxidation in semen of two avian species: chicken and goose. The experiment was conducted on Greenleg Partridge roosters and White Koluda(®) ganders, each represented by 10 mature males. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma. In gander spermatozoa, the amount of MDA was 10 times greater (Pantioxidant enzymes had greater (Pactivity in goose than chicken sperm. Catalase activity was detected in seminal plasma and spermatozoa from both studied species for the first time. In seminal plasma, the activity of GPx was two times greater (Pactivity was less (Pactivity of antioxidant defense and LPO. The greater amount of lipid peroxidation and greater activity of antioxidant enzymes in goose semen might suggest that spermatozoa were under greater oxidative stress and the enzymes were not utilized for the protection of functionally and structurally impaired cells. In turn, in fresh chicken semen a lesser activity of antioxidant enzymes accompanied with a lesser lipid peroxidation amount and good semen quality could indicate that fowl spermatozoa were under oxidative stress, but the enzymes were employed to protect and maintain sperm quality. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Combined effects of urinary phytoestrogens metabolites and polymorphisms in metabolic enzyme gene on idiopathic male infertility.

    Science.gov (United States)

    Qin, Yufeng; Du, Guizhen; Chen, Minjian; Hu, Weiyue; Lu, Chuncheng; Wu, Wei; Hang, Bo; Zhou, Zuomin; Wang, Xinru; Xia, Yankai

    2014-08-01

    Phytoestrogens are plant-derived compounds that may interact with estrogen receptors and mimic estrogenic effects. It remains unclear whether the individual variability in metabolizing phytoestrogens contributes to phytoestrogens-induced beneficial or detrimental effects. Our aim was to determine whether there is any interaction between metabolic rates (MR) of phytoestrogens and genetic polymorphisms in related xenobiotic metabolizing enzyme genes. MR was used to assess phytoestrogen exposure and individual metabolic ability. The amount of phytoestrogens in urine was measured by ultra-high performance liquid chromatography-tandem mass spectrometry in 600 idiopathic infertile male patients and 401 controls. Polymorphisms were genotyped using the SNPstream platform combined with the Taqman method. Prototypes and metabolites of secoisolariciresinol (SEC) have inverse effects on male reproduction. It was found that low MR of SEC increased the risk of male infertility (OR 2.49, 95 % CI 1.78, 3.48, P trend = 8.00 × 10(-8)). Novel interactions were also observed between the MR of SEC and rs1042389 in CYP2B6, rs1048943 in CYP1A1, and rs1799931 in NAT2 on male infertility (P inter = 1.06 × 10(-4), 1.14 × 10(-3), 3.55 × 10(-3), respectively). By analyzing the relationships between urinary phytoestrogen concentrations, their metabolites and male infertility, we found that individual variability in metabolizing SEC contributed to the interpersonal differences in SEC's effects on male reproduction.

  18. Strain differences in the induction of hepatic microsomal cytochrome P450 and drug-metabolizing enzymes by climbazole in rats

    National Research Council Canada - National Science Library

    Michiya Suzuki; Yasuna Kobayashi; Takashi Sunagawa; Takiko Oguro; Shogo Tokuyama; Toshinori Yamamoto; Takemi Yoshida

    2000-01-01

    .... Climbazole is one of imidazole-ring containing antifungal agents. However, there is not enough data concerned with their effect on hepatic drug metabolizing enzymes with respect to the strain differences...

  19. Genetic variants of methyl metabolizing enzymes and epigenetic regulators: Associations with promoter CpG island hypermethylation in colorectal cancer

    NARCIS (Netherlands)

    Vogel, S. de; Wouters, K.A.D.; Gottschalk, R.W.H.; Schooten, F.J. van; Goeij, A.F.P.M. de; Bruïne, A.P. de; Goldbohm, R.A.; Brandt, P.A. van den; Weijenberg, M.P.; Engeland, M. van

    2009-01-01

    Aberrant DNA methylation affects carcinogenesis of colorectal cancer. Folate metabolizing enzymes may influence the bioavailability of methyl groups, whereas DNA and histone methyltransferases are involved in epigenetic regulation of gene expression. We studied associations of genetic variants of fo

  20. Effects of Arctium lappa aqueous extract on lipid profile and hepatic enzyme levels of sucrose-induced metabolic syndrome in female rats

    Directory of Open Access Journals (Sweden)

    Akram Ahangarpour

    Full Text Available ABSTRACT Arctium lappa is known to have antioxidant and antidiabetic effects in traditional medicine. Objectives: The aim of this paper was to study the effects of A. lappa root extract (AE on lipid profile and hepatic enzyme levels in sucrose-induced metabolic syndrome (MS in female rats. The study used 40 adult female Wistar rats weighing 150 g-250 g randomly divided into five groups: control, metabolic syndrome (MS, metabolic syndrome+AE at 50,100, 200 mg/kg. MS was induced by administering 50% sucrose in drinking water for 6 weeks. AE was intra-peritoneally administered daily at doses of 50,100, and 200 mg/kg for two sequential weeks at the end of the fourth week in metabolic syndrome rats. Twenty-four hours after the last administration of AE, blood was collected and centrifuged, and then the serum was used for the measurement of lipid profile and hepatic enzyme. Serum glucose, insulin, fasting insulin resistance index, body weight, water intake, lipid profile, and hepatic enzymes were significantly increased although food intake was decreased in MS rats compared to the control rats. The lipids and liver enzymes were reduced by AE extracts in the MS group. This study showed that the A. lappa root aqueous extract exhibits a hypolipidemic activity of hyperlipidemic rats. This activity is practically that of a triple-impact antioxidant, hypolipidemic, and hepatoprotective.

  1. CART modulates beta-amyloid metabolism-associated enzymes and attenuates memory deficits in APP/PS1 mice.

    Science.gov (United States)

    Yin, Kailin; Jin, Jiali; Zhu, Xiaolei; Yu, Linjie; Wang, Sulei; Qian, Lai; Han, Lijuan; Xu, Yun

    2017-10-01

    Cocaine- and amphetamine-regulated transcript (CART) peptide has been demonstrated to exert neuroprotective effects in stroke and some neurodegeneration diseases. In current study, we investigated the protective effects and underlying mechanisms of CART in APP/PS1 mice. The protein levels of CART, soluble Aβ1-40 and Aβ1-42 were measured in the hippocampus of APP/PS1 mice by enzyme-linked immunosorbent assay. We determined the mRNA and protein levels of Aβ metabolism-associated enzymes including neprilysin (NEP), insulin-degrading enzyme (IDE), receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP-1) in the hippocampus of APP/PS1 mice using real-time PCR and western blotting. Spatial memory was measured in APP/PS1 mice using the Morris water maze. The phosphorylation of AKT, ERK, p38, and JNK was determined using western blotting. The levels of soluble Aβ1-40 and Aβ1-42 were significantly decreased in the hippocampus of APP/PS1 mice after CART treatment. CART modulated the levels of NEP, IDE, RAGE, and LRP-1. In addition, CART inhibited the MAPK pathways and activated the AKT pathway, whereas inhibition of the AKT pathway decreased the levels of IDE and LRP-1. Furthermore, CART attenuated spatial memory deficits in the APP/PS1 mice. CART decreases the levels of soluble Aβ in the hippocampus of APP/PS1 mice by modulating the expression of Aβ metabolism-associated enzymes, which may be associated with the MAPK and AKT pathways.

  2. Extraction of Active Enzymes from "Hard-to-Break-Cells"

    DEFF Research Database (Denmark)

    Ottaviani, Alessio; Tesauro, Cinzia; Fjelstrup, S

    life stages of microorganisms (e.g. spores from bacteria or fungi) is hampered by the lack of efficient lysis protocols that preserve the activity and integrity of the cellular content. Presented herein is a flexible scheme to screen lysis protocols for active enzyme extraction. We also report a gentle...

  3. Soil Enzyme Activities under Agroforestry Systems in Northern Jiangsu Province

    Institute of Scientific and Technical Information of China (English)

    Wan Fuxu; Chen Ping

    2004-01-01

    The authors presented the enzyme characteristics of catalase, sucrase, urease and alkaline phosphatase under agroforestry systems in northern Jiangsu Province. The results show that soil enzyme activities reduce gradually from top to bottom layer of the soil profile, and the fluctuations of catalase and urease are smaller than those of sucrase and alkaline phosphatase. Soil enzyme activities differe significantly in different samples, and the order is arranged as poplar-crop intercropping segment (A, D) > paulownia-crop intercropping segment (B, C) > CK. Furthermore, soil enzyme activities increase with intercropping age. On the other hand, in the same plot, there are closer relationships between enzymes in the soil samples. Catalase, alkaline phosphatase and urease are negatively related, while alkaline phosphatase and urease are positively related (except in samples B and C). In addition, the enzyme activities have a close relationship with the fertilizers. Catalase is positively correlated with the soil pH value (r = 0.854, 0.804, 0.078 and 0.082, respectively), and is negatively correlated with total N (r = -0.201, -0.529, -0.221 and -0.821, respectively), total P (r = -0.143, -0.213, -0.362 and -0.751, respectively) and available P (r = -0.339, -0.351, -0.576, and -0.676, respectively). Sucrase, urease and alkaline phosphatase are negatively correlated with the pH value, while positively correlated with the other fertilizers (r ≈ 1). The authors suggest that enzyme activity will be a great potential as an indicator of soil quality.

  4. Antioxidant enzymes activities in obese Tunisian children

    Directory of Open Access Journals (Sweden)

    Sfar Sonia

    2013-01-01

    Full Text Available Abstract Background The oxidant stress, expected to increase in obese adults, has an important role in the pathogenesis of many diseases. It results when free radical formation is greatly increased or protective antioxidant mechanisms are compromised. The main objective of this study is to evaluate the antioxidant response to obesity-related stress in healthy children. Methods A hundred and six healthy children (54 obese and 52 controls, aged 6–12 years old, participated in this study. The collected data included anthropometric measures, blood pressure, fasting glucose, total cholesterol, triglycerides and enzymatic antioxidants (Superoxide dismutase: SOD, Catalase: CAT and Glutathione peroxidase: GPx. Results The first step antioxidant response, estimated by the SOD activity, was significantly higher in obese children compared with normal-weight controls (p  Conclusions The obesity-related increase of the oxidant stress can be observed even in the childhood period. In addition to the complications of an increased BMI, obesity itself can be considered as an independent risk factor of free radical production resulting in an increased antioxidant response.

  5. Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut.

    Science.gov (United States)

    Araji, Soha; Grammer, Theresa A; Gertzen, Ross; Anderson, Stephen D; Mikulic-Petkovsek, Maja; Veberic, Robert; Phu, My L; Solar, Anita; Leslie, Charles A; Dandekar, Abhaya M; Escobar, Matthew A

    2014-03-01

    The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut.

  6. Chemoproteomic profiling of host and pathogen enzymes active in cholera.

    Science.gov (United States)

    Hatzios, Stavroula K; Abel, Sören; Martell, Julianne; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A; Qadri, Firdausi; Ryan, Edward T; Davis, Brigid M; Weerapana, Eranthie; Waldor, Matthew K

    2016-04-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human choleric stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, and genetic disruption of all four proteases increased the abundance of intelectin, an intestinal lectin, and its binding to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting that it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialog in an animal model of infection.

  7. Chemoproteomic profiling of host and pathogen enzymes active in cholera

    Science.gov (United States)

    Hatzios, Stavroula K.; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A.; Qadri, Firdausi; Ryan, Edward T.; Davis, Brigid M.; Weerapana, Eranthie; Waldor, Matthew K.

    2016-01-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human cholera stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, while genetic disruption of all four proteases increased the abundance and binding of an intestinal lectin—intelectin—to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialogue in an animal model of infection. PMID:26900865

  8. Evolutionary transitions in enzyme activity of ant fungus gardens.

    Science.gov (United States)

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

    2010-07-01

    Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

  9. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A. (UMASS, MED)

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  10. Does diet influence salivary enzyme activities in elephant species?

    Science.gov (United States)

    Boehlke, Carolin; Pötschke, Sandra; Behringer, Verena; Hannig, Christian; Zierau, Oliver

    2017-01-01

    Asian elephants (Elephas maximus) and African elephants (Loxodonta africana) are herbivore generalists; however, Asian elephants might ingest a higher proportion of grasses than Africans. Although some studies have investigated nutrition-specific morphological adaptations of the two species, broader studies on salivary enzymes in both elephant species are lacking. This study focuses on the comparison of salivary enzymes activity profiles in the two elephant species; these enzymes are relevant for protective and digestive functions in humans. We aimed to determine whether salivary amylase (sAA), lysozyme (sLYS), and peroxidase (sPOD) activities have changed in a species-specific pattern during evolutionary separation of the elephant genera. Saliva samples of 14 Asian and eight African elephants were collected in three German zoos. Results show that sAA and sLYS are salivary components of both elephant species in an active conformation. In contrast, little to no sPOD activity was determined in any elephant sample. Furthermore, sAA activity was significantly higher in Asian compared with African elephants. sLYS and sPOD showed no species-specific differences. The time of food provision until sample collection affected only sAA activity. In summary, the results suggest several possible factors modulating the activity of the mammal-typical enzymes, such as sAA, sLYS, and sPOD, e.g., nutrition and sampling procedure, which have to be considered when analyzing differences in saliva composition of animal species.

  11. A DNA enzyme with N-glycosylase activity

    Science.gov (United States)

    Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

  12. De novo active sites for resurrected Precambrian enzymes

    Science.gov (United States)

    Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

    2017-07-01

    Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

  13. The effects of inhibitors on organic acids and activities of metabolism-related enzymes in aluminum-treated roots of rape%抑制剂对铝胁迫下油菜根系代谢有机酸和相关酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    王志颖; 刘鹏; 徐艳

    2013-01-01

    Under hydroponic conditions, activities of four metabolism-related enzymes were investigated in roots of two rape cultivars Huayou 2790 ( Al-sensitive) and Zhongshuang No. 7 (Al-resistant) , treated with different aluminum (Al) concentrations ofO, 50, 100, 200 μmol·L-1 combined with an inhibitor Mersal (5 μmol·L-1 ) , respectively. The results showed that the root secretion of citric acid, malic acid, total organic acid and activities of metabolism-related enzymes increased to some extents under short-term and low concentration treatments, but started to gradually decrease with the increased time and Al concentrations. Compared to the Al treatment only, combination treatments with Al and Mersal significantly reduced citric acid content, activities of Citrate acid ( CS ) , Malate dehydrogenase ( MDH ) , and Aconitase ( ACO ) , while enhanced malic acid content, Phosphoenolpyruvate carboxylase (PEPCase) enzyme activity. These results suggested that the inhibitor Mersal aggravated Al toxicity and diminished the role of organic acids in alleviating Al toxicity in the rape cultivars, especially in the sensitive cultivar Huayou 2790. The oil rape can adjust the root exudation, increase the secretion of malic acid, etc. , and regulate the PEPCase enzyme activity to respond to low concentrations and short-time Al stress, but with the increase of Al concentrations and treatment time, aluminum toxicity was further deepened.%采用水培法,研究两个油菜品种华油2790(铝敏感型)和中双7号(耐铝型),在4个铝浓度(0、50、100、200μmol· L-1)和外源抑制剂Mersal(5.μmol·L-1)复合处理下,根系分泌柠檬酸、苹果酸、总有机酸和4种根系代谢相关酶的活性变化.实验结果显示:短期、低浓度的铝胁迫使油菜根系分泌的柠檬酸、苹果酸、总有机酸含量,以及代谢相关酶活性总体上有所增加,但随着时间的延长和铝浓度的增加,均有所降低.加入Mersal后,与单铝处理相比,柠檬酸

  14. Peroxisome Proliferator Activated Receptors and Lipoprotein Metabolism

    Directory of Open Access Journals (Sweden)

    Sander Kersten

    2008-01-01

    Full Text Available Plasma lipoproteins are responsible for carrying triglycerides and cholesterol in the blood and ensuring their delivery to target organs. Regulation of lipoprotein metabolism takes place at numerous levels including via changes in gene transcription. An important group of transcription factors that mediates the effect of dietary fatty acids and certain drugs on plasma lipoproteins are the peroxisome proliferator activated receptors (PPARs. Three PPAR isotypes can be distinguished, all of which have a major role in regulating lipoprotein metabolism. PPARα is the molecular target for the fibrate class of drugs. Activation of PPARα in mice and humans markedly reduces hepatic triglyceride production and promotes plasma triglyceride clearance, leading to a clinically significant reduction in plasma triglyceride levels. In addition, plasma high-density lipoprotein (HDL-cholesterol levels are increased upon PPARα activation in humans. PPARγ is the molecular target for the thiazolidinedione class of drugs. Activation of PPARγ in mice and human is generally associated with a modest increase in plasma HDL-cholesterol and a decrease in plasma triglycerides. The latter effect is caused by an increase in lipoprotein lipase-dependent plasma triglyceride clearance. Analogous to PPARα, activation of PPARβ/δ leads to increased plasma HDL-cholesterol and decreased plasma triglyceride levels. In this paper, a fresh perspective on the relation between PPARs and lipoprotein metabolism is presented. The emphasis is on the physiological role of PPARs and the mechanisms underlying the effect of synthetic PPAR agonists on plasma lipoprotein levels.

  15. Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Gokarn, R.R.; Eiteman, M.A.; Altman, E.

    2000-05-01

    Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99a-pyc than by cells which overproduced PPC(JCL1242/pPC201, ppc{sup +}), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc{sup +}) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc{sup +} strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc{sup +} strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.

  16. Enzyme Activities in Perfluorooctanoic Acid (PFOA)-Polluted Soils

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei; LIN Kuang-Fei; YANG Sha-Sha; ZHANG Meng

    2013-01-01

    Perfluorooctanoic acid (PFOA) is a popular additive of the chemical industry; its effect on activities of important soil enzymes is not well understood.A laboratory incubation experiment was carried out to analyze the PFOA-induced changes in soil urease,catalase,and phosphatase activities.During the entire incubation period,the activities of the three soil enzymes generally declined with increasing PFOA concentration,following certain dose-response relationships.The values of EC10,the contaminant concentration at which the biological activity is inhibited by 10%,of PFOA for the soil enzyme activity calculated from the modeling equation of the respective dose-response curve suggested a sensitivity order of phosphatase > catalase > urease.The effect of PFOA on soil enzyme activities provided a basic understanding of the eco-toxicological effect of PFOA in the environment.Results of this study supported using soil phosphatase as a convenient biomarker for ecological risk assessment of PFOA-polluted soils.

  17. 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.

  18. Influence of long-term fertilization on soil enzyme activities

    Directory of Open Access Journals (Sweden)

    Alina Dora SAMUEL

    2009-05-01

    Full Text Available Soil enzyme activities (actual and potential dehydrogenase, catalase, acid and alkaline phosphatase were determined in the 0–10, 10–20, and 20–30 cm layers of a brown luvic soil submitted to a complex fertilization experiment with different types of green manure. It was found that each activity decreased with increasing sampling depth. It should be emphasized that greenmanuring of maize led to a significant increase in each of the five enzymatic activities determined. The enzymatic indicators of soil quality calculated from the values of enzymatic activities showed the order: lupinus + rape + oat > lupinus > vetch + oat + ryegrass > lupinus + oat + vetch > unfertilized plot. This order means that by determination of enzymatic activities valuable information can be obtained regarding fertility status of soils. There were significant correlations of soil enzyme activities with chemical properties.

  19. Characterization of human cytochrome P450 enzymes involved in the metabolism of cyamemazine.

    Science.gov (United States)

    Arbus, Christophe; Benyamina, Amine; Llorca, Pierre-Michel; Baylé, Franck; Bromet, Norbert; Massiere, Frédéric; Garay, Ricardo P; Hameg, Ahcène

    2007-12-01

    Recombinant human liver microsomal enzymes of the cytochrome P450 family (CYP1A2, CYP2A6, CYP3A4, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1) were used to determine the metabolic fate of the antipsychotic anxiolytic agent cyamemazine. An LC/MS-MS tandem methodology was developed specifically for identifying the presence of cyamemazine and its metabolites in reaction media. All P450 enzymes investigated, with the exception of CYP2A6 and CYP2E1, degraded cyamemazine, albeit to a different extent, with CYP1A2, CYP2C8 and CYP2C19 being the most efficient (>80%). However, in microsomes prepared from native human hepatocytes, only relatively specific competitors (inhibitors and/or substrates) of CYP1A2, CYP2C8, CYP2C9 and CYP3A4 reduced notably the degradation cyamemazine. The main routes of cyamemazine biotransformation are N-mono-demethylation (CYP1A2, CYP3A4 and CYP2C8) and mono-oxidation (either S-oxidized or hydroxylated derivatives which could not be discriminated because characterized by the same mass value) by CYP1A2 and CYP2C9. Secondary metabolic routes yields N,N-di-demethylated and N-demethylated mono-oxidized products. Thus, under in vitro conditions, cyamemazine is extensively degraded by at least four distinct P450 enzymes, into two primary hydrophilic metabolites. These results suggest that cyamemazine detoxification process is unlikely to be significantly impaired by co-administration of therapeutic agents that are substrates of the CYP metabolic system.

  20. In Vivo Exposure of Kaempferol Is Driven by Phase II Metabolic Enzymes and Efflux Transporters.

    Science.gov (United States)

    Zheng, Liang; Zhu, Lijun; Zhao, Min; Shi, Jian; Li, Yuhuan; Yu, Jia; Jiang, Huangyu; Wu, Jinjun; Tong, Yunli; Liu, Yuting; Hu, Ming; Lu, Linlin; Liu, Zhongqiu

    2016-09-01

    Kaempferol is a well-known flavonoid; however, it lacks extensive pharmacokinetic studies. Phase II metabolic enzymes and efflux transporters play an important role in the disposition of flavonoids. This study aimed to investigate the mechanism by which phase II metabolic enzymes and efflux transporters determine the in vivo exposure of kaempferol. Pharmacokinetic analysis in Sprague-Dawley rats revealed that kaempferol was mostly biotransformed to conjugates, namely, kaempferol-3-glucuronide (K-3-G), kaempferol-7-glucuronide (K-7-G), and kaempferol-7-sulfate, in plasma. K-3-G represented the major metabolite. Compared with that in wild-type mice, pharmacokinetics in knockout FVB mice demonstrated that the absence of multidrug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP) significantly increased the area under the curve (AUC) of the conjugates. The lack of MRP1 resulted in a much lower AUC of the conjugates. Intestinal perfusion in rats revealed that the glucuronide conjugates were mainly excreted in the small intestine, but 7-sulfate was mainly excreted in the colon. In Caco-2 monolayers, K-7-G efflux toward the apical (AP) side was significantly higher than K-3-G efflux. In contrast, K-3-G efflux toward the basolateral (BL) side was significantly higher than K-7-G efflux. The BL-to-AP efflux was significantly reduced in the presence of the MRP2 inhibitor LTC4. The AP-to-BL efflux was significantly decreased in the presence of the BL-side MRPs inhibitor MK571. The BCRP inhibitor Ko143 decreased the glucuronide conjugate efflux. Therefore, kaempferol is mainly exposed as K-3-G in vivo, which is driven by phase II metabolic enzymes and efflux transporters (i.e., BCRP and MRPs).

  1. In vivo cytochrome P450 drug metabolizing enzyme characterization using surface-enhanced Raman spectroscopy

    Science.gov (United States)

    Li, Yanfang; Bachmann, Kenneth A.; Cameron, Brent D.

    2003-07-01

    The development of a rapid, inexpensive, and accurate in vivo phenotyping methodology for characterizing drug-metabolizing phenotypes with reference to the cytochrome P450 (CYP450) enzymes would be very beneficial. In terms of application, in the wake of the human genome project, considerable interest is focused on the development of new drugs whose uses will be tailored to specific genetic polymorphisms, and on the individualization of dosing regimens that are also tailored to meet individual patient needs depending upon genotype. In this investigation, chemical probes for CYP450 enzymes were characterized and identified with Raman spectroscopy. Furthermore, gold-based metal colloid clusters were utilized to generate surface enhanced Raman spectra for each of the chemical probes. Results will be presented demonstrating the ability of SERS to identify minute quantities of these probes on the order needed for in vivo application.

  2. Effects of prolonged recombinant human erythropoietin administration on muscle membrane transport systems and metabolic marker enzymes

    DEFF Research Database (Denmark)

    Juel, C; Thomsen, J J; Rentsch, R L;

    2007-01-01

    Adaptations to chronic hypoxia involve changes in membrane transport proteins. The underlying mechanism of this response may be related to concomitant occurring changes in erythropoietin (Epo) levels. We therefore tested the direct effects of recombinant human erythropoietin (rHuEpo) treatment...... on the expression of muscle membrane transport proteins. Likewise, improvements in performance may involve upregulation of metabolic enzymes. Since Epo is known to augment performance we tested the effect of rHuEpo on some marker enzymes that are related to aerobic capacity. For these purposes eight subjects...... received 5,000 IU rHuEpo every second day for 14 days, and subsequently a single dose of 5,000 IU weekly for 12 weeks. Muscle biopsies were obtained before and after 14 weeks of rHuEpo treatment. The treatment increased hematocrit (from 44.7 to 48.8%), maximal oxygen uptake by 8.1%, and submaximal...

  3. Neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-HSD type 4

    Directory of Open Access Journals (Sweden)

    Wise Petra M

    2010-04-01

    Full Text Available Abstract Background Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E2 is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4, an enzyme that converts E2 to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ and females (ZW because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E2 in their brains than females. Results Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain. Conclusions Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E2 metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms.

  4. Neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17beta-HSD type 4.

    Science.gov (United States)

    London, Sarah E; Itoh, Yuichiro; Lance, Valentin A; Wise, Petra M; Ekanayake, Preethika S; Oyama, Randi K; Arnold, Arthur P; Schlinger, Barney A

    2010-04-01

    Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E2) is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17beta-hydroxysteroid dehydrogenase type 4 (HSD17B4), an enzyme that converts E2 to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ) and females (ZW) because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E2 in their brains than females. Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain. Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E2 metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms.

  5. Neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-HSD type 4

    Science.gov (United States)

    2010-01-01

    Background Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E2) is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4), an enzyme that converts E2 to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ) and females (ZW) because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E2 in their brains than females. Results Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain. Conclusions Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E2 metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms. PMID:20359329

  6. Difference in the distribution pattern of substrate enzymes in the metabolic network of Escherichia coli, according to chaperonin requirement

    Directory of Open Access Journals (Sweden)

    Niwa Tatsuya

    2011-06-01

    Full Text Available Abstract Background Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE in Escherichia coli, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear. Results We investigated the distribution of chaperonin substrate enzymes in the metabolic network using network analysis techniques as a first step towards revealing this relationship, and found that as chaperonin requirement increases, substrate enzymes are more laterally distributed in the metabolic. In addition, comparative genome analysis showed that the chaperonin-dependent substrates were less conserved, suggesting that these substrates were acquired later on in evolutionary history. Conclusions This result implies the expansion of metabolic networks due to this chaperonin, and it supports the existing hypothesis of acceleration of evolution by chaperonins. The distribution of chaperonin substrate enzymes in the metabolic network is inexplicable because it does not seem to be associated with individual protein features such as protein abundance, which has been observed characteristically in chaperonin substrates in previous works. However, it becomes clear by considering this expansion process due to chaperonin. This finding provides new insights into metabolic evolution and the roles of chaperonins in living systems.

  7. Expression of two drug-metabolizing cytochrome P450-enzymes in human salivary glands

    DEFF Research Database (Denmark)

    Kragelund, C; Hansen, C; Torpet, L A

    2008-01-01

    : Formalin-fixed paraffin-embedded specimens from parotid (10), submandibular (7) and labial (10) salivary glands were examined immunohistochemically and by in situ hybridization for expression of CYP1A2 and CYP3A4 protein and mRNA. RESULTS: CYP1A2 and CYP3A4 protein and mRNA were detected in ductal......OBJECTIVE: The oral cavity is constantly lubricated by saliva and even small amounts of xenobiotics and / or their metabolites in the saliva may affect the oral mucosa. Our aim was therefore to clarify if xenobiotic metabolizing enzymes CYP1A2 and CYP3A4 are expressed in salivary glands. METHODS...... and seromucous / serous acinar cells in all gland types although to a varying degree and intensity. Mucous acinar cells were positive to a lesser extent. CONCLUSION: The results indicate a xenobiotic metabolizing capability of salivary glands. This may have implications for development of oral mucosal disease...

  8. [Enzyme activity of an actinomycete producer of carotenes and macrotetrolides].

    Science.gov (United States)

    Nefelova, M V; Sverdlova, A N

    1982-01-01

    The activity of pyruvate dehydrogenase and dehydrogenases of the tricarboxylic acid cycle was assayed in the mycelium of Streptomyces chrysomallus var. Carotenoides growing under different conditions of the medium. The activity of the enzymes increased when acetic, citric and succinic acids were added at different periods of the growth. Moreover, addition of the acids increased the time of intensive functioning of the dehydrogenases whose activity abruptly decreased after 60 h of the growth under the control conditions.

  9. Gene expression of regulatory enzymes involved in the intermediate metabolism of sheep subjected to feed restriction.

    Science.gov (United States)

    van Harten, S; Brito, R; Almeida, A M; Scanlon, T; Kilminster, T; Milton, J; Greeff, J; Oldham, C; Cardoso, L A

    2013-03-01

    The effect of feed restriction on gene expression of regulatory enzymes of intermediary metabolism was studied in two sheep breeds (Australian Merino and Dorper) subjected to two nutritional treatments: feed restriction (85% of daily maintenance requirements) and control (ad libitum feeding), during 42 days. The experimental animals (ram lambs) were divided into four groups, n = 5 (Australian Merino control (MC), Australian Merino Restriction (MR), Dorper control (DC) and Dorper Restriction (DR)). After the trial, animals were sacrificed and samples were taken from liver tissue to quantify glucose levels and gene expression of relevant intermediary metabolism enzymes (phosphofructokinase (PFK), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, glucose-6-phosphatase, glycogen synthase (GS), fatty acid synthase (FAS), glutamate dehydrogenase (GDH) and carbamoyl phosphate synthase (CPS)) through real-time PCR. During the experimental period, the MR animals lost 12.6% in BW compared with 5.3% lost by the Dorper lambs. MC and DC rams gained, respectively, 8.8% and 14% during the same period. Within the Dorper breed, restricted feed animals revealed a significant decrease over controls in the transcription of PFK (1.95-fold) and PK (2.26-fold), both glycolytic enzymes. The gluconeogenesis showed no change in the feed restricted animals of both breeds. DR feed group presented a significant decrease over the homologous Merino sheep group on GS. In both experimental breeds, FAS mRNA expression was decreased in restricted feed groups. GDH expression was decreased only in the DR animals (1.84-fold) indicating a reduced catabolism of amino acids in these animals. Finally, CPS was significantly (P enzymes and hepatic glucose production of Dorper sheep to feed restriction concurring with the BW results in the experimental groups.

  10. 氮、磷、钾对烤烟碳氮代谢关键酶活性及其经济效益的影响%Effects of N,P, K on Activity of Key Enzymes for Carbon and Nitrogen Metabolism and Economic Benefit of Flue-cured Tobacco

    Institute of Scientific and Technical Information of China (English)

    赵宪凤; 刘卫群; 王树会

    2012-01-01

    为探讨氮、磷、钾肥对烤烟碳氮代谢关键酶活性及其经济效益影响,采用田间小区试验,测定了缺氮、缺磷、缺钾和氮磷钾正常(CK)处理烤烟不同生育时期碳氮代谢关键酶即硝酸还原酶(NR)、谷氨酰胺合成酶(GS)、蔗糖磷酸合成酶(SPS))、蔗糖合成酶(SS)和转化酶(INV)的活性变化.结果表明:在烟叶的氮代谢过程中,NR活性主要在烟叶生长发育前期起作用,与CK相比,氮、磷、钾素缺乏分别使烟叶NR活性下降35.9%,13.4%,19.5%,但移栽后85 d无差异显著性;在碳代谢过程中,SPS,SS和INV活性主要在烟叶生育中期起作用,缺氮能显著降低碳代谢关键酶活性,而缺磷、钾素则表现不明显,很可能与试验田磷钾含量较高有关.缺氮处理对烟叶的产量、产值影响最大,与CK处理相比,分别降低了19.7%,13.2%,缺磷、钾素处理与CK差异不显著.因此,烤烟为达到优质高产,首先要确保氮肥供应充足,同时要适当控制磷钾肥用量.%The purpose of the paper was to study the effect of N, P, K fertilizer on the activeness of key enzymes involved in C and N metabolism during leaf development and the economic benefit of flue-cured tobacco. Four different treatments (i. e. nitrogen, phosphorus, potassium deficiency and CK) were designed in a field experiment to study the enzyme activeness of nitrate reductase ( NR ) , Glutamina synthetase ( GS ) , sucrose phosphate syhthase (SPS) , sucrose synthase ( SS) and invertase (Inv). The results showed that NR was majored at the early growth stage of flue-cured tobacco. Compared with CK,the NR activity of N,P and K lack treatments decreased by 35. 9% , 13.4% and 19. 5% , respectively, but no significant difference was detected after 85 d of transplanting. In the process of carbon metabolism,the key enzymes SPS.SS and INV played a major role at the middle growth stage. N deficiency could decrease significantly the activeness of key enzyme, while P

  11. Transcriptional co-regulation of secondary metabolism enzymes in Arabidopsis: functional and evolutionary implications.

    Science.gov (United States)

    Gachon, Claire M M; Langlois-Meurinne, Mathilde; Henry, Yves; Saindrenan, Patrick

    2005-05-01

    The combined knowledge of the Arabidopsis genome and transcriptome now allows to get an integrated view of the dynamics and evolution of metabolic pathways in plants. We used publicly available sets of microarray data obtained in a wide range of different stress and developmental conditions to investigate the co-expression of genes encoding enzymes of secondary metabolism pathways, in particular indoles, phenylpropanoids, and flavonoids. We performed hierarchical clustering of gene expression profiles and found that major enzymes of each pathway display a clear and robust co-expression throughout all the conditions studied. Moreover, detailed analysis evidenced that some genes display co-regulation in particular physiological conditions only, certainly reflecting their modular recruitment into stress- or developmentally regulated biosynthetic pathways. The combination of these microarray data with sequence analysis allows to draw very precise hypotheses on the function of otherwise uncharacterized genes. To illustrate this approach, we focused our analysis on secondary metabolism glycosyltransferases (UGTs), a multigenic family involved in the conjugation of small molecules to sugars like glucose. We propose that UGT74B1 and UGT74C1 may be involved in aromatic and aliphatic glucosinolates synthesis, respectively. We also suggest that UGT75C1 may function as an anthocyanin-5-O-glucosyltransferase in planta. Therefore, this data-mining approach appears very powerful for the functional prediction of unknown genes, and could be transposed to virtually any other gene family. Finally, we suggest that analysis of expression pattern divergence of duplicated genes also provides some insight into the mechanisms of metabolic pathway evolution.

  12. Sedentary Activity Associated With Metabolic Syndrome Independent of Physical Activity

    OpenAIRE

    Bankoski, Andrea; Tamara B. Harris; McClain, James J.; Brychta, Robert J.; Caserotti, Paolo; Chen, Kong Y.; Berrigan, David; Troiano, Richard P.; Koster, Annemarie

    2011-01-01

    OBJECTIVE This study examined the association between objectively measured sedentary activity and metabolic syndrome among older adults. RESEARCH DESIGN AND METHODS Data were from 1,367 men and women, aged ≥60 years who participated in the 2003–2006 National Health and Nutrition Examination Survey (NHANES). Sedentary time during waking hours was measured by an accelerometer (5 min. A sedentary break was defined as an interruption in sedentary time (≥100 counts per minute). Metabolic syndrome ...

  13. Early feeding to modify digestive enzyme activity in broiler chickens

    Directory of Open Access Journals (Sweden)

    Milagro León T.

    2014-09-01

    Full Text Available Objective. To evaluate the effect on digestive enzyme activity in broiler chickens by providing food in the first 48 hrs. after birth. Materials and methods. After incubating 300 fertile eggs from Hubbard breeding and immediately after hatching, the chicks were randomly assigned to treatments: fasting (from hatching to 48 hrs.; Hydrated Balanced Food (HBF from birth to 48 hrs.; commercial hydrating supplement (CHS from birth to 48 hrs. The diets were provided ad libitum. After 48 hrs. a commercial diet was fed. At birth and at 48 and 72 hrs. of age 30 chicks/treatment were sacrificed to determine the enzyme activity of maltase, sucrase, alkaline phosphatase, phytase, a-amylase, trypsin and lipase in samples of duodenal or pancreatic homogenate. Results. The supply of HBF or CHS during the first 48 hrs. of life increased the activity of maltase, sucrase and phytase in the first 3 days of life, with values between 1.2 and up to 4-fold compared to the control (p<0.05. Chickens that fasted for the first 48 hrs. had higher activity of the pancreatic enzymes a-amylase, trypsin, and lipase at 72 hrs. of life (p<0.05. Conclusions. The food supply in the first 48 hrs. after hatching increases the duodenal enzyme activity in the intestinal brush border during the first 3 days of age in broiler chickens.

  14. Identification of glyoxalase 1 polymorphisms associated with enzyme activity.

    Science.gov (United State