Science.gov

Sample records for key metabolic enzyme

  1. Metabolism of aflatoxins: key enzymes and interindividual as well as interspecies differences.

    PubMed

    Dohnal, Vlastimil; Wu, Qinghua; Kuča, Kamil

    2014-09-01

    Aflatoxins are potent hepatocarcinogen in animal models and suspected carcinogen in humans. The most important aflatoxin in terms of toxic potency and occurrence is aflatoxin B1 (AFB1). In this review, we mainly summarized the key metabolizing enzymes of AFB1 in animals and humans. Moreover, the interindividual and the interspecies differences in AFB1 metabolism are highly concerned. In human liver, CYP3A4 plays an important role in biotransforming AFB1 to the toxic product AFB1-8,9-epoxide. In human lung, CYP2A13 has a significant activity in metabolizing AFB1 to AFB1-8,9-epoxide and AFM1-8,9-epoxide. The epoxide of AFB1-8,9-epoxide could conjugate with glutathione to reduce the toxicity by glutathione-S-transferase (GST). In poultry species, CYP2A6, CYP3A37, CYP1A5, and CYP1A1 are responsible for bioactivation of AFB1. There are interindividual variations in the rate of activation of aflatoxins in various species, and there are also differences between children and adults. The age and living regions are important factors affecting resistance of species to AFB1. The rate of AFB1-8,9-epoxide formation and its conjugation with glutathione are key parameters in interspecies and interindividual differences in sensitivity to the toxic effect of AFB1. This review provides an important information for key metabolizing enzymes and the global metabolism of aflatoxins in different species.

  2. [Controlling arachidonic acid metabolic network: from single- to multi-target inhibitors of key enzymes].

    PubMed

    Liu, Ying; Chen, Zheng; Shang, Er-chang; Yang, Kun; Wei, Deng-guo; Zhou, Lu; Jiang, Xiao-lu; He, Chong; Lai, Lu-hua

    2009-03-01

    Inflammatory diseases are common medical conditions seen in disorders of human immune system. There is a great demand for anti-inflammatory drugs. There are major inflammatory mediators in arachidonic acid metabolic network. Several enzymes in this network have been used as key targets for the development of anti-inflammatory drugs. However, specific single-target inhibitors can not sufficiently control the network balance and may cause side effects at the same time. Most inflammation induced diseases come from the complicated coupling of inflammatory cascades involving multiple targets. In order to treat these complicated diseases, drugs that can intervene multi-targets at the same time attracted much attention. The goal of this review is mainly focused on the key enzymes in arachidonic acid metabolic network, such as phospholipase A2, cyclooxygenase, 5-lipoxygenase and eukotriene A4 hydrolase. Advance in single target and multi-targe inhibitors is summarized.

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

    PubMed

    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.

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

    PubMed

    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

  5. Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood.

    PubMed

    Gaspar, Paula; Al-Bayati, Firas A Y; Andrew, Peter W; Neves, Ana Rute; Yesilkaya, Hasan

    2014-12-01

    Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression.

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

    NASA Technical Reports Server (NTRS)

    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.

  7. Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans.

    PubMed

    Anaganti, Narasimha; Basu, Bhakti; Gupta, Alka; Joseph, Daisy; Apte, Shree Kumar

    2015-01-01

    Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD50 = 40 μM tellurite, 40 min exposure). The organism reduced 70% of 40 μM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status. The level of ROS and protein carbonyl contents increased while the cellular reduction potential substantially decreased following tellurite exposure. Cellular thiols levels initially increased (within 30 min) of tellurite exposure but decreased at later time points. At proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes (superoxide dismutase and thioredoxin reductase), and protein folding chaperones (DnaK, EF-Ts, and PPIase) displayed increased abundance in tellurite-stressed cells. However, remarkably decreased levels of key metabolic enzymes (aconitase, transketolase, 3-hydroxy acyl-CoA dehydrogenase, acyl-CoA dehydrogenase, electron transfer flavoprotein alpha, and beta) involved in carbon and energy metabolism were observed upon tellurite stress. The results demonstrate that depletion of reduction potential in intensive tellurite reduction with impaired energy metabolism lead to tellurite toxicity in D. radiodurans.

  8. Impact of androgenic/antiandrogenic compounds (AAC) on human sex steroid metabolizing key enzymes.

    PubMed

    Alléra, A; Lo, S; King, I; Steglich, F; Klingmüller, D

    2004-12-01

    Various pesticides, industrial pollutants and synthetic compounds, to which human populations are exposed, are known or suspected to interfere with endogenous sex hormone functions. Such interference potentially affect the development and expression of the male and female reproductive system or both. Chemicals in this class are thus referred to as endocrine disruptors (ED). This emphazises on the relevance of screening ED for a wide range of sex hormone-mimicking effects. These compounds are believed to exert influence on hormonal actions predominantly by (i) interfering with endogenous steroids in that they functionally interact with plasma membrane-located receptors as well as with nuclear receptors both for estrogens and androgens or (ii) affecting the levels of sex hormones as a result of their impact on steroid metabolizing key enzymes. Essential sex hormone-related enzymes within the endocrine system of humans are aromatase, 5alpha-reductase 2 as well as specific sulfotransferases and sulfatases (so-called phase I and phase II enzymes, respectively). Using suitable human tissues and human cancer cell lines (placenta, prostate, liver and JEG-3, lymph node carcinoma of prostate (LnCaP) cells) we investigated the impact of 10 widely used chemicals suspected of acting as ED with androgenic or antiandrogenic activity (so-called AAC) on the activity of these sex hormone metabolizing key enzymes in humans. In addition, the respective effects of six substances were also studied as positive controls due to their well-known specific hormonal agonistic/antagonistic activities. The aim of this report and subsequent investigations is to improve human health risk assessment for AAC and other ED.

  9. Structure and function of sphingosine-1-phosphate lyase, a key enzyme of sphingolipid metabolism.

    PubMed

    Bourquin, Florence; Riezman, Howard; Capitani, Guido; Grütter, Markus G

    2010-08-11

    Sphingosine-1-phosphate lyase (SPL), a key enzyme of sphingolipid metabolism, catalyzes the irreversible degradation of sphingoid base phosphates. Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. There, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SPL is a pivotal enzyme regulating S1P intracellular concentrations and a promising drug target for the design of immunosuppressants. We structurally and functionally characterized yeast SPL (Dpl1p) and its first prokaryotic homolog, from Symbiobacterium thermophilum. The Dpl1p structure served as a basis for a very reliable model of Homo sapiens SPL. The above results, together with in vitro and in vivo studies of SPL mutants, reveal which residues are involved in activity and substrate binding and pave the way to studies aimed at controlling the activity of this pivotal enzyme.

  10. Levels of Key Enzymes of Methionine-Homocysteine Metabolism in Preeclampsia

    PubMed Central

    Pérez-Sepúlveda, Alejandra; España-Perrot, Pedro P.; Fernández B, Ximena; Ahumada, Verónica; Bustos, Vicente; Arraztoa, José Antonio; Dobierzewska, Aneta; Figueroa-Diesel, Horacio; Rice, Gregory E.; Illanes, Sebastián E.

    2013-01-01

    Objective. To evaluate the role of key enzymes in the methionine-homocysteine metabolism (MHM) in the physiopathology of preeclampsia (PE). Methods. Plasma and placenta from pregnant women (32 controls and 16 PE patients) were analyzed after informed consent. Protein was quantified by western blot. RNA was obtained with RNA purification kit and was quantified by reverse transcritase followed by real-time PCR (RT-qPCR). Identification of the C677T and A1298C methylenetetrahydrofolate reductase (MTHFR) single-nucleotide polymorphisms (SNPs) and A2756G methionine synthase (MTR) SNP was performed using PCR followed by a high-resolution melting (HRM) analysis. S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) were measured in plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). The SNP association analysis was carried out using Fisher's exact test. Statistical analysis was performed using a Mann-Whitney test. Results. RNA expression of MTHFR and MTR was significantly higher in patients with PE as compared with controls. Protein, SAM, and SAH levels showed no significant difference between preeclamptic patients and controls. No statistical differences between controls and PE patients were observed with the different SNPs studied. Conclusion. The RNA expression of MTHFR and MTR is elevated in placentas of PE patients, highlighting a potential compensation mechanism of the methionine-homocysteine metabolism in the physiopathology of this disease. PMID:24024209

  11. New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria.

    PubMed

    Selmer, Thorsten; Pierik, Antonio J; Heider, Johann

    2005-10-01

    During the last decade, an increasing number of new enzymes containing glycyl radicals in their active sites have been identified and biochemically characterised. These include benzylsuccinate synthase (Bss), 4-hydroxyphenylacetate decarboxylase (Hpd) and the coenzyme B12-independent glycerol dehydratase (Gdh). These are involved in metabolic pathways as different as anaerobic toluene metabolism, fermentative production of p-cresol and glycerol fermentation. Some features of these newly discovered enzymes are described and compared with those of the previously known glycyl radical enzymes pyruvate formate-lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd). Among the new enzymes, Bss and Hpd share the presence of small subunits, the function of which in the catalytic mechanisms is still enigmatic, and both enzymes contain metal centres in addition to the glycyl radical prosthetic group. The activating enzymes of the novel systems also deviate from the standard type, containing at least one additional Fe-S cluster. Finally, the available whole-genome sequences of an increasing number of strictly or facultative anaerobic bacteria revealed the presence of many more hitherto unknown glycyl radical enzyme (GRE) systems. Recent studies suggest that the particular types of these enzymes represent the ends of different evolutionary lines, which emerged early in evolution and diversified to yield remarkably versatile biocatalysts for chemical reactions that are otherwise difficult to perform in anoxic environments.

  12. Characterization of Genes Encoding Key Enzymes Involved in Anthocyanin Metabolism of Kiwifruit during Storage Period

    PubMed Central

    Li, Boqiang; Xia, Yongxiu; Wang, Yuying; Qin, Guozheng; Tian, Shiping

    2017-01-01

    ‘Hongyang’ is a red fleshed kiwifruit with high anthocyanin content. In this study, we mainly investigated effects of different temperatures (25 and 0°C) on anthocyanin biosynthesis in harvested kiwifruit, and characterized the genes encoding key enzymes involved in anthocyanin metabolism, as well as evaluated the mode of the action, by which low temperature regulates anthocyanin accumulation in ‘Hongyang’ kiwifruit during storage period. The results showed that low temperature could effectively enhance the anthocyanin accumulation of kiwifruit in the end of storage period (90 days), which related to the increase in mRNA levels of ANS1, ANS2, DRF1, DRF2, and UGFT2. Moreover, the transcript abundance of MYBA1-1 and MYB5-1, the genes encoding an important component of MYB–bHLH–WD40 (MBW) complex, was up-regulated, possibly contributing to the induction of specific anthocyanin biosynthesis genes under the low temperature. To further investigate the roles of AcMYB5-1/5-2/A1-1 in regulation of anthocyanin biosynthesis, genes encoding the three transcription factors were transiently transformed in Nicotiana benthamiana leaves. Overexpression of AcMYB5-1/5-2/A1-1 activated the gene expression of NtANS and NtDFR in tobacco. Our results suggested that low temperature storage could stimulate the anthocyanin accumulation in harvested kiwifruit via regulating several structural and regulatory genes involved in anthocyanin biosynthesis. PMID:28344589

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

    PubMed

    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.

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

    PubMed

    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.

  15. Shared origins of a key enzyme during the evolution of C4 and CAM metabolism

    PubMed Central

    Christin, Pascal-Antoine; Arakaki, Monica; Osborne, Colin P.; Bräutigam, Andrea; Sage, Rowan F.; Hibberd, Julian M.; Kelly, Steven; Covshoff, Sarah; Wong, Gane Ka-Shu; Hancock, Lillian; Edwards, Erika J.

    2014-01-01

    CAM and C4 photosynthesis are two key plant adaptations that have evolved independently multiple times, and are especially prevalent in particular groups of plants, including the Caryophyllales. We investigate the origin of photosynthetic PEPC, a key enzyme of both the CAM and C4 pathways. We combine phylogenetic analyses of genes encoding PEPC with analyses of RNA sequence data of Portulaca, the only plants known to perform both CAM and C4 photosynthesis. Three distinct gene lineages encoding PEPC exist in eudicots (namely ppc-1E1, ppc-1E2 and ppc-2), one of which (ppc-1E1) was recurrently recruited for use in both CAM and C4 photosynthesis within the Caryophyllales. This gene is present in multiple copies in the cacti and relatives, including Portulaca. The PEPC involved in the CAM and C4 cycles of Portulaca are encoded by closely related yet distinct genes. The CAM-specific gene is similar to genes from related CAM taxa, suggesting that CAM has evolved before C4 in these species. The similar origin of PEPC and other genes involved in the CAM and C4 cycles highlights the shared early steps of evolutionary trajectories towards CAM and C4, which probably diverged irreversibly only during the optimization of CAM and C4 phenotypes. PMID:24638902

  16. Characterisation of genes encoding key enzymes involved in sugar metabolism of apple fruit in controlled atmosphere storage.

    PubMed

    Zhu, Zhu; Liu, Ruiling; Li, Boqiang; Tian, Shiping

    2013-12-15

    Sugars are essential contributors to fruit flavour. Controlled atmosphere (CA) storage has been proved to be beneficial for maintaining harvested fruit quality. To explore regulatory mechanism of sugar metabolism in fruit stored in CA condition, we cloned several genes, encoding key enzymes, involved in sugar metabolism in apple fruit, and analyzed sugar contents, along with gene expression and enzyme activities in fruits stored in air and CA. The results indicated that CA could maintain higher contents of sugars, including sucrose, fructose and glucose. Expression levels of key genes, such as sucrose synthase (SS), sucrose phosphate synthase (SPS), fructokinase (FK) and hexokinase (HK), were shown to be correlated with the corresponding enzyme activities. We found that activities of neutral invertase (NI), vacuolar invertase (VI), FK and HK were inhibited, but SPS activity was promoted in apple fruit stored in CA, suggesting that CA storage could enhance sucrose synthesis and delay hydrolysis of sucrose and hexose. These findings provided molecular evidence to explain why higher sugar levels in harvested fruit are maintained under CA storage.

  17. Key Residues Controlling Phenacetin Metabolism By Human Cytochrome P450 2A Enzymes

    SciTech Connect

    DeVore, N.M.; Smith, B.D.; Urban, M.J.; Scott, E.E.

    2009-05-14

    Although the human lung cytochrome P450 2A13 (CYP2A13) and its liver counterpart cytochrome P450 2A6 (CYP2A6) are 94% identical in amino acid sequence, they metabolize a number of substrates with substantially different efficiencies. To determine differences in binding for a diverse set of cytochrome P450 2A ligands, we have measured the spectral binding affinities (K{sub D}) for nicotine, phenethyl isothiocyanate (PEITC), coumarin, 2{prime}-methoxyacetophenone (MAP), and 8-methoxypsoralen. The differences in the K{sub D} values for CYP2A6 versus CYP2A13 ranged from 74-fold for 2{prime}-methoxyacetophenone to 1.1-fold for coumarin, with CYP2A13 demonstrating the higher affinity. To identify active site amino acids responsible for the differences in binding of MAP, PEITC, and coumarin, 10 CYP2A13 mutant proteins were generated in which individual amino acids from the CYP2A6 active site were substituted into CYP2A13 at the corresponding position. Titrations revealed that substitutions at positions 208, 300, and 301 individually had the largest effects on ligand binding. The collective relevance of these amino acids to differential ligand selectivity was verified by evaluating binding to CYP2A6 mutant enzymes that incorporate several of the CYP2A13 amino acids at these positions. Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein (I208S/I300F/G301A/S369G) with binding affinities for MAP and PEITC much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding. The structure-based quantitative structure-activity relationship analysis using COMBINE successfully modeled the observed mutant-ligand trends and emphasized steric roles for active site residues including four substituted amino acids and an adjacent conserved Leu{sup 370}.

  18. An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

    PubMed Central

    Romagnoli, G; Verhoeven, M D; Mans, R; Fleury Rey, Y; Bel-Rhlid, R; van den Broek, M; Maleki Seifar, R; Ten Pierick, A; Thompson, M; Müller, V; Wahl, S A; Pronk, J T; Daran, J M

    2014-01-01

    Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. PMID:24912400

  19. Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism.

    PubMed Central

    Ochs, D; Kaletta, C; Entian, K D; Beck-Sickinger, A; Poralla, K

    1992-01-01

    The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coli that express this enzyme in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found. Images PMID:1729216

  20. Tyrosol, a phenolic compound, ameliorates hyperglycemia by regulating key enzymes of carbohydrate metabolism in streptozotocin induced diabetic rats.

    PubMed

    Chandramohan, Ramasamy; Pari, Leelavinothan; Rathinam, Ayyasamy; Sheikh, Bashir Ahmad

    2015-03-05

    The present study was designed to evaluate the effects of tyrosol, a phenolic compound, on the activities of key enzymes of carbohydrate metabolism in the control and streptozotocin-induced diabetic rats. Diabetes mellitus was induced in rats by a single intraperitoneal injection of streptozotocin (40 mg/kg body weight). Experimental rats were administered tyrosol 1 ml intra gastrically at the doses of 5, 10 and 20mg/kg body weight and glibenclamide 1 ml at a dose of 600 μg/kg body weight once a day for 45 days. At the end of the experimental period, diabetic control rats exhibited significant (p<0.05) increase in plasma glucose, glycosylated hemoglobin with significant (p<0.05) decrease in plasma insulin, total hemoglobin and body weight. The activities of key enzymes of carbohydrate metabolism such as phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase were significantly (p<0.05) increased and the activities of hexokinase and glucose-6-phosphate dehydrogenase were significantly (p<0.05) decreased in the liver and kidney of diabetic control rats. Further, antioxidants were lowered in diabetic control rats. A significant (p<0.05) decline in glycogen level in the liver and muscle and glycogen synthase activity in the liver and a significant (p<0.05) increase in the activity of liver glycogen phosphorylase were observed in diabetic control rats compared to normal control rats. Oral administration of tyrosol to diabetic rats reversed all the above mentioned biochemical parameters to near normal in a dose dependent manner. Tyrosol at a dose of 20mg/kg body weight showed the highest significant effect than the other two doses. Immunohistochemical staining of pancreas revealed that tyrosol treated diabetic rats showed increased insulin immunoreactive β-cells, which confirmed the biochemical findings. The observed results were compared with glibenclamide, a standard oral hypoglycemic drug. The results of the present study suggest

  1. [Effects of applying biogas liquid manure on the key source-sink metabolism enzymes and grain yield of summer maize].

    PubMed

    Lü, Shu-Min; Qu, Xiao-Fei; Wang, Lin-Hua; Liang, Shu-Rong; Wang, Jun-Zhong; Zhao, Hui-Jie

    2010-02-01

    15000, 22500, and 30000 kg x hm(-2) of biogas liquid manure were applied to maize hybrid Zhengdan 958 to study their effects on the key source-sink metabolism enzymes and yield components of the summer maize. Compared with CK and applying nitrogen fertilizer, the application of biogas liquid manure not only increased the aboveground biomass, leaf area index (LAI) and chlorophyll content, but also enhanced the activities of nitrate reductase (NR), glutamine synthetase (GS), and sucrose phosphate synthetase (SPS) in leaves and the sucrose synthetase (SS) in grains. The yield parameters such as ear diameter, ear length, grain rows per ear, grains per row, kernels per ear, 1000-kernel mass, and grain yield per unit area were also increased significantly. Of the three test application rates, 22500 kg x hm(-2) (7500 kg x hm(-2) applied at jointing, big trumpet, and tasseling stages, respectively) had the best effects on enhancing the above mentioned enzyme activities and grain yield, with the yield reached 14006.7 kg x hm(-2) and being 40.7% higher than the control.

  2. Immunogold Localization of Key Metabolic Enzymes in the Anammoxosome and on the Tubule-Like Structures of Kuenenia stuttgartiensis

    PubMed Central

    de Almeida, Naomi M.; Neumann, Sarah; Mesman, Rob J.; Ferousi, Christina; Keltjens, Jan T.; Jetten, Mike S. M.; van Niftrik, Laura

    2015-01-01

    ABSTRACT Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite as the terminal electron acceptor to form dinitrogen gas in the absence of oxygen. Anammox bacteria have a compartmentalized cell plan with a central membrane-bound “prokaryotic organelle” called the anammoxosome. The anammoxosome occupies most of the cell volume, has a curved membrane, and contains conspicuous tubule-like structures of unknown identity and function. It was suggested previously that the catalytic reactions of the anammox pathway occur in the anammoxosome, and that proton motive force was established across its membrane. Here, we used antibodies raised against five key enzymes of the anammox catabolism to determine their cellular location. The antibodies were raised against purified native hydroxylamine oxidoreductase-like protein kustc0458 with its redox partner kustc0457, hydrazine dehydrogenase (HDH; kustc0694), hydroxylamine oxidase (HOX; kustc1061), nitrite oxidoreductase (NXR; kustd1700/03/04), and hydrazine synthase (HZS; kuste2859-61) of the anammox bacterium Kuenenia stuttgartiensis. We determined that all five protein complexes were exclusively located inside the anammoxosome matrix. Four of the protein complexes did not appear to form higher-order protein organizations. However, the present data indicated for the first time that NXR is part of the tubule-like structures, which may stretch the whole length of the anammoxosome. These findings support the anammoxosome as the locus of catabolic reactions of the anammox pathway. IMPORTANCE Anammox bacteria are environmentally relevant microorganisms that contribute significantly to the release of fixed nitrogen in nature. Furthermore, the anammox process is applied for nitrogen removal from wastewater as an environment-friendly and cost-effective technology. These microorganisms feature a unique cellular organelle, the anammoxosome, which was proposed to contain the energy metabolism of the cell and

  3. Russelioside B, a pregnane glycoside ameliorates hyperglycemia in streptozotocin induced diabetic rats by regulating key enzymes of glucose metabolism.

    PubMed

    Abdel-Sattar, Essam; El-Maraghy, Shohda A; El-Dine, Riham Salah; Rizk, Sherine M

    2016-05-25

    An alternative strategy to treat diabetes mellitus is the use of various natural agents possessing hypoglycemic effect. Caralluma quadrangula has been used in Saudi traditional medicine in cases of thirst and hunger and for the treatment of diabetes. The present study was designed to evaluate the improving effect of russelioside B, a pregnane glycoside isolated from Caralluma quadrangula on glucose metabolism in the liver of streptozotocin-induced diabetic rats. Diabetes mellitus was induced in rats by a single intraperitoneal injection of streptozotocin (50 mg/kg body weight). Experimental rats were administered russelioside B at a dose of 50 mg/kg body weight once a day for 30 days. The results showed that RB improved the fasting serum glucose level, glycated hemoglobin percent, serum insulin level and lipid profile. A significant improvement was observed upon the administration of russelioside B on the activities of the key enzymes of carbohydrate metabolism (glucokinase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, and glycogen phosphorylase) in the liver of diabetic rats. Further, russelioside B administration to diabetic rats reverted gene expression of glucokinase, glucose-6-phosphatase, glycogen synthase and glycogen synthase kinase-3β to near normal levels. In conclusion, russelioside B possess antidiabetic and antihyperlipidemic effect in streptozotocin induced diabetic rats. Hence, administration of russelioside B may represent a potentially useful strategy for the management of diabetes.

  4. Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer

    NASA Astrophysics Data System (ADS)

    Nilsson, Roland; Jain, Mohit; Madhusudhan, Nikhil; Sheppard, Nina Gustafsson; Strittmatter, Laura; Kampf, Caroline; Huang, Jenny; Asplund, Anna; Mootha, Vamsi K.

    2014-01-01

    Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.

  5. Antidiabetic efficacy of citronellol, a citrus monoterpene by ameliorating the hepatic key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats.

    PubMed

    Srinivasan, Subramani; Muruganathan, Udaiyar

    2016-04-25

    Diabetes mellitus is a clinically complex disease characterized by chronic hyperglycemia with metabolic disturbances. During diabetes, endogenous hepatic glucose production is increased as a result of impaired activities of the key enzymes of carbohydrate metabolism. The purpose of the present study was to evaluate the antidiabetic efficacy of citronellol, a citrus monoterpene in streptozotocin (STZ)-induced diabetic rats. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (40 mg/kg b.w). STZ induced diabetic rats received citronellol orally at the doses of 25, 50, and 100 mg/kg b.w for 30 days. In this study the levels of plasma glucose, insulin, hemoglobin (Hb), glycated hemoglobin (HbA1C), glycogen, and the activities of carbohydrate metabolic enzymes, liver and kidney markers were evaluated. Oral administration of citronellol (50 mg/kg) for 30 days dose dependently improved the levels of insulin, Hb and hepatic glycogen with significant decrease in glucose and HbA1C levels. The altered activities of carbohydrate metabolic enzymes, hepatic and kidney markers were restored to near normal. Citronellol supplement was found to be effective in preserving the normal histological appearance of hepatic cells and insulin-positive β-cells in STZ-rats. Our results suggest that administration of citronellol attenuates the hyperglycemia in the STZ-induced diabetic rats by ameliorating the key carbohydrate metabolic enzymes and could be developed as a functional and nutraceutical ingredient in combating diabetes mellitus.

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

    PubMed

    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.

  7. Protein homeostasis disorders of key enzymes of amino acids metabolism: mutation-induced protein kinetic destabilization and new therapeutic strategies.

    PubMed

    Pey, Angel L

    2013-12-01

    Many inborn errors of amino acids metabolism are caused by single point mutations affecting the ability of proteins to fold properly (i.e., protein homeostasis), thus leading to enzyme loss-of-function. Mutations may affect protein homeostasis by altering intrinsic physical properties of the polypeptide (folding thermodynamics, and rates of folding/unfolding/misfolding) as well as the interaction of partially folded states with elements of the protein homeostasis network (such as molecular chaperones and proteolytic machineries). Understanding these mutational effects on protein homeostasis is required to develop new therapeutic strategies aimed to target specific features of the mutant polypeptide. Here, I review recent work in three different diseases of protein homeostasis associated to inborn errors of amino acids metabolism: phenylketonuria, inherited homocystinuria and primary hyperoxaluria type I. These three different genetic disorders involve proteins operating in different cell organelles and displaying different structural complexities. Mutations often decrease protein kinetic stability of the native state (i.e., its half-life for irreversible denaturation), which can be studied using simple kinetic models amenable to biophysical and biochemical characterization. Natural ligands and pharmacological chaperones are shown to stabilize mutant enzymes, thus supporting their therapeutic application to overcome protein kinetic destabilization. The role of molecular chaperones in protein folding and misfolding is also discussed as well as their potential pharmacological modulation as promising new therapeutic approaches. Since current available treatments for these diseases are either burdening or only successful in a fraction of patients, alternative treatments must be considered covering studies from protein structure and biophysics to studies in animal models and patients.

  8. Effect of schistosomal infection and its treatment on some key enzymes of glucose metabolism in mice livers.

    PubMed

    Ahmed, S A; Gad, M Z

    1995-12-01

    Three antischistosomal drugs, praziquantel (CAS 55268-74-1, EMBAY 8440, Prz), oxamniquine (CAS 21738-42-1, Oxa) and oltipraz (CAS 64224-21-1, Olt) were examined for their ability to reverse the disturbances in carbohydrate metabolism induced by Schistosoma mansoni (S. mansoni) infection. The infected mice were screened every 2 weeks for 16 weeks for their body and liver weights in addition to assessment of the activities of liver pyruvate kinase (PK), phosphofructokinase (PFK) (glycolysis), citrate synthase (CS) (Krebs' cycle) glycogen phosphorylase (GP) (glycogenolysis), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) (hexose monophosphate shunt). Results of the study showed that infection with S. mansoni caused the following changes in mice livers: 1. significant increase in liver weights from the 6th week of infection, which coincided with schistosomal egg deposition, whereas body weights were reduced, 2. remarkable increase in the activities of PK and PFK from the 4th week of infection, 3. marked reduction in CS, GP, G6PDH and 6PGDH. These results lead to the conclusion that glycolysis is largely stimulated in the livers of infected mice on the expense of other metabolic pathways of glucose utilization. Administration of Prz to infected mice caused normalization of all measured enzyme activities almost from the 2nd week of infection, whereas liver and body weights were improved from the 10th week. Oxa was less effective in these regards while Olt was the least. These data support the selection of Prz as a drug of choice for S. mansoni infection.

  9. Modulatory effect of green tea extract on hepatic key enzymes of glucose metabolism in streptozotocin and high fat diet induced diabetic rats.

    PubMed

    Sundaram, Ramalingam; Naresh, Rajendran; Shanthi, Palanivelu; Sachdanandam, Panchanatham

    2013-05-15

    The study was undertaken to evaluate the antidiabetic effect of green tea extract on carbohydrate metabolic key enzymes in control and streptozotocin high fat diet -induced diabetic rats. The daily oral treatment of green tea extract (300 mg/kg body weight) to diabetic rats for 30 days resulted in a significant reduction in the levels of plasma glucose, glycosylated hemoglobin (HbA1c) and increase in the levels of insulin and hemoglobin. The altered activities of the key enzymes of carbohydrate metabolism such as hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase and glycogen phosphorylase in liver of diabetic rats were significantly reverted to near normal levels by the administration of green tea extract. Further, green tea extract administration to diabetic rats improved muscle and hepatic glycogen content suggesting the antihyperglycemic potential of green tea extract in diabetic rats. The obtained results were compared with metformin, a standard oral hypoglycemic drug. Thus, this study indicates that the administration of green tea extract to diabetic rats resulted in alterations in the metabolism of glucose with subsequent reduction in plasma glucose levels.

  10. Characterization of a NADH-dependent glutamate dehydrogenase mutant of Arabidopsis demonstrates the key role of this enzyme in root carbon and nitrogen metabolism.

    PubMed

    Fontaine, Jean-Xavier; Tercé-Laforgue, Thérèse; Armengaud, Patrick; Clément, Gilles; Renou, Jean-Pierre; Pelletier, Sandra; Catterou, Manuella; Azzopardi, Marianne; Gibon, Yves; Lea, Peter J; Hirel, Bertrand; Dubois, Frédéric

    2012-10-01

    The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism.

  11. Homocysteine and the C677T Gene Polymorphism of Its Key Metabolic Enzyme MTHFR Are Risk Factors of Early Renal Damage in Hypertension in a Chinese Han Population

    PubMed Central

    Yun, Lin; Xu, Rui; Li, Guohua; Yao, Yucai; Li, Jiamin; Cong, Dehong; Xu, Xingshun; Zhang, Lihua

    2015-01-01

    Abstract The combined hyperhomocysteinemia condition is a feature of the Chinese hypertensive population. This study used the case-control method to investigate the association between plasma homocysteine and the C677T gene polymorphism of its key metabolic enzyme, 5, 10-methylenetetrahydrofolate reductase (MTHFR), and early renal damage in a hypertensive Chinese Han population. A total of 379 adult essential hypertensive patients were selected as the study subjects. The personal information, clinical indicators, and the C677T gene polymorphism of MTHFR were texted. This study used the urine microalbumin/urine creatinine ratio (UACR) as a grouping basis: the hypertension without renal damage group (NRD group) and the hypertension combined with early renal damage group (ERD group). Early renal damage in the Chinese hypertensive population was associated with body weight, systolic pressure, diastolic pressure, urea nitrogen, serum creatinine, cystatin C, uric acid, aldosterone, and glomerular filtration rate. The homocysteine level and the UACR in the TT genotype group were higher than those in the CC genotype group. The binary logistic regression analysis results showed that after sex and age were adjusted, the MTHFR C677T gene polymorphism was correlated with early renal damage in hypertension in both the recessive model and in the additive model. Plasma homocysteine and the C677T gene polymorphism of its key metabolic enzyme MTHFR might be independent risk factors of early renal damage in the hypertensive Chinese Han population. PMID:26717388

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

    PubMed

    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.

  13. In vitro inhibitory potential of Cynara scolymus, Silybum marianum, Taraxacum officinale, and Peumus boldus on key enzymes relevant to metabolic syndrome.

    PubMed

    Villiger, Angela; Sala, Filippo; Suter, Andy; Butterweck, Veronika

    2015-01-15

    Boldocynara®, a proprietary dietary supplement product consisting of the plants Cynara scolymus, Silybum marianum, Taraxacum officinale, and Peumus boldus, used to promote functions of the liver and the gallbladder. It was the aim of the present study to look from a different perspective at the product by investigating the in vitro potential of Boldocynara® as a combination product and its individual extracts on key enzymes relevant to metabolic syndrome. Peumus boldus extract exhibited pronounced inhibitory activities on α-glucosidase (80% inhibition at 100 µg/ml, IC50: 17.56 µg/ml). Silybum marianum had moderate pancreatic lipase (PL) inhibitory activities (30% at 100 µg/ml) whereas Cynara scolymus showed moderate ACE inhibitory activity (31% at 100 µg/ml). The combination had moderate to weak effects on the tested enzymes. In conclusion, our results indicate some moderate potential of the dietary supplement Boldocynara® and its single ingredients for the prevention of metabolic disorders.

  14. Effects of inhibitors of key enzymes of sphingolipid metabolism on insulin-induced glucose uptake and glycogen synthesis in liver cells of old rats.

    PubMed

    Babenko, N A; Kharchenko, V S

    2015-01-01

    Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

  15. Synthetic metabolism: metabolic engineering meets enzyme design.

    PubMed

    Erb, Tobias J; Jones, Patrik R; Bar-Even, Arren

    2017-01-30

    Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future.

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

    PubMed

    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.

  17. Effect of a compost and its water-soluble fractions on key enzymes of nitrogen metabolism in maize seedlings.

    PubMed

    Vaccaro, Silvia; Muscolo, Adele; Pizzeghello, Diego; Spaccini, Riccado; Piccolo, Alessandro; Nardi, Serenella

    2009-12-09

    The growing concern on long-term productivity of agroecosystems has emphasized the need to develop management strategies to maintain and protect soil resources, particularly soil organic matter (SOM). Among these, the composting process allows both recycling of the increasing amount of organic waste materials and restoration of the content of organic matter in soil. A sequential chemical fractionation into structurally unbound (SU), weakly bound (WB) and strongly bound (SB) compounds was applied to a bulk compost, and its soluble fractions were extracted in water, either after oxidation of compost suspension with an oxygen flux (TEA), or without oxidation but separated into hydrophilic (HiDOM) and hydrophobic (HoDOM) components. The ratio of hydrophilic over hydrophobic compounds decreased in the order HiDOM > TEA > compost > HoDOM, while TEA and compost showed the largest content of SU and WB components, respectively. Such chemically characterized bulk compost and fractions were tested on maize seedlings grown in sand and in hydroponic conditions, and the effects on plant growth and nitrogen metabolism were measured. The structurally complex bulk compost and the hydrophobic HoDOM fraction negatively affected plant growth, whereas the hydrophilic and less-structured fractions (HiDOM and TEA) showed large positive effects on both growth and enzymatic activities of plants. These results suggest that composted organic matter can become useful to stimulate plant growth if the content of potentially bioavailable hydrophilic and poorly structured components is large. These components may be progressively separated from the compost matrix and contribute to the dynamics of natural organic matter in soil.

  18. Orphan enzymes in ether lipid metabolism.

    PubMed

    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.

  19. Metabolic regulation via enzyme filamentation

    PubMed Central

    Aughey, Gabriel N.; Liu, Ji-Long

    2016-01-01

    Abstract Determining the mechanisms of enzymatic regulation is central to the study of cellular metabolism. Regulation of enzyme activity via polymerization-mediated strategies has been shown to be widespread, and plays a vital role in mediating cellular homeostasis. In this review, we begin with an overview of the filamentation of CTP synthase, which forms filamentous structures termed cytoophidia. We then highlight other important examples of the phenomenon. Moreover, we discuss recent data relating to the regulation of enzyme activity by compartmentalization into cytoophidia. Finally, we hypothesize potential roles for enzyme filament formation in the regulation of metabolism, development and disease. PMID:27098510

  20. A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

    PubMed Central

    Führing, Jana Indra; Cramer, Johannes Thomas; Schneider, Julia; Baruch, Petra; Gerardy-Schahn, Rita; Fedorov, Roman

    2015-01-01

    In mammals, UDP-glucose pyrophosphorylase (UGP) is the only enzyme capable of activating glucose-1-phosphate (Glc-1-P) to UDP-glucose (UDP-Glc), a metabolite located at the intersection of virtually all metabolic pathways in the mammalian cell. Despite the essential role of its product, the molecular basis of UGP function is poorly understood. Here we report the crystal structure of human UGP in complex with its product UDP-Glc. Beyond providing first insight into the active site architecture, we describe the substrate binding mode and intermolecular interactions in the octameric enzyme that are crucial to its activity. Importantly, the quaternary mechanism identified for human UGP in this study may be common for oligomeric sugar-activating nucleotidyltransferases. Elucidating such mechanisms is essential for understanding nucleotide sugar metabolism and opens the perspective for the development of drugs that specifically inhibit simpler organized nucleotidyltransferases in pathogens. PMID:25860585

  1. Drug-metabolizing enzymes: mechanisms and functions.

    PubMed

    Sheweita, S A

    2000-09-01

    Drug-metabolizing enzymes are called mixed-function oxidase or monooxygenase and containing many enzymes including cytochrome P450, cytochrome b5, and NADPH-cytochrome P450 reductase and other components. The hepatic cytochrome P450s (Cyp) are a multigene family of enzymes that play a critical role in the metabolism of many drugs and xenobiotics with each cytochrome isozyme responding differently to exogenous chemicals in terms of its induction and inhibition. For example, Cyp 1A1 is particularly active towards polycyclic aromatic hydrocarbons (PAHs), activating them into reactive intermediates those covalently bind to DNA, a key event in the initiation of carcinogenesis. Likewise, Cyp 1A2 activates a variety of bladder carcinogens, such as aromatic amines and amides. Also, some forms of cytochrome P450 isozymes such as Cyp 3A and 2E1 activate the naturally occurring carcinogens (e.g. aflatoxin B1) and N-nitrosamines respectively into highly mutagenic and carcinogenic agents. The carcinogenic potency of PAHs, and other carcinogens and the extent of binding of their ultimate metabolites to DNA and proteins are correlated with the induction of cytochrome P450 isozymes. Phase II drug-metabolizing enzymes such as glutathione S-transferase, aryl sulfatase and UDP-glucuronyl transferase inactivate chemical carcinogens into less toxic or inactive metabolites. Many drugs change the rate of activation or detoxification of carcinogens by changing the activities of phases I and II drug-metabolizing enzymes. The balance of detoxification and activation reactions depends on the chemical structure of the agents, and is subjected to many variables that are a function of this structure, or genetic background, sex, endocrine status, age, diet, and the presence of other chemicals. It is important to realize that the enzymes involved in carcinogen metabolism are also involved in the metabolism of a variety of substrates, and thus the introduction of specific xenobiotics may change

  2. Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities.

    PubMed

    Lauer, Mariana Machado; de Oliveira, Camila Bento; Yano, Natalia Lie Inocencio; Bianchini, Adalto

    2012-11-01

    The estuarine crab Neohelice granulata was exposed (96 h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2 ppt salinity, 1 mg Cu/L; isosmotic crabs: 30 ppt salinity, 5 mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2 ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30 ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30 ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes

  3. Long-term feeding a plant-based diet devoid of marine ingredients strongly affects certain key metabolic enzymes in the rainbow trout liver.

    PubMed

    Véron, Vincent; Panserat, Stéphane; Le Boucher, Richard; Labbé, Laurent; Quillet, Edwige; Dupont-Nivet, Mathilde; Médale, Françoise

    2016-04-01

    Incorporation of a plant blend in the diet can affect growth parameters and metabolism in carnivorous fish. We studied for the first time the long-term (1 year) metabolic response of rainbow trout fed from first feeding with a plant-based diet totally devoid of marine ingredients. Hepatic enzymes were analyzed at enzymatic and molecular levels, at 3, 8 and 24 h after the last meal to study both the short-term effects of the last meal and long-term effects of the diet. The results were compared with those of fish fed a control diet of fish meal and fish oil. Growth, feed intake, feed efficiency and protein retention were lower in the group fed the plant-based diet. Glucokinase and pyruvate kinase activity were lower in the livers of trout fed the plant-based diet which the proportion of starch was lower than in the control diet. Glutamate dehydrogenase was induced by the plant-based diet, suggesting an imbalance of amino acids and a possible link with the lower protein retention observed. Gene expression of delta 6 desaturase was higher in fish fed the plant-based diet, probably linked to a high dietary level of linolenic acid and the absence of long-chain polyunsaturated fatty acids in vegetable oils. Hydroxymethylglutaryl-CoA synthase expression was also induced by plant-based diet because of the low rate of cholesterol in the diet. Changes in regulation mechanisms already identified through short-term nutritional experiments (<12 weeks) suggest that metabolic responses are implemented at short term and remain in the long term.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  7. Contributions of human enzymes in carcinogen metabolism.

    PubMed

    Rendic, Slobodan; Guengerich, F Peter

    2012-07-16

    Considerable support exists for the roles of metabolism in modulating the carcinogenic properties of chemicals. In particular, many of these compounds are pro-carcinogens that require activation to electrophilic forms to exert genotoxic effects. We systematically analyzed the existing literature on the metabolism of carcinogens by human enzymes, which has been developed largely in the past 25 years. The metabolism and especially bioactivation of carcinogens are dominated by cytochrome P450 enzymes (66% of bioactivations). Within this group, six P450s--1A1, 1A2, 1B1, 2A6, 2E1, and 3A4--accounted for 77% of the P450 activation reactions. The roles of these P450s can be compared with those estimated for drug metabolism and should be considered in issues involving enzyme induction, chemoprevention, molecular epidemiology, interindividual variations, and risk assessment.

  8. Contributions of Human Enzymes in Carcinogen Metabolism

    PubMed Central

    Rendic, Slobodan; Guengerich, F. Peter

    2012-01-01

    Considerable support exists for roles of metabolism in modulating the carcinogenic properties of chemicals. In particular, many of these compounds are procarcinogens that require activation to electrophilic forms to exert genotoxic effects. We systematically analyzed the existing literature on metabolism of carcinogens by human enzymes, which has been developed largely in the past 25 years. The metabolism and especially bioactivation of carcinogens are dominated by cytochrome P450 enzymes (66% of bioactivations). Within this group, six P450s—1A1, 1A2, 1B1, 2A6, 2E1, and 3A4—accounted for 77% of the P450 activation reactions. The roles of these P450s can be compared with those estimated for drug metabolism and should be considered in issues involving enzyme induction, chemoprevention, molecular epidemiology, inter-individual variations, and risk assessment. PMID:22531028

  9. Imaging enzymes at work: metabolic mapping by enzyme histochemistry.

    PubMed

    Van Noorden, Cornelis J F

    2010-06-01

    For the understanding of functions of proteins in biological and pathological processes, reporter molecules such as fluorescent proteins have become indispensable tools for visualizing the location of these proteins in intact animals, tissues, and cells. For enzymes, imaging their activity also provides information on their function or functions, which does not necessarily correlate with their location. Metabolic mapping enables imaging of activity of enzymes. The enzyme under study forms a reaction product that is fluorescent or colored by conversion of either a fluorogenic or chromogenic substrate or a fluorescent substrate with different spectral characteristics. Most chromogenic staining methods were developed in the latter half of the twentieth century but still find new applications in modern cell biology and pathology. Fluorescence methods have rapidly evolved during the last decade. This review critically evaluates the methods that are available at present for metabolic mapping in living animals, unfixed cryostat sections of tissues, and living cells, and refers to protocols of the methods of choice.

  10. Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection.

    PubMed

    Marden, James H

    2013-12-01

    Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes.

  11. Biotransformation of cobicistat: metabolic pathways and enzymes

    PubMed Central

    Wang, Pengcheng; Shehu, Amina I.; Liu, Ke; Lu, Jie; Ma, Xiaochao

    2017-01-01

    Background Cobicistat (COBI) is a pharmacoenhancer for antiretroviral therapy. Objective The current study was designed to profile the metabolic pathways of COBI and to determine the enzymes that contribute to COBI metabolism. Method We screened COBI metabolites in mice and human liver microsomes. We also used cDNA-expressed human cytochromes P450 (CYPs) to explore the role of human enzymes in COBI metabolism. Results Twenty new and three known metabolites of COBI were identified in mouse urine and feces. These new metabolic pathways of COBI include glycine conjugation, N-acetyl cysteine conjugation, morpholine ring-opening, and thiazole ring-opening. Twelve of COBI metabolites were further confirmed in mouse and human liver microsomes, including nine new metabolites. Consistent with the previous report, CYP3A4 and CYP2D6 were determined as the major enzymes that contribute to COBI metabolism. Conclusion This study provided a full map of COBI metabolism. These results can be used to manage CYP-mediated drug-drug interactions and adverse drug reactions that are associated with COBI-containing regimens in human. PMID:26935921

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

    PubMed

    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.

  13. P450 enzymes of estrogen metabolism.

    PubMed

    Martucci, C P; Fishman, J

    1993-01-01

    Endogenous and exogenous estrogens undergo extensive oxidative metabolism by specific cytochrome P450 enzymes. Certain drugs and xenobiotics have been found to be potent inducers of estrogen hydroxylating enzymes with C-2 hydroxylase induction being greater than that of C-16 hydroxylase. Oxygenated estrogen metabolites have different biological activities, with C-2 metabolites having limited or no activity and C-4 and C-16 metabolites having similar potency to estradiol. Pathophysiological roles for some of the oxygenated estrogen metabolites have been proposed, e.g. 16 alpha-hydroxyestrone and 4-hydroxyestrone. These reactive estrogens are capable of damaging cellular proteins and DNA and may be carcinogenic in specific cells.

  14. Metabolic profiling reveals key metabolic features of renal cell carcinoma.

    PubMed

    Catchpole, Gareth; Platzer, Alexander; Weikert, Cornelia; Kempkensteffen, Carsten; Johannsen, Manfred; Krause, Hans; Jung, Klaus; Miller, Kurt; Willmitzer, Lothar; Selbig, Joachim; Weikert, Steffen

    2011-01-01

    Recent evidence suggests that metabolic changes play a pivotal role in the biology of cancer and in particular renal cell carcinoma (RCC). Here, a global metabolite profiling approach was applied to characterize the metabolite pool of RCC and normal renal tissue. Advanced decision tree models were applied to characterize the metabolic signature of RCC and to explore features of metastasized tumours. The findings were validated in a second independent dataset. Vitamin E derivates and metabolites of glucose, fatty acid, and inositol phosphate metabolism determined the metabolic profile of RCC. α-tocopherol, hippuric acid, myoinositol, fructose-1-phosphate and glucose-1-phosphate contributed most to the tumour/normal discrimination and all showed pronounced concentration changes in RCC. The identified metabolic profile was characterized by a low recognition error of only 5% for tumour versus normal samples. Data on metastasized tumours suggested a key role for metabolic pathways involving arachidonic acid, free fatty acids, proline, uracil and the tricarboxylic acid cycle. These results illustrate the potential of mass spectroscopy based metabolomics in conjunction with sophisticated data analysis methods to uncover the metabolic phenotype of cancer. Differentially regulated metabolites, such as vitamin E compounds, hippuric acid and myoinositol, provide leads for the characterization of novel pathways in RCC.

  15. Expression of Enzymes that Metabolize Medications

    NASA Technical Reports Server (NTRS)

    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.

  16. Genes Encoding Enzymes Involved in Ethanol Metabolism

    PubMed Central

    Hurley, Thomas D.; Edenberg, Howard J.

    2012-01-01

    The effects of beverage alcohol (ethanol) on the body are determined largely by the rate at which it and its main breakdown product, acetaldehyde, are metabolized after consumption. The main metabolic pathway for ethanol involves the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Seven different ADHs and three different ALDHs that metabolize ethanol have been identified. The genes encoding these enzymes exist in different variants (i.e., alleles), many of which differ by a single DNA building block (i.e., single nucleotide polymorphisms [SNPs]). Some of these SNPs result in enzymes with altered kinetic properties. For example, certain ADH1B and ADH1C variants that are commonly found in East Asian populations lead to more rapid ethanol breakdown and acetaldehyde accumulation in the body. Because acetaldehyde has harmful effects on the body, people carrying these alleles are less likely to drink and have a lower risk of alcohol dependence. Likewise, an ALDH2 variant with reduced activity results in acetaldehyde buildup and also has a protective effect against alcoholism. In addition to affecting drinking behaviors and risk for alcoholism, ADH and ALDH alleles impact the risk for esophageal cancer. PMID:23134050

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

    PubMed

    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.

  18. Molecular mechanism of induction of key enzymes related to lipogenesis.

    PubMed

    Noguchi, T; Iritani, N; Tanaka, T

    1992-06-01

    Key enzymes related to lipogenesis in the liver are induced by a high glucose diet or insulin and suppressed by starvation, diabetes, or glucagon. Most of these enzymes are also induced by dietary fructose, even in diabetic liver. This regulation occurs at the posttranscriptional level as well as at the transcriptional level. We studied extensively the molecular mechanism of induction of L-type pyruvate kinase (LPK). The transcription of the LPK gene in the liver was stimulated by insulin and inhibited by glucagon. This insulin action required ongoing protein synthesis and metabolism of glucose and was enhanced by glucocorticoid. On the other hand, the mechanism of induction of the LPK by dietary fructose depended on plasma insulin levels. Dietary fructose stimulated transcription of the LPK gene in normal rats, whereas it acted mainly at the posttranscriptional level in diabetic rats. These fructose effects were attributable to a common metabolite of fructose and glycerol. The induction of LPK mRNA by dietary glucose was impaired in the liver of Wistar fatty rats, a model of obese non-insulin-dependent diabetes mellitus, but fructose-induced accumulation of the mRNA was not. Studies on transgenic mice indicated that the 5'-flanking region up to -3 kb of the LPK gene contained all cis-acting elements necessary for tissue-specific expression of LPK and its stimulation by diets and insulin. Further analysis using a transient expression assay revealed the presence of three cis-acting elements necessary for expression of LPK in hepatocytes in the region up to -170 kb. However, these elements alone were not sufficient for dietary and hormonal regulation of this enzyme when analyzed in transgenic mice.

  19. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  20. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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...

  1. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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...

  2. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

  3. Truffles contain endocannabinoid metabolic enzymes and anandamide.

    PubMed

    Pacioni, Giovanni; Rapino, Cinzia; Zarivi, Osvaldo; Falconi, Anastasia; Leonardi, Marco; Battista, Natalia; Colafarina, Sabrina; Sergi, Manuel; Bonfigli, Antonella; Miranda, Michele; Barsacchi, Daniela; Maccarrone, Mauro

    2015-02-01

    Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.

  4. Enzyme Recruitment and Its Role in Metabolic Expansion

    PubMed Central

    2015-01-01

    Although more than 109 years have passed since the existence of the last universal common ancestor, proteins have yet to reach the limits of divergence. As a result, metabolic complexity is ever expanding. Identifying and understanding the mechanisms that drive and limit the divergence of protein sequence space impact not only evolutionary biologists investigating molecular evolution but also synthetic biologists seeking to design useful catalysts and engineer novel metabolic pathways. Investigations over the past 50 years indicate that the recruitment of enzymes for new functions is a key event in the acquisition of new metabolic capacity. In this review, we outline the genetic mechanisms that enable recruitment and summarize the present state of knowledge regarding the functional characteristics of extant catalysts that facilitate recruitment. We also highlight recent examples of enzyme recruitment, both from the historical record provided by phylogenetics and from enzyme evolution experiments. We conclude with a look to the future, which promises fruitful consequences from the convergence of molecular evolutionary theory, laboratory-directed evolution, and synthetic biology. PMID:24483367

  5. Enzyme recruitment and its role in metabolic expansion.

    PubMed

    Schulenburg, Cindy; Miller, Brian G

    2014-02-11

    Although more than 10(9) years have passed since the existence of the last universal common ancestor, proteins have yet to reach the limits of divergence. As a result, metabolic complexity is ever expanding. Identifying and understanding the mechanisms that drive and limit the divergence of protein sequence space impact not only evolutionary biologists investigating molecular evolution but also synthetic biologists seeking to design useful catalysts and engineer novel metabolic pathways. Investigations over the past 50 years indicate that the recruitment of enzymes for new functions is a key event in the acquisition of new metabolic capacity. In this review, we outline the genetic mechanisms that enable recruitment and summarize the present state of knowledge regarding the functional characteristics of extant catalysts that facilitate recruitment. We also highlight recent examples of enzyme recruitment, both from the historical record provided by phylogenetics and from enzyme evolution experiments. We conclude with a look to the future, which promises fruitful consequences from the convergence of molecular evolutionary theory, laboratory-directed evolution, and synthetic biology.

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

    PubMed Central

    Yang, Ming; Su, Huizhong; Soga, Tomoyoshi; Kranc, Kamil R; Pollard, Patrick J

    2014-01-01

    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. PMID:27774472

  7. Substrate Specificity and Diastereoselectivity of Strictosidine Glucosidase, a Key Enzyme in Monoterpene Indole Alkaloid Biosynthesis

    PubMed Central

    Yerkes, Nancy; Wu, Jia; McCoy, Elizabeth; Galan, M. Carmen; Chen, Shi; O’Connor, Sarah E.

    2008-01-01

    Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted. PMID:18061449

  8. Substrate specificity and diastereoselectivity of strictosidine glucosidase, a key enzyme in monoterpene indole alkaloid biosynthesis.

    PubMed

    Yerkes, Nancy; Wu, Jia Xin; McCoy, Elizabeth; Galan, M Carmen; Chen, Shi; O'Connor, Sarah E

    2008-05-15

    Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady-state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted.

  9. Hepatic Xenobiotic Metabolizing Enzyme Gene Expression ...

    EPA Pesticide Factsheets

    BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been carried out through life stages in any species. RESULTS: Using full-genome arrays, the mRNA expression of all XMETs and their regulatory proteins was examined during fetal (gestation day (GD) 19), neonatal (postnatal day (PND) 7), prepubescent (PND32), middle age (12 months), and old age (18 and 24 months) in the C57BL/6J (C57) mouse liver and compared to adults. Fetal and neonatal life stages exhibited dramatic differences in XMET mRNA expression compared to the relatively minor effects of old age. The total number of XMET probe sets that differed from adults was 636, 500, 84, 5, 43, and 102 for GD19, PND7, PND32, 12 months, 18 months and 24 months, respectively. At all life stages except PND32, under-expressed genes outnumbered over-expressed genes. The altered XMETs included those in all of the major metabolic and transport phases including introduction of reactive or polar groups (Phase I), conjugation (Phase II) and excretion (Phase III). In the fetus and neonate, parallel increases in expression were noted in the dioxin receptor, Nrf2 components and their regulated genes while nuclear receptors and regulated genes were generally down-regulated. Suppression of male-specific XMETs w

  10. Expression of Enzymes that Metabolize Medications

    NASA Technical Reports Server (NTRS)

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

    2011-01-01

    INTRODUCTION: Increased exposure to radiation is one physiological stressor associated with spaceflight and it is feasible to conduct ground experiments using known radiation exposures. The health of the liver, especially the activity rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. While radiation is known to alter normal physiological function, how radiation affects liver metabolism of administered medications is unclear. Crew health could be affected if the actions of medications used in spaceflight deviated from expectations formed during terrestrial medication use. This study is an effort to identify liver metabolic enzymes whose expression is altered by spaceflight or by radiation exposures that mimic features of the spaceflight environment. METHODS: Using procedures approved by the Animal Care and Use Committee, mice were exposed to either 137Cs (controls, 50 mGy, 6Gy, or 50 mGy + 6Gy separated by 24 hours) or 13 days of spaceflight on STS 135. Animals were anesthetized and sacrificed at several time points (4 hours, 24 hours or 7 days) after their last radiation exposure, or within 6 hours of return to Earth for the STS 135 animals. Livers were removed immediately and flash-frozen in liquid nitrogen. Tissue was homogenized, RNA extracted, purified and quality-tested. Complementary DNA was prepared from high-quality RNA samples, and used in RT-qPCR experiments to determine relative expression of a wide variety of genes involved in general metabolism and drug metabolism. RESULTS: Results of the ground radiation exposure experiments indicated 65 genes of the 190 tested were significantly affected by at least one of the radiation doses. Many of the affected genes are involved in the metabolism of drugs with hydrophobic or steroid-like structures, maintenance of redox homeostasis and repair of DNA damage. Most affected genes returned to near control expression levels by 7 days post

  11. Chemical and functional characterization of seed, pulp and skin powder from chilto (Solanum betaceum), an Argentine native fruit. Phenolic fractions affect key enzymes involved in metabolic syndrome and oxidative stress.

    PubMed

    Orqueda, María Eugenia; Rivas, Marisa; Zampini, Iris Catiana; Alberto, María Rosa; Torres, Sebastian; Cuello, Soledad; Sayago, Jorge; Thomas-Valdes, Samanta; Jiménez-Aspee, Felipe; Schmeda-Hirschmann, Guillermo; Isla, María Inés

    2017-02-01

    The aim of this work was to assess the nutritional and functional components of powder obtained by lyophilization of whole fruits, seeds, pulp and skin from chilto (Solanum betaceum Cav) cultivated in the ecoregion of Yungas, Argentina. The powders have low carbohydrate and sodium content and are a source of vitamin C, carotenoid, phenolics, potassium and fiber. The HPLC-ESI-MS/MS analysis of the fractions enriched in phenolics allowed the identification of 12 caffeic acid derivatives and related phenolics, 10 rosmarinic acid derivatives and 7 flavonoids. The polyphenols enriched extracts before and after simulated gastroduodenal digestion inhibited enzymes associated with metabolic syndrome, including α-glucosidase, amylase and lipase and exhibited antioxidant activity by different mechanisms. None of the analyzed fruit powders showed acute toxicity or genotoxicity. The powders from the three parts of S. betaceum fruit may be a potential functional food and the polyphenol enriched extract of seed and skin may have nutraceutical properties.

  12. Enzymes of fructose metabolism in human liver

    PubMed Central

    Heinz, Fritz; Lamprecht, Walther; Kirsch, Joachim

    1968-01-01

    The enzyme activities involved in fructose metabolism were measured in samples of human liver. On the basis of U/g of wet-weight the following results were found: ketohexokinase, 1.23; aldolase (substrate, fructose-1-phosphate), 2.08; aldolase (substrate, fructose-1,6-diphosphate), 3.46; triokinase, 2.07; aldehyde dehydrogenase (substrate, D-glyceraldehyde), 1.04; D-glycerate kinase, 0.13; alcohol dehydrogenase (nicotinamide adenine dinucleotide [NAD]) substrate, D-glyceraldehyde), 3.1; alcohol dehydrogenase (nicotinamide adenine dinucleotide phosphate [NADP]) (substrate, D-glyceraldehyde), 3.6; and glycerol kinase, 0.62. Sorbitol dehydrogenases (25.0 U/g), hexosediphosphatase (4.06 U/g), hexokinase (0.23 U/g), and glucokinase (0.08 U/g) were also measured. Comparing these results with those of the rat liver it becomes clear that the activities of alcohol dehydrogenases (NAD and NADP) in rat liver are higher than those in human liver, and that the values of ketohexokinase, sorbitol dehydrogenases, and hexosediphosphatase in human liver are lower than those values found in rat liver. Human liver contains only traces of glycerate kinase. The rate of fructose uptake from the blood, as described by other investigators, can be based on the activity of ketohexokinase reported in the present paper. In human liver, ketohexokinase is present in a four-fold activity of glucokinase and hexokinase. This result may explain the well-known fact that fructose is metabolized faster than glucose. PMID:4385849

  13. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section 862.3360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... drug metabolizing enzyme. This device is used as an aid in determining treatment choice...

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

    PubMed

    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. Ketone Body Metabolic Enzyme OXCT1 Regulates Prostate Cancer Chemoresistance

    DTIC Science & Technology

    2015-12-01

    and cellular energy homeostasis . Analysis of patient data indicated that higher OXCT1 levels are associated with docetaxel chemotherapy resistance...knock down induced metabolic inefficiency upon docetaxel treatment Since OXCT1 is a metabolic enzyme involved in energy homeostasis , next, to...ketone body metabolism and cellular energy homeostasis . Analysis of our previous data from patient needle biopsy samples indicated that higher

  16. Interplay of drug metabolizing enzymes with cellular transporters.

    PubMed

    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.

  17. Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana.

    PubMed

    Strand, A; Zrenner, R; Trevanion, S; Stitt, M; Gustafsson, P; Gardeström, P

    2000-09-01

    Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi-limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose-1,5-bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose-P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end-product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP-glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards

  18. Enzyme clustering accelerates processing of intermediates through metabolic channeling

    PubMed Central

    Castellana, Michele; Wilson, Maxwell Z.; Xu, Yifan; Joshi, Preeti; Cristea, Ileana M.; Rabinowitz, Joshua D.; Gitai, Zemer; Wingreen, Ned S.

    2015-01-01

    We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation. PMID:25262299

  19. Key Applications of Plant Metabolic Engineering

    PubMed Central

    Lau, Warren; Fischbach, Michael A.; Osbourn, Anne; Sattely, Elizabeth S.

    2014-01-01

    Great strides have been made in plant metabolic engineering over the last two decades, with notable success stories including Golden rice. Here, we discuss the field's progress in addressing four long-standing challenges: creating plants that satisfy their own nitrogen requirement, so reducing or eliminating the need for nitrogen fertilizer; enhancing the nutrient content of crop plants; engineering biofuel feed stocks that harbor easy-to-access fermentable saccharides by incorporating self-destructing lignin; and increasing photosynthetic efficiency. We also look to the future at emerging areas of research in this field. PMID:24915445

  20. Key enzymes catalyzing glycerol to 1,3-propanediol.

    PubMed

    Jiang, Wei; Wang, Shizhen; Wang, Yuanpeng; Fang, Baishan

    2016-01-01

    Biodiesel can replace petroleum diesel as it is produced from animal fats and vegetable oils, and it produces about 10 % (w/w) glycerol, which is a promising new industrial microbial carbon, as a major by-product. One of the most potential applications of glycerol is its biotransformation to high value chemicals such as 1,3-propanediol (1,3-PD), dihydroxyacetone (DHA), succinic acid, etc., through microbial fermentation. Glycerol dehydratase, 1,3-propanediol dehydrogenase (1,3-propanediol-oxydoreductase), and glycerol dehydrogenase, which were encoded, respectively, by dhaB, dhaT, and dhaD and with DHA kinase are encompassed by the dha regulon, are the three key enzymes in glycerol bioconversion into 1,3-PD and DHA, and these are discussed in this review article. The summary of the main research direction of these three key enzyme and methods of glycerol bioconversion into 1,3-PD and DHA indicates their potential application in future enzymatic research and industrial production, especially in biodiesel industry.

  1. Undercover: gene control by metabolites and metabolic enzymes

    PubMed Central

    van der Knaap, Jan A.; Verrijzer, C. Peter

    2016-01-01

    To 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 expression response remain poorly understood. There is a growing awareness that central components of intermediary metabolism are cofactors or cosubstrates of chromatin-modifying enzymes. As such, their concentrations constitute a potential regulatory interface between the metabolic and chromatin states. In addition, there is increasing evidence for a direct involvement of classic metabolic enzymes in gene expression control. These dual-function proteins may provide a direct link between metabolic programing and the control of gene expression. Here, we discuss our current understanding of the molecular mechanisms connecting metabolism to gene expression and their implications for development and disease. PMID:27881599

  2. Bedaquiline metabolism: enzymes and novel metabolites.

    PubMed

    Liu, Ke; Li, Feng; Lu, Jie; Liu, Shinlan; Dorko, Kenneth; Xie, Wen; Ma, Xiaochao

    2014-05-01

    Bedaquiline is a recently approved drug for the treatment of multidrug-resistant tuberculosis. Adverse cardiac and hepatic drug reactions to bedaquiline have been noted in clinical practice. The current study investigated bedaquiline metabolism in human hepatocytes using a metabolomic approach. Bedaquiline N-demethylation via CYP3A4 was confirmed as the major pathway in bedaquiline metabolism. In addition to CYP3A4, we found that both CYP2C8 and CYP2C19 contributed to bedaquiline N-demethylation. The Km values of CYP2C8, CYP2C19, and CYP3A4 in bedaquiline N-demethylation were 13.1, 21.3, and 8.5 µM, respectively. We also identified a novel metabolic pathway of bedaquiline that produced an aldehyde intermediate. In summary, this study extended our knowledge of bedaquiline metabolism, which can be applied to predict and prevent drug-drug interactions and adverse drug reactions associated with bedaquiline.

  3. Spermidine/spermine-N(1)-acetyltransferase: a key metabolic regulator.

    PubMed

    Pegg, Anthony E

    2008-06-01

    Spermidine/spermine-N(1)-acetyltransferase (SSAT) regulates cellular polyamine content. Its acetylated products are either excreted from the cell or oxidized by acetylpolyamine oxidase. Since polyamines play critical roles in normal and neoplastic growth and in ion channel regulation, SSAT is a key enzyme in these processes. SSAT is very highly regulated. Its content is adjusted in response to alterations in polyamine content to maintain polyamine homeostasis. Certain polyamine analogs can mimic the induction of SSAT and cause a loss of normal polyamines. This may have utility in cancer chemotherapy. SSAT activity is also induced via a variety of other stimuli, including toxins, hormones, cytokines, nonsteroidal anti-inflammatory agents, natural products, and stress pathways, and by ischemia-reperfusion injury. These increases are initiated by alterations in Sat1 gene transcription reinforced by alterations at the other regulatory steps, including protein turnover, mRNA processing, and translation. Transgenic manipulation of SSAT activity has revealed that SSAT activity links polyamine metabolism to lipid and carbohydrate metabolism by means of alterations in the content of acetyl-CoA and ATP. A high level of SSAT stimulates flux through the polyamine biosynthetic pathway, since biosynthetic enzymes are induced in response to the fall in polyamines. This sets up a futile cycle in which ATP is used to generate S-adenosylmethionine for polyamine biosynthesis and acetyl-CoA is consumed in the acetylation reaction. A variety of other effects of increased SSAT activity include death of pancreatic cells, blockage of regenerative tissue growth, behavioral changes, keratosis follicularis spinulosa decalvans, and hair loss. These are very likely due to changes in polyamine and putrescine levels, although increased oxidative stress via the oxidation of acetylated polyamines may also contribute. Recently, it was found that the SSAT protein and/or a related protein, thialysine

  4. Targeting protozoan parasite metabolism: glycolytic enzymes in the therapeutic crosshairs.

    PubMed

    Harris, M T; Mitchell, W G; Morris, J C

    2014-01-01

    Glycolysis is an important metabolic pathway for most organisms, including protozoan parasites. Many of these primitive eukaryotes have streamlined their metabolism, favoring glycolysis for generating ATP in the glucose-rich environments in which they reside. Therefore, the enzymes involved in hexose metabolism could prove to be attractive targets for therapeutic development. This hypothesis is supported by a number of chemical and genetic validation studies. Additionally, the peculiar biochemistry of many of the components, along with limited protein sequence identity emphasizes the likelihood of developing compounds that selectively inhibit the parasite enzymes. In this review, we examine the status of target validation at the genetic and/or chemical levels from the protozoan parasites. While the proteins from some species have been interrogated to the point that well-defined lead compounds have been identified with activities against both enzyme and parasite growth, progress in other systems has to date been limited.

  5. Review of aerobic glycolysis and its key enzymes – new targets for lung cancer therapy

    PubMed Central

    Li, Xue-bing; Gu, Jun-dong; Zhou, Qing-hua

    2015-01-01

    Most tumor cells show different metabolic pathways than normal cells. Even under the conditions of sufficient oxygen, they produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, which is known as aerobic glycolysis or the Warburg effect. Lung cancer is a malignant tumor with one of the highest incidence and mortality rates in the world at present. However, the exact mechanisms underlying lung cancer development remain unclear. The three key enzymes of glycolysis are hexokinase, phosphofructokinase, and pyruvate kinase. Lactate dehydrogenase catalyzes the transfer of pyruvate to lactate. All four enzymes have been reported to be overexpressed in tumors, including lung cancer, and can be regulated by many oncoproteins to promote tumor proliferation, migration, and metastasis with dependence or independence of glycolysis. The discovery of aerobic glycolysis in the 1920s has provided new means and potential therapeutic targets for lung cancer. PMID:26273330

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

    PubMed

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

    PubMed

    Zhang, Qingwei; Song, Xiaomin; Bartels, Dorothea

    2016-12-15

    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.

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

    PubMed Central

    Zhang, Qingwei; Song, Xiaomin; Bartels, Dorothea

    2016-01-01

    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. PMID:28248249

  9. Enzyme Regulation& Catalysis in Carbon Fixation Metabolism

    SciTech Connect

    Miziorko, Henry M

    2004-12-14

    The overall long term goal of this program is the elucidation of molecular events in carbon assimilation. It has become axiomatic that control of flux through metabolic pathways is effectively imposed at irreversible reactions situated early in those pathways. The current focal point of this project is phosphoribulokinase (PRK), which catalyzes formation of the carbon dioxide acceptor, ribulose 1,5-bisphosphate. This reaction represents an early irreversible step unique to Calvin's reductive pentose phosphate pathway. Predictably, the PRK reaction represents an important control point in carbon fixation, regulated by a light dependent thiol/disulfide exchange in eukaryotes and by allosteric effectors in prokaryotes. Characterization of naturally occurring mutants as well as gene knockout experiments substantiate the importance of PRK to in vivo control of carbon assimilation in both prokaryotes and eukaryotes. Thus, given the potential impact of enhancement or inhibition of PRK activity on energy (biomass/biofuel) production, elucidation of the molecular events that account for PRK activity is a significant scientific goal.

  10. Experiment K-7-21: Effect of Microgravity on 1: Metabolic Enzymes of Type 1 and Type 2 Muscle Fibers, and on 2: Metabolic Enzymes, Neurotransmitter Amino Acids, and Neurotransmitter Associated Enzymes in Selected Regions of the Central Nervous System. Part 2; The Distribution of Selected Enzymes and Amino Acids in the Hippocampal Formation

    NASA Technical Reports Server (NTRS)

    Lowry, O. H.; Krasnov, I.; Ilyina-Kakueva, E. I.; Nemeth, P. M.; McDougal, D. B., Jr.; Choksi, R.; Carter, J. G.; Chi, M. M. Y.; Manchester, J. K.; Pusateri, M. E.

    1994-01-01

    Six key metabolic enzymes plus glutaminase and glutamate decarboxylase, as well as glutamate, aspartate and GABA, were measured in 11 regions of the hippocampal formation of synchronous, flight and tail suspension rats. Major differences were observed in the normal distribution patterns of each enzyme and amino acid, but no substantive effects of either microgravity or tail suspension on these patterns were clearly demonstrated.

  11. How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion.

    PubMed

    Brown, Neil Andrew; Ries, Laure Nicolas Annick; Goldman, Gustavo Henrique

    2014-11-01

    The utilisation of lignocellulosic plant biomass as an abundant, renewable feedstock for green chemistries and biofuel production is inhibited by its recalcitrant nature. In the environment, lignocellulolytic fungi are naturally capable of breaking down plant biomass into utilisable saccharides. Nonetheless, within the industrial context, inefficiencies in the production of lignocellulolytic enzymes impede the implementation of green technologies. One of the primary causes of such inefficiencies is the tight transcriptional control of lignocellulolytic enzymes via carbon catabolite repression. Fungi coordinate metabolism, protein biosynthesis and secretion with cellular energetic status through the detection of intra- and extra-cellular nutritional signals. An enhanced understanding of the signals and signalling pathways involved in regulating the transcription, translation and secretion of lignocellulolytic enzymes is therefore of great biotechnological interest. This comparative review describes how nutrient sensing pathways regulate carbon catabolite repression, metabolism and the utilisation of alternative carbon sources in Saccharomyces cerevisiae and ascomycete fungi.

  12. Engineering of Metabolic Pathways by Artificial Enzyme Channels

    PubMed Central

    Pröschel, Marlene; Detsch, Rainer; Boccaccini, Aldo R.; Sonnewald, Uwe

    2015-01-01

    Application of industrial enzymes for production of valuable chemical compounds has greatly benefited from recent developments in Systems and Synthetic Biology. Both, in vivo and in vitro systems have been established, allowing conversion of simple into complex compounds. Metabolic engineering in living cells needs to be balanced which is achieved by controlling gene expression levels, translation, scaffolding, compartmentation, and flux control. In vitro applications are often hampered by limited protein stability/half-life and insufficient rates of substrate conversion. To improve stability and catalytic activity, proteins are post-translationally modified and arranged in artificial metabolic channels. Within the review article, we will first discuss the supramolecular organization of enzymes in living systems and second summarize current and future approaches to design artificial metabolic channels by additive manufacturing for the efficient production of desired products. PMID:26557643

  13. Key Building Blocks via Enzyme-Mediated Synthesis

    NASA Astrophysics Data System (ADS)

    Fischer, Thomas; Pietruszka, Jörg

    Biocatalytic approaches to valuable building blocks in organic synthesis have emerged as an important tool in the last few years. While first applications were mainly based on hydrolases, other enzyme classes such as oxidoreductases or lyases moved into the focus of research. Nowadays, a vast number of biotransformations can be found in the chemical and pharmaceutical industries delivering fine chemicals or drugs. The mild reaction conditions, high stereo-, regio-, and chemoselectivities, and the often shortened reaction pathways lead to economical and ecological advantages of enzymatic conversions. Due to the enormous number of enzyme-mediated syntheses, the present chapter is not meant to be a complete review, but to deliver comprehensive insights into well established enzymatic systems and recent advances in the application of enzymes in natural product synthesis. Furthermore, it is focused on the most frequently used enzymes or enzyme classes not covered elsewhere in the present volume.

  14. Optimal control of metabolic networks with saturable enzyme kinetics.

    PubMed

    Oyarzuun, D A

    2011-03-01

    This note addresses the optimal control of non-linear metabolic networks by means of time-dependent enzyme synthesis rates. The authors consider networks with general topologies described by a control-affine dynamical system coupled with a linear model for enzyme synthesis and degradation. The problem formulation accounts for transitions between two metabolic equilibria, which typically arise in metabolic adaptations to environmental changes, and the minimisation of a quadratic functional that weights the cost/benefit relation between the transcriptional effort required for enzyme synthesis and the transition to the new phenotype. Using a linear time-variant approximation of the non-linear dynamics, the problem is recast as a sequence of linear-quadratic problems, the solution of which involves a sequence of differential Lyapunov equations. The authors provide conditions for convergence to an approximate solution of the original problem, which are naturally satisfied by a wide class of models for saturable enzyme kinetics. As a case study the authors use the method to examine the robustness of an optimal just-in-time gene expression pattern with respect to heterogeneity in the biosynthetic costs of individual proteins.

  15. Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

    PubMed Central

    O’Connell, Jeremy D.; Zhao, Alice; Ellington, Andrew D.; Marcotte, Edward M.

    2013-01-01

    Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable. PMID:23057741

  16. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme

    PubMed Central

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

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

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

    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.

  19. Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation

    PubMed Central

    Petrovska, Ivana; Nüske, Elisabeth; Munder, Matthias C; Kulasegaran, Gayathrie; Malinovska, Liliana; Kroschwald, Sonja; Richter, Doris; Fahmy, Karim; Gibson, Kimberley; Verbavatz, Jean-Marc; Alberti, Simon

    2014-01-01

    One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell. DOI: http://dx.doi.org/10.7554/eLife.02409.001 PMID:24771766

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

    PubMed Central

    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

  1. Activity of anandamide (AEA) metabolic enzymes in rat placental bed.

    PubMed

    Fonseca, B M; Battista, N; Correia-da-Silva, G; Rapino, C; Maccarrone, M; Teixeira, N A

    2014-11-01

    Endocannabinoids are endogenous lipid mediators, with anandamide (AEA) being the first member identified. It is now widely accepted that AEA influences early pregnancy events and its levels, which primarily depend on its synthesis by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and degradation by a fatty acid amide hydrolase (FAAH), must be tightly regulated. Previous studies demonstrated that AEA levels require in situ regulation of these respective metabolic enzymes, and thus, any disturbance in AEA levels may impact maternal remodeling processes occurring during placental development. In this study, the activities of the AEA-metabolic enzymes that result in the establishment of proper local AEA levels during rat gestation were examined. Here, we demonstrate that during placentation NAPE-PLD and FAAH activities change in a temporal manner. Our findings suggest that NAPE-PLD and FAAH create the appropriate AEA levels required for tissue remodeling in the placental bed, a process essential to pregnancy maintenance.

  2. Radiation Exposure Alters Expression of Metabolic Enzyme Genes In Mice

    NASA Technical Reports Server (NTRS)

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

    2010-01-01

    Most 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. Because of the importance of the liver in drug metabolism it is important to understand the effects of spaceflight on the enzymes of the liver. Exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments. This study is an effort to examine the effects of adaptive mechanisms that may be triggered by early exposure to low radiation doses. Using procedures approved by the JSC Animal Care & Use Committee, C57 male mice were exposed to Cs-137 in groups: controls, low dose (50 mGy), high dose (6Gy) and a fourth group that received both radiation doses separated by 24 hours. Animals were anesthetized and sacrificed 4 hours after their last radiation exposure. Livers were removed immediately and flash-frozen in liquid nitrogen. Tissue was homogenized, RNA extracted and purified (Absolutely RNA, Agilent). Quality of RNA samples was evaluated (Agilent Bioanalyzer 2100). Complementary DNA was prepared from high-quality RNA samples, and used to run RT-qPCR screening arrays for DNA Repair and Drug Metabolism (SuperArray, SABiosciences/Qiagen; BioRad Cfx96 qPCR System). Of 91 drug metabolism genes examined, expression of 7 was altered by at least one treatment condition. Genes that had elevated expression include those that metabolize promethazine and steroids (4-8-fold), many that reduce oxidation products, and one that reduces heavy metal exposure (greater than 200-fold). Of the 91 DNA repair and general metabolism genes examined, expression of 14 was altered by at least one treatment condition. These gene expression changes are likely homeostatic and could lead to development of new radioprotective countermeasures.

  3. Clinically Relevant Genetic Variations in Drug Metabolizing Enzymes

    PubMed Central

    Pinto, Navin; Dolan, M. Eileen

    2011-01-01

    In the field of pharmacogenetics, we currently have a few markers to guide physicians as to the best course of therapy for patients. For the most part, these genetic variants are within a drug metabolizing enzyme that has a large effect on the degree or rate at which a drug is converted to its metabolites. For many drugs, response and toxicity are multi-genic traits and understanding relationships between a patient's genetic variation in drug metabolizing enzymes and the efficacy and/or toxicity of a medication offers the potential to optimize therapies. This review will focus on variants in drug metabolizing enzymes with predictable and relatively large impacts on drug efficacy and/or toxicity; some of these drug/gene variant pairs have impacted drug labels by the United States Food and Drug Administration. The challenges in identifying genetic markers and implementing clinical changes based on known markers will be discussed. In addition, the impact of next generation sequencing in identifying rare variants will be addressed. PMID:21453273

  4. Enzyme kinetics of oxidative metabolism: cytochromes P450.

    PubMed

    Korzekwa, Ken

    2014-01-01

    The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 3 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapter 4 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

  5. Enzymes To Die For: Exploiting Nucleotide Metabolizing Enzymes for Cancer Gene Therapy

    PubMed Central

    Ardiani, Andressa; Johnson, Adam J.; Ruan, Hongmei; Sanchez-Bonilla, Marilyn; Serve, Kinta; Black, Margaret E.

    2012-01-01

    Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects. PMID:22384805

  6. Hepatotoxicity of piperazine designer drugs: up-regulation of key enzymes of cholesterol and lipid biosynthesis.

    PubMed

    Arbo, Marcelo Dutra; Melega, Simone; Stöber, Regina; Schug, Markus; Rempel, Eugen; Rahnenführer, Jörg; Godoy, Patricio; Reif, Raymond; Cadenas, Cristina; de Lourdes Bastos, Maria; Carmo, Helena; Hengstler, Jan G

    2016-12-01

    The piperazine derivatives most frequently consumed for recreational purposes are 1-benzylpiperazine, 1-(3,4-methylenedioxybenzyl) piperazine, 1-(3-trifluoromethylphenyl) piperazine and 1-(4-methoxyphenyl) piperazine. Generally, they are consumed as capsules, tablets or pills but also in powder or liquid forms. Currently, the precise mechanism by which piperazine designer drugs induce hepatotoxicity and whether they act by a common pathway is unclear. To answer this question, we performed a gene array study with rat hepatocytes incubated with the four designer drugs. Non-cytotoxic concentrations were chosen that neither induce a decrease in reduced glutathione or ATP depletion. Analysis of the gene array data showed a large overlap of gene expression alterations induced by the four drugs. This 'piperazine designer drug consensus signature' included 101 up-regulated and 309 down-regulated probe sets (p < 0.05; FDR adjusted). In the up-regulated genes, GO groups of cholesterol biosynthesis represented a dominant overrepresented motif. Key enzymes of cholesterol biosynthesis up-regulated by all four piperazine drugs include sterol C4-methyloxidase, isopentyl-diphosphate-Δ-isomerase, Cyp51A1, squalene epoxidase and farnesyl diphosphate synthase. Additionally, glycoprotein transmembrane nmb, which participates in cell adhesion processes, and fatty acid desaturase 1, an enzyme that regulates unsaturation of fatty acids, were also up-regulated by the four piperazine designer drugs. Regarding the down-regulated probe sets, only one gene was common to all four piperazine derivatives, the betaine-homocysteine-S-methyltransferase 2. Analysis of transcription factor binding sites of the 'piperazine designer drug consensus signature' identified the sterol regulatory element binding protein (SREBP-1) as strongly overrepresented in the up-regulated genes. SREBP transcription factors are known to regulate multiple genes of cholesterol metabolism. In conclusion, the present

  7. CYP450 Enzyme-Mediated Metabolism of TCAS and Its Inhibitory and Induced Effects on Metabolized Enzymes in Vitro.

    PubMed

    Shen, Guolin; Wang, Cheng; Zhou, Lili; Li, Lei; Chen, Huiming; Yu, Wenlian; Li, Haishan

    2015-09-02

    In this study, we investigated the enzymes catalyzing the phase I metabolism of thiacalixarene (TCAS) based on in vitro system including cDNA-expressed P450 enzymes, human liver microsomes plus inhibitors and monoclonal antibodies. In addition, the inhibitory potential of TCAS on major CYP450 drug metabolizing enzymes (CYP1A2, CYP2C9, CYP2B6, CYP2D6 and CYP3A4) was assessed. The results showed that CYP1A2 and CYP2C9 mediated TCAS hydroxylation. IC50 values for TCAS in rat and human liver microsomes were greater than 50 µM, and it demonstrated a weak inhibition of rat and human CYP450 enzymes. Finally, sandwiched hepatocytes were used to evaluate the induction of CYP1A and CYP3A to define the function of TCAS in vivo. The results showed that incubation of TCAS at different concentrations for 72 h failed to induce CYP1A and CYP3A. However, incubation of the cells with 50 and 100 µM TCAS caused a profound decrease in the activities of CYP1A and CYP3A, which was probably due to cytotoxic effects, suggesting that exposure to TCAS might be a health concern.

  8. In vivo enzyme activity in inborn errors of metabolism

    SciTech Connect

    Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D. )

    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.

  9. Regulation of amino acid metabolic enzymes and transporters in plants.

    PubMed

    Pratelli, Réjane; Pilot, Guillaume

    2014-10-01

    Amino acids play several critical roles in plants, from providing the building blocks of proteins to being essential metabolites interacting with many branches of metabolism. They are also important molecules that shuttle organic nitrogen through the plant. Because of this central role in nitrogen metabolism, amino acid biosynthesis, degradation, and transport are tightly regulated to meet demand in response to nitrogen and carbon availability. While much is known about the feedback regulation of the branched biosynthesis pathways by the amino acids themselves, the regulation mechanisms at the transcriptional, post-transcriptional, and protein levels remain to be identified. This review focuses mainly on the current state of our understanding of the regulation of the enzymes and transporters at the transcript level. Current results describing the effect of transcription factors and protein modifications lead to a fragmental picture that hints at multiple, complex levels of regulation that control and coordinate transport and enzyme activities. It also appears that amino acid metabolism, amino acid transport, and stress signal integration can influence each other in a so-far unpredictable fashion.

  10. Alteration of drug metabolizing enzymes in sulphite oxidase deficiency

    PubMed Central

    Tutuncu, Begum; Kuçukatay, Vural; Arslan, Sevki; Sahin, Barbaros; Semiz, Asli; Sen, Alaattin

    2012-01-01

    The aim of this study was to investigate the possible effects of sulphite oxidase (SOX, E.C. 1.8.3.1) deficiency on xenobiotic metabolism. For this purpose, SOX deficiency was produced in rats by the administration of a low molybdenum diet with concurrent addition of 200 ppm tungsten to their drinking water. First, hepatic SOX activity in deficient groups was measured to confirm SOX deficiency. Then, aminopyrine N-demethylase, aniline 4-hydroxylase, aromatase, caffeine N-demethylase, cytochrome b5 reductase, erythromycin N-demethylase, ethoxyresorufin O-deethylase, glutathione S-transferase, N-nitrosodimethylamine N-demethylase and penthoxyresorufin O-deethylase activities were determined to follow changes in the activity of drug metabolizing enzymes in SOX-deficient rats. Our results clearly demonstrated that SOX deficiency significantly elevated A4H, caffeine N-demethylase, erythromycin N-demethylase and N-nitrosodimethylamine N-demethylase activities while decreasing ethoxyresorufin O-deethylase and aromatase activities. These alterations in drug metabolizing enzymes can contribute to the varying susceptibility and response of sulphite-sensitive individuals to different drugs and/or therapeutics used for treatments. PMID:22798713

  11. Key Players in Choline Metabolic Reprograming in Triple-Negative Breast Cancer

    PubMed Central

    Iorio, Egidio; Caramujo, Maria José; Cecchetti, Serena; Spadaro, Francesca; Carpinelli, Giulia; Canese, Rossella; Podo, Franca

    2016-01-01

    Triple-negative breast cancer (TNBC), defined as lack of estrogen and progesterone receptors in the absence of protein overexpression/gene amplification of human epidermal growth factor receptor 2, is still a clinical challenge despite progress in breast cancer care. 1H magnetic resonance spectroscopy allows identification and non-invasive monitoring of TNBC metabolic aberrations and elucidation of some key mechanisms underlying tumor progression. Thus, it has the potential to improve in vivo diagnosis and follow-up and also to identify new targets for treatment. Several studies have shown an altered phosphatidylcholine (PtdCho) metabolism in TNBCs, both in patients and in experimental models. Upregulation of choline kinase-alpha, an enzyme of the Kennedy pathway that phosphorylates free choline (Cho) to phosphocholine (PCho), is a major contributor to the increased PCho content detected in TNBCs. Phospholipase-mediated PtdCho headgroup hydrolysis also contributes to the build-up of a PCho pool in TNBC cells. The oncogene-driven PtdCho cycle appears to be fine tuned in TNBC cells in at least three ways: by modulating the choline import, by regulating the activity or expression of specific metabolic enzymes, and by contributing to the rewiring of the entire metabolic network. Thus, only by thoroughly dissecting these mechanisms, it will be possible to effectively translate this basic knowledge into further development and implementation of Cho-based imaging techniques and novel classes of therapeutics. PMID:27747192

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

    PubMed

    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.

  13. Drug metabolizing enzyme systems in the houbara bustard (Chlamydotis undulata).

    PubMed

    Bailey, T A; John, A; Mensah-Brown, E P; Garner, A; Samour, J; Raza, H

    1998-10-01

    This study compared catalytic and immunochemical properties of drug metabolizing phase I and II enzyme systems in houbara bustard (Chlamydotis undulata) liver and kidney and rat liver. P450 content in bustard liver (0.34 +/- 0.03 nmol mg-1 protein) was 50% lower than that of rat liver (0.70 +/- 0.02 nmol mg-1 protein). With the exception of aniline hydroxylase activity, monooxygenase activities using aminopyrine, ethoxyresorufin and ethoxycoumarin as substrates were all significantly lower than corresponding rat liver enzymes. As found in mammalian systems the P450 activities in the bird liver were higher than in the kidney. Immunohistochemical analysis of microsomes using antibodies to rat hepatic P450 demonstrated that bustard liver and kidney express P4502C11 homologous protein; no appreciable cross-reactivity was observed in bustards using antibodies to P4502E1, 1A1 or 1A2 isoenzymes. Glutathione content and glutathione S-transferase (GST) activity in bustard liver were comparable with those of rat liver. GST activity in the kidney was 65% lower than the liver. Western blotting of liver and kidney cytosol with human GST isoenzyme-specific antibodies revealed that the expression of alpha-class of antibodies exceeds mu in the bustard. In contrast, the pi-class of GST was not detected in the bustard liver. This data demonstrates that hepatic and renal microsomes from the bustard have multiple forms of phase I and phase II enzymes. The multiplicity and tissue specific expression of xenobiotic metabolizing enzymes in bustards may play a significant role in determining the pharmacokinetics of drugs and susceptibility of the birds to various environmental pollutants and toxic insults.

  14. The RNA world and the origin of metabolic enzymes

    PubMed Central

    Ralser, Markus

    2014-01-01

    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. PMID:25109990

  15. Pharmacogenetics of drug-metabolizing enzymes in US Hispanics

    PubMed Central

    Duconge, Jorge; Cadilla, Carmen L.; Ruaño, Gualberto

    2015-01-01

    Although the Hispanic population is continuously growing in the United States, they are underrepresented in pharmacogenetic studies. This review addresses the need for compiling available pharmacogenetic data in US Hispanics, discussing the prevalence of clinically relevant polymorphisms in pharmacogenes encoding for drug-metabolizing enzymes. CYP3A5*3 (0.245–0.867) showed the largest frequency in a US Hispanic population. A higher prevalence of CYP2C9*3, CYP2C19*4, and UGT2B7 IVS1+985 A>Gwas observed in US Hispanic vs. non-Hispanic populations. We found interethnic and intraethnic variability in frequencies of genetic polymorphisms for metabolizing enzymes, which highlights the need to define the ancestries of participants in pharmacogenetic studies. New approaches should be integrated in experimental designs to gain knowledge about the clinical relevance of the unique combination of genetic variants occurring in this admixed population. Ethnic subgroups in the US Hispanic population may harbor variants that might be part of multiple causative loci or in linkage-disequilibrium with functional variants. Pharmacogenetic studies in Hispanics should not be limited to ascertain commonly studied polymorphisms that were originally identified in their parental populations. The success of the Personalized Medicine paradigm will depend on recognizing genetic diversity between and within US Hispanics and the uniqueness of their genetic backgrounds. PMID:25431893

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

    PubMed

    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.

  17. Fermentation, Respiration & Enzyme Specificity: A Simple Device & Key Experiments with Yeast.

    ERIC Educational Resources Information Center

    Reinking, Larry N.; And Others

    1994-01-01

    Using graphs and diagrams, the authors describe a simple fermentation chamber and provide key experiments that can be used in the classroom to give students meaningful insight into metabolic processes. (ZWH)

  18. Enzyme activities of D-glucose metabolism in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Tsai, C S; Shi, J L; Beehler, B W; Beck, B

    1992-12-01

    The activities of key enzymes that are members of D-glucose metabolic pathways in Schizosaccharomyces pombe undergoing respirative, respirofermentative, and fermentative metabolisms are monitored. The steady-state activities of glycolytic enzymes, except phosphofructokinase, decrease with a reduced efficiency in D-glucose utilization by yeast continuous culture. On the other hand, the enzymic activities of pentose monophosphate pathway reach the maximum when the cell mass production of the cultures is optimum. Enzymes of tricarboxylate cycle exhibit the maximum activities at approximately the washout rate. The steady-state activity of pyruvate dehydrogenase complex increases rapidly when D-glucose is efficiently utilized. By comparison, the activity of pyruvate decarboxylase begins to increase only when ethanol production occurs. Depletion of dissolved oxygen suppresses the activity of pyruvate dehydrogenase complex but facilitates that of pyruvate decarboxylase. Acetate greatly enhances the acetyl CoA synthetase activity. Similarly, ethanol stimulates alcohol dehydrogenase and aldehyde dehydrogenase activities. Evidence for the existence of alcohol dehydrogenase isozymes in the fission yeast is presented.

  19. Monitoring the metabolic status of geobacter species in contaminated groundwater by quantifying key metabolic proteins with Geobacter-specific antibodies.

    PubMed

    Yun, Jiae; Ueki, Toshiyuki; Miletto, Marzia; Lovley, Derek R

    2011-07-01

    Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of subsurface microorganisms are required before bioremediation practitioners will adopt molecular diagnosis of the bioremediation community as a routine practice for guiding the development of bioremediation strategies. Quantifying gene transcripts can diagnose important aspects of microbial physiology during bioremediation but is technically challenging and does not account for the impact of translational modifications on protein abundance. An alternative strategy is to directly quantify the abundance of key proteins that might be diagnostic of physiological state. To evaluate this strategy, an antibody-based quantification approach was developed to investigate subsurface Geobacter communities. The abundance of citrate synthase corresponded with rates of metabolism of Geobacter bemidjiensis in chemostat cultures. During in situ bioremediation of uranium-contaminated groundwater the quantity of Geobacter citrate synthase increased with the addition of acetate to the groundwater and decreased when acetate amendments stopped. The abundance of the nitrogen-fixation protein, NifD, increased as ammonium became less available in the groundwater and then declined when ammonium concentrations increased. In a petroleum-contaminated aquifer, the abundance of BamB, an enzyme subunit involved in the anaerobic degradation of mono-aromatic compounds by Geobacter species, increased in zones in which Geobacter were expected to play an important role in aromatic hydrocarbon degradation. These results suggest that antibody-based detection of key metabolic proteins, which should be readily adaptable to standardized kits, may be a feasible method for diagnosing the metabolic state of microbial communities responsible for bioremediation, aiding in the rational design of bioremediation strategies.

  20. Monitoring the Metabolic Status of Geobacter Species in Contaminated Groundwater by Quantifying Key Metabolic Proteins with Geobacter-Specific Antibodies▿

    PubMed Central

    Yun, Jiae; Ueki, Toshiyuki; Miletto, Marzia; Lovley, Derek R.

    2011-01-01

    Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of subsurface microorganisms are required before bioremediation practitioners will adopt molecular diagnosis of the bioremediation community as a routine practice for guiding the development of bioremediation strategies. Quantifying gene transcripts can diagnose important aspects of microbial physiology during bioremediation but is technically challenging and does not account for the impact of translational modifications on protein abundance. An alternative strategy is to directly quantify the abundance of key proteins that might be diagnostic of physiological state. To evaluate this strategy, an antibody-based quantification approach was developed to investigate subsurface Geobacter communities. The abundance of citrate synthase corresponded with rates of metabolism of Geobacter bemidjiensis in chemostat cultures. During in situ bioremediation of uranium-contaminated groundwater the quantity of Geobacter citrate synthase increased with the addition of acetate to the groundwater and decreased when acetate amendments stopped. The abundance of the nitrogen-fixation protein, NifD, increased as ammonium became less available in the groundwater and then declined when ammonium concentrations increased. In a petroleum-contaminated aquifer, the abundance of BamB, an enzyme subunit involved in the anaerobic degradation of mono-aromatic compounds by Geobacter species, increased in zones in which Geobacter were expected to play an important role in aromatic hydrocarbon degradation. These results suggest that antibody-based detection of key metabolic proteins, which should be readily adaptable to standardized kits, may be a feasible method for diagnosing the metabolic state of microbial communities responsible for bioremediation, aiding in the rational design of bioremediation strategies. PMID:21551286

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

    PubMed

    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.

  2. Cortisol-Metabolizing Enzymes in Polycystic Ovary Syndrome

    PubMed Central

    Blumenfeld, Zeev; Kaidar, Gabi; Zuckerman-Levin, Nehama; Dumin, Elena; Knopf, Carlos; Hochberg, Ze’ev

    2016-01-01

    OBJECTIVE The aim of this study was to assess the activity of cortisol-metabolizing enzymes in women with polycystic ovary syndrome (PCOS), using a fully quantitative gas chromatography/mass spectrometry (GCMS) method. DESIGN We investigated the glucocorticoid degradation pathways that include 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, 5α-reductase (5α-R) and 5β-reductase (5β-R), 3α-hydroxysteroid dehydrogenase, and 20α- and 20β-hydroxysteroid dehydrogenase (20α-HSD and 20β-HSD, respectively) in young nonobese women with PCOS, using a fully quantitative GCMS method. SETTING This study was conducted in a tertiary referral hospital in Israel. PATIENTS This study group consisted of 13 young women, aged 20.1 ± 2.8 years (mean ± SD), with the body mass index (BMI) of 22.6 ± 3.7 kg/m2, diagnosed with PCOS according to the Rotterdam criteria. The control group consisted of 14 healthy young women matched for weight, height, and BMI. INTERVENTIONS Urine samples were analyzed using GCMS. We measured urinary steroid metabolites that represent the products and substrates of the study enzymes and calculated the product/substrate ratios to represent enzyme activity. MAIN OUTCOME MEASURES The calculation of enzymatic activity, based on glucocorticoid degradation metabolites, was done by GCMS in PCOS vs. controls. RESULTS All glucocorticoid degradation metabolites were higher in the PCOS group than in controls. Of the adrenal enzymes, the activities of 21-hydroxylase and 17α-hydroxylase were reduced, whereas the activity of 17,20-lyase was enhanced in PCOS. Of the degradation enzymes, the activity of 11β-HSD type 1 was reduced in women with PCOS only when calculated from cortoles and cortolones ratios. The activities of 5α-R/5β-R were increased only when calculating the 11-hydroxy metabolites of androgens. The activity of 20α-HSD was elevated in the patients with PCOS and its relation with the substrate levels was lost. CONCLUSIONS We confirm PCOS

  3. Specific point mutations in key redox enzymes are associated with chemoresistance in epithelial ovarian cancer.

    PubMed

    Fletcher, Nicole M; Belotte, Jimmy; Saed, Mohammed G; Memaj, Ira; Diamond, Michael P; Morris, Robert T; Saed, Ghassan M

    2017-01-01

    Oxidative stress plays an important role in the pathophysiology of ovarian cancer. Resistance to chemotherapy presents a significant challenge for ovarian cancer treatment. Specific single nucleotide polymorphisms (SNPs) in key redox enzymes have been associated with ovarian cancer survival and progression. The objective of this study was to determine whether chemotherapy induces point mutations in key redox enzymes that lead to the acquisition of chemoresistance in epithelial ovarian cancer (EOC). Human EOC cell lines and their chemoresistant counterpart were utilized for this study. Specific SNPs in key redox enzymes were analyzed by TaqMan SNP Genotyping. Activities and levels of key redox enzymes were determined by real-time RT-PCR, ELISA and a greiss assay. Point mutations in key redox enzymes were introduced into sensitive EOC cells via the CRISPR/Cas9 system. Cell viability and IC50 for cisplatin were determined by the MTT Cell Proliferation Assay. Data was analyzed with SPSS using Student's two-tailed t-tests and One-way ANOVA followed by Dunnett's or Tukey's post hoc tests, p<0.05. Here, we demonstrate that chemoresistant EOC cells are characterized by a further enhancement in oxidative stress as compared to sensitive counterparts. Additionally, chemoresistant EOC cells manifested specific point mutations, which are associated with altered enzymatic activity, in key redox enzymes that are not detected in sensitive counterparts. Supplementation of an antioxidant was able to successfully sensitize EOC cells to chemotherapeutics. Causality was established by the induction of these point mutations in sensitive EOC cells, which resulted in a significant increase in the level of chemoresistance. These findings indicate that chemotherapy induces specific point mutations in key redox enzymes that contribute to the acquisition of chemoresistance in EOC cells, highlighting a potential novel mechanism. Identification of targets for chemoresistance with either

  4. Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques.

    PubMed Central

    King, S. P.; Lunn, J. E.; Furbank, R. T.

    1997-01-01

    Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning. PMID:12223695

  5. Mercaptosuccinate Dioxygenase, a Cysteine Dioxygenase Homologue, from Variovorax paradoxus Strain B4 Is the Key Enzyme of Mercaptosuccinate Degradation

    PubMed Central

    Brandt, Ulrike; Schürmann, Marc; Steinbüchel, Alexander

    2014-01-01

    The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mm, and a specific activity (Vmax) of 20.0 μmol min−1 mg−1 corresponding to a kcat of 7.7 s−1. Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase. PMID:25228698

  6. Mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, from Variovorax paradoxus strain B4 is the key enzyme of mercaptosuccinate degradation.

    PubMed

    Brandt, Ulrike; Schürmann, Marc; Steinbüchel, Alexander

    2014-10-31

    The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mM, and a specific activity (Vmax) of 20.0 μmol min(-1) mg(-1) corresponding to a kcat of 7.7 s(-1). Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase.

  7. Engineering specialized metabolic pathways--is there a room for enzyme improvements?

    PubMed

    Bar-Even, Arren; Salah Tawfik, Dan

    2013-04-01

    Recent advances in enzyme engineering enable dramatic improvements in catalytic efficiency and/or selectivity, as well as de novo engineering of enzymes to catalyze reactions where natural enzymes are not available. Can these capabilities be utilized to transform biosynthesis pathways? Metabolic engineering is traditionally based on combining existing enzymes to give new, or modified, pathways, within a new context and/or organism. How efficient, however, are the individual enzyme components? Is there room to improve pathway performance by enzyme engineering? We discuss the differences between enzymes in central versus specialized, or secondary metabolism and highlight unique features of specialized metabolism enzymes participating in the synthesis of natural products. We argue that, for the purpose of metabolic engineering, the catalytic efficiency and selectivity of many enzymes can be improved with the aim of achieving higher rates, yields and product purities. We also note the relative abundance of spontaneous reactions in specialized metabolism, and the potential advantage of engineering enzymes that will catalyze these steps. Specialized metabolism therefore offers new opportunities to integrate enzyme and pathway engineering, thereby achieving higher metabolic efficiencies, enhanced production rates and improved product purities.

  8. Motility, ATP levels and metabolic enzyme activity of sperm from bluegill (Lepomis macrochirus).

    PubMed

    Burness, Gary; Moyes, Christopher D; Montgomerie, Robert

    2005-01-01

    Male bluegill displays one of two life history tactics. Some males (termed "parentals") delay reproduction until ca. 7 years of age, at which time they build nests and actively courts females. Others mature precociously (sneakers) and obtain fertilizations by cuckolding parental males. In the current study, we studied the relations among sperm motility, ATP levels, and metabolic enzyme activity in parental and sneaker bluegill. In both reproductive tactics, sperm swimming speed and ATP levels declined in parallel over the first 60 s of motility. Although sneaker sperm initially had higher ATP levels than parental sperm, by approximately 30 s postactivation, no differences existed between tactics. No differences were noted between tactics in swimming speed, percent motility, or the activities of key metabolic enzymes, although sperm from parentals had a higher ratio of creatine phosphokinase (CPK) to citrate synthase (CS). In both tactics, with increasing CPK and CS activity, sperm ATP levels increased at 20 s postactivation, suggesting that capacities for phosphocreatine hydrolysis and aerobic metabolism may influence interindividual variation in rates of ATP depletion. Nonetheless, there was no relation between sperm ATP levels and either swimming speed or percent of sperm that were motile. This suggests that interindividual variation in ATP levels may not be the primary determinant of variation in sperm swimming performance in bluegill.

  9. Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

    PubMed

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

    Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

  10. A computational analysis of protein interactions in metabolic networks reveals novel enzyme pairs potentially involved in metabolic channeling.

    PubMed

    Huthmacher, Carola; Gille, Christoph; Holzhütter, Hermann-Georg

    2008-06-07

    Protein-protein interactions are operative at almost every level of cell structure and function as, for example, formation of sub-cellular organelles, packaging of chromatin, muscle contraction, signal transduction, and regulation of gene expression. Public databases of reported protein-protein interactions comprise hundreds of thousands interactions, and this number is steadily growing. Elucidating the implications of protein-protein interactions for the regulation of the underlying cellular or extra-cellular reaction network remains a great challenge for computational biochemistry. In this work, we have undertaken a systematic and comprehensive computational analysis of reported enzyme-enzyme interactions in the metabolic networks of the model organisms Escherichia coli and Saccharomyces cerevisiae. We grouped all enzyme pairs according to the topological distance that the catalyzed reactions have in the metabolic network and performed a statistical analysis of reported enzyme-enzyme interactions within these groups. We found a higher frequency of reported enzyme-enzyme interactions within the group of enzymes catalyzing reactions that are adjacent in the network, i.e. sharing at least one metabolite. As some of these interacting enzymes have already been implicated in metabolic channeling our analysis may provide a useful screening for candidates of this phenomenon. To check for a possible regulatory role of interactions between enzymes catalyzing non-neighboring reactions, we determined potentially regulatory enzymes using connectivity in the network and absolute change of Gibbs free energy. Indeed a higher portion of reported interactions pertain to such potentially regulatory enzymes.

  11. Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

    PubMed

    Chen, Hongzhang; Shao, Meixue; Li, Hongqiang

    2014-03-05

    The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation.

  12. Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase–an Essential Enzyme of the Polyamine Metabolism

    PubMed Central

    Krossa, Sebastian; Faust, Annette; Ober, Dietrich; Scheidig, Axel J.

    2016-01-01

    The highly conserved bacterial homospermidine synthase (HSS) is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila and Pseudomonas aeruginosa; The unique usage of NAD(H) as a prosthetic group is a common feature of bacterial HSS, eukaryotic HSS and deoxyhypusine synthase (DHS). The structure of the bacterial enzyme does not possess a lysine residue in the active center and thus does not form an enzyme-substrate Schiff base intermediate as observed for the DHS. In contrast to the DHS the active site is not formed by the interface of two subunits but resides within one subunit of the bacterial HSS. Crystal structures of Blastochloris viridis HSS (BvHSS) reveal two distinct substrate binding sites, one of which is highly specific for putrescine. BvHSS features a side pocket in the direct vicinity of the active site formed by conserved amino acids and a potential substrate discrimination, guiding, and sensing mechanism. The proposed reaction steps for the catalysis of BvHSS emphasize cation-π interaction through a conserved Trp residue as a key stabilizer of high energetic transition states. PMID:26776105

  13. Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase-an Essential Enzyme of the Polyamine Metabolism.

    PubMed

    Krossa, Sebastian; Faust, Annette; Ober, Dietrich; Scheidig, Axel J

    2016-01-18

    The highly conserved bacterial homospermidine synthase (HSS) is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila and Pseudomonas aeruginosa; The unique usage of NAD(H) as a prosthetic group is a common feature of bacterial HSS, eukaryotic HSS and deoxyhypusine synthase (DHS). The structure of the bacterial enzyme does not possess a lysine residue in the active center and thus does not form an enzyme-substrate Schiff base intermediate as observed for the DHS. In contrast to the DHS the active site is not formed by the interface of two subunits but resides within one subunit of the bacterial HSS. Crystal structures of Blastochloris viridis HSS (BvHSS) reveal two distinct substrate binding sites, one of which is highly specific for putrescine. BvHSS features a side pocket in the direct vicinity of the active site formed by conserved amino acids and a potential substrate discrimination, guiding, and sensing mechanism. The proposed reaction steps for the catalysis of BvHSS emphasize cation-π interaction through a conserved Trp residue as a key stabilizer of high energetic transition states.

  14. Acute metabolic effects of ammonia on the enzymes of glutamate metabolism in isolated astroglial cells.

    PubMed

    Subbalakshmi, G Y; Murthy, C R

    1983-01-01

    Enzymes of glutamate metabolism were studied in the astrocytes isolated from rats injected with a large dose of ammonium acetate and compared with those isolated from controls. The activities of glutamate dehydrogenase (GDH) and glutaminase decreased while those of glutamine synthetase (GS) and aspartate aminotransferase (AAT) increased both in convulsive and comatose states. The activity of alanine aminotransferase (A1AT) increased only in convulsive state. The results suggested that glutamate required for the formation of glutamine in astrocytes might have its origin in nerve endings and the depletion of citric acid cycle intermediates might occur in nerve endings at least in acute ammonia toxicity.

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

    PubMed Central

    Sangwan, Punesh; Joshi, U. N.

    2014-01-01

    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. PMID:24744916

  16. Life-history evolution and the microevolution of intermediary metabolism: activities of lipid-metabolizing enzymes in life-history morphs of a wing-dimorphic cricket.

    PubMed

    Zera, Anthony J; Zhao, Zhangwu

    2003-03-01

    Although a considerable amount of information is available on the ecology, genetics, and physiology of life-history traits, much more limited data are available on the biochemical and genetic correlates of life-history variation within species. Specific activities of five enzymes of lipid biosynthesis and two enzymes of amino acid catabolism were compared among lines selected for flight-capable (LW[f]) versus flightless (SW) morphs of the cricket Gryllus firmus. These morphs, which exist in natural populations, differ genetically in ovarian growth (100-400% higher in SW) and aspects of flight capability including the size of wings and flight muscles, and the concentration of triglyceride flight fuel (40% greater in LW[f]). Consistently higher activity of each enzyme in LW(f) versus SW-selected lines, and strong co-segregation between morph and enzyme activity, demonstrated genetically based co-variance between wing morph and enzyme activity. Developmental profiles of enzyme activities strongly paralleled profiles of triglyceride accumulation during adulthood and previous measures of in vivo lipid biosynthesis. These data strongly imply that genetically based elevation in activities of lipogenic enzymes, and enzymes controlling the conversion of amino acids into lipids, is an important cause underlying the elevated accumulation of triglyceride in the LW(f) morph, a key biochemical component of the trade-off between elevated early fecundity and flight capability. Global changes in lipid and amino-acid metabolism appear to have resulted from microevolutionary alteration of regulators of metabolism. Finally, strong genotype x environment (diet) interactions were observed for most enzyme activities. Future progress in understanding the functional causes of life-history evolution requires a more detailed synthesis of the fields of life-history evolution and metabolic biochemistry. Wing polymorphism is a powerful experimental model in such integrative studies.

  17. [Effect of T-activin on enzymes in the metabolism of xenobiotics].

    PubMed

    Arion, V Ia; Khromenkov, Iu I; Tagirova, A K; Karaulov, A V; Breusov, Iu N

    1987-01-01

    Effect of T-activin on some xenobiotic metabolizing enzymes and several immunity parametres in secondary immune deficiency (subacute and chronic benzene intoxication) were investigated. T-activin was shown to regulate the xenobiotic metabolizing enzyme system. The compound decreased the enzymatic activity in subacute benzene intoxication, approaching their values up to the control level. In chronic benzene intoxications, which were accompanied by decrease in xenobiotic metabolizing enzymes activity, except of glutathione-S-transferase, T-activin stimulated these enzymes. At the same time, T-activin increased some immunological parameters in these models. Thymus-tropic peptides appear to be the bioregulators of some in vivo systems.

  18. Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants1[OPEN

    PubMed Central

    Zhang, Peifen; Kim, Taehyong; Banf, Michael; Chavali, Arvind K.; Nilo-Poyanco, Ricardo; Bernard, Thomas

    2017-01-01

    Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can be used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters. PMID:28228535

  19. Effects of microsomal enzyme induction on paracetamol metabolism in man.

    PubMed Central

    Prescott, L F; Critchley, J A; Balali-Mood, M; Pentland, B

    1981-01-01

    1 The metabolism of paracetamol after a single oral dose of 20 mg/kg was compared in fifteen patients with microsomal enzyme induction taking anticonvulsants or rifampicin and twelve healthy volunteers. 2 Induction was confirmed by measurement of the plasma antipyrine half-life (mean 6.4 h in the patients compared with 12.8 h in the volunteers). 3 The glucuronide conjugation of paracetamol was enhanced in the induced patients as shown by lower plasma paracetamol concentrations, a shorter paracetamol half-life, higher paracetamol glucuronide concentrations and an increased ratio of the area under the plasma concentration time curves of the glucuronide to the unchanged drug. There were no significant differences in sulphate conjugation. 4 There was a corresponding change in the pattern of urinary metabolite excretion. The induced patients excreted significantly less unchanged drug and sulphate conjugate and more glucuronide conjugate than the healthy volunteers. 5 The urinary excretion of the mercapturic acid and cysteine conjugated of paracetamol was the same in both groups. 6 Conversion of paracetamol to its potentially hepatotoxic metabolite does not seem to be increased in patients induced with anticonvulsants or rifampicin. There would seem to be no contraindication to the use of these drugs in combination. PMID:7306429

  20. Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

    PubMed Central

    Ding, Dongqin; Liu, Yongfei; Xu, Yiran; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

    2016-01-01

    L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product. PMID:27558633

  1. An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

    PubMed

    Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

    2015-12-07

    As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

  2. Freeze-thaw effects on metabolic enzymes in wood frog organs.

    PubMed

    Cowan, K J; Storey, K B

    2001-08-01

    To determine whether episodes of natural freezing and thawing altered the metabolic makeup of wood frog (Rana sylvatica) organs, the maximal activities of 28 enzymes of intermediary metabolism were assessed in six organs (brain, heart, kidney, liver, skeletal muscle, gut) of control (5 degrees C acclimated), frozen (24 h at -3 degrees C), and thawed (24 h back at 5 degrees C) frogs. The enzymes assessed represented pathways including glycolysis, gluconeo-genesis, amino acid metabolism, fatty acid metabolism, the TCA cycle, and adenylate metabolism. Organ-specific responses seen included (a) the number of enzymes affected by freeze-thaw (1 in gut ranging to 17 in heart), (b) the magnitude and direction of response (most often enzyme activities decreased during freezing and rebounded with thawing but, liver showed freeze-specific increases in several enzymes), and (c) the response to freezing versus thawing (enzyme activities in gut and kidney changed during freezing, whereas most enzymes in skeletal muscle responded to thawing). Overall, the data show that freeze-thaw implements selected changes to the maximal activities of various enzymes of intermediary metabolism and that these may aid organ-specific responses that alter fuel use during freeze-thaw, support cryoprotectant metabolism, and aid organ endurance of freeze-induced ischemia.

  3. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  4. Multivalent inhibitors for carbohydrate-processing enzymes: beyond the "lock-and-key" concept.

    PubMed

    Gouin, Sébastien G

    2014-09-08

    During the last decades, tremendous chemical efforts have been dedicated to design monovalent inhibitors of carbohydrate-processing enzymes, with comparatively few rewards in terms of marketed drugs. Recently, an alternative to the traditional "lock and key" approach has emerged. Multivalency, a widely used strategy for lectin inhibition, has been successfully applied to specific glycosidases and glycosyltransferases.

  5. A QUANTITATIVE MODEL FOR XENOBIOTIC METABOLIZING ENZYME (XME) INDUCTION REGULATED BY THE PREGNANE X RECEPTOR (PXR)

    EPA Science Inventory

    The nuclear receptor, PXR, is an integral part of the regulation of hepatic metabolism. It has been shown to regulate specific CYPs (phase I drug-metabolizing enzymes) as well as certain phase II drug metabolism activities, including UDP-glucuronosyl transferase (UGT), sulfotran...

  6. Regulation of Squalene Synthase, a Key Enzyme of Sterol Biosynthesis, in Tobacco1

    PubMed Central

    Devarenne, Timothy P.; Ghosh, Anirban; Chappell, Joe

    2002-01-01

    Squalene synthase (SS) represents a putative branch point in the isoprenoid biosynthetic pathway capable of diverting carbon flow specifically to the biosynthesis of sterols and, hence, is considered a potential regulatory point for sterol metabolism. For example, when plant cells grown in suspension culture are challenged with fungal elicitors, suppression of sterol biosynthesis has been correlated with a reduction in SS enzyme activity. The current study sought to correlate changes in SS enzyme activity with changes in the level of the corresponding protein and mRNA. Using an SS-specific antibody, the initial suppression of SS enzyme activity in elicitor-challenged cells was not reflected by changes in the absolute level of the corresponding polypeptide, implicating a post-translational control mechanism for this enzyme activity. In comparison, the absolute level of the SS mRNA did decrease approximately 5-fold in the elicitor-treated cells, which is suggestive of decreased transcription of the SS gene. Study of SS in intact plants was also initiated by measuring the level of SS enzyme activity, the level of the corresponding protein, and the expression of SS gene promoter-reporter gene constructs in transgenic plants. SS enzyme activity, polypeptide level, and gene expression were all localized predominately to the shoot apical meristem, with much lower levels observed in leaves and roots. These later results suggest that sterol biosynthesis is localized to the apical meristems and that apical meristems may be a source of sterols for other plant tissues. PMID:12114564

  7. Structure and Function of Human Xylulokinase, an Enzyme with Important Roles in Carbohydrate Metabolism*

    PubMed Central

    Bunker, Richard D.; Bulloch, Esther M. M.; Dickson, James M. J.; Loomes, Kerry M.; Baker, Edward N.

    2013-01-01

    d-Xylulokinase (XK; EC 2.7.1.17) catalyzes the ATP-dependent phosphorylation of d-xylulose (Xu) to produce xylulose 5-phosphate (Xu5P). In mammals, XK is the last enzyme in the glucuronate-xylulose pathway, active in the liver and kidneys, and is linked through its product Xu5P to the pentose-phosphate pathway. XK may play an important role in metabolic disease, given that Xu5P is a key regulator of glucose metabolism and lipogenesis. We have expressed the product of a putative human XK gene and identified it as the authentic human d-xylulokinase (hXK). NMR studies with a variety of sugars showed that hXK acts only on d-xylulose, and a coupled photometric assay established its key kinetic parameters as Km(Xu) = 24 ± 3 μm and kcat = 35 ± 5 s−1. Crystal structures were determined for hXK, on its own and in complexes with Xu, ADP, and a fluorinated inhibitor. These reveal that hXK has a two-domain fold characteristic of the sugar kinase/hsp70/actin superfamily, with glycerol kinase as its closest relative. Xu binds to domain-I and ADP to domain-II, but in this open form of hXK they are 10 Å apart, implying that a large scale conformational change is required for catalysis. Xu binds in its linear keto-form, sandwiched between a Trp side chain and polar side chains that provide exquisite hydrogen bonding recognition. The hXK structure provides a basis for the design of specific inhibitors with which to probe its roles in sugar metabolism and metabolic disease. PMID:23179721

  8. Simultaneous prediction of enzyme orthologs from chemical transformation patterns for de novo metabolic pathway reconstruction

    PubMed Central

    Tabei, Yasuo; Yamanishi, Yoshihiro; Kotera, Masaaki

    2016-01-01

    Motivation: Metabolic pathways are an important class of molecular networks consisting of compounds, enzymes and their interactions. The understanding of global metabolic pathways is extremely important for various applications in ecology and pharmacology. However, large parts of metabolic pathways remain unknown, and most organism-specific pathways contain many missing enzymes. Results: In this study we propose a novel method to predict the enzyme orthologs that catalyze the putative reactions to facilitate the de novo reconstruction of metabolic pathways from metabolome-scale compound sets. The algorithm detects the chemical transformation patterns of substrate–product pairs using chemical graph alignments, and constructs a set of enzyme-specific classifiers to simultaneously predict all the enzyme orthologs that could catalyze the putative reactions of the substrate–product pairs in the joint learning framework. The originality of the method lies in its ability to make predictions for thousands of enzyme orthologs simultaneously, as well as its extraction of enzyme-specific chemical transformation patterns of substrate–product pairs. We demonstrate the usefulness of the proposed method by applying it to some ten thousands of metabolic compounds, and analyze the extracted chemical transformation patterns that provide insights into the characteristics and specificities of enzymes. The proposed method will open the door to both primary (central) and secondary metabolism in genomics research, increasing research productivity to tackle a wide variety of environmental and public health matters. Availability and Implementation: Contact: maskot@bio.titech.ac.jp PMID:27307627

  9. Drug Metabolizing Enzyme and Transporter Gene Variation, Nicotine Metabolism, Prospective Abstinence, and Cigarette Consumption.

    PubMed

    Bergen, Andrew W; Michel, Martha; Nishita, Denise; Krasnow, Ruth; Javitz, Harold S; Conneely, Karen N; Lessov-Schlaggar, Christina N; Hops, Hyman; Zhu, Andy Z X; Baurley, James W; McClure, Jennifer B; Hall, Sharon M; Baker, Timothy B; Conti, David V; Benowitz, Neal L; Lerman, Caryn; Tyndale, Rachel F; Swan, Gary E

    2015-01-01

    The Nicotine Metabolite Ratio (NMR, ratio of trans-3'-hydroxycotinine and cotinine), has previously been associated with CYP2A6 activity, response to smoking cessation treatments, and cigarette consumption. We searched for drug metabolizing enzyme and transporter (DMET) gene variation associated with the NMR and prospective abstinence in 2,946 participants of laboratory studies of nicotine metabolism and of clinical trials of smoking cessation therapies. Stage I was a meta-analysis of the association of 507 common single nucleotide polymorphisms (SNPs) at 173 DMET genes with the NMR in 449 participants of two laboratory studies. Nominally significant associations were identified in ten genes after adjustment for intragenic SNPs; CYP2A6 and two CYP2A6 SNPs attained experiment-wide significance adjusted for correlated SNPs (CYP2A6 PACT=4.1E-7, rs4803381 PACT=4.5E-5, rs1137115, PACT=1.2E-3). Stage II was mega-regression analyses of 10 DMET SNPs with pretreatment NMR and prospective abstinence in up to 2,497 participants from eight trials. rs4803381 and rs1137115 SNPs were associated with pretreatment NMR at genome-wide significance. In post-hoc analyses of CYP2A6 SNPs, we observed nominally significant association with: abstinence in one pharmacotherapy arm; cigarette consumption among all trial participants; and lung cancer in four case:control studies. CYP2A6 minor alleles were associated with reduced NMR, CPD, and lung cancer risk. We confirmed the major role that CYP2A6 plays in nicotine metabolism, and made novel findings with respect to genome-wide significance and associations with CPD, abstinence and lung cancer risk. Additional multivariate analyses with patient variables and genetic modeling will improve prediction of nicotine metabolism, disease risk and smoking cessation treatment prognosis.

  10. Primacy of cardiac chymase over angiotensin converting enzyme as an angiotensin-(1-12) metabolizing enzyme

    PubMed Central

    Ahmad, Sarfaraz; Varagic, Jasmina; VonCannon, Jessica L.; Groban, Leanne; Collawn, James F.; Dell'Italia, Louis J.; Ferrario, Carlos M.

    2016-01-01

    We showed previously that rat angiotensin-(1-12) [Ang-(1-12)] is metabolized by chymase and angiotensin converting enzyme (ACE) to generate Angiotensin II (Ang II). Here, we investigated the affinity of cardiac chymase and ACE enzymes for Ang-(1-12) and Angiotensin I (Ang I) substrates. Native plasma membranes (PMs) isolated from heart and lung tissues of adult spontaneously hypertensive rats (SHR) were incubated with radiolabeled 125I-Ang-(1-12) or 125I-Ang I, in the absence or presence of a chymase or ACE inhibitor (chymostatin and lisinopril, respectively). Products were quantitated by HPLC connected to an in-line flow-through gamma detector. The rate of 125I-Ang II formation from 125I-Ang-(1-12) by chymase was significantly higher (heart: 7.0 ± 0.6 fmol/min/mg; lung: 33 ± 1.2 fmol/min/mg, P < 0.001) when compared to 125I-Ang I substrate (heart: 0.8 ± 0.1 fmol/min/mg; lung: 2.1 ± 0.1 fmol/min/mg). Substrate affinity of 125I-Ang-(1-12) for rat cardiac chymase was also confirmed using excess unlabeled Ang-(1-12) or Ang I (0–250 µM). The rate of 125I-Ang II formation was significantly lower using unlabeled Ang-(1-12) compared to unlabeled Ang I substrate. Kinetic data showed that rat chymase has a lower Km (64 ± 6.3 µM vs 142 ± 17 µM), higher Vmax (13.2 ± 1.3 µM/min/mg vs 1.9 ± 0.2 µM/min/mg) and more than 15-fold higher catalytic efficiency (ratio of Vmax/Km) for Ang-(1-12) compared to Ang I substrate, respectively. We also investigated ACE mediated hydrolysis of 125I-Ang-(1-12) and 125I-Ang I in solubilized membrane fractions of the SHR heart and lung. Interestingly, no significant difference in 125I-Ang II formation by ACE was detected using either substrate, 125I-Ang-(1-12) or 125I-Ang I, both in the heart (1.8 ± 0.2 fmol/min/mg and 1.8 ± 0.3 fmol/min/mg, respectively) and in the lungs (239 ± 25 fmol/min/mg and 248 ± 34 fmol/min/mg, respectively). Compared to chymase, ACE-mediated Ang-(1-12) metabolism in the heart was several fold lower

  11. Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism

    PubMed Central

    2015-01-01

    Background Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. Results ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids

  12. Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes.

    PubMed

    Baraibar, Martin; Hyzewicz, Janek; Rogowska-Wrzesinska, Adelina; Bulteau, Anne-Laure; Prip-Buus, Carina; Butler-Browne, Gillian; Friguet, Bertrand

    2014-10-01

    Accumulation of damaged macromolecules, including irreversibly oxidized proteins, is a hallmark of cellular and organismal ageing. Failure of protein homesotasis is a major contributor to the age-related accumulation of damaged proteins. In skeletal muscle, tissue maintenance and regeneration is assured by resident adult stem cells known as satellite cells. During senescence their replication and differentiation is compromised contributing to sarcopenia. In this study we have addressed the impact of oxidatively modified proteins in the impaired metabolism of senescent human satellite cells. By using a targeted proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during replicative senescence of satellite cells. Inactivation of the proteasome in aged cells appeared as a key contributor to the accumulation of such damaged proteins. Untargeted metabolomic profiling and functional analyses indicated glucose metabolism impairment in senescent cells, although mitochondrial respiration remained unaffected. A metabolic shift leading to increased mobilization of non-carbohydrate substrates as branched chain amino acids or long chain fatty acids was observed in senescent cells. In addition, phospho-and glycerolipids metabolism was altered. Increased levels of acyl-carnitines indicated augmented turnover of storage and membrane lipids for energy production. Such changes reflect alterations in membrane composition and dysregulation of sphingolipids signaling during senescence. This study establishes a new concept connecting oxidative protein modifications with the altered cellular metabolism associated with the senescent phenotype. In addition, these findings highlight the molecular mechanisms implicated in satellite cells dysfunction during ageing, paving the road for future therapeutic interventions

  13. Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing

    PubMed Central

    Kola, Vijaya Sudhakara Rao; Renuka, P.; Madhav, Maganti Sheshu; Mangrauthia, Satendra K.

    2015-01-01

    RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21–24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed. PMID:25954206

  14. Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing.

    PubMed

    Kola, Vijaya Sudhakara Rao; Renuka, P; Madhav, Maganti Sheshu; Mangrauthia, Satendra K

    2015-01-01

    RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21-24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed.

  15. The gut microbiota: a key regulator of metabolic diseases.

    PubMed

    Yang, Jin-Young; Kweon, Mi-Na

    2016-10-01

    The prevalence of obesity and type 2 diabetes, two closely linked metabolic disorders, is increasing worldwide. Over the past decade, the connection between these disorders and the microbiota of the gut has become a major focus of biomedical research, with recent studies demonstrating the fundamental role of intestinal microbiota in the regulation and pathogenesis of metabolic disorders. Because of the complexity of the microbiota community, however, the underlying molecular mechanisms by which the gut microbiota is associated with metabolic disorders remain poorly understood. In this review, we summarize recent studies that investigate the role of the microbiota in both human subjects and animal models of disease and discuss relevant therapeutic targets for future research. [BMB Reports 2016; 49(10): 536-541].

  16. The gut microbiota: a key regulator of metabolic diseases

    PubMed Central

    Yang, Jin-Young; Kweon, Mi-Na

    2016-01-01

    The prevalence of obesity and type 2 diabetes, two closely linked metabolic disorders, is increasing worldwide. Over the past decade, the connection between these disorders and the microbiota of the gut has become a major focus of biomedical research, with recent studies demonstrating the fundamental role of intestinal microbiota in the regulation and pathogenesis of metabolic disorders. Because of the complexity of the microbiota community, however, the underlying molecular mechanisms by which the gut microbiota is associated with metabolic disorders remain poorly understood. In this review, we summarize recent studies that investigate the role of the microbiota in both human subjects and animal models of disease and discuss relevant therapeutic targets for future research. [BMB Reports 2016; 49(10): 536-541] PMID:27530685

  17. Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

    PubMed Central

    Jenney, Francis E.; McTernan, Patrick M.; Adams, Michael W. W.

    2010-01-01

    Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins. PMID:20463892

  18. Anandamide and decidual remodelling: COX-2 oxidative metabolism as a key regulator.

    PubMed

    Almada, M; Piscitelli, F; Fonseca, B M; Di Marzo, V; Correia-da-Silva, G; Teixeira, N

    2015-11-01

    Recently, endocannabinoids have emerged as signalling mediators in reproduction. It is widely accepted that anandamide (AEA) levels must be tightly regulated, and that a disturbance in AEA levels may impact decidual stability and regression. We have previously characterized the endocannabinoid machinery in rat decidual tissue and reported the pro-apoptotic action of AEA on rat decidual cells. Cyclooxygenase-2 (COX-2) is an inducible enzyme that plays a crucial role in early pregnancy, and is also a key modulator in the crosstalk between endocannabinoids and prostaglandins. On the other hand, AEA-oxidative metabolism by COX-2 is not merely a mean to inactivate its action, but it yields the formation of a new class of mediators, named prostaglandin-ethanolamides, or prostamides. In this study we found that AEA-induced apoptosis in decidual cells involves COX-2 metabolic pathway. AEA induced COX-2 expression through p38 MAPK, resulting in the formation of prostamide E2 (PME2). Our findings also suggest that AEA-induced effect is associated with NF-kB activation. Finally, we describe the involvement of PME2 in the induction of the intrinsic apoptotic pathway in rat decidual cells. Altogether, our findings highlight the role of COX-2 as a gatekeeper in the uterine environment and clarify the impact of the deregulation of AEA levels on the decidual remodelling process.

  19. Citric-acid cycle key enzyme activities during in vitro growth and metacyclogenesis of Leishmania infantum promastigotes.

    PubMed

    Louassini, M; Foulquié, M; Benítez, R; Adroher, J

    1999-08-01

    The activities of 5 key regulatory enzymes in most energetic systems, namely citrate synthase (EC 4.1.3.7, CS), NADP-specific isocitrate dehydrogenase (EC 1.1.1.42, ICDH), succinate dehydrogenase (EC 1.3.99.1, SDH), L-malate dehydrogenase (EC 1.1.1.37, MDH), and decarboxylating malic enzyme (EC 1.1.1.40, ME), were measured during the growth and metacyclogenesis of a cutaneous (CL) and a visceral (VL) strain of Leishmania infantum. As occurs with other Leishmania species, infective promastigotes were present along all phases of growth, but their percentages were higher at the early stationary phase for VL and the end of the same phase for CL. High CS and SDH activities were detected in both strains, as compared with other trypanosomatids, bringing more evidence for an actively functional citric-acid cycle in L. infantum. Both strains showed higher levels of CS, ICDH, and MDH and lower SDH and ME activities when more metacyclic promastigotes were present, but in VL these changes paralleled an increase in glucose consumption, whereas in CL these changes coincided with an NH3 hyperproduction. This suggests that the energy metabolism during L. infantum growth and metacyclogenesis is affected by regulated enzymes that probably respond to changes in the culture medium in the levels of glucose and amino acids.

  20. Enzyme and metabolic engineering for the production of novel biopolymers: crossover of biological and chemical processes.

    PubMed

    Matsumoto, Ken'ichiro; Taguchi, Seiichi

    2013-12-01

    The development of synthetic biology has transformed microbes into useful factories for producing valuable polymers and/or their precursors from renewable biomass. Recent progress at the interface of chemistry and biology has enabled the production of a variety of new biopolymers with properties that substantially differ from their petroleum-derived counterparts. This review touches on recent trials and achievements in the field of biopolymer synthesis, including chemo-enzymatically synthesized aliphatic polyesters, wholly biosynthesized lactate-based polyesters, polyhydroxyalkanoates and other unusual bacterially synthesized polyesters. The expanding diversities in structure and the material properties of biopolymers are key for exploring practical applications. The enzyme and metabolic engineering approaches toward this goal are discussed by shedding light on the successful case studies.

  1. Fungal colonization and enzyme-mediated metabolism of waste coal by Neosartorya fischeri strain ECCN 84.

    PubMed

    Sekhohola, Lerato Mary; Isaacs, Michelle Louise; Cowan, Ashton Keith

    2014-01-01

    Colonization and oxidative metabolism of South African low-rank discard coal by the fungal strain ECCN 84 previously isolated from a coal environment and identified as Neosartorya fischeri was investigated. Results show that waste coal supported fungal growth. Colonization of waste coal particles by N. fischeri ECCN 84 was associated with the formation of compact spherical pellets or sclerotia-like structures. Dissection of the pellets from liquid cultures revealed a nucleus of "engulfed" coal which when analyzed by energy dispersive X-ray spectroscopy showed a time-dependent decline in weight percentage of elemental carbon and an increase in elemental oxygen. Proliferation of peroxisomes in hyphae attached to coal particles and increased extracellular laccase activity occurred after addition of waste coal to cultures of N. fischeri ECCN 84. These results support a role for oxidative enzyme action in the biodegradation of coal and suggest that extracellular laccase is a key component in this process.

  2. Temperature Features of Enzymes Affecting Crassulacean acid Metabolism

    PubMed Central

    Brandon, P. C.

    1967-01-01

    Enzymes involved in malic acid production via a pathway with 2 carboxylation reactions and in malic acid conversion via total oxidation have been demonstrated in mitochondria of Bryophyllum tubiflorum Harv. Activation of the mitochondria by Tween 40 was necessary to reveal part of the enzyme activities. The temperature behavior of the enzymes has been investigated, revealing optimal activity of acid-producing enzymes at 35°. Even at 53° the optimum for acid-converting enzymes was not yet reached. From the simultaneous action of acid-producing and acid-converting enzyme systems the overall result at different temperatures was established. Up to 15° the net result was a malic acid production. Moderate temperatures brought about a decrease in this accumulation, which was partly accompanied by a shift to isocitrate production, while at higher temperatures total oxidation of the acids exceeded the production. PMID:16656606

  3. Microbial responses to membrane cleaning using sodium hypochlorite in membrane bioreactors: Cell integrity, key enzymes and intracellular reactive oxygen species.

    PubMed

    Han, Xiaomeng; Wang, Zhiwei; Wang, Xueye; Zheng, Xiang; Ma, Jinxing; Wu, Zhichao

    2016-01-01

    Sodium hypochlorite (NaClO) is a commonly used reagent for membrane cleaning in membrane bioreactors (MBRs), while it, being a kind of disinfectant (oxidant), may impair viability of microbes or even totally inactivate them upon its diffusion into mixed liquor during membrane cleaning. In this study, we systematically examine the effects of NaClO on microorganisms in terms of microbial cell integrity, metabolism behaviours (key enzymes), and intracellular reactive oxygen species (ROS) under various NaClO concentrations. Different proportions of microbial cells in activated sludge were damaged within several minutes dependent on NaClO dosages (5-50 mg/g-SS), and correspondingly organic matters were released to bulk solution. Inhibition of key enzymes involved in organic matter biodegradation, nitrification and denitrification was observed in the presence of NaClO above 1 mg/g-SS, and thus organic matter and nitrogen removal efficiencies were decreased. It was also demonstrated that intracellular ROS production was increased with the NaClO dosage higher than 1 mg/g-SS, which likely induced further damage to microbial cells.

  4. Effects of boron deficiency on major metabolites, key enzymes and gas exchange in leaves and roots of Citrus sinensis seedlings.

    PubMed

    Lu, Yi-Bin; Yang, Lin-Tong; Li, Yan; Xu, Jing; Liao, Tian-Tai; Chen, Yan-Bin; Chen, Li-Song

    2014-06-01

    Boron (B) deficiency is a widespread problem in many crops, including Citrus. The effects of B-deficiency on gas exchange, carbohydrates, organic acids, amino acids, total soluble proteins and phenolics, and the activities of key enzymes involved in organic acid and amino acid metabolism in 'Xuegan' [Citrus sinensis (L.) Osbeck] leaves and roots were investigated. Boron-deficient leaves displayed excessive accumulation of nonstructural carbohydrates and much lower CO2 assimilation, demonstrating feedback inhibition of photosynthesis. Dark respiration, concentrations of most organic acids [i.e., malate, citrate, oxaloacetate (OAA), pyruvate and phosphoenolpyruvate] and activities of enzymes [i.e., phosphoenolpyruvate carboxylase (PEPC), NAD-malate dehydrogenase, NAD-malic enzyme (NAD-ME), NADP-ME, pyruvate kinase (PK), phosphoenolpyruvate phosphatase (PEPP), citrate synthase (CS), aconitase (ACO), NADP-isocitrate dehydrogenase (NADP-IDH) and hexokinase] involved in glycolysis, the tricarboxylic acid (TCA) cycle and the anapleurotic reaction were higher in B-deficient leaves than in controls. Also, total free amino acid (TFAA) concentration and related enzyme [i.e., NADH-dependent glutamate 2-oxoglutarate aminotransferase (NADH-GOGAT) and glutamate OAA transaminase (GOT)] activities were enhanced in B-deficient leaves. By contrast, respiration, concentrations of nonstructural carbohydrates and three organic acids (malate, citrate and pyruvate), and activities of most enzymes [i.e., PEPC, NADP-ME, PK, PEPP, CS, ACO, NAD-isocitrate dehydrogenase, NADP-IDH and hexokinase] involved in glycolysis, the TCA cycle and the anapleurotic reaction, as well as concentration of TFAA and activities of related enzymes (i.e., nitrate reductase, NADH-GOGAT, glutamate pyruvate transaminase and glutamine synthetase) were lower in B-deficient roots than in controls. Interestingly, leaf and root concentration of total phenolics increased, whereas that of total soluble protein decreased

  5. Acute Liver Injury Induces Nucleocytoplasmic Redistribution of Hepatic Methionine Metabolism Enzymes

    PubMed Central

    Delgado, Miguel; Garrido, Francisco; Pérez-Miguelsanz, Juliana; Pacheco, María; Partearroyo, Teresa; Pérez-Sala, Dolores

    2014-01-01

    Abstract Aims: The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. Results: Using the rat model of d-galactosamine intoxication, severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities, and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine treatment induced hepatic nuclear accumulation of methionine adenosyltransferase (MAT) α1 and S-adenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear MAT activity. Acetaminophen (APAP) intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells that overexpress tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and APAP that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased glutathione reduced form/glutathione oxidized form ratios and was prevented by N-acetylcysteine (NAC) and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by NAC in galactosamine-treated cells. Innovation: Cytoplasmic and nuclear changes in proteins that regulate the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. Conclusion: Altogether these results demonstrate galactosamine- and APAP-induced nuclear accumulation of methionine metabolism enzymes as active oligomers and unveil the implication of

  6. Effect of oxytocin on serum biochemistry, liver enzymes, and metabolic hormones in lactating Nili Ravi buffaloes.

    PubMed

    Iqbal, Zafar; ur Rahman, Zia; Muhammad, Faqir; Akhtar, Masood; Awais, Mian Muhammad; Khaliq, Tanweer; Nasir, Amar; Nadeem, Muhammad; Khan, Kinza; Arshad, Hafiz Muhammad; Basit, Muhammad Abdul

    2015-01-01

    Studies reporting the effects of oxytocin on the health of lactating animals are lacking and still no such data is available on Nili Ravi buffalo, the most prominent Asian buffalo breed. The present study was conducted to investigate the effect of oxytocin on physiological and metabolic parameters of lactating Nili Ravi buffaloes. Healthy lactating buffaloes (n = 40) of recent calving were selected from a commercial dairy farm situated in the peri-urban area of district Faisalabad, Pakistan. These buffaloes were randomly allocated to two equal groups viz experimental and control, comprising 20 animals each. Twice-a-day (morning and evening) milking practice was followed. The experimental and control buffaloes were administered subcutaneously with 3 mL of oxytocin (10 IU/mL) and normal saline respectively, prior to each milking. Serum biochemical profile including glucose, total cholesterol (tChol), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), total proteins (TP), C-reactive protein (CRP), liver enzymes aspartate transaminase (AST), alanine transaminase (ALT), and metabolic hormones triiodothyronine (T₃) and thyroxine (T₄) were studied. Results revealed significantly higher (P ≤ 0.01) levels of glucose, total cholesterol, LDL-C, triglycerides, total proteins, and C-reactive protein in experimental (oxytocin-injected) lactating buffaloes compared to control group. Liver enzymes AST and ALT as well as serum T₄ concentration was significantly higher (P ≤ 0.01) in oxytocin-injected lactating buffaloes as compared to control animals. It was concluded that oxytocin had the key role in increasing the metabolic parameters and hormones, resulting in the optimization of production. But, at the same time, it may pose a threat to the animal health.

  7. Potent inhibition of retinoic acid metabolism enzyme(s) by novel azolyl retinoids.

    PubMed

    Njar, V C; Nnane, I P; Brodie, A M

    2000-09-04

    Novel (+/-)-4-azolyl retinoic acid analogues 4, 5, 7 and 8 have been designed and synthesized and have been shown to be powerful inhibitors of hamster microsomal all-trans-retinoic acid 4-hydroxylase enzyme(s). (+/-)-4-(1H-Imidazol-1-yl)retinoic acid (4) is the most potent inhibitor of this enzyme reported to date.

  8. Gallium nitrate is efficacious in murine models of tuberculosis and inhibits key bacterial Fe-dependent enzymes.

    PubMed

    Olakanmi, Oyebode; Kesavalu, Banurekha; Pasula, Rajamouli; Abdalla, Maher Y; Schlesinger, Larry S; Britigan, Bradley E

    2013-12-01

    Acquiring iron (Fe) is critical to the metabolism and growth of Mycobacterium tuberculosis. Disruption of Fe metabolism is a potential approach for novel antituberculous therapy. Gallium (Ga) has many similarities to Fe. Biological systems are often unable to distinguish Ga(3+) from Fe(3+). Unlike Fe(3+), Ga(3+) cannot be physiologically reduced to Ga(2+). Thus, substituting Ga for Fe in the active site of enzymes may render them nonfunctional. We previously showed that Ga inhibits growth of M. tuberculosis in broth and within cultured human macrophages. We now report that Ga(NO3)3 shows efficacy in murine tuberculosis models. BALB/c SCID mice were infected intratracheally with M. tuberculosis, following which they received daily intraperitoneal saline, Ga(NO3)3, or NaNO3. All mice receiving saline or NaNO3 died. All Ga(NO3)3-treated mice survived. M. tuberculosis CFU in the lungs, liver, and spleen of the NaNO3-treated or saline-treated mice were significantly higher than those in Ga-treated mice. When BALB/c mice were substituted for BALB/c SCID mice as a chronic (nonlethal) infection model, Ga(NO3)3 treatment significantly decreased lung CFU. To assess the mechanism(s) whereby Ga inhibits bacterial growth, the effect of Ga on M. tuberculosis ribonucleotide reductase (RR) (a key enzyme in DNA replication) and aconitase activities was assessed. Ga decreased M. tuberculosis RR activity by 50 to 60%, but no additional decrease in RR activity was seen at Ga concentrations that completely inhibited mycobacterial growth. Ga decreased aconitase activity by 90%. Ga(NO3)3 shows efficacy in murine M. tuberculosis infection and leads to a decrease in activity of Fe-dependent enzymes. Additional work is warranted to further define Ga's mechanism of action and to optimize delivery forms for possible therapeutic uses in humans.

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

    PubMed Central

    2013-01-01

    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. PMID:23398642

  10. Expression and Regulation of Drug Transporters and Metabolizing Enzymes in the Human Gastrointestinal Tract.

    PubMed

    Drozdzik, M; Oswald, S

    2016-01-01

    Orally administered drugs must pass through the intestinal wall and then through the liver before reaching systemic circulation. During this process drugs are subjected to different processes that may determine the therapeutic value. The intestinal barrier with active drug metabolizing enzymes and drug transporters in enterocytes plays an important role in the determination of drug bioavailability. Accumulating information demonstrates variable distribution of drug metabolizing enzymes and transporters along the human gastrointestinal tract (GI), that creates specific barrier characteristics in different segments of the GI. In this review, expression of drug metabolizing enzymes and transporters in the healthy and diseased human GI as well as their regulatory aspects: genetic, miRNA, DNA methylation are outlined. The knowledge of unique interplay between drug metabolizing enzymes and transporters in specific segments of the GI tract allows more precise definition of drug release sites within the GI in order to assure more complete bioavailability and prediction of drug interactions.

  11. Comparative genomic and phylogenetic investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes characterized in several bacteria and eukaryotic organisms. We report a comprehensive phylogenetic analysis employing an exhaustive dataset of NAT-homologous sequences recovered through inspection of 2445 genomes. We describe ...

  12. Xenobiotic metabolizing enzyme (XME) expression in aging humans.

    EPA Science Inventory

    In the presence of foreign compounds, metabolic homeostasis of the organism is maintained by the liver’s ability to detoxify and eliminate these xenobiotics. This is accomplished, in part, by the expression of XMEs, which metabolize xenobiotics and determine whether exposure will...

  13. Activity of key enzymes in glucose catabolism during the growth and metacyclogenesis of Leishmania infantum.

    PubMed

    Louassini, M; Foulquié, M R; Benítez, R; Adroher, F J

    1999-04-01

    This paper follows the development in the activity of the key enzymes of glycolysis and dehydrogenases of the pentose phosphate shunt throughout the in vitro growth and metacyclogenesis of two human strains of Leishmania infantum - one visceral (VL) and the other cutaneous (CL) - together with changes in the glucose, ammonium, and proton concentrations in the culture medium. In the first stage, ammonium was generated and no glucose was consumed. Later on, all the glucose was consumed and, finally, ammonium was generated again. The ammonium concentration increased 16- and 21-fold in cultures of VL and CL strains, respectively. The activities of the glycosomal enzymes hexokinase and phosphofructokinase differed in each strain, always being higher in CL than in VL and increasing throughout the culture period in CL while decreasing in VL. This probably indicates a different capability to adapt to the culture medium conditions. The activities of the pentose phosphate shunt enzymes examined indicate that 6-phosphogluconate dehydrogenase is possibly a rate-limiting enzyme for this pathway. Pyruvate kinase is a cytosolic control enzyme of glycolysis in trypanosomatids, and its activity decreased throughout the growth and differentiation of both strains of L. infantum, as occurs in other trypanosomatids. It was also observed that glucose catabolism was more active in the cutaneous strain than in the visceral one.

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

    PubMed

    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.

  15. The presence of amorpha-4, 11-diene synthase, a key enzyme in artemisinin production in ten Artemisia species

    PubMed Central

    Hosseini, R.; Yazdani, N.; Garoosi, GA.

    2011-01-01

    Background and the purpose of the study Artemisinin is one of the most effective medicine against malaria, which is produced naturally by Artemisia annua in low yield. It is produced in a metabolic pathway, in which several genes and gene products are involved. One of the key genes in this pathway is am1, which encodes amorpha-4, 11-diene synthase (ADS), a key enzyme in artemisinin biosynthesis pathway. The aim of this study was to determine the presence of this gene in ten Artemisia species in order to increase the yield of production of Artemisinin. Methods The experiments were carried out using PCR. Specific primers were designed based on the published am1 gene sequence obtained from A. annua (NCBI, accession number AF327527). Results The amplification of this gene by the specific primers was considered as a positive sign for the potentiality of artemisinin production. Since the entire am1 gene was not amplified in any of the 10 species used, four parts of the gene, essential in ADS enzyme function, corresponding to a) pair site of Arg10-Pro12 in the first 100 amino acids, b) aspartate rich motif (DDXXD), c) active site final lid and d) active site including farnesyl diphosphate (FDP) ionization sites and catalytic site in the ADS enzyme, were investigated. Major conclusion The sequence corresponding to ADS active site was amplified only in A. annua, A. aucheri and A. chamaemelifolia. The negative results obtained with other species could be due to some sequence alteration, such as point mutations or INDELs. We propose A. aucheri and A. chamaemelifolia as two potential candidate species for further characterization, breeding and transferring am1 gene for artemisinin overproduction. PMID:22615678

  16. High doses of atorvastatin and simvastatin induce key enzymes involved in VLDL production.

    PubMed

    Roglans, Núria; Verd, Joan C; Peris, Cristina; Alegret, Marta; Vázquez, Manuel; Adzet, Tomás; Díaz, Cristina; Hernández, Gonzalo; Laguna, Juan C; Sánchez, Rosa M

    2002-05-01

    Treatments with high doses of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors may induce the expression of sterol regulatory element binding protein (SREBP)-target genes, causing different effects from those attributed to the reduction of hepatic cholesterol content. The aim of this study was to investigate the effects of high doses of statins on the key enzymes involved in VLDL production in normolipidemic rats. To examine whether the effects caused by statin treatment are a consequence of HMG-CoA reductase inhibition, we tested the effect of atorvastatin on these enzymes in mevalonate-fed rats. Atorvastatin and simvastatin enhanced not only HMG-CoA reductase but also the expression of the SREBP-2 gene itself. As a result of the overexpression of SREBP-2 caused by the statin treatment, genes regulated basically by SREBP-1, as FA synthase and acetyl-coenzyme A carboxylase, were also induced and their mRNA levels increased. DAG acyltransferase and microsomal TG transfer protein mRNA levels as well as phosphatidate phosphohydrolase activity were increased by both statins. Simvastatin raised liver cholesterol content, ACAT mRNA levels, and CTP:phosphocholine cytidylyltransferase activity, whereas it reduced liver DAG and phospholipid content. Mevalonate feeding reversed all changes induced by the atorvastatin treatment. These results show that treatment with high doses of statins induces key enzymes controlling rat liver lipid synthesis and VLDL assembly, probably as a result of SREBP-2 overexpression. Despite the induction of the key enzymes involved in VLDL production, both statins markedly reduced plasma TG levels, suggesting that different mechanisms may be involved in the hypotriglyceridemic effect of statins at high or low doses.

  17. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    PubMed

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

  18. Metabolic engineering is key to a sustainable chemical industry.

    PubMed

    Murphy, Annabel C

    2011-08-01

    The depletion of fossil fuel stocks will prohibit their use as the main feedstock of future industrial processes. Biocatalysis is being increasingly used to reduce fossil fuel reliance and to improve the sustainability, efficiency and cost of chemical production. Even with their current small market share, biocatalyzed processes already generate approximately US$50 billion and it has been estimated that they could be used to produce up to 20% of fine chemicals by 2020. Until the advent of molecular biological technologies, the compounds that were readily accessible from renewable biomass were restricted to naturally-occurring metabolites. However, metabolic engineering has considerably broadened the range of compounds now accessible, providing access to compounds that cannot be otherwise reliably sourced, as well as replacing established chemical processes. This review presents the case for continued efforts to promote the adoption of biocatalyzed processes, highlighting successful examples of industrial chemical production from biomass and/or via biocatalyzed processes. A selection of emerging technologies that may further extend the potential and sustainability of biocatalysis are also presented. As the field matures, metabolic engineering will be increasingly crucial in maintaining our quality of life into a future where our current resources and feedstocks cannot be relied upon.

  19. Molecular thermodynamics of metabolism: quantum thermochemical calculations for key metabolites.

    PubMed

    Hadadi, N; Ataman, M; Hatzimanikatis, V; Panayiotou, C

    2015-04-28

    The present work is the first of a series of papers aiming at a coherent and unified development of the thermodynamics of metabolism and the rationalization of feasibility analysis of metabolic pathways. The focus in this part is on high-level quantum chemical calculations of the thermochemical quantities of relatively heavy metabolites such as amino acids/oligopeptides, nucleosides, saccharides and their derivatives in the ideal gas state. The results of this study will be combined with the corresponding hydration/solvation results in subsequent parts of this work in order to derive the desired thermochemical quantities in aqueous solutions. The above metabolites exist in a vast conformational/isomerization space including rotational conformers, tautomers or anomers exhibiting often multiple or cooperative intramolecular hydrogen bonding. We examine the challenges posed by these features for the reliable estimation of thermochemical quantities. We discuss conformer search, conformer distribution and averaging processes. We further consider neutral metabolites as well as protonated and deprotonated metabolites. In addition to the traditional presentation of gas-phase acidities, basicities and proton affinities, we also examine heats and free energies of ionic species. We obtain simple linear relations between the thermochemical quantities of ions and the formation quantities of their neutral counterparts. Furthermore, we compare our calculations with reliable experimental measurements and predictive calculations from the literature, when available. Finally, we discuss the next steps and perspectives for this work.

  20. Analysis of the key enzymes of butyric and acetic acid fermentation in biogas reactors

    PubMed Central

    Gabris, Christina; Bengelsdorf, Frank R; Dürre, Peter

    2015-01-01

    This study aimed at the investigation of the mechanisms of acidogenesis, which is a key process during anaerobic digestion. To expose possible bottlenecks, specific activities of the key enzymes of acidification, such as acetate kinase (Ack, 0.23–0.99 U mg−1 protein), butyrate kinase (Buk, < 0.03 U mg−1 protein) and butyryl-CoA:acetate-CoA transferase (But, 3.24–7.64 U mg−1 protein), were determined in cell free extracts of biogas reactor content from three different biogas reactors. Furthermore, the detection of Ack was successful via Western blot analysis. Quantification of corresponding functional genes encoding Buk (buk) and But (but) was not feasible, although an amplification was possible. Thus, phylogenetic trees were constructed based on respective gene fragments. Four new clades of possible butyrate-producing bacteria were postulated, as well as bacteria of the genera Roseburia or Clostridium identified. The low Buk activity was in contrast to the high specific But activity in the analysed samples. Butyrate formation via Buk activity does barely occur in the investigated biogas reactor. Specific enzyme activities (Ack, Buk and But) in samples drawn from three different biogas reactors correlated with ammonia and ammonium concentrations (NH3 and NH4+-N), and a negative dependency can be postulated. Thus, high concentrations of NH3 and NH4+-N may lead to a bottleneck in acidogenesis due to decreased specific acidogenic enzyme activities. PMID:26086956

  1. Enzymes Involved in Pyrophosphate and Calcium Metabolism as Targets for Anti-scuticociliate Chemotherapy.

    PubMed

    Mallo, Natalia; Lamas, Jesús; DeFelipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-07-01

    Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca(2+) homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the in vitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+-PPases (H+-PPases). CQ and ART inhibited the in vitro growth of ciliates with IC50 values of respectively 74 ± 9 μM and 80 ± 8 μM. CQ inhibits the H+ translocation (with an IC50 of 13.4 ± 0.2 μM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+-PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca(2+) induced by ATP, indicating an effect on the Ca(2+) -ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca(2+) metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis.

  2. Experiment K-6-21. Effect of microgravity on 1) metabolic enzymes of type 1 and type 2 muscle fibers and on 2) metabolic enzymes, neutransmitter amino acids, and neurotransmitter associated enzymes in motor and somatosensory cerebral cortex. Part 1: Metabolic enzymes of individual muscle fibers; part 2: metabolic enzymes of hippocampus and spinal cord

    NASA Technical Reports Server (NTRS)

    Lowry, O.; Mcdougal, D., Jr.; Nemeth, Patti M.; Maggie, M.-Y. Chi; Pusateri, M.; Carter, J.; Manchester, J.; Norris, Beverly; Krasnov, I.

    1990-01-01

    The individual fibers of any individual muscle vary greatly in enzyme composition, a fact which is obscured when enzyme levels of a whole muscle are measured. The purpose of this study was therefore to assess the changes due to weightless on the enzyme patterns composed by the individual fibers within the flight muscles. In spite of the limitation in numbers of muscles examined, it is apparent that: (1) that the size of individual fibers (i.e., their dry weight) was reduced about a third, (2) that this loss in dry mass was accompanied by changes in the eight enzymes studied, and (3) that these changes were different for the two muscles, and different for the two enzyme groups. In the soleus muscle the absolute amounts of the three enzymes of oxidative metabolism decreased about in proportion to the dry weight loss, so that their concentration in the atrophic fibers was almost unchanged. In contrast, there was little loss among the four enzymes of glycogenolysis - glycolysis so that their concentrations were substantially increased in the atrophic fibers. In the TA muscle, these seven enzymes were affected in just the opposite direction. There appeared to be no absolute loss among the oxidative enzymes, whereas the glycogenolytic enzymes were reduced by nearly half, so that the concentrations of the first metabolic group were increased within the atrophic fibers and the concentrations of the second group were only marginally decreased. The behavior of hexokinase was exceptional in that it did not decrease in absolute terms in either type of muscle and probably increased as much as 50 percent in soleus. Thus, their was a large increase in concentration of this enzyme in the atrophied fibers of both muscles. Another clear-cut finding was the large increase in the range of activities of the glycolytic enzymes among individual fibers of TA muscles. This was due to the emergence of TA fibers with activities for enzymes of this group extending down to levels as low as

  3. Metaproteomic Analysis of a Chemosynthetic Hydrothermal Vent Community Reveals Insights into Key-Metabolic Processes

    NASA Astrophysics Data System (ADS)

    Steen, I.; Stokke, R.; Lanzen, A.; Pedersen, R.; Øvreås, L.; Urich, T.

    2010-12-01

    internal sulfur cycle within the community. The community contained expressed enzymes of a variety of carbon metabolism pathways. Key enzymes of the reverse TCA cycle for fixation of CO2 and the Wood-Ljungdahl pathway for oxidation of acetyl-CoA and / or the fixation of CO2 were found. Key enzymes of aerobic and anaerobic methane-oxidation pathways were identified as well, namely particulate methane monooxygenase and methyl-Coenzyme M reductase. Various house-keeping gene-products, like cold- and heat shock proteins as well as ribosomal proteins and ATP synthases were identified. This approach has a future potential of broadening our understanding of environmental complexity and regulation in response to geochemical constraints. [1] Dupierris, V., Masselon, C., Court, M., Kieffer-Jaquinod, S., and Bruley, C. (2009) A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa. Bioinformatics 25, 1980-1981.

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

    PubMed

    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.

  5. Role of cytochrome P-450 and related enzymes in the pulmonary metabolism of xenobiotics.

    PubMed Central

    Philpot, R M; Smith, B R

    1984-01-01

    The lung metabolizes a wide variety of xenobiotics and, in the process, forms products that may be more or less toxic than the parent compound. The consequence of metabolism, activation or detoxication, is a function of the nature of the substrate and of the characteristics and concentrations of the enzymes involved. As a result, the biotransformation of xenobiotics can lead to their excretion or to the formation of reactive products that produce deleterious effects by binding covalently to tissue macromolecules. Among the enzymes that metabolize xenobiotics, those associated with the cytochrome P-450-dependent monooxygenase system are probably the most important. The route by which a given substrate is metabolized in a tissue or cell is, to a great extent, determined by the types and concentrations of cytochrome P-450 isozymes present. We are just beginning to understand the distribution of these enzymes in lung and to appreciate the species and cellular differences that exist. PMID:6376107

  6. Computational Prediction of Metabolism: Sites, Products, SAR, P450 Enzyme Dynamics, and Mechanisms

    PubMed Central

    2012-01-01

    Metabolism of xenobiotics remains a central challenge for the discovery and development of drugs, cosmetics, nutritional supplements, and agrochemicals. Metabolic transformations are frequently related to the incidence of toxic effects that may result from the emergence of reactive species, the systemic accumulation of metabolites, or by induction of metabolic pathways. Experimental investigation of the metabolism of small organic molecules is particularly resource demanding; hence, computational methods are of considerable interest to complement experimental approaches. This review provides a broad overview of structure- and ligand-based computational methods for the prediction of xenobiotic metabolism. Current computational approaches to address xenobiotic metabolism are discussed from three major perspectives: (i) prediction of sites of metabolism (SOMs), (ii) elucidation of potential metabolites and their chemical structures, and (iii) prediction of direct and indirect effects of xenobiotics on metabolizing enzymes, where the focus is on the cytochrome P450 (CYP) superfamily of enzymes, the cardinal xenobiotics metabolizing enzymes. For each of these domains, a variety of approaches and their applications are systematically reviewed, including expert systems, data mining approaches, quantitative structure–activity relationships (QSARs), and machine learning-based methods, pharmacophore-based algorithms, shape-focused techniques, molecular interaction fields (MIFs), reactivity-focused techniques, protein–ligand docking, molecular dynamics (MD) simulations, and combinations of methods. Predictive metabolism is a developing area, and there is still enormous potential for improvement. However, it is clear that the combination of rapidly increasing amounts of available ligand- and structure-related experimental data (in particular, quantitative data) with novel and diverse simulation and modeling approaches is accelerating the development of effective tools for

  7. Deep sequencing of Lotus corniculatus L. reveals key enzymes and potential transcription factors related to the flavonoid biosynthesis pathway.

    PubMed

    Wang, Ying; Hua, Wenping; Wang, Jian; Hannoufa, Abdelali; Xu, Ziqin; Wang, Zhezhi

    2013-04-01

    Lotus corniculatus L. is used worldwide as a forage crop due to its abundance of secondary metabolites and its ability to grow in severe environments. Although the entire genome of L. corniculatus var. japonicus R. is being sequenced, the differences in morphology and production of secondary metabolites between these two related species have led us to investigate this variability at the genetic level, in particular the differences in flavonoid biosynthesis. Our goal is to use the resulting information to develop more valuable forage crops and medicinal materials. Here, we conducted Illumina/Solexa sequencing to profile the transcriptome of L. corniculatus. We produced 26,492,952 short reads that corresponded to 2.38 gigabytes of total nucleotides. These reads were then assembled into 45,698 unigenes, of which a large number associated with secondary metabolism were annotated. In addition, we identified 2,998 unigenes based on homology with L. japonicus transcription factors (TFs) and grouped them into 55 families. Meanwhile, a comparison of four tag-based digital gene expression libraries, built from the flowers, pods, leaves, and roots, revealed distinct patterns of spatial expression of candidate unigenes in flavonoid biosynthesis. Based on these results, we identified many key enzymes from L. corniculatus which were different from reference genes of L. japonicus, and five TFs that are potential enhancers in flavonoid biosynthesis. Our results provide initial genetics resources that will be valuable in efforts to manipulate the flavonoid metabolic pathway in plants.

  8. Polymorphisms of xenobiotic-metabolizing enzymes and susceptibility to cancer.

    PubMed Central

    Hirvonen, A

    1999-01-01

    The variation in individual responses to exogenous agents is exceptionally wide. It is because of this large diversity of responsiveness that risk factors to environmentally induced diseases have been difficult to pinpoint, particularly at low exposure levels. Opportunities now exist for studies of host factors in cancer or other diseases in which an environmental component can be presumed. Many of the studies have shown an elevated disease proneness for individuals carrying the potential at-risk alleles of metabolic genes, but a number of controversial results have also been reported. This article is an overview of the data published to date on metabolic genotypes related to individual susceptibility to cancer. PMID:10229705

  9. Key Roles of Glutamine Pathways in Reprogramming the Cancer Metabolism

    PubMed Central

    Michalak, Krzysztof Piotr; Maćkowska-Kędziora, Agnieszka; Sobolewski, Bogusław; Woźniak, Piotr

    2015-01-01

    Glutamine (GLN) is commonly known as an important metabolite used for the growth of cancer cells but the effects of its intake in cancer patients are still not clear. However, GLN is the main substrate for DNA and fatty acid synthesis. On the other hand, it reduces the oxidative stress by glutathione synthesis stimulation, stops the process of cancer cachexia, and nourishes the immunological system and the intestine epithelium, as well. The current paper deals with possible positive effects of GLN supplementation and conditions that should be fulfilled to obtain these effects. The analysis of GLN metabolism suggests that the separation of GLN and carbohydrates in the diet can minimize simultaneous supply of ATP (from glucose) and NADPH2 (from glutamine) to cancer cells. It should support to a larger extent the organism to fight against the cancer rather than the cancer cells. GLN cannot be considered the effective source of ATP for cancers with the impaired oxidative phosphorylation and pyruvate dehydrogenase inhibition. GLN intake restores decreased levels of glutathione in the case of chemotherapy and radiotherapy; thus, it facilitates regeneration processes of the intestine epithelium and immunological system. PMID:26583064

  10. Interaction of 3,4-methylenedioxymethamphetamine and methamphetamine during metabolism by in vitro human metabolic enzymes and in rats.

    PubMed

    Kuwayama, Kenji; Tsujikawa, Kenji; Miyaguchi, Hajime; Kanamori, Tatsuyuki; Iwata, Yuko T; Inoue, Hiroyuki

    2012-07-01

    Illicit amphetamine-type stimulant (ATS) tablets commonly contain one or more active ingredients, which have hallucinogenic and/or stimulant effects. Because components such as 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (MA) in ATS tablets have similar chemical structures, they could be metabolized by common metabolic enzymes. To investigate potential metabolic interactions of ATS tablet components, we studied the in vitro metabolism of MDMA and MA using human metabolic enzymes. MDMA and MA were mainly metabolized by cytochrome P450 2D6 (CYP2D6) and mutually inhibited the production of their main metabolites. In vivo experiments were also performed using intravenous administration of MDMA, MA, or their mixture to rats. The plasma concentrations of MDMA and MA after co-administration were higher than those after administration of MDMA or MA alone. The results in this study imply that multiple components in ATS tablets can interact to mutually inhibit their metabolism and potentially enhance the toxicity of each component.

  11. Neonicotinoid metabolism: compounds, substituents, pathways, enzymes, organisms, and relevance.

    PubMed

    Casida, John E

    2011-04-13

    Neonicotinoids are one of the three principal insecticide chemotypes. The seven major commercial neonicotinoids are readily biodegraded by metabolic attack at their N-heterocyclylmethyl moiety, heterocyclic or acyclic spacer, and N-nitroimine, nitromethylene, or N-cyanoimine tip. Phase I metabolism is largely dependent on microsomal CYP450 isozymes with situ selectivity in hydroxylation, desaturation, dealkylation, sulfoxidation, and nitro reduction. Cytosolic aldehyde oxidase is a nitroreductase for some neonicotinoids. Phase II metabolism involves methylation, acetylation, and formation of glucuronide, glucoside, amino acid, and sulfate- and glutathione-derived conjugates. Some neonicotinoids act as proinsecticides with metabolism to more potent nicotinic agonists. Pest resistance is more commonly due to synergist-reversible CYP450 detoxification than to nAChR mutants or variants. Metabolites in some cases contribute to mammalian hepatotoxicity and carcinogenesis and in others to enhanced plant vigor and stress shields. These relationships explain much of neonicotinoid comparative toxicology and provide the basis for continued and improved safety and effectiveness of this chemotype.

  12. Metabolic cold adaptation in fishes occurs at the level of whole animal, mitochondria and enzyme.

    PubMed

    White, Craig R; Alton, Lesley A; Frappell, Peter B

    2012-05-07

    Metabolic cold adaptation (MCA), the hypothesis that species from cold climates have relatively higher metabolic rates than those from warm climates, was first proposed nearly 100 years ago and remains one of the most controversial hypotheses in physiological ecology. In the present study, we test the MCA hypothesis in fishes at the level of whole animal, mitochondria and enzyme. In support of the MCA hypothesis, we find that when normalized to a common temperature, species with ranges that extend to high latitude (cooler climates) have high aerobic enzyme (citrate synthase) activity, high rates of mitochondrial respiration and high standard metabolic rates. Metabolic compensation for the global temperature gradient is not complete however, so when measured at their habitat temperature species from high latitude have lower absolute rates of metabolism than species from low latitudes. Evolutionary adaptation and thermal plasticity are therefore insufficient to completely overcome the acute thermodynamic effects of temperature, at least in fishes.

  13. Hydrogenosome Metabolism Is the Key Target for Antiparasitic Activity of Resveratrol against Trichomonas vaginalis

    PubMed Central

    Mallo, Natalia; Lamas, Jesús

    2013-01-01

    Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 μM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 μM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 μM). At doses of 50 to 100 μM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs. PMID:23478970

  14. Hydrogenosome metabolism is the key target for antiparasitic activity of resveratrol against Trichomonas vaginalis.

    PubMed

    Mallo, Natalia; Lamas, Jesús; Leiro, José M

    2013-06-01

    Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 μM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 μM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 μM). At doses of 50 to 100 μM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs.

  15. Expression of xenobiotic and steroid hormone metabolizing enzymes in human breast carcinomas.

    PubMed

    Haas, Susanne; Pierl, Christiane; Harth, Volker; Pesch, Beate; Rabstein, Sylvia; Brüning, Thomas; Ko, Yon; Hamann, Ute; Justenhoven, Christina; Brauch, Hiltrud; Fischer, Hans-Peter

    2006-10-15

    The potential to metabolize endogenous and exogenous substances may influence breast cancer development and tumor growth. Therefore, the authors investigated the protein expression of Glutathione S-transferase (GST) isoforms and cytochrome P450 (CYP) known to be involved in the metabolism of steroid hormones and endogenous as well as exogenous carcinogens in breast cancer tissue to obtain new information on their possible role in tumor progression. Expression of GST pi, mu, alpha and CYP1A1/2, 1A2, 3A4/5, 1B1, 2E1 was assessed by immunohistochemistry for primary breast carcinomas of 393 patients from the German GENICA breast cancer collection. The percentages of positive tumors were 50.1 and 44.5% for GST mu and CYP2E1, and ranged from 13 to 24.7% for CYP1A2, GST pi, CYP1A1/2, CYP3A4/5, CYP1B1. GST alpha was expressed in 1.8% of tumors. The authors observed the following associations between strong protein expression and histopathological characteristics: GST expression was associated with a better tumor differentiation (GST mu, p = 0.018) and with reduced lymph node metastasis (GST pi, p = 0.02). In addition, GST mu expression was associated with a positive estrogen receptor and progesterone receptor status (p < 0.001). CYP3A4/5 expression was associated with a positive nodal status (p = 0.018). Expression of CYP1B1 was associated with poor tumor differentiation (p = 0.049). Our results demonstrate that the majority of breast carcinomas expressed xenobiotic and drug metabolizing enzymes. They particularly suggest that GST mu and pi expression may indicate a better prognosis and that strong CYP3A4/5 and CYP1B1 expression may be key features of nonfavourable prognosis.

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

    PubMed

    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.

  17. Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum: presence of a mitochondrial NADPH dehydrogenase.

    PubMed

    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.

  18. Ketone Body Metabolic Enzyme OXCT1 Regulates Prostate Cancer Chemoresistance

    DTIC Science & Technology

    2014-10-01

    was upregulated in a subset of patients and the upregulation was associated with chemotherapy resistance. In vitro analysis showed that OXCT1 was...hypothesis that OXCT1 plays important role prostate cancer chemotherapy sensitivity. 15. SUBJECT TERMS chemosensitivity, OXCT1, docetaxel...prostate cancer resistance to docetaxel-based chemotherapy has never been tested. OXCT1 encodes the rate limiting enzyme converting ketone bodies to

  19. [Effect of domestication of the silver fox on the main enzymes of serotonin metabolism and serotonin receptors].

    PubMed

    Popova, N K; Kulikov, A V; Avgustinovich, D F; Voĭtenko, N N; Trut, L N

    1997-03-01

    In silver foxes significant alterations in the activities of basic enzymes of neurotransmitter serotonin metabolism as well as in the densities of receptors caused by selection for the absence of the aggressive defensive reaction to man were demonstrated. In the midbrain and hypothalamus of animals selected for the absence of aggressive behavior, the activity of tryptophan hydroxylase, the key enzyme of serotonin biosynthesis, was found to be remarkably higher than in animals selected for highly aggressive behavior. Domesticated animals were characterized by low activity of the main enzyme of serotonin catabolism, monoamine oxidase type A, increased Michaelis constant km, and an unchanged maximum reaction rate (Vmax). No changes in the specific binding of [3H]-ketanserin and [3H]-8-OH-DPAT in the frontal cortex of domesticated foxes were revealed; however, in the hypothalamus, the low values of Bmax for the [3H]-8-OH-DPAT specific binding were observed, indicating the decreased density of the 5-HT1A receptors. It is assumed that the transformation of a wild aggressive animal into a domesticated one taking place during directional selection is caused by hereditary alterations favored by artificial selection in the activity of the main enzymes of serotonin metabolism and serotonin receptors.

  20. Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides

    NASA Astrophysics Data System (ADS)

    Nguyen, G. T. T.; Erlenkamp, G.; Jäck, O.; Küberl, A.; Bott, M.; Fiorani, F.; Gohlke, H.; Groth, G.

    2016-06-01

    Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2‧,3‧,4‧,3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2‧,3‧,4‧-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15–45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme.

  1. Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides

    PubMed Central

    Nguyen, G. T. T.; Erlenkamp, G.; Jäck, O.; Küberl, A.; Bott, M.; Fiorani, F.; Gohlke, H.; Groth, G.

    2016-01-01

    Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2′,3′,4′,3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2′,3′,4′-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15–45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme. PMID:27263468

  2. Sensitivity of bacterioplankton nitrogen metabolism to eutrophication in sub-tropical coastal waters of Key West, Florida.

    PubMed

    Hoch, Matthew P; Dillon, Kevin S; Coffin, Richard B; Cifuentes, Luis A

    2008-05-01

    Expression of intracellular ammonium assimilation enzymes were used to assess the response of nitrogen (N) metabolism in bacterioplankton to N-loading of sub-tropical coastal waters of Key West, Florida. Specific activities of glutamine synthetase (GS) and total glutamate dehydrogenase (GDHT) were measured on the bacterial size fraction (<0.8 microm) to assess N-deplete versus N-replete metabolic states, respectively. Enzyme results were compared to concentrations of dissolved organic matter and nutrients and to the biomass and production of phytoplankton and bacteria. Concentrations of dissolved inorganic N (DIN), dissolved organic N (DON), and dissolved organic carbon (DOC) positively correlated with specific activities of GDHT and negatively correlated with that of GS. Total dissolved N (TDN) concentration explained 81% of variance in bacterioplankton GDHT:GS activity ratio. The GDHT:GS ratio, TDN, DOC, and bacterial parameters decreased in magnitude along a tidally dynamic trophic gradient from north of Key West to south at the reef tract, which is consistent with the combined effects of localized coastal eutrophication and tidal exchange of seawater from the Southwest Florida Shelf and Florida Strait. The N-replete bacterioplankton north of Key West can regenerate ammonium which sustains primary production transported south to the reef. The range in GDHT:GS ratios was 5-30 times greater than that for commonly used indicators of planktonic eutrophication, which emphasizes the sensitivity of bacterioplankton N-metabolism to changes in N-bioavailability caused by nutrient pollution in sub-tropical coastal waters and utility of GDHT:GS ratio as an bioindicator of N-replete conditions.

  3. Relevance of induction of human drug-metabolizing enzymes: pharmacological and toxicological implications

    PubMed Central

    PARK, B. K.; KITTERINGHAM, N. R.; PIRMOHAMED, M.; TUCKER, G. T.

    1996-01-01

    1Human drug-metabolizing systems can be induced, or activated, by a large number of exogenous agents including drugs, alcohol, components in the diet and cigarette smoke, as well as by endogenous factors. 2Such perturbation of enzyme activity undoubtedly contributes to both intra- and inter-individual variation both with respect to the rate and route of metabolism for a particular drug. Induction may, in theory, either attenuate the pharmacological response or exacerbate the toxicity of a particular drug, or both. 3The clinical impact of enzyme induction will depend upon the number of different enzyme isoforms affected and the magnitude of the inductive response within an individual, and also on the therapeutic indices of the affected substrates. 4The toxicological implications will be determined either by any change in the route of metabolism, or by a disturbance of the balance between activation and detoxication processes, which may be isozyme selective. PMID:8799511

  4. CO2 Metabolism in Corn Roots. II. Intracellular Distribution of Enzymes 1

    PubMed Central

    Danner, Jean; Ting, Irwin P.

    1967-01-01

    Three enzymes assumed to mediate CO2 metabolism in corn root tips, P-enolpyruvate carboxylase, malic dehydrogenase, and the malic enzyme, were extracted to determine their relative specific activities and their partitioning between soluble and particulate fractions. The data indicated that the intracellular location of these 3 enzymes is nonparticulate and thus these enzymatic reactions of CO2 metabolism are apparently nonparticulate. The soluble malic dehydrogenase fraction differed from the particulate fraction in several kinetic properties, viz., response to the thionicotinamide analog of nicotinamide-adenine dinucleotide, oxaloacetate substrate inhibition at pH 8.3, and Km's for nicotinamide-adenine dinucleotide and l-malate. It was concluded that the soluble-malic dehydrogenase differed from the particulate forms in both structure and function. The soluble malic dehydrogenase is apparently involved in CO2 metabolism. PMID:16656561

  5. EnzDP: improved enzyme annotation for metabolic network reconstruction based on domain composition profiles.

    PubMed

    Nguyen, Nam-Ninh; Srihari, Sriganesh; Leong, Hon Wai; Chong, Ket-Fah

    2015-10-01

    Determining the entire complement of enzymes and their enzymatic functions is a fundamental step for reconstructing the metabolic network of cells. High quality enzyme annotation helps in enhancing metabolic networks reconstructed from the genome, especially by reducing gaps and increasing the enzyme coverage. Currently, structure-based and network-based approaches can only cover a limited number of enzyme families, and the accuracy of homology-based approaches can be further improved. Bottom-up homology-based approach improves the coverage by rebuilding Hidden Markov Model (HMM) profiles for all known enzymes. However, its clustering procedure relies firmly on BLAST similarity score, ignoring protein domains/patterns, and is sensitive to changes in cut-off thresholds. Here, we use functional domain architecture to score the association between domain families and enzyme families (Domain-Enzyme Association Scoring, DEAS). The DEAS score is used to calculate the similarity between proteins, which is then used in clustering procedure, instead of using sequence similarity score. We improve the enzyme annotation protocol using a stringent classification procedure, and by choosing optimal threshold settings and checking for active sites. Our analysis shows that our stringent protocol EnzDP can cover up to 90% of enzyme families available in Swiss-Prot. It achieves a high accuracy of 94.5% based on five-fold cross-validation. EnzDP outperforms existing methods across several testing scenarios. Thus, EnzDP serves as a reliable automated tool for enzyme annotation and metabolic network reconstruction. Available at: www.comp.nus.edu.sg/~nguyennn/EnzDP .

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

    PubMed

    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.

  7. Dietary n-6 PUFA deprivation downregulates arachidonate but upregulates docosahexaenoate metabolizing enzymes in rat brain

    PubMed Central

    Kim, Hyung-Wook; Rao, Jagadeesh S; Rapoport, Stanley I.; Igarashi, Miki

    2010-01-01

    Background Dietary n-3 polyunsaturated fatty acid (PUFA) deprivation increases expression of arachidonic acid (AA 20:4n-6)-selective cytosolic phospholipase A2 (cPLA2) IVA and cyclooxygenase (COX)-2 in rat brain, while decreasing expression of docosahexaenoic acid (DHA 22:6n-3)-selective calcium-independent iPLA2 VIA. Assuming that these enzyme changes represented brain homeostatic responses to deprivation, we hypothesized that dietary n-6 PUFA deprivation would produce changes in the opposite directions. Methods Brain expression of PUFA-metabolizing enzymes and their transcription factors was quantified in male rats fed an n-6 PUFA adequate or deficient diet for 15 weeks post-weaning. Results The deficient compared with adequate diet increased brain mRNA, protein and activity of iPLA2 VIA and 15-lipoxygenase (LOX), but decreased cPLA2 IVA and COX-2 expression. The brain protein level of the iPLA2 transcription factor SREBP-1 was elevated, while protein levels were decreased for AP-2α and NF-κB p65, cPLA2 and COX-2 transcription factors, respectively. Conclusions With dietary n-6 PUFA deprivation, rat brain PUFA metabolizing enzymes and some of their transcription factors change in a way that would homeostatically dampen reductions in brain n-6 PUFA concentrations and metabolism, while n-3 PUFA metabolizing enzyme expression is increased. The changes correspond to reported in vitro enzyme selectivities for AA compared with DHA. (198 words) PMID:21070866

  8. Biotransformation of anthelmintics and the activity of drug-metabolizing enzymes in the tapeworm Moniezia expansa.

    PubMed

    Prchal, Lukáš; Bártíková, Hana; Bečanová, Aneta; Jirásko, Robert; Vokřál, Ivan; Stuchlíková, Lucie; Skálová, Lenka; Kubíček, Vladimír; Lamka, Jiří; Trejtnar, František; Szotáková, Barbora

    2015-04-01

    The sheep tapeworm Moniezia expansa is very common parasite, which affects ruminants such as sheep, goats as well as other species. The benzimidazole anthelmintics albendazole (ABZ), flubendazole (FLU) and mebendazole (MBZ) are often used to treat the infection. The drug-metabolizing enzymes of helminths may alter the potency of anthelmintic treatment. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (ABZ, MBZ and FLU) in M. expansa. Activities of biotransformation enzymes were determined in subcellular fractions. Metabolites of the anthelmintics were detected and identified using high performance liquid chromatography/ultra-violet/VIS/fluorescence or ultra-high performance liquid chromatography/mass spectrometry. Reduction of MBZ, FLU and oxidation of ABZ were proved as well as activities of various metabolizing enzymes. Despite the fact that the conjugation enzymes glutathione S-transferase, UDP-glucuronosyl transferase and UDP-glucosyl transferase were active in vitro, no conjugated metabolites of anthelmintics were identified either ex vivo or in vitro. The obtained results indicate that sheep tapeworm is able to deactivate the administered anthelmintics, and thus protects itself against their action.

  9. Sulfatases and radical SAM enzymes: emerging themes in glycosaminoglycan metabolism and the human microbiota.

    PubMed

    Benjdia, Alhosna; Berteau, Olivier

    2016-02-01

    Humans live in a permanent association with bacterial populations collectively called the microbiota. In the last 10 years, major advances in our knowledge of the microbiota have shed light on its critical roles in human physiology. The microbiota has also been shown to be a major factor in numerous pathologies including obesity or inflammatory disorders. Despite tremendous progresses, our understanding of the key functions of the human microbiota and the molecular basis of its interactions with the host remain still poorly understood. Among the factors involved in host colonization, two enzymes families, sulfatases and radical S-adenosyl-L-methionine enzymes, have recently emerged as key enzymes.

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

    PubMed

    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.

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

    PubMed Central

    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

  12. [Regulation of key enzymes of L-alanine biosynthesis by Brevibacterium flavum producer strains].

    PubMed

    Melkonian, L O; Avetisova, G E; Ambartsumian, A A; Chakhalian, A Kh; Sagian, A S

    2013-01-01

    The mechanisms of L-alanine overproduction by Brevibacterium flavum producer strains were studied. It was shown that beta-CI-L-alanine is an inhibitor of some key enzymes involved in the synthesis of L-alanine, including alanine transaminase and valine-pyruvate transaminase. Two highly active B. flavum GL1 and GL1 8 producer strains, which are resistant to the inhibitory effect of beta-Cl-L-alanine, were obtained using a parental B. flavum AA5 producer strain, characterized by a reduced activity of alanine racemase (>or=98%). It was demonstrated that the increased L-alanine synthesis efficiency observed in the producer strains developed in this work is associated with the absence of inhibition of alanine transaminase by the end product of the biosynthesis reaction, as well as with the effect of derepression of both alanine transaminase and valine-pyruvate transaminase synthesis by the studied compound.

  13. T-2 toxin inhibits gene expression and activity of key steroidogenesis enzymes in mouse Leydig cells.

    PubMed

    Yang, Jian Ying; Zhang, Yong Fa; Meng, Xiang Ping; Li, Yuan Xiao; Ma, Kai Wang; Bai, Xue Fei

    2015-08-01

    T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of the testosterone secretion in the primary Leydig cells derived from the mouse testis. The previous study demonstrated the effects of T-2 toxin through direct decrease of the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis. In this study, we further examined the direct biological effects of T-2 toxin on steroidogenesis production, primarily in Leydig cells of mice. Mature mouse Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining. To examine T-2 toxin-induced testosterone secretion decrease, we measured the transcription levels of 3 key steroidogenic enzymes and 5 enzyme activities including 3β-HSD-1, P450scc, StAR, CYP17A1, and 17β-HSD in T-2 toxin/human chorionicgonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7) M, 10(-8) M and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with the decreases in the levels of transcription of 3β-HSD-1, P450scc, and StAR (P<0.05) and also in enzyme activities of 3β-HSD-1, P450scc, StAR, CYP17A1, and 17β-HSD (P<0.05).

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

    PubMed

    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. Fumarate-Mediated Inhibition of Erythrose Reductase, a Key Enzyme for Erythritol Production by Torula corallina

    PubMed Central

    Lee, Jung-Kul; Koo, Bong-Seong; Kim, Sang-Yong

    2002-01-01

    Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu2+ has been thoroughly reported, but the mechanism by which Cu2+ increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu2+ enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu2+ concentration. This suggests that supplemental Cu2+ reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina. PMID:12200310

  16. Catalase is a key enzyme in seed recovery from ageing during priming.

    PubMed

    Kibinza, Serge; Bazin, Jérémie; Bailly, Christophe; Farrant, Jill M; Corbineau, Françoise; El-Maarouf-Bouteau, Hayat

    2011-09-01

    Ageing induces seed deterioration expressed as the loss of seed vigour and/or viability. Priming treatment, which consists in soaking of seeds in a solution of low water potential, has been shown to reinvigorate aged seeds. We investigate the importance of catalase in oxidation protection during accelerated ageing and repair during subsequent priming treatment of sunflower (Helianthus annuus L.) seeds. Seeds equilibrated to 0.29g H2Og(-1) dry matter (DM) were aged at 35°C for different durations and then primed by incubation for 7 days at 15°C in a solution of polyethylene glycol 8000 at -2MPa. Accelerated ageing affected seed germination and priming treatment reversed partially the ageing effect. The inhibition of catalase by the addition of aminotriazol during priming treatment reduced seed repair indicating that catalase plays a key role in protection and repair systems during ageing. Ageing was associated with H2O2 accumulation as showed by biochemical quantification and CeCl3 staining. Catalase was reduced at the level of gene expression, protein content and affinity. Interestingly, priming induced catalase synthesis by activating expression and translation of the enzyme. Immunocytolocalization of catalase showed that the enzyme co-localized with H2O2 in the cytosol. These results clearly indicate that priming induce the synthesis of catalase which is involved in seed recovery during priming.

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

    PubMed

    Mueller, Edith E; Mayr, Johannes A; Zimmermann, Franz A; Feichtinger, René G; Stanger, Olaf; Sperl, Wolfgang; Kofler, Barbara

    2012-01-20

    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 ρ(0) cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in ρ(0) cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.

  18. Case study 3. Application of basic enzyme kinetics to metabolism studies: real-life examples.

    PubMed

    Li, Yongmei; McCabe, Michelle; Podila, Lalitha; Tracy, Timothy S; Tweedie, Donald J

    2014-01-01

    An appreciation of the principles of enzyme kinetics can be applied in a number of drug metabolism applications. The concept for this chapter arose from a simple discussion on selecting appropriate time points to most efficiently assess metabolite profiles in a human Phase 1a clinical study (Subheading 4). By considering enzyme kinetics, a logical approach to the issue was derived. The dialog was an important learning opportunity for the participants in the discussion, and we have endeavored to capture this experience with other questions related to determination of K m and V max parameters, a consideration of the value of hepatocytes versus liver microsomes and enzyme inhibition parameters.

  19. Phenylalanine 4-monooxygenase and the role of endobiotic metabolism enzymes in xenobiotic biotransformation.

    PubMed

    Steventon, Glyn B; Mitchell, Stephen C

    2009-10-01

    Phenylalanine 4-monooxygenase is the key enzyme in the sulfoxidation of the thioether drug S-carboxymethyl-l-cysteine and its thioether metabolites, S-methyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine and N-acetyl-S-methyl-l-cysteine in humans, and a number of other mammalian species. The kinetics constants of the sulfoxidation reaction (K(m), V(max) and CL(E)) have been investigated in cytosolic fractions derived from rat and human liver, in cytosolic fractions of HepG2 cells and using both human and mouse cDNA expressed phenylalanine 4-monooxygenase. Differences in K(m), V(max) and CL(E) of S-carboxymethyl-l-cysteine have been seen in HepG2 cells and human and mouse cDNA expressed phenylalanine 4-monooxygenase when compared to both rat and human hepatic cytosolic fractions. The association of the genetic polymorphism in the sulfoxidation of S-carboxymethyl-l-cysteine is highlighted with particular reference to this biotransformation reaction as being a biomarker of disease susceptibility in Parkinson's, Alzheimer's and motor neurone diseases and in rheumatoid arthritis. The possible underlying molecular genetics of the sulfoxidation polymorphism is also discussed in relation to the known allelic frequencies of phenylalanine 4-monooxygenase. Finally, the new found role phenylalanine 4-monooxygenase plays in xenobiotic metabolism is discussed.

  20. Enzymes involved in crotonate metabolism in Syntrophomonas wolfei

    SciTech Connect

    McInerney, M.J.; Wofford, N.Q.

    1992-12-31

    Cell-free extracts of Syntrophomonas wolfei subsp. wolfei grown with crotonate in pure culture or in coculture with Methanospirillum hungatei contained crotonyl-coenzyme A (CoA):acetate CoA-transferase activity. This activity was not detected in cell-free extracts from the butyrate-grown coculture which suggests that the long lag times observed before S. wolfei grew with crotonate were initially due to the inability to activate crotonate. Cell-free extracts of S. wolfei grown in pure culture contained high specific activities of hydrogenase and very low levels of formate dehydrogenase. The low levels suggest a biosynthetic rather than a catabolic role for the latter enzyme when S. wolfei is grown in pure culture. CO dehydrogenase activity was not detected. S. wolfei can form butyrate using a CoA transferase activity, but not by a phosphotransbutyrylase or enoate reductase activity. A c-type cytochrome was detected in S. wolfei grown in pure culture or in coculture indicating the presence of an electron transport system. This is a characteristic which separates S. wolfei from other known crotonate-using bacteria.

  1. Subcellular Distribution of Enzymes of Glycolate Metabolism in the Alga Cyanidium caldarium1

    PubMed Central

    Gross, Wolfgang; Beevers, Harry

    1989-01-01

    The intracellular distribution of enzymes capable of catalyzing the reactions from phosphoglycolate to glycerate in the bluegreen colored eucaryotic alga Cyanidium caldarium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes glycolate oxidase and glutamate-glyoxylate aminotransferase along with catalase were present in the peroxisomal fraction (density: 1.23 grams per cubic centimeter). Serine hydroxymethyltransferase was found in the mitochondrial fraction (density: 1.18 grams per cubic centimeter). In contrast to the observations in green leaves of higher plants, the enzymes for the conversion of serine to glycerate (serine-glyoxylate aminotransferase and hydroxypyruvate reductase) were found only in the soluble fraction of the gradient. The partial characterization of enzymes from Cyanidium participating in glycolate metabolism revealed only slight differences from the corresponding enzymes from higher plants. The phylogenetic implications of the observed similarities between the enigmatic alga Cyanidium and higher plants are discussed. PMID:16666880

  2. Identification and Functional Analysis of Delta-9 Desaturase, a Key Enzyme in PUFA Synthesis, Isolated from the Oleaginous Diatom Fistulifera

    PubMed Central

    Muto, Masaki; Kubota, Chihiro; Tanaka, Masayoshi; Satoh, Akira; Matsumoto, Mitsufumi; Yoshino, Tomoko; Tanaka, Tsuyoshi

    2013-01-01

    Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of Δ9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 Δ9 desaturase genes (fD9desA, fD9desB, fD9desC and fD9desD) from the oleaginous diatom Fistulifera were newly isolated and functionally characterized. The putative Δ9 acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical Δ9 desaturase. Furthermore, the function of these Δ9 desaturases was confirmed in the Saccharomyces cerevisiae ole1 gene deletion mutant (Δole1). All the putative Δ9 acyl-CoA desaturases showed Δ9 desaturation activity for C16∶0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18∶0 fatty acids. This study represents the first functional analysis of Δ9 desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain. PMID:24039966

  3. In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner.

    PubMed

    Sachdeva, Gairik; Garg, Abhishek; Godding, David; Way, Jeffrey C; Silver, Pamela A

    2014-08-01

    Co-localization of biochemical processes plays a key role in the directional control of metabolic fluxes toward specific products in cells. Here, we employ in vivo scaffolds made of RNA that can bind engineered proteins fused to specific RNA binding domains. This allows proteins to be co-localized on RNA scaffolds inside living Escherichia coli. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in one or two dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of substrates.

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

    PubMed

    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.

  5. Elasticity analysis and design for large metabolic responses produced by changes in enzyme activities.

    PubMed Central

    Ortega, Fernando; Acerenza, Luis

    2002-01-01

    Metabolic control analysis has been extensively used to describe how the sensitivity properties of the component enzymes in a metabolic pathway (represented by the elasticity coefficients) determine the way in which metabolic variables respond (described by the control coefficients). Similarly, metabolic control design addresses the inverse problem of obtaining the sensitivity properties of the component enzymes that are required for the system to show a pre-established pattern of responses. These formalisms, including what is called elasticity analysis and design, were developed for small, strictly speaking infinitesimal, changes. Here we extend them to large metabolic responses. The new approach can be applied to simple two-step pathways or to any arbitrary metabolic system divided into two groups linked by one intermediate. General expressions that relate control and elasticity coefficients for large changes are derived. Concentration and flux connectivity relationships are obtained. The relationships for large changes indicate that the pattern of responses is not necessarily the same as the one obtained with the traditional infinitesimal approach, in some cases the patterns being qualitatively different. The general analysis is used to study the control of ketogenesis in rat liver mitochondria, starting from data available in the literature. The control profile of the pathway subject to large changes shows both quantitative and qualitative differences from the one obtained from an analysis that is performed with infinitesimal coefficients. This exemplifies the type of errors that may be introduced when drawing conclusions about large metabolic responses from results obtained with an infinitesimal treatment. PMID:12084013

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

    PubMed

    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.

  7. Activities of xenobiotic metabolizing enzymes in rat placenta and liver in vitro.

    PubMed

    Fabian, Eric; Wang, Xinyi; Engel, Franziska; Li, Hequn; Landsiedel, Robert; van Ravenzwaay, Bennard

    2016-06-01

    In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling.

  8. Phylogenetic and biological investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and eukaryotic organisms. The role of NATs in fungal biology has only recently been investigated. The NAT1 (FDB2) gene of Fusarium verticillioides was the first NAT cloned and character...

  9. Comparative investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated. The NAT1 gene of Gibberella moniliformis was the first NAT cloned and characterized from fun...

  10. Hepatic Xenobiotic Metabolizing Enzyme Gene Expression Through the Life Stages of the Mouse

    EPA Science Inventory

    BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been ca...

  11. Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

    PubMed

    Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

    2015-02-01

    Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes.

  12. Coordinated Changes in Xenobiotic Metabolizing Enzyme Gene Expression in Aging Male Rats

    EPA Science Inventory

    In order to gain better insight on aging and susceptibility, we characterized the expression of xenobiotic metabolizing enzymes (XMEs) from the livers of rats to evaluate the change in capacity to respond to xenobiotics across the adult lifespan. Gene expression profiles for XMEs...

  13. IDENTIFICATION OF CHANGES IN XENOBIOTIC METABOLISM ENZYME EXPRESSION DURING AGING USING COMPREHENSIVE TRANSCRIPT PROFILING

    EPA Science Inventory

    Aging leads to changes in the expression of enzymes and transporters important in the metabolism and fate of xenobiotics in liver, kidney and intestine. Most notable are the changes in a number of CYP and xenobiotic transporter genes regulated by the nuclear receptors PXR, CAR an...

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

    PubMed Central

    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

  15. Sex-specific basal and hypoglycemic patterns of in vivo caudal dorsal vagal complex astrocyte glycogen metabolic enzyme protein expression.

    PubMed

    Tamrakar, Pratistha; Shrestha, Prem; Briski, Karen P

    2014-10-24

    Astrocytes contribute to neurometabolic stability through uptake, catabolism, and storage of glucose. These cells maintain the major brain glycogen reservoir, which is a critical fuel supply to neurons during glucose deficiency and increased brain activity. We used a combinatory approach incorporating immunocytochemistry, laser microdissection, and Western blotting to investigate the hypothesis of divergent expression of key enzymes regulating glycogen metabolism and glycolysis during in vivo normo- and/or hypoglycemia in male versus female hindbrain astrocytes. Glycogen synthase (GS) and glycogen phosphorylase (GP) levels were both enhanced in dorsal vagal complex astrocytes from vehicle-injected female versus male controls, with incremental increase in GS exceeding GP. Insulin-induced hypoglycemia (IIH) diminished GS and increased glycogen synthase kinase-3-beta (GSK3β) expression in both sexes, but decreased phosphoprotein phosphatase-1 (PP1) levels only in males. Astrocyte GP content was elevated by IIH in male, but not female rats. Data reveal sex-dependent sensitivity of these enzyme proteins to lactate as caudal hindbrain repletion of this energy substrate fully or incompletely reversed hypoglycemic inhibition of GS and prevented hypoglycemic augmentation of GSK3β and GP in females and males, respectively. Sex dimorphic patterns of glycogen branching and debranching enzyme protein expression were also observed. Levels of the rate-limiting glycolytic enzyme, phosphofructokinase, were unaffected by IIH with or without lactate repletion. Current data demonstrating sex-dependent basal and hypoglycemic patterns of hindbrain astrocyte glycogen metabolic enzyme expression imply that glycogen volume and turnover during glucose sufficiency and shortage may vary accordingly.

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

    USGS Publications Warehouse

    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.

  17. Comparative metabolism as a key driver of wildlife species sensitivity to human and veterinary pharmaceuticals

    PubMed Central

    Hutchinson, Thomas H.; Madden, Judith C.; Naidoo, Vinny; Walker, Colin H.

    2014-01-01

    Human and veterinary drug development addresses absorption, distribution, metabolism, elimination and toxicology (ADMET) of the Active Pharmaceutical Ingredient (API) in the target species. Metabolism is an important factor in controlling circulating plasma and target tissue API concentrations and in generating metabolites which are more easily eliminated in bile, faeces and urine. The essential purpose of xenobiotic metabolism is to convert lipid-soluble, non-polar and non-excretable chemicals into water soluble, polar molecules that are readily excreted. Xenobiotic metabolism is classified into Phase I enzymatic reactions (which add or expose reactive functional groups on xenobiotic molecules), Phase II reactions (resulting in xenobiotic conjugation with large water-soluble, polar molecules) and Phase III cellular efflux transport processes. The human–fish plasma model provides a useful approach to understanding the pharmacokinetics of APIs (e.g. diclofenac, ibuprofen and propranolol) in freshwater fish, where gill and liver metabolism of APIs have been shown to be of importance. By contrast, wildlife species with low metabolic competency may exhibit zero-order metabolic (pharmacokinetic) profiles and thus high API toxicity, as in the case of diclofenac and the dramatic decline of vulture populations across the Indian subcontinent. A similar threat looms for African Cape Griffon vultures exposed to ketoprofen and meloxicam, recent studies indicating toxicity relates to zero-order metabolism (suggesting P450 Phase I enzyme system or Phase II glucuronidation deficiencies). While all aspects of ADMET are important in toxicity evaluations, these observations demonstrate the importance of methods for predicting API comparative metabolism as a central part of environmental risk assessment. PMID:25405970

  18. Colon cancer chemopreventive efficacy of silibinin through perturbation of xenobiotic metabolizing enzymes in experimental rats.

    PubMed

    Sangeetha, Nagarajan; Viswanathan, Periyaswamy; Balasubramanian, Thangavel; Nalini, Namasivayam

    2012-01-15

    Our findings reported so far demonstrate that silibinin modulates gut microbial enzymes, colonic oxidative stress and Wnt/β-catenin signaling, to exert its antiproliferative effect against 1,2 di-methylhydrazine (DMH) induced colon carcinogenesis. Since xenobiotic metabolizing enzymes play a crucial role in carcinogen activation and metabolism, we aimed to explore the effect of silibinin on xenobiotic metabolizing enzymes during DMH induced colon carcinogenesis. Male albino rats were randomly divided into six groups. Group 1 served as control and group 2 rats received 50mg/kg body weight of silibinin p.o. every day. Groups 3-6 rats were given DMH at a dose of (20mg/kg body weight subcutaneously) once a week for 15 weeks to induce colonic tumors. In addition to DMH, group 4 (initiation), group 5 (post-initiation) and group 6 (entire period) rats received silibinin (50mg/kg body weight, p.o., everyday) at different time points during the experimental period of 32 weeks. Rats exposed to DMH alone showed increased activities of phase I enzymes (cytochrome b5, cytochrome b5 reductase, cytochromeP450, cytochromeP450 reductase, cytochromP4502E1) and decreased activities of phase II enzymes (Uridine diphospho glucuronyl transferase, Glutathione-S-transferase and DT-Diaphorase) in the liver and colonic mucosa as compared to control rats. Silibinin supplementation modulates the xenobiotic metabolizing enzymes favoring carcinogen detoxification. Evaluation of lipid peroxidation and antioxidants status showed that silibinin supplementation counteracts DMH induced hepatic and circulatory oxidative stress. Tumor burden in experimental animals was assessed both macroscopically and microscopically in the colon tissues. Our findings emphasize the potential chemopreventive action of silibinin against DMH induced colon carcinogenesis.

  19. Connecting metabolism and reproduction: roles of central energy sensors and key molecular mediators.

    PubMed

    Roa, Juan; Tena-Sempere, Manuel

    2014-11-01

    It is well established that pubertal activation of the reproductive axis and maintenance of fertility are critically dependent on the magnitude of body energy reserves and the metabolic state of the organism. Hence, conditions of impaired energy homeostasis often result in deregulation of puberty and reproduction, whereas gonadal dysfunction can be associated with the worsening of the metabolic profile and, eventually, changes in body weight. While much progress has taken place in our knowledge about the neuroendocrine mechanisms linking metabolism and reproduction, our understanding of how such dynamic interplay happens is still incomplete. As paradigmatic example, much has been learned in the last two decades on the reproductive roles of key metabolic hormones (such as leptin, insulin and ghrelin), their brain targets and the major transmitters and neuropeptides involved. Yet, the molecular mechanisms whereby metabolic information is translated and engages into the reproductive circuits remain largely unsolved. In this work, we will summarize recent developments in the characterization of the putative central roles of key cellular energy sensors, such as mTOR, in this phenomenon, and will relate these with other molecular mechanisms likely contributing to the brain coupling of energy balance and fertility. In doing so, we aim to provide an updated view of an area that, despite still underdeveloped, may be critically important to fully understand how reproduction and metabolism are tightly connected in health and disease.

  20. Homologues of xenobiotic metabolizing N-acetyltransferases in plant-associated fungi: Novel functions for an old enzyme family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified...

  1. Metabolic Pathways of Inhaled Glucocorticoids by the CYP3A Enzymes

    PubMed Central

    Moore, Chad D.; Roberts, Jessica K.; Orton, Christopher R.; Murai, Takahiro; Fidler, Trevor P.; Reilly, Christopher A.; Ward, Robert M.

    2013-01-01

    Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and Δ6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ6-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography–mass spectrometry and NMR analysis. PMID:23143891

  2. Key role of an ADP - ribose - dependent transcriptional regulator of NAD metabolism for fitness and virulence of Pseudomonas aeruginosa.

    PubMed

    Okon, Elza; Dethlefsen, Sarah; Pelnikevich, Anna; Barneveld, Andrea van; Munder, Antje; Tümmler, Burkhard

    2017-01-01

    NAD is an essential co-factor of redox reactions and metabolic conversions of NAD-dependent enzymes. NAD biosynthesis in the opportunistic pathogen Pseudomonas aeruginosa has yet not been experimentally explored. The in silico search for orthologs in the P. aeruginosa PAO1 genome identified the operon pncA - pncB1-nadE (PA4918-PA4920) to encode the nicotinamidase, nicotinate phosporibosyltransferase and Nad synthase of salvage pathway I. The functional role of the preceding genes PA4917 and PA4916 was resolved by the characterization of recombinant protein. PA4917 turned out to encode the nicotinate mononucleotide adenylyltransferase NadD2 and PA4916 was determined to encode the transcriptional repressor NrtR that binds to an intergenic sequence between nadD2 and pncA. Complex formation between the catalytically inactive Nudix protein NrtR and its DNA binding site was suppressed by the antirepressor ADP-ribose. NrtR plasposon mutagenesis abrogated virulence of P. aeruginosa TBCF10839 in a murine acute airway infection model and constrained its metabolite profile. When grown together with other isogenic plasposon mutants, the nrtR knock-out was most compromised in competitive fitness to persist in nutrient-rich medium in vitro or murine airways in vivo. This example demonstrates how tightly metabolism and virulence can be intertwined by key elements of metabolic control.

  3. Understanding central carbon metabolism of rapidly proliferating mammalian cells based on analysis of key enzymatic activities in GS-CHO cell lines.

    PubMed

    Zou, Wu; Al-Rubeai, Mohamed

    2016-09-01

    The central carbon metabolism (glycolysis, the pentose phosphate pathway [PPP], and the tricarboxylic acid [TCA] cycle) plays an essential role in the supply of biosynthetic precursors and energy. How the central carbon metabolism changes with the varying growth rates in the in vitro cultivation of rapidly proliferating mammalian cells, such as cancer cells and continuous cell lines for recombinant protein production, remains elusive. Based on relationships between the growth rate and the activity of seven key enzymes from six cell clones, this work reports finding an important metabolic characteristic in rapidly proliferating glutamine synthetase-Chinese hamster ovary cells. The key enzymatic activity involved in the TCA cycle that is responsible for the supply of energy became elevated as the growth rate exhibited increases, while the activity of key enzymes in metabolic pathways (glycolysis and the PPP), responsible for the supply of biosynthetic precursors, tended to decrease-suggesting that rapidly proliferating cells still depended predominantly on the TCA cycle rather than on aerobic glycolysis for their energetic demands. Meanwhile, the growth-limiting resource was most likely biosynthetic substrates rather than energy provision. In addition, the multifaceted role of glucose-6-phosphate isomerase (PGI) was confirmed, based on a significant correlation between PGI activity and the percentage of G2/M-phase cells.

  4. Expression in human prostate of drug- and carcinogen-metabolizing enzymes: association with prostate cancer risk.

    PubMed Central

    Agúndez, J. A.; Martínez, C.; Olivera, M.; Gallardo, L.; Ladero, J. M.; Rosado, C.; Prados, J.; Rodriguez-Molina, J.; Resel, L.; Benítez, J.

    1998-01-01

    The role of two common polymorphisms of enzymes involved in the metabolism of drugs and carcinogens was studied in relation to prostate cancer. The gene encoding one of these enzymes (NAT2) is located in an area where frequent allelic loss occurs in prostate cancer. Mutations at the genes CYP2D6 and NAT2 were analysed by allele-specific polymerase chain reaction and restriction mapping in DNA from 94 subjects with prostate cancer and 160 male healthy control subjects. Eleven prostate specimens were analysed for genotype and enzymatic activities NAT2, CYP2D6 and CYP3A by using the enzyme-specific substrates sulphamethazine and dextromethorphan. Enzyme activities with substrate specificities corresponding to NAT2, CYP2D6 and CYP3A are present in human prostate tissue, with mean +/-s.d. activities of 4.8+/-4.4 pmol min(-1) mg(-1) protein, 156+/-91 and 112+/-72 nmol min(-1) mg(-1) protein respectively. The Km values for the prostate CYP2D6 and CYP3A enzyme activities corresponded to that of liver CYP2D6 and CYP3A activities, and the CYP2D6 enzyme activity is related to the CYP2D6 genotype. The N-acetyltransferase, in contrast, had a higher Km than NAT2 and was independent of the NAT2 genotype. The CYP2D6 and CYP3A enzymes, and an N-acetyltransferase activity that is independent of the regulation of the NAT2 gene, are expressed in human prostate tissue. The presence of carcinogen-metabolizing enzymes in human prostate with a high interindividual variability may be involved in the regulation of local levels of carcinogens and mutagens and may underlie interindividual differences in cancer susceptibility. Images Figure 1 PMID:9823980

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

    PubMed

    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.

  6. Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a 
Detoxifying Enzyme

    PubMed Central

    Battelli, Maria Giulia; Polito, Letizia; Bortolotti, Massimo; Bolognesi, Andrea

    2016-01-01

    The enzyme xanthine oxidoreductase (XOR) catalyzes the last two steps of purine catabolism in the highest uricotelic primates. XOR is an enzyme with dehydrogenase activity that, in mammals, may be converted into oxidase activity under a variety of pathophysiologic conditions. XOR activity is highly regulated at the transcriptional and post-translational levels and may generate reactive oxygen and nitrogen species, which trigger different consequences, ranging from cytotoxicity to inflammation. The low specificity for substrates allows XOR to metabolize a number of endogenous metabolites and a variety of exogenous compounds, including drugs. The present review focuses on the role of XOR as a drug-metabolizing enzyme, specifically for drugs with anticancer, antimicrobial, antiviral, immunosuppressive or vasodilator activities, as well as drugs acting on metabolism or inducing XOR expression. XOR has an activating role that is essential to the pharmacological action of quinone drugs, cyadox, antiviral nucleoside analogues, allopurinol, nitrate and nitrite. XOR activity has a degradation function toward thiopurine nucleotides, pyrazinoic acid, methylxanthines and tolbutamide, whose half-life may be prolonged by the use of XOR inhibitors. In conclusion, to avoid potential drug interaction risks, such as a toxic excess of drug bioavailability or a loss of drug efficacy, caution is suggested in the use of XOR inhibitors, as in the case of hyperuricemic patients affected by gout or tumor lysis syndrome, when it is necessary to simultaneously administer therapeutic substances that are activated or degraded by the drug-metabolizing activity of XOR. PMID:27458036

  7. Enzyme

    MedlinePlus

    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 ...

  8. Type 3 adenylyl cyclase: a key enzyme mediating the cAMP signaling in neuronal cilia

    PubMed Central

    Qiu, Liyan; LeBel, Robert P; Storm, Daniel R; Chen, Xuanmao

    2016-01-01

    Cilia are rigid, centriole-derived, microtubule-based organelles present in a majority of vertebrate cells including neurons. They are considered the cellular “antennae” attuned for detecting a range of extracellular signals including photons, odorants, morphogens, hormones and mechanical forces. The ciliary microenvironment is distinct from most actin-based subcellular structures such as microvilli or synapses. In the nervous system, there is no evidence that neuronal cilia process any synaptic structure. Apparently, the structural features of neuronal cilia do not allow them to harbor any synaptic connections. Nevertheless, a large number of G protein-coupled receptors (GPCRs) including odorant receptors, rhodopsin, Smoothened, and type 6 serotonin receptor are found in cilia, suggesting that these tiny processes largely depend on metabotropic receptors and their tuned signals to impact neuronal functions. The type 3 adenylyl cyclase (AC3), widely known as a cilia marker, is highly and predominantly expressed in olfactory sensory cilia and primary cilia throughout the brain. We discovered that ablation of AC3 in mice leads to pleiotropic phenotypes including anosmia, failure to detect mechanical stimulation of airflow, cognitive deficit, obesity, and depression-like behaviors. Multiple lines of human genetic evidence also demonstrate that AC3 is associated with obesity, major depressive disorder (MDD), sarcoidosis, and infertility, underscoring its functional importance. Here we review recent progress on AC3, a key enzyme mediating the cAMP signaling in neuronal cilia. PMID:27785336

  9. Effects of cerium on key enzymes of carbon assimilation of spinach under magnesium deficiency.

    PubMed

    Yuguan, Ze; Min, Zhou; Luyang, Luo; Zhe, Ji; Chao, Liu; Sitao, Yin; Yanmei, Duan; Na, Li; Fashui, Hong

    2009-11-01

    The mechanism of the fact that cerium improves the photosynthesis of plants under magnesium deficiency is poorly understood. The main aim of the study was to determine the role of cerium in the amelioration of magnesium deficiency effects in CO(2) assimilation of spinach. Spinach plants were cultivated in Hoagland's solution. They were subjected to magnesium deficiency and to cerium chloride administered in the magnesium-present Hoagland's media and magnesium-deficient Hoagland's media. The results showed that the chlorophyll synthesis and oxygen evolution was destroyed, and the activities of Rubisco carboxylasae and Rubisco activase and the expression of Rubisco large subunit (rbcL), Rubisco small subunit (rbcS), and Rubisco activase subunit (rca) were significantly inhibited, then plant growth was inhibited by magnesium deficiency. However, cerium promotes the chlorophyll synthesis, the activities of two key enzymes in CO(2) assimilation, and the expression of rbcL, rbcS, and rca, thus leading to the enhancement of spinach growth under magnesium-deficient conditions.

  10. Spore Germination and Carbon Metabolism in Fusarium solani V. Changes in Anaerobic Metabolism and Related Enzyme Activities during Development 1

    PubMed Central

    Cochrane, Vincent W.; Cochrane, Jean C.

    1966-01-01

    Macroconidia of Fusarium solani f. phascoli have no detectable capacity to respire glucose anaerobically; germinated spores and mycelium, on the other hand, ferment glucose, although slowly. Extracts of ungerminated spores contain hexokinase, phosphohexoisomerase, phosphofructokinase, aldolase, triose phosphate dehydrogenase, triose phosphate isomerase, phosphoglyceric kinase, enolase, phosphoglyceric mutase, pyruvate kinase, and pyruvate decarboxylase. It follows, therefore, that the appearance of fermentative capacity during spore germination cannot be ascribed to the de novo synthesis of any of these enzymes. During germination and mycelial development the specific activity of all of the enzymes named except phosphohexoisomerase and aldolase increases 2- to 8-fold. Specific activity of all of the enzymes is substantially higher than the fermentative capacity of intact cells, i.e., none is limiting to anaerobic respiration. The enzymatic assay data are consistent with a conclusion reached earlier on the basis of studies of aerobic glucose metabolism, that the process of germination involves an acceleration of pre-existing metabolic systems rather than an appearance of new pathways. PMID:16656324

  11. Application of the Key Events Dose-response Framework to Folate Metabolism.

    PubMed

    Hu, Jing; Wang, Bing; Sahyoun, Nadine R

    2016-06-10

    Folate is a vitamin that plays a role as a cofactor and coenzyme in many essential reactions. These reactions are interrelated and any change in folate homeostasis could affect other reactions. With food fortified with folic acid, and use of multivitamin, unmetabolized folic acid (UMFA) has been detected in blood circulation, particularly among older adults. This has raised concern about the potential harmful effect of high folic acid intake and UMFA on health conditions such as cognitive dysfunction and cancer. To examine what is known about folate metabolism and the release of circulating UMFA, the Key Events Dose-Response Framework (KEDRF) was used to review each of the major key events, dose-response characteristics and homeostatic mechanisms of folate metabolism. The intestine, liver and kidneys each play essential roles in regulating body folate homeostasis. But the determining event in folate metabolism leading to the release of UMFA in circulation appears to be the saturation of dihydrofolate reductase in the liver. However, at each of the key events in folate metabolism, limited information is available on threshold, homeostatic regulation and intracellular effects of folic acid. More studies are needed to fill in the knowledge gaps for quantitatively characterizing the dose-effect relationship especially in light of the call for extending folate fortification to other foods.

  12. Increased oxygen radical-dependent inactivation of metabolic enzymes by liver microsomes after chronic ethanol consumption

    SciTech Connect

    Dicker, E.; Cederbaum, A.I. )

    1988-10-01

    Enzymatic and nonenzymatic mixed-function oxidase systems have been shown to generate an oxidant that catalyzes the inactivation of glutamine synthetase and other metabolic enzymes. Recent studies have shown that microsomes isolated from rats chronically fed ethanol generate reactive oxygen intermediates at elevated rates compared with controls. Microsomes from rats fed ethanol were found to be more effective than control microsomes in catalyzing the inactivation of enzymes added to the incubation system. The enzymes studied were alcohol dehydrogenase, lactic dehydrogenase, and pyruvate kinase. The inactivation process by both types of microsomal preparations was sensitive to catalase and glutathione plus glutathione peroxidase, but was not affected by superoxide dismutase or hydroxyl radical scavengers. Iron was required for the inactivation of added enzymes; microsomes from the rats fed ethanol remained more effective than control microsomes in catalyzing the inactivation of enzymes in the absence or presence of several ferric complexes. The inactivation of enzymes was enhanced by the addition of menadione or paraquat to the microsomes, and rates of inactivation were higher with the microsomes from the ethanol-fed rats. The enhanced generation of reactive oxygen intermediates and increased inactivation of enzymes by microsomes may contribute toward the hepatotoxic effects associated with ethanol consumption.

  13. PLP-dependent enzymes as entry and exit gates of sphingolipid metabolism

    PubMed Central

    Bourquin, Florence; Capitani, Guido; Grütter, Markus Gerhard

    2011-01-01

    Sphingolipids are membrane constituents as well as signaling molecules involved in many essential cellular processes. Serine palmitoyltransferase (SPT) and sphingosine-1-phosphate lyase (SPL), both PLP (pyridoxal 5′-phosphate)-dependent enzymes, function as entry and exit gates of the sphingolipid metabolism. SPT catalyzes the condensation of serine and a fatty acid into 3-keto-dihydrosphingosine, whereas SPL degrades sphingosine-1-phosphate (S1P) into phosphoethanolamine and a long-chain aldehyde. The recently solved X-ray structures of prokaryotic homologs of SPT and SPL combined with functional studies provide insight into the structure–function relationship of the two enzymes. Despite carrying out different reactions, the two enzymes reveal striking similarities in the overall fold, topology, and residues crucial for activity. Unlike their eukaryotic counterparts, bacterial SPT and SPL lack a transmembrane helix, making them targets of choice for biochemical characterization because the use of detergents can be avoided. Both human enzymes are linked to severe diseases or disorders and might therefore serve as targets for the development of therapeutics aiming at the modulation of their activity. This review gives an overview of the sphingolipid metabolism and of the available biochemical studies of prokaryotic SPT and SPL, and discusses the major similarities and differences to the corresponding eukaryotic enzymes. PMID:21710479

  14. Stereochemical course, isotope effects, and enzyme inhibitor studies of glaucine metabolism in fungi

    SciTech Connect

    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.

  15. Ornithine Aminotransferase, an Important Glutamate-Metabolizing Enzyme at the Crossroads of Multiple Metabolic Pathways

    PubMed Central

    Ginguay, Antonin; Cynober, Luc; Curis, Emmanuel; Nicolis, Ioannis

    2017-01-01

    Ornithine δ-aminotransferase (OAT, E.C. 2.6.1.13) catalyzes the transfer of the δ-amino group from ornithine (Orn) to α-ketoglutarate (aKG), yielding glutamate-5-semialdehyde and glutamate (Glu), and vice versa. In mammals, OAT is a mitochondrial enzyme, mainly located in the liver, intestine, brain, and kidney. In general, OAT serves to form glutamate from ornithine, with the notable exception of the intestine, where citrulline (Cit) or arginine (Arg) are end products. Its main function is to control the production of signaling molecules and mediators, such as Glu itself, Cit, GABA, and aliphatic polyamines. It is also involved in proline (Pro) synthesis. Deficiency in OAT causes gyrate atrophy, a rare but serious inherited disease, a further measure of the importance of this enzyme. PMID:28272331

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

    PubMed

    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.

  17. Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida

    PubMed Central

    2012-01-01

    Background Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. Results We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. Conclusions We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin. PMID:22272603

  18. Preparation of Metabolically Active Staphylococcus aureus Protoplasts by Use of the Aeromonas hydrophila Lytic Enzyme

    PubMed Central

    Coles, N. W.; Gross, R.

    1973-01-01

    Stable, metabolically active protoplasts of Staphylococcus aureus have been prepared by the use of a staphylolytic enzyme produced by Aeromonas hydrophila. Respiratory and glycolytic rates and synthesis of nucleic acids, protein, and lipid in these protoplasts, stabilized variously in 1.1 M sucrose, 0.37 M sodium succinate, or 0.37 M sodium sulfate, have been shown to be comparable with the same parameters in intact cells under the same conditions. Images PMID:4728270

  19. Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine.

    PubMed

    Calvo, Ana C; Pey, Angel L; Miranda-Vizuete, Antonio; Døskeland, Anne P; Martinez, Aurora

    2011-02-15

    TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.

  20. Importance of influx and efflux systems and xenobiotic metabolizing enzymes in intratumoral disposition of anticancer agents.

    PubMed

    Rochat, B

    2009-08-01

    In this review, intratumoral drug disposition will be integrated into the wide range of resistance mechanisms to anticancer agents with particular emphasis on targeted protein kinase inhibitors. Six rules will be established: 1. There is a high variability of extracellular/intracellular drug level ratios; 2. There are three main systems involved in intratumoral drug disposition that are composed of SLC, ABC and XME enzymes; 3. There is a synergistic interplay between these three systems; 4. In cancer subclones, there is a strong genomic instability that leads to a highly variable expression of SLC, ABC or XME enzymes; 5. Tumor-expressed metabolizing enzymes play a role in tumor-specific ADME and cell survival and 6. These three systems are involved in the appearance of resistance (transient event) or in the resistance itself. In addition, this article will investigate whether the overexpression of some ABC and XME systems in cancer cells is just a random consequence of DNA/chromosomal instability, hypo- or hypermethylation and microRNA deregulation, or a more organized modification induced by transposable elements. Experiments will also have to establish if these tumor-expressed enzymes participate in cell metabolism or in tumor-specific ADME or if they are only markers of clonal evolution and genomic deregulation. Eventually, the review will underline that the fate of anticancer agents in cancer cells should be more thoroughly investigated from drug discovery to clinical studies. Indeed, inhibition of tumor expressed metabolizing enzymes could strongly increase drug disposition, specifically in the target cells resulting in more efficient therapies.

  1. Dynamic expression of retinoic acid synthesizing and metabolizing enzymes in the developing mouse inner ear

    PubMed Central

    Romand, Raymond; Kondo, Takako; Fraulob, Valérie; Petkovich, Martin; Dollé, Pascal; Hashino, Eri

    2008-01-01

    Retinoic acid signaling plays essential roles in morphogenesis and neural development through transcriptional regulation of downstream target genes. It is believed that the balance between the activities of synthesizing and metabolizing enzymes determines the amount of active retinoic acid to which a developing tissue is exposed. In this study, we investigated spatio-temporal expression patterns of four synthesizing enzymes, the retinaldehyde dehydrogenases 1, 2, 3 and 4 (Raldh1, Raldh2, Raldh3 and Raldh4) and two metabolizing enzymes (Cyp26A1 and Cyp26B1) in the embryonic and postnatal mouse inner ear using quantitative RT-PCR, in situ hybridization and Western blot analysis. Quantitative RT-PCR analysis and Western blot data revealed that the expression of CYP26s was much higher than that of Raldhs at early embryonic ages, but that Cyp26 expression was down-regulated during embryonic development. Conversely, the expression levels of Raldh2 and -3 increased during development and were significantly higher than the Cyp26 levels at postnatal day 20. At this age, Raldh3 was expressed predominantly in the cochlea, while Raldh2 was present in the vestibular end organ. At early embryonic stages as observed by in situ hybridization, the synthesizing enzymes were expressed only in the dorsoventral epithelium of the otocyst, while the metabolizing enzymes were present mainly in mesenchymal cells surrounding the otic epithelium. At later stages, Raldh2, Raldh3 and Cyp26B1 were confined to the stria vascularis, spiral ganglion and supporting cells in the cochlear and vestibular epithelia, respectively. The downregulation of Cyp26s and the upregulation of Raldhs after birth during inner ear maturation suggests tissue changes in the sensitivity to retinoic acid concentrations. PMID:16615129

  2. The effect of enzyme inhibition on the metabolism and activation of tacrine by human liver microsomes.

    PubMed Central

    Spaldin, V; Madden, S; Pool, W F; Woolf, T F; Park, B K

    1994-01-01

    1. Tacrine (1,2,3,4-tetrahydro-9-aminoacridine-hydrochloride: THA) underwent metabolism in vitro by a panel (n = 12) of human liver microsomes genotyped for CYP2D6, in the presence of NADPH, to both protein-reactive and stable metabolites. 2. There was considerable variation in the extent of THA metabolism amongst human livers. Protein-reactive metabolite formation showed a 10-fold variation (0.6 +/- 0.1%-5.2 +/- 0.8% of incubated radioactivity mg-1 protein) whilst stable metabolites showed a 3-fold variation (24.3 +/- 1.7%-78.6 +/- 2.6% of incubated radioactivity). 3. Using cytochrome P450 isoform specific inhibitors CYP1A2 was identified as the major enzyme involved in all routes of THA metabolism. 4. There was a high correlation between aromatic and alicyclic hydroxylation (r = 0.92, P < 0.0001) consistent with these biotransformations being catalysed by the same enzymes. 5. Enoxacin (ENOX), cimetidine (CIM) and chloroquine (CQ) inhibited THA metabolism by a preferential decrease in the bioactivation to protein-reactive, and hence potentially toxic, species. The inhibitory potency of ENOX and CIM was increased significantly upon pre-incubation with microsomes and NADPH. 6. Covalent binding correlated with 7-OH-THA formation before (r = 0.792, P < 0.0001) and after (r = 0.73, P < 0.0001) inhibition by CIM, consistent with a two-step mechanism in the formation of protein-reactive metabolite(s) via a 7-OH intermediate. 7. The use of enzyme inhibitors may provide a useful tool for examining the relationship between the metabolism and toxicity of THA in vivo. PMID:7946932

  3. Antioxidative properties and inhibition of key enzymes relevant to type-2 diabetes and hypertension by essential oils from black pepper.

    PubMed

    Oboh, Ganiyu; Ademosun, Ayokunle O; Odubanjo, Oluwatoyin V; Akinbola, Ifeoluwa A

    2013-01-01

    The antioxidant properties and effect of essential oil of black pepper (Piper guineense) seeds on α -amylase, α -glucosidase (key enzymes linked to type-2 diabetes), and angiotensin-I converting enzyme (ACE) (key enzyme linked to hypertension) were assessed. The essential oil was obtained by hydrodistillation and dried with anhydrous Na2SO4, and the phenolic content, radical [1,1-diphenyl-2 picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and nitric oxide (NO)] scavenging abilities as well as the ferric reducing antioxidant property (FRAP) and Fe(2+)-chelating ability of the essential oil were investigated. Furthermore, the effect on α -amylase, α -glucosidase, and ACE enzyme activities was also investigated. The characterization of the constituents was done using GC. The essential oil scavenged DPPH∗, NO∗, and ABTS∗ and chelated Fe(2+). α -Pinene, β -pinene, cis-ocimene, myrcene, allo-ocimene, and 1,8-cineole were among the constituents identified by GC. The essential oil inhibited α -amylase, α -glucosidase, and ACE enzyme activities in concentration-dependent manners, though exhibiting a stronger inhibition of α -glucosidase than α -amylase activities. Conclusively, the phenolic content, antioxidant activity, and inhibition of α -amylase, α -glucosidase, and angiotensin-1 converting enzyme activities by the essential oil extract of black pepper could be part of the mechanism by which the essential oil could manage and/or prevent type-2 diabetes and hypertension.

  4. Dietary chemoprevention strategies for induction of phase II xenobiotic-metabolizing enzymes in lung carcinogenesis: A review

    PubMed Central

    Tan, Xiang-Lin; Spivack, Simon D.

    2013-01-01

    Lung cancer is the leading cause of cancer mortality for men and women in the United States and is a growing worldwide problem. Protection against lung cancer is associated with higher dietary intake of fruits and vegetables, according to recent large epidemiologic studies. One strategy for lung cancer chemoprevention focuses on the use of agents to modulate the metabolism and disposition of tobacco, environmental and endogenous carcinogens through upregulation of detoxifying phase II enzymes. We summarize the substantial evidence that suggests that induction of phase II enzymes, particularly the glutathione S-transferases, plays a direct role in chemoprotection against lung carcinogenesis. The engagement of the Keap1–Nrf2 complex regulating the antioxidant response element (ARE) signaling pathway has been identified as a key molecular target of chemopreventive phase II inducers in several systems. Monitoring of phase II enzyme induction has led to identification of novel chemopreventive agents such as the isothiocyanate sulforaphane, and the 1,2-dithiole-3-thiones. However, no agents have yet demonstrated clear benefit in human cell systems, or in clinical trials. Alternative strategies include: (a) using intermediate cancer biomarkers for the endpoint in human trials; (b) high-throughput small molecule discovery approaches for induced expression of human phase II genes; and (c) integrative approaches that consider pharmacogenetics, along with pharmacokinetics and pharmacodynamics in target lung tissue. These approaches may lead to a more effective strategy of tailored chemoprevention efforts using compounds with proven human activity. PMID:19185948

  5. Dietary chemoprevention strategies for induction of phase II xenobiotic-metabolizing enzymes in lung carcinogenesis: A review.

    PubMed

    Tan, Xiang-Lin; Spivack, Simon D

    2009-08-01

    Lung cancer is the leading cause of cancer mortality for men and women in the United States and is a growing worldwide problem. Protection against lung cancer is associated with higher dietary intake of fruits and vegetables, according to recent large epidemiologic studies. One strategy for lung cancer chemoprevention focuses on the use of agents to modulate the metabolism and disposition of tobacco, environmental and endogenous carcinogens through upregulation of detoxifying phase II enzymes. We summarize the substantial evidence that suggests that induction of phase II enzymes, particularly the glutathione S-transferases, plays a direct role in chemoprotection against lung carcinogenesis. The engagement of the Keap1-Nrf2 complex regulating the antioxidant response element (ARE) signaling pathway has been identified as a key molecular target of chemopreventive phase II inducers in several systems. Monitoring of phase II enzyme induction has led to identification of novel chemopreventive agents such as the isothiocyanate sulforaphane, and the 1,2-dithiole-3-thiones. However, no agents have yet demonstrated clear benefit in human cell systems, or in clinical trials. Alternative strategies include: (a) using intermediate cancer biomarkers for the endpoint in human trials; (b) high-throughput small molecule discovery approaches for induced expression of human phase II genes; and (c) integrative approaches that consider pharmacogenetics, along with pharmacokinetics and pharmacodynamics in target lung tissue. These approaches may lead to a more effective strategy of tailored chemoprevention efforts using compounds with proven human activity.

  6. Oxidative bioelectrocatalysis: From natural metabolic pathways to synthetic metabolons and minimal enzyme cascades.

    PubMed

    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.

  7. Effect of urethane, dimethylnitrosamine, paraquat, and butylated hydroxytoluene on the activities of glycolytic key enzymes in mouse lung

    SciTech Connect

    Arany, I.; Rady, P.; Bojan, I.; Kertai, P.

    1981-12-01

    Effects of carcinogens and noncarcinogenic pulmonary toxicants on the activities of glycolytic key enzymes in the mouse lung were investigated. The carcinogens urethane (URTH) and dimethylnitrosamine (DMN) permanently enhanced, and the noncarcinogenic pulmonary toxicants paraquat (PAR) and butylated hydroxytoluene (BHT) temporarily, enhanced the activities of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) in the lungs of mice.

  8. Key intestinal genes involved in lipoprotein metabolism are downregulated in dyslipidemic men with insulin resistance.

    PubMed

    Couture, Patrick; Tremblay, André J; Kelly, Isabelle; Lemelin, Valéry; Droit, Arnaud; Lamarche, Benoît

    2014-01-01

    Insulin resistance (IR) is associated with elevated plasma levels of triglyceride-rich lipoproteins (TRLs) of intestinal origin. However, the mechanisms underlying the overaccumulation of apolipoprotein (apo)B-48-containing TRLs in individuals with IR are not yet fully understood. This study examined the relationships between apoB-48-containing TRL kinetics and the expression of key intestinal genes and proteins involved in lipid/lipoprotein metabolism in 14 obese nondiabetic men with IR compared with 10 insulin-sensitive (IS) men matched for waist circumference. The in vivo kinetics of TRL apoB-48 were assessed using a primed-constant infusion of L-[5,5,5-D₃]leucine for 12 h with the participants in a constantly fed state. The expression of key intestinal genes and proteins involved in lipid/lipoprotein metabolism was assessed by performing real-time PCR quantification and LC-MS/MS on duodenal biopsy specimens. The TRL apoB-48 pool size and production rate were 102% (P < 0.0001) and 87% (P = 0.01) greater, respectively, in the men with IR versus the IS men. On the other hand, intestinal mRNA levels of sterol regulatory element binding factor-2, hepatocyte nuclear factor-4α, and microsomal triglyceride transfer protein were significantly lower in the men with IR than in the IS men. These data indicate that IR is associated with intestinal overproduction of lipoproteins and significant downregulation of key intestinal genes involved in lipid/lipoprotein metabolism.

  9. Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver

    PubMed Central

    Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao

    2015-01-01

    Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. PMID:26400395

  10. Metabolic stability: main enzymes involved and best tools to assess it.

    PubMed

    Laine, Romuald

    2008-11-01

    The introduction of in vitro tools to predict clearance in the early discovery process has led to new ways of working. Combined with metabolite identification, such tools have allowed design of compounds with low clearance. Encouraged by the success of such an approach and by the better knowledge of the enzyme involved in the metabolism, in vitro teams have begun to develop a plethora of assays to assess the metabolic clearance, understand the route of metabolism, and predict the human clearance. Although the diversity of assays may have allowed a more thorough approach to addressing specific issues, in the time of budget constrictions, limited access to resources and materials in vitro teams have now to decide what are the 'must have' and 'nice to have' assays to enable them to help as efficiently as possible projects at the discovery stage. Reducing the number of assays and focusing on the most relevant ones is an option to consider. Knowledge of the main enzymes involved in the drug metabolism should help to select the most relevant in vitro tools. Although the systems presented here have their merits, the author proposes that hepatocytes should be considered as the in vitro tool of choice.

  11. Mouse Genetics Suggests Cell-Context Dependency for Myc-Regulated Metabolic Enzymes during Tumorigenesis

    PubMed Central

    Nilsson, Lisa M.; Kreutzer, Christiane; Pretsch, Walter; Bornkamm, Georg W.; Nilsson, Jonas A.

    2012-01-01

    c-Myc (hereafter called Myc) belongs to a family of transcription factors that regulates cell growth, cell proliferation, and differentiation. Myc initiates the transcription of a large cast of genes involved in cell growth by stimulating metabolism and protein synthesis. Some of these, like those involved in glycolysis, may be part of the Warburg effect, which is defined as increased glucose uptake and lactate production in the presence of adequate oxygen supply. In this study, we have taken a mouse-genetics approach to challenge the role of select Myc-regulated metabolic enzymes in tumorigenesis in vivo. By breeding λ-Myc transgenic mice, Apc Min mice, and p53 knockout mice with mouse models carrying inactivating alleles of Lactate dehydrogenase A (Ldha), 3-Phosphoglycerate dehydrogenase (Phgdh) and Serine hydroxymethyltransferase 1 (Shmt1), we obtained offspring that were monitored for tumor development. Very surprisingly, we found that these genes are dispensable for tumorigenesis in these genetic settings. However, experiments in fibroblasts and colon carcinoma cells expressing oncogenic Ras show that these cells are sensitive to Ldha knockdown. Our genetic models reveal cell context dependency and a remarkable ability of tumor cells to adapt to alterations in critical metabolic pathways. Thus, to achieve clinical success, it will be of importance to correctly stratify patients and to find synthetic lethal combinations of inhibitors targeting metabolic enzymes. PMID:22438825

  12. Novel role of a triglyceride-synthesizing enzyme: DGAT1 at the crossroad between triglyceride and cholesterol metabolism

    PubMed Central

    Sachdev, Vinay; Leopold, Christina; Bauer, Raimund; Patankar, Jay V.; Iqbal, Jahangir; Obrowsky, Sascha; Boverhof, Renze; Doktorova, Marcela; Scheicher, Bernhard; Goeritzer, Madeleine; Kolb, Dagmar; Turnbull, Andrew V.; Zimmer, Andreas; Hoefler, Gerald; Hussain, M. Mahmood; Groen, Albert K.; Kratky, Dagmar

    2016-01-01

    Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1−/−) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1−/− and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia. PMID:27344248

  13. Metabolomic strategies for the identification of new enzyme functions and metabolic pathways

    PubMed Central

    Prosser, Gareth A; Larrouy-Maumus, Gerald; de Carvalho, Luiz Pedro S

    2014-01-01

    Recent technological advances in accurate mass spectrometry and data analysis have revolutionized metabolomics experimentation. Activity-based and global metabolomic profiling methods allow simultaneous and rapid screening of hundreds of metabolites from a variety of chemical classes, making them useful tools for the discovery of novel enzymatic activities and metabolic pathways. By using the metabolome of the relevant organism or close species, these methods capitalize on biological relevance, avoiding the assignment of artificial and non-physiological functions. This review discusses state-of-the-art metabolomic approaches and highlights recent examples of their use for enzyme annotation, discovery of new metabolic pathways, and gene assignment of orphan metabolic activities across diverse biological sources. PMID:24829223

  14. DMET™ (Drug Metabolism Enzymes and Transporters): a pharmacogenomic platform for precision medicine

    PubMed Central

    Arbitrio, Mariamena; Martino, Maria Teresa Di; Scionti, Francesca; Agapito, Giuseppe; Guzzi, Pietro Hiram; Cannataro, Mario

    2016-01-01

    In the era of personalized medicine, high-throughput technologies have allowed the investigation of genetic variations underlying the inter-individual variability in drug pharmacokinetics/pharmacodynamics. Several studies have recently moved from a candidate gene-based pharmacogenetic approach to genome-wide pharmacogenomic analyses to identify biomarkers for selection of patient-tailored therapies. In this aim, the identification of genetic variants affecting the individual drug metabolism is relevant for the definition of more active and less toxic treatments. This review focuses on the potentiality, reliability and limitations of the DMET™ (Drug Metabolism Enzymes and Transporters) Plus as pharmacogenomic drug metabolism multi-gene panel platform for selecting biomarkers in the final aim to optimize drugs use and characterize the individual genetic background. PMID:27304055

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

    PubMed

    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.

  16. Effects of liver diseases on drug-metabolizing enzymes: implications for drug fate alterations and nano-therapeutic openings.

    PubMed

    Lu, L; Shi, J; Li, Q; Peng, X; Dong, L; Li, Y; Dai, P; Wang, Y; Guo, E; Zhou, F; Liu, Z

    2014-01-01

    Metabolizing and eliminating toxic chemicals in the liver are key processes in the body's defense system. Drug-metabolizing enzymes (DMEs) play central roles in such processes. The activity and expression of several key DMEs are changed in various liver diseases and thus lead to significantly altered drug disposition. This phenomenon severely affects the pharmacotherapy of clinical medications in terms of the safety and efficacy of drug responses. This review highlights liver physiological functions, altered DMEs, and altered drug disposition in liver diseases. Moreover, the implications of changes in DMEs on the fate of clinically relevant drugs are also discussed. Pregnane X receptor and constitutive androstane receptor are two liver-enriched nuclear receptors originally defined as xenobiotic sensors that affect regulation of DMEs. Altered regulation of DMEs in liver diseases contributes to the development of powerful in vitro and in vivo tools to predict drug responses and options for improved drug delivery and development. Although a number of treatment drugs are available for liver diseases, they are limited by their low drug concentration in the target site, presence of side effects, and instability in the human body. The nanoparticle drug delivery system has recently attracted research attention because of its potential to offer solutions to current obstacles that involve the use of therapeutic drugs for liver diseases. Conclusively, this review aims to improve understanding on the regulation of DMEs in liver diseases and on corresponding implications in drug disposition, including novel therapeutic medications.

  17. The Stabilisation of Enzymes — a Key Factor in the Practical Application of Biocatalysis

    NASA Astrophysics Data System (ADS)

    Martinek, K.; Berezin, I. B.

    1980-05-01

    The present state of the problem of the practical application of biocatalysis is examined and physicochemical approaches whereby the denaturation of enzymes under the influence of elevated temperatures, extreme pH values, and organic solvents can be suppressed are analysed. The general principles of the stabilisation of enzymes are formulated. The bibliography includes 225 references.

  18. Multi-Omics Reveals that Lead Exposure Disturbs Gut Microbiome Development, Key Metabolites, and Metabolic Pathways.

    PubMed

    Gao, Bei; Chi, Liang; Mahbub, Ridwan; Bian, Xiaoming; Tu, Pengcheng; Ru, Hongyu; Lu, Kun

    2017-03-16

    Lead exposure remains a global public health issue, and the recent Flint water crisis has renewed public concern about lead toxicity. The toxicity of lead has been well established in a variety of systems and organs. The gut microbiome has been shown to be highly involved in many critical physiological processes, including food digestion, immune system development, and metabolic homeostasis. However, despite the key role of the gut microbiome in human health, the functional impact of lead exposure on the gut microbiome has not been studied. The aim of this study is to define gut microbiome toxicity induced by lead exposure in C57BL/6 mice using multiomics approaches, including 16S rRNA sequencing, whole genome metagenomics sequencing, and gas chromatography-mass spectrometry (GC-MS) metabolomics. 16S rRNA sequencing revealed that lead exposure altered the gut microbiome trajectory and phylogenetic diversity. Metagenomics sequencing and metabolomics profiling showed that numerous metabolic pathways, including vitamin E, bile acids, nitrogen metabolism, energy metabolism, oxidative stress, and the defense/detoxification mechanism, were significantly disturbed by lead exposure. These perturbed molecules and pathways may have important implications for lead toxicity in the host. Taken together, these results demonstrated that lead exposure not only altered the gut microbiome community structures/diversity but also greatly affected metabolic functions, leading to gut microbiome toxicity.

  19. Genetic polymorphisms in human drug-metabolizing enzymes: potential uses of reverse genetics to identify genes of toxicological relevance.

    PubMed

    Puga, A; Nebert, D W; McKinnon, R A; Menon, A G

    1997-03-01

    The human mind was engaged with fundamental questions on the nature of heredity long before the study of genetics became a scientific discipline. Many traits, such as height, eye color, blood pressure, or cancer susceptibility, have been known to run in families, although the genes or combination of genes that underlie these observable characteristics remain unknown in most cases. Differences in susceptibility to environmental agents in humans are likewise determined by variations in genetic background--genetic polymorphisms. In this article, we review the current status of studies on human polymorphisms in drug-metabolizing enzymes and discuss various approaches to the analysis of genetic polymorphisms. We expect that in the near future, novel methods in genetic analysis of human populations will be likely to play a key role in the identification of genes of toxicological relevance.

  20. Detection of sugar accumulation and expression levels of correlative key enzymes in winter wheat (Triticum aestivum) at low temperatures.

    PubMed

    Zeng, Yan; Yu, Jing; Cang, Jing; Liu, Lijie; Mu, Yongchao; Wang, Junhong; Zhang, Da

    2011-01-01

    Carbohydrate accumulation is common in frost-resistant plants, and many enzymes participate in this process. The sugar content and expression levels of metabolic enzymes related to sugar biosynthesis in response to drops in temperature were measured in two cultivars of winter wheat (Triticum aestivum) with different cold tolerances. The results indicate that the two cultivars examined, Dongnongdongmai 1 and Jimai 22, accumulated high levels of carbohydrate before November 4 (above 0°C), and that accumulation decreased as temperatures fell. However, this decrease was more modest in Dongnongdongmai 1, which had a higher sugar content. Sucrose and fructose were the main soluble sugars, indicating an important role in freezing tolerance. Gene expression studies revealed that expression of the genes encoding chloroplastic enzymes was significantly upregulated in the tillering nodes. Expression upregulation of TaSS and TaTPT may be helpful for sugar accumulation before November 4.

  1. Impact of dioxin-type induction of drug-metabolizing enzymes on the metabolism of endo- and xenobiotics.

    PubMed

    Schrenk, D

    1998-04-15

    The induction of a number of drug-metabolizing enzymes is among the best-understood biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related agonists of the aryl hydrocarbon receptor (AhR). Among the cytochrome P450s (CYPs), the genes encoding CYP1A1, 1A2, and 1B1 are responsive to AhR agonists, i.e. their expression is inducible in various mammalian tissues and organs as well as in many types of cell lines and primary cells in culture. In addition, an aldehyde dehydrogenase, an NADPH-quinone-oxidoreductase, and the phase II conjugating enzymes glutathione-S-transferase (GST) Ya and UDP-glucuronosyltransferase 1A1 have been identified as responsive to AhR agonists. Induced expression of these members of the AhR gene battery is thought to be aimed at an improved elimination of the inducing agent and its metabolites. However, the identity of the physiological ligand(s) of the AhR is still obscure. The consequences of induced expression of AhR-regulated genes encoding drug-metabolizing enzymes have been investigated in human populations, e.g. in smokers, and in various experimental models. A prominent example of increased adverse effects due to the induction of CYP1A isozymes is the metabolic activation of carcinogenic aromatic amines and polycyclic aromatic hydrocarbons. An increasing amount of data is also available on the impact of dioxin-type induction on the metabolism of drugs, food constituents, and endogenous substrates. For example, the hepatic clearance of the drug theophylline, which is widely used in asthma therapy, is enhanced significantly in smokers. Increased glucuronidation of thyroxine in rats treated with TCDD or other potent AhR agonists is thought to result in hypothyroxinemia and its biological consequences, such as sustained hyperplasia of the thyroid, bearing a higher risk of thyroid cancer. The relevance of these observations for humans exposed to dioxin-type inducers is discussed.

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

    PubMed

    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.

  3. Gestational Age-Dependent Changes in Gene Expression of Metabolic Enzymes and Transporters in Pregnant Mice

    PubMed Central

    Shuster, Diana L.; Bammler, Theo K.; Beyer, Richard P.; MacDonald, James W.; Tsai, Jesse M.; Farin, Frederico M.; Hebert, Mary F.; Thummel, Kenneth E.

    2013-01-01

    Pregnancy-induced changes in drug pharmacokinetics can be explained by changes in expression of drug-metabolizing enzymes and transporters and/or normal physiology. In this study, we determined gestational age-dependent expression profiles for all metabolic enzyme and transporter genes in the maternal liver, kidney, small intestine, and placenta of pregnant mice by microarray analysis. We specifically examined the expression of genes important for xenobiotic, bile acid, and steroid hormone metabolism and disposition, namely, cytochrome P450s (Cyp), UDP-glucuronosyltranserases (Ugt), sulfotransferases (Sult), and ATP-binding cassette (Abc), solute carrier (Slc), and solute carrier organic anion (Slco) transporters. Few Ugt and Sult genes were affected by pregnancy. Cyp17a1 expression in the maternal liver increased 3- to 10-fold during pregnancy, which was the largest observed change in the maternal tissues. Cyp1a2, most Cyp2 isoforms, Cyp3a11, and Cyp3a13 expression in the liver decreased on gestation days (gd) 15 and 19 compared with nonpregnant controls (gd 0). In contrast, Cyp2d40, Cyp3a16, Cyp3a41a, Cyp3a41b, and Cyp3a44 in the liver were induced throughout pregnancy. In the placenta, Cyp expression on gd 10 and 15 was upregulated compared with gd 19. Notable changes were also observed in Abc and Slc transporters. Abcc3 expression in the liver and Abcb1a, Abcc4, and Slco4c1 expression in the kidney were downregulated on gd 15 and 19. In the placenta, Slc22a3 (Oct3) expression on gd 10 was 90% lower than that on gd 15 and 19. This study demonstrates important gestational age-dependent expression of metabolic enzyme and transporter genes, which may have mechanistic relevance to drug disposition in human pregnancy. PMID:23175668

  4. CO2-fixing enzymes and phosphoenolpyruvate metabolism in the fish parasite Hysterothylacium aduncum (Ascaridoidea, Anisakidae).

    PubMed

    Malagón, David; Benítez, Rocio; Valero, Adela; Adroher, Francisco Javier

    2009-07-23

    CO2 stimulates the development of many of the intestinal helminths that are able to fix CO2 by means of phosphoenolpyruvate carboxykinase (PEPCK), such as Hysterothylacium aduncum. We determined the activity of CO2-fixing enzymes such as PEPCK and phosphoenolpyruvate carboxylase (PEPC), although no significant activity was detected for pyruvate carboxylase or carboxylating-malic enzyme. The former act on phosphoenolpyruvate (PEP) to yield oxalacetate. In the helminths studied, PEP has a vital role in glucidic metabolism. Consequently, we determined the activity of other enzymes involved in the crossroad of PEP, such as pyruvate kinase (PK), lactate dehydrogenase and malate dehydrogenase. All enzymes detected showed significant variations in activity during the in vitro development of the parasite from the third larval stage to mature adult. Fixing of CO2 by PEPCK decreased during development (from 228 to 115 nmol min(-1) mg(-1) protein), while that by PEPC increased (from 19 to 46 nmol min(-1) mg(-1) protein). This enzyme, which is rare in animals, could play a part in detecting levels of free phosphate, releasing it from PEP when required for processes such as glycogenolysis, glycolysis and adenosine 5'-triphosphate (ATP) synthesis. PK, which showed increasing activity during development up to immature adult (from 56 to 82 nmol min(-1) mg(-1) protein), could act in combination with PEPC to obtain energy in the cytosol (in the form of ATP) and in the mitochondria (possible destination of the pyruvate formed), compensating for the decrease in activity of PEPCK.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26378592

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

    PubMed

    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.

  7. Efficient production of optically pure L-lactic acid from food waste at ambient temperature by regulating key enzyme activity.

    PubMed

    Li, Xiang; Chen, Yinguang; Zhao, Shu; Chen, Hong; Zheng, Xiong; Luo, Jinyang; Liu, Yanan

    2015-03-01

    Bio-production of optically pure L-lactic acid from food waste has attracted much interest as it can treat organic wastes with simultaneous recovery of valuable by-products. However, the yield of L-lactic acid was very low and no optically pure L-lactic acid was produced in the literature due to (1) the lower activity of enzymes involved in hydrolysis and L-lactic acid generation, and (2) the participation of other enzymes related to D-lactic acid and acetic and propionic acids production. In this paper, a new strategy was reported for effective production of optically pure L-lactic acid from food waste at ambient temperature, i.e. via regulating key enzyme activity by sewage sludge supplement and intermittent alkaline fermentation. It was found that not only optically pure L-lactic acid was produced, but the yield was enhanced by 2.89-fold. The mechanism study showed that the activities of enzymes relevant to food waste hydrolysis and lactic acid production were enhanced, and the key enzymes related to volatile fatty acids and D-lactic acid generations were severally decreased or inhibited. Also, the microbes responsible for L-lactic acid production were selectively proliferated. Finally, the pilot-scale continuous experiment was conducted to testify the feasibility of this new technique.

  8. Human microsomal carbonyl reducing enzymes in the metabolism of xenobiotics: well-known and promising members of the SDR superfamily.

    PubMed

    Skarydová, Lucie; Wsól, Vladimír

    2012-05-01

    The best known, most widely studied enzyme system in phase I biotransformation is cytochrome P450 (CYP), which participates in the metabolism of roughly 9 of 10 drugs in use today. The main biotransformation isoforms of CYP are associated with the membrane of the endoplasmatic reticulum (ER). Other enzymes that are also active in phase I biotransformation are carbonyl reducing enzymes. Much is known about the role of cytosolic forms of carbonyl reducing enzymes in the metabolism of xenobiotics, but their microsomal forms have been mostly poorly studied. The only well-known microsomal carbonyl reducing enzyme taking part in the biotransformation of xenobiotics is 11β-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase superfamily. Physiological roles of microsomal carbonyl reducing enzymes are better known than their participation in the metabolism of xenobiotics. This review is a summary of the fragmentary information known about the roles of the microsomal forms. Besides 11β-hydroxysteroid dehydrogenase 1, it has been reported, so far, that retinol dehydrogenase 12 participates only in the detoxification of unsaturated aldehydes formed upon oxidative stress. Another promising group of microsomal biotransformation carbonyl reducing enzymes are some members of 17β-hydroxysteroid dehydrogenases. Generally, it is clear that this area is, overall, quite unexplored, but carbonyl reducing enzymes located in the ER have proven very interesting. The study of these enzymes could shed new light on the metabolism of several clinically used drugs or they could become an important target in connection with some diseases.

  9. Impaired energy metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes.

    PubMed

    Baraibar, Martín A; Hyzewicz, Janek; Rogowska-Wrzesinska, Adelina; Bulteau, Anne-Laure; Prip-Buus, Carina; Butler-Browne, Gillian; Friguet, Bertrand

    2016-12-04

    Accumulation of oxidized proteins is a hallmark of cellular and organismal aging. Adult muscle stem cell (or satellite cell) replication and differentiation is compromised with age contributing to sarcopenia. However, the molecular events related to satellite cell dysfunction during aging are not completely understood. In the present study we have addressed the potential impact of oxidatively modified proteins on the altered metabolism of senescent human satellite cells. By using a modified proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during the replicative senescence of satellite cells. Inactivation of the proteasome appeared to be a likely contributor to the accumulation of such damaged proteins. Metabolic and functional analyses revealed an impaired glucose metabolism in senescent cells. A metabolic shift leading to increased mobilization of non-carbohydrate substrates such as branched chain amino acids or long chain fatty acids was observed. Increased levels of acyl-carnitines indicated an increased turnover of storage and membrane lipids for energy production. Taken together, these results support a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts.

  10. Disturbances to neurotransmitter levels and their metabolic enzyme activity in a freshwater planarian exposed to cadmium.

    PubMed

    Wu, Jui-Pin; Li, Mei-Hui; Chen, Jhih-Sheng; Chung, Szu-Yao; Lee, Hui-Ling

    2015-03-01

    Using specific neurobehaviors as endpoints, previous studies suggested that planarian neurotransmission systems could be targets of Cd neurotoxicity. However, direct evidence for disturbed neurotransmission systems by Cd in treated planarians is still lacking. In planarians, dopamine (DA) and serotonin (5-HT) play critical roles in neuromuscular function, but little is known about their metabolic degradation. Therefore, in this study, we attempted to determine the appearances of DA, 5-HT, and their metabolic products in the freshwater planarian Dugesia japonica, characterize the activity of enzymes involved in their metabolism, and investigate the effects of Cd on planarian 5-HTergic and DAergic neurotransmission systems. Only DA, 5-HT, and 5-hydroxyindole-3-acetic acid (5-HIAA) were found in planarian tissues. Further enzymatic study revealed the activity of planarian monoamine oxidase (MAO) but not catechol-O-methyl transferase (COMT). These findings suggest that planarian MAO catalyzes the metabolism of 5-HT into 5-HIAA. However, DA metabolites from the MAO-involved metabolic pathway were not found, which might be due to a lack of COMT activity. Finally, in Cd-treated planarians, tissue levels of 5-HT and DA were decreased and MAO activity altered, suggesting that planarian neurotransmission systems are disturbed following Cd treatment.

  11. Carbohydrate metabolism in Archaea: current insights into unusual enzymes and pathways and their regulation.

    PubMed

    Bräsen, Christopher; Esser, Dominik; Rauch, Bernadette; Siebers, Bettina

    2014-03-01

    The metabolism of Archaea, the third domain of life, resembles in its complexity those of Bacteria and lower Eukarya. However, this metabolic complexity in Archaea is accompanied by the absence of many "classical" pathways, particularly in central carbohydrate metabolism. Instead, Archaea are characterized by the presence of unique, modified variants of classical pathways such as the Embden-Meyerhof-Parnas (EMP) pathway and the Entner-Doudoroff (ED) pathway. The pentose phosphate pathway is only partly present (if at all), and pentose degradation also significantly differs from that known for bacterial model organisms. These modifications are accompanied by the invention of "new," unusual enzymes which cause fundamental consequences for the underlying regulatory principles, and classical allosteric regulation sites well established in Bacteria and Eukarya are lost. The aim of this review is to present the current understanding of central carbohydrate metabolic pathways and their regulation in Archaea. In order to give an overview of their complexity, pathway modifications are discussed with respect to unusual archaeal biocatalysts, their structural and mechanistic characteristics, and their regulatory properties in comparison to their classic counterparts from Bacteria and Eukarya. Furthermore, an overview focusing on hexose metabolic, i.e., glycolytic as well as gluconeogenic, pathways identified in archaeal model organisms is given. Their energy gain is discussed, and new insights into different levels of regulation that have been observed so far, including the transcript and protein levels (e.g., gene regulation, known transcription regulators, and posttranslational modification via reversible protein phosphorylation), are presented.

  12. Impaired energy metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes

    PubMed Central

    Baraibar, Martín A.; Hyzewicz, Janek; Rogowska-Wrzesinska, Adelina; Bulteau, Anne-Laure; Prip-Buus, Carina; Butler-Browne, Gillian; Friguet, Bertrand

    2016-01-01

    Accumulation of oxidized proteins is a hallmark of cellular and organismal aging. Adult muscle stem cell (or satellite cell) replication and differentiation is compromised with age contributing to sarcopenia. However, the molecular events related to satellite cell dysfunction during aging are not completely understood. In the present study we have addressed the potential impact of oxidatively modified proteins on the altered metabolism of senescent human satellite cells. By using a modified proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during the replicative senescence of satellite cells. Inactivation of the proteasome appeared to be a likely contributor to the accumulation of such damaged proteins. Metabolic and functional analyses revealed an impaired glucose metabolism in senescent cells. A metabolic shift leading to increased mobilization of non-carbohydrate substrates such as branched chain amino acids or long chain fatty acids was observed. Increased levels of acyl-carnitines indicated an increased turnover of storage and membrane lipids for energy production. Taken together, these results support a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts. PMID:27922824

  13. Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation.

    PubMed

    Ermler, U; Grabarse, W; Shima, S; Goubeaud, M; Thauer, R K

    1997-11-21

    Methyl-coenzyme M reductase (MCR), the enzyme responsible for the microbial formation of methane, is a 300-kilodalton protein organized as a hexamer in an alpha2beta2gamma2 arrangement. The crystal structure of the enzyme from Methanobacterium thermoautotrophicum, determined at 1.45 angstrom resolution for the inactive enzyme state MCRox1-silent, reveals that two molecules of the nickel porphinoid coenzyme F430 are embedded between the subunits alpha, alpha', beta, and gamma and alpha', alpha, beta', and gamma', forming two identical active sites. Each site is accessible for the substrate methyl-coenzyme M through a narrow channel locked after binding of the second substrate coenzyme B. Together with a second structurally characterized enzyme state (MCRsilent) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.

  14. The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis

    SciTech Connect

    Viola, Ronald E.; Faehnle, Christopher R.; Blanco, Julio; Moore, Roger A.; Liu, Xuying; Arachea, Buenafe T.; Pavlovsky, Alexander G.

    2013-02-28

    The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate {beta}-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

  15. Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus.

    PubMed

    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. Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

    PubMed

    Dulermo, Thierry; Lazar, Zbigniew; Dulermo, Rémi; Rakicka, Magdalena; Haddouche, Ramedane; Nicaud, Jean-Marc

    2015-09-01

    The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

  17. Pharmacogenetic profile of xenobiotic enzyme metabolism in survivors of the Spanish toxic oil syndrome.

    PubMed Central

    Ladona, M G; Izquierdo-Martinez, M; Posada de la Paz, M P; de la Torre, R; Ampurdanés, C; Segura, J; Sanz, E J

    2001-01-01

    In 1981, the Spanish toxic oil syndrome (TOS) affected more than 20,000 people, and over 300 deaths were registered. Assessment of genetic polymorphisms on xenobiotic metabolism would indicate the potential metabolic capacity of the victims at the time of the disaster. Thus, impaired metabolic pathways may have contributed to the clearance of the toxicant(s) leading to a low detoxification or accumulation of toxic metabolites contributing to the disease. We conducted a matched case-control study using 72 cases (54 females, 18 males) registered in the Official Census of Affected Patients maintained by the Spanish government. Controls were nonaffected siblings (n =72) living in the same household in 1981 and nonaffected nonrelatives (n = 70) living in the neighborhood at that time, with no ties to TOS. Genotype analyses were performed to assess the metabolic capacity of phase I [cytochrome P450 1A1 (CYP1A1), CYP2D6] and phase II [arylamine N-acetyltransferase-2 (NAT2), GSTM1 (glutathione S-transferase M1) and GSTT1] enzyme polymorphisms. The degree of association of the five metabolic pathways was estimated by calculating their odds ratios (ORs) using conditional logistic regression analysis. In the final model, cases compared with siblings (72 pairs) showed no differences either in CYP2D6 or CYP1A1 polymorphisms, or in conjugation enzyme polymorphisms, whereas cases compared with the unrelated controls (70 pairs) showed an increase in NAT2 defective alleles [OR = 6.96, 95% confidence interval (CI), 1.46-33.20] adjusted by age and sex. Glutathione transferase genetic polymorphisms (GSTM1, GSTT1) showed no association with cases compared with their siblings or unrelated controls. These findings suggest a possible role of impaired acetylation mediating susceptibility in TOS. PMID:11335185

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

    NASA Technical Reports Server (NTRS)

    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. Seasonal changes in thermal environment and metabolic enzyme activity in the diamondback terrapin (Malaclemys terrapin).

    PubMed

    Williard, Amanda Southwood; Harden, Leigh Anne

    2011-04-01

    Diamondback terrapins experience broad fluctuations in temperature on both a daily and seasonal basis in their estuarine environment. We measured metabolic enzyme activity in terrapin muscle tissue to assess thermal dependence and the role of temperature in seasonal metabolic downregulation in this species. Activity of lactate dehydrogenase (LDH), pyruvate kinase (PK), citrate synthase (CS), and cytochrome c oxidase (CCO) was assayed at 10, 20, 30, and 40 °C for tissue collected during summer and winter. The Q(10) for enzyme activity varied between 1.31 and 2.11 within the temperature range at which terrapins were active (20-40 °C). The Q(10) for LDH, CS, and CCO varied between 1.39 and 1.76 and between 10 and 20 °C, but PK exhibited heightened thermal sensitivity within this lower temperature range, with a Q(10) of 2.90 for summer-collected tissue and 5.55 for winter-collected tissue. There was no significant effect of season on activity of LDH or PK, but activity of CS and CCO was significantly lower in winter-collected tissue compared with summer-collected tissue. Results indicate that temperature effects contribute to seasonal metabolic downregulation and dormancy in terrapins, but other environmental factors (i.e. oxygen availability), as well as seasonal shifts in blood biochemistry and circulating hormones may also play an important role.

  20. Heterogeneous composition of key metabolic gene clusters in a vent mussel symbiont population

    PubMed Central

    Ikuta, Tetsuro; Takaki, Yoshihiro; Nagai, Yukiko; Shimamura, Shigeru; Tsuda, Miwako; Kawagucci, Shinsuke; Aoki, Yui; Inoue, Koji; Teruya, Morimi; Satou, Kazuhito; Teruya, Kuniko; Shimoji, Makiko; Tamotsu, Hinako; Hirano, Takashi; Maruyama, Tadashi; Yoshida, Takao

    2016-01-01

    Chemosynthetic symbiosis is one of the successful systems for adapting to a wide range of habitats including extreme environments, and the metabolic capabilities of symbionts enable host organisms to expand their habitat ranges. However, our understanding of the adaptive strategies that enable symbiotic organisms to expand their habitats is still fragmentary. Here, we report that a single-ribotype endosymbiont population in an individual of the host vent mussel, Bathymodiolus septemdierum has heterogeneous genomes with regard to the composition of key metabolic gene clusters for hydrogen oxidation and nitrate reduction. The host individual harbours heterogeneous symbiont subpopulations that either possess or lack the gene clusters encoding hydrogenase or nitrate reductase. The proportions of the different symbiont subpopulations in a host appeared to vary with the environment or with the host's development. Furthermore, the symbiont subpopulations were distributed in patches to form a mosaic pattern in the gill. Genomic heterogeneity in an endosymbiont population may enable differential utilization of diverse substrates and confer metabolic flexibility. Our findings open a new chapter in our understanding of how symbiotic organisms alter their metabolic capabilities and expand their range of habitats. PMID:26418631

  1. DHN melanin biosynthesis in the plant pathogenic fungus Botrytis cinerea is based on two developmentally regulated key enzyme (PKS)-encoding genes.

    PubMed

    Schumacher, Julia

    2016-02-01

    Botrytis cinerea is the causal agent of gray mold disease in various plant species and produces grayish macroconidia and/or black sclerotia at the end of the infection cycle. It has been suggested that the pigmentation is due to the accumulation of 1,8-dihydroxynaphthalene (DHN) melanin. To unravel its basis and regulation, the putative melanogenic and regulatory genes were identified and functionally characterized. Unlike other DHN melanin-producing fungi, B. cinerea and other Leotiomycetes contain two key enzyme (PKS)-encoding enzymes. Bcpks12 and bcpks13 are developmentally regulated and are required for melanogenesis in sclerotia and conidia respectively. BcYGH1 converts the BcPKS13 product and contributes thereby to conidial melanogenesis. In contrast, enzymes acting downstream in conversion of the PKS products (BcBRN2, BcSCD1 and BcBRN1) are required for both, sclerotial and conidial melanogenesis, suggesting that DHN melanogenesis in B. cinerea follows a non-linear pathway that is rather unusual for secondary metabolic pathways. Regulation of the melanogenic genes involves three pathway-specific transcription factors (TFs) that are clustered with bcpks12 or bcpks13 and other developmental regulators such as light-responsive TFs. Melanogenic genes are dispensable in vegetative mycelia for proper growth and virulence. However, DHN melanin is considered to contribute to the longevity of the reproduction structures.

  2. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement.

    PubMed Central

    Mulquiney, P J; Kuchel, P W

    1999-01-01

    Over the last 25 years, several mathematical models of erythrocyte metabolism have been developed. Although these models have identified the key features in the regulation and control of erythrocyte metabolism, many important aspects remain unexplained. In particular, none of these models have satisfactorily accounted for 2,3-bisphosphoglycerate (2,3-BPG) metabolism. 2,3-BPG is an important modulator of haemoglobin oxygen affinity, and hence an understanding of the regulation of 2,3-BPG concentration is important for understanding blood oxygen transport. A detailed, comprehensive, and hence realistic mathematical model of erythrocyte metabolism is presented that can explain the regulation and control of 2,3-BPG concentration and turnover. The model is restricted to the core metabolic pathways, namely glycolysis, the 2,3-BPG shunt and the pentose phosphate pathway (PPP), and includes membrane transport of metabolites, the binding of metabolites to haemoglobin and Mg(2+), as well as pH effects on key enzymic reactions and binding processes. The model is necessarily complex, since it is intended to describe the regulation and control of 2,3-BPG metabolism under a wide variety of physiological and experimental conditions. In addition, since H(+) and blood oxygen tension are important external effectors of 2,3-BPG concentration, it was important that the model take into account the large array of kinetic and binding phenomena that result from changes in these effectors. Through an iterative loop of experimental and simulation analysis many values of enzyme-kinetic parameters of the model were refined to yield close conformity between model simulations and 'real' experimental data. This iterative process enabled a single set of parameters to be found which described well the metabolic behaviour of the erythrocyte under a wide variety of conditions. PMID:10477269

  3. A novel "complement-metabolism-inflammasome axis" as a key regulator of immune cell effector function.

    PubMed

    Arbore, Giuseppina; Kemper, Claudia

    2016-07-01

    The inflammasomes are intracellular multiprotein complexes that induce and regulate the generation of the key pro-inflammatory cytokines IL-1β and IL-18 in response to infectious microbes and cellular stress. The activation of inflammasomes involves several upstream signals including classic pattern or danger recognition systems such as the TLRs. Recently, however, the activation of complement receptors, such as the anaphylatoxin C3a and C5a receptors and the complement regulator CD46, in conjunction with the sensing of cell metabolic changes, for instance increased amino acid influx and glycolysis (via mTORC1), have emerged as additional critical activators of the inflammasome. This review summarizes recent advances in our knowledge about complement-mediated inflammasome activation, with a specific focus on a novel "complement - metabolism - NLRP3 inflammasome axis."

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

    NASA Astrophysics Data System (ADS)

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

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

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

    SciTech Connect

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

    2008-04-02

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

  6. Metabolism of (+)-terpinen-4-ol by cytochrome P450 enzymes in human liver microsomes.

    PubMed

    Haigou, Risa; Miyazawa, Mitsuo

    2012-01-01

    We examined the in vitro metabolism of (+)-terpinen-4-ol by human liver microsomes and recombinant enzymes. The biotransformation of (+)-terpinen-4-ol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Terpinen-4-ol was found to be oxidized to (+)-(1R,2S,4S)-1,2-epoxy-p-menthan-4-ol, (+)-(1S,2R,4S)-1,2-epoxy-p-menthan-4-ol, and (4S)-p-menth-1-en-4,8-diol by human liver microsomal P450 enzymes. The identities of (+)-terpinen-4-ol metabolites were determined through the relative abundance of mass fragments and retention times on GC-MS. Of 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6, and CYP3A4 were found to catalyze the oxidation of (+)-terpinen-4-ol. Based on several lines of evidence, CYP2A6 and CYP3A4 were determined to be major enzymes involved in the oxidation of (+)-terpinen-4-ol by human liver microsomes. First, of the 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6 and CYP3A4 catalyzed oxidation of (+)-terpinen-4-ol. Second, oxidation of (+)-terpinen-4-ol was inhibited by (+)-menthofuran and ketoconazole, inhibitors known to be specific for these enzymes. Finally, there was a good correlation between CYP2A6 and CYP3A4 activities and (+)-terpinen-4-ol oxidation activities in the 10 human liver microsomes.

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

    PubMed

    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 < 0.01). Meanwhile, the mRNA abundance of oxidative and intermediate fibers was increased in Jinhua pigs, whereas the glycolytic fibers were more highly expressed in the Landrace (P < 0.01). In addition, Jinhua pigs possessed greater oxidative capacity than that of the Landrace (P < 0.05). These results suggested that the increased expression of the oxidative and intermediate fibers and greater activities of oxidative enzymes in Jinhua pigs were related to meat quality as indicated by a greater intramuscular fat and reduced drip loss. Based on these results, we conclude that muscle fiber composition and postmortem muscle metabolism can explain, in part, the variation of meat quality in Jinhua and Landrace pigs. These results may provide valuable information for understanding the molecular mechanism responsible for breed specific differences in growth performance

  8. In Vivo Exposure of Kaempferol Is Driven by Phase II Metabolic Enzymes and Efflux Transporters.

    PubMed

    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).

  9. Calcium-regulated nuclear enzymes: potential mediators of phytochrome-induced changes in nuclear metabolism?

    NASA Technical Reports Server (NTRS)

    Roux, S. J.

    1992-01-01

    Calcium ions have been proposed to serve as important regulatory elements in stimulus-response coupling for phytochrome responses. An important test of this hypothesis will be to identify specific targets of calcium action that are required for some growth or development process induced by the photoactivated form of phytochrome (Pfr). Initial studies have revealed that there are at least two enzymes in pea nuclei that are stimulated by Pfr in a Ca(2+)-dependent fashion, a calmodulin-regulated nucleoside triphosphatase and a calmodulin-independent but Ca(2+)-dependent protein kinase. The nucleoside triphosphatase appears to be associated with the nuclear envelope, while the protein kinase co-purifies with a nuclear fraction highly enriched for chromatin. This short review summarizes the latest findings on these enzymes and relates them to what is known about Pfr-regulated nuclear metabolism.

  10. In vivo cytochrome P450 drug metabolizing enzyme characterization using surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    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.

  11. A metabolic node in action: chorismate-utilizing enzymes in microorganisms.

    PubMed

    Dosselaere, F; Vanderleyden, J

    2001-01-01

    The shikimate pathway has been described as a metabolic tree with many branches that led to the synthesis of an extensive range of products. This pathway is present only in bacteria, fungi, and plants. While there is only little difference in the sequence of the chemical reactions of the pathway, significant differences exist in terms of organization and regulation. In the main trunk of the shikimate pathway, D-erythrose 4-phosphate and phosphoenolpyruvate are converted via shikimate to chorismate. Chorismate is the common precursor for the biosynthesis of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, but also for other products as diverse as folate cofactors, benzoid and naphthoid coenzymes, phenazines, and siderophores. Five chorismate-utilizing enzymes have been characterized in microorganisms: chorismate mutase, anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and chorismate pyruvate-lyase. In this review these enzymes are discussed in terms of the corresponding gene structures and regulation, nucleotide and protein sequences, protein structures, and reaction mechanisms. The main emphasis is on transcriptional and posttranslational regulatory mechanisms, in view of how a microbial cell exploits its chorismate pool in diverse anabolic pathways. Comparison of the chorismate-utilizing enzymes has shown that some of them share sequence similarity, suggesting divergent evolution and commonality in reaction mechanisms. However, other chorismate-utilizing enzymes are examples of convergent evolution toward similar reaction capabilities.

  12. Systems biology approaches to enzyme kinetics: analyzing network models of drug metabolism.

    PubMed

    Finn, Nnenna A; Kemp, Melissa L

    2014-01-01

    Intracellular drug metabolism involves transport, bioactivation, conjugation, and other biochemical steps. The dynamics of these steps are each dependent on a number of other cellular factors that can ultimately lead to unexpected behavior. In this review, we discuss the confounding processes and coupled reactions within bioactivation networks that require a systems-level perspective in order to fully understand the time-varying behavior. When converting known in vitro characteristics of drug-enzyme interactions into descriptions of cellular systems, features such as substrate availability, cell-to-cell variability, and intracellular redox state deserve special focus. An example of doxorubicin bioactivation is used for discussing points of consideration when constructing and analyzing network models of drug metabolism.

  13. Characterization of benidipine and its enantiomers' metabolism by human liver cytochrome P450 enzymes.

    PubMed

    Yoon, Yune-Jung; Kim, Kwon-Bok; Kim, Hyunmi; Seo, Kyung-Ah; Kim, Ho-Sook; Cha, In-June; Kim, Eun-Young; Liu, Kwang-Hyeon; Shin, Jae-Gook

    2007-09-01

    Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)-alpha and (+)-alpha isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CL(int)) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-alpha isomer was similar to those from the (+)-alpha isomer (1.9 +/- 0.1 versus 2.3 +/- 2.3 microl/min/pmol P450 and 0.5 +/- 0.2 versus 0.6 +/- 0.6 microl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CL(int) values of CYP3A4-mediated metabolite formation from (-)-alpha isomer were similar to those from (+)-alpha isomer (17.7 versus 14.4 microl/min/pmol P450, respectively). The total CL(int) values of CYP3A5-mediated metabolite formation from (-)-alpha isomer were also similar to those from (+)-alpha isomer (8.3 versus 11.0 microl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors.

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

    NASA Astrophysics Data System (ADS)

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

    We have examined, in the livers of rats carried aboard the Cosmos 936 biosatellite, the activities of about 30 enzymes concerned with carbohydrate and lipid metabolism. In addition to the enzyme studies, the levels of glycogen and of the individual fatty acids in hepatic lipids were determined. Livers from flight and ground control rats at recovery (R0) and 25 days after recovery (R25) were used for these analyses. For all parameters measured, the most meaningful comparisons are those made between flight stationary (FS) and flight centrifuged (FC) animals at R0. When these two groups of flight rats were compared at R0, statistically significant decreases in the activity levels of glycogen phosphorylase, α-glycerol phosphate acyl transferase, diglyceride acyl transferase, aconitase and 6-phosphogluconate dehydrogenase and an increase in the palmitoyl CoA desaturase were noted in the weightless group (FS). The significance of these findings was strengthened by the fact that all enzyme activities showing alterations at R0 returned to normal 25 days postflight. When liver glycogen and total fatty acids of the two sets of flight animals were determined, significant differences that could be attributed to reduced gravity were observed. The weightless group (FS) at R0 contained, on the average, more than twice the amount of glycogen than did the centrifuged controls (FC) and a remarkable shift in the ratio of palmitate to palmitoleate was noted. These metabolic alterations, both in enzyme levels and in hepatic constituents, appear to be characteristic of the weightless condition. Our data seem to justify the conclusion that centrifugation during flight is equivalent to terrestrial gravity.

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

    PubMed

    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.

  16. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data

    PubMed Central

    Barker, Brandon E.; Smallbone, Kieran; Myers, Christopher R.; Xi, Hongwei; Locasale, Jason W.; Gu, Zhenglong

    2015-01-01

    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 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. PMID:26381164

  17. Analysis of celery (Apium graveolens) mannitol dehydrogenase (Mtd) promoter regulation in Arabidopsis suggests roles for MTD in key environmental and metabolic responses.

    PubMed

    Zamski, E; Guo, W W; Yamamoto, Y T; Pharr, D M; Williamson, J D

    2001-11-01

    Of the growing list of promising genes for plant improvement, some of the most versatile appear to be those involved in sugar alcohol metabolism. Mannitol, one of the best characterized sugar alcohols, is a significant photosynthetic product in many higher plants. The roles of mannitol as both a metabolite and an osmoprotectant in celery (Apium graveolens) are well documented. However, there is growing evidence that 'metabolites' can also have key roles in other environmental and developmental responses in plants. For instance, in addition to its other properties, mannitol is an antioxidant and may have significant roles in plant-pathogen interactions. The mannitol catabolic enzyme mannitol dehydrogenase (MTD) is a prime modulator of mannitol accumulation in plants. Because the complex regulation of MTD is central to the balanced integration of mannitol metabolism in celery, its study is crucial in clarifying the physiological role(s) of mannitol metabolism in environmental and metabolic responses. In this study we used transformed Arabidopsis to analyze the multiple environmental and metabolic responses of the Mtd promoter. Our data show that all previously described changes in Mtd RNA accumulation in celery cells mirrored changes in Mtd transcription in Arabidopsis. These include up-regulation by salicylic acid, hexokinase-mediated sugar down-regulation, and down-regulation by salt, osmotic stress and ABA. In contrast, the massive up-regulation of Mtd expression in the vascular tissues of salt-stressed Arabidopsis roots suggests a possible role for MTD in mannitol translocation and unloading and its interrelation with sugar metabolism.

  18. Effects of naturally occurring coumarins on hepatic drug-metabolizing enzymes inmice

    SciTech Connect

    Kleiner, Heather E. Xia, Xiaojun; Sonoda, Junichiro; Zhang, Jun; Pontius, Elizabeth; Abey, Jane; Evans, Ronald M.; Moore, David D.; DiGiovanni, John

    2008-10-15

    Cytochromes P450 (P450s) and glutathione S-transferases (GSTs) constitute two important enzyme families involved in carcinogen metabolism. Generally, P450s play activation or detoxifying roles while GSTs act primarily as detoxifying enzymes. We previously demonstrated that oral administration of the linear furanocoumarins, isopimpinellin and imperatorin, modulated P450 and GST activities in various tissues of mice. The purpose of the present study was to compare a broader range of naturally occurring coumarins (simple coumarins, and furanocoumarins of the linear and angular type) for their abilities to modulate hepatic drug-metabolizing enzymes when administered orally to mice. We now report that all of the different coumarins tested (coumarin, limettin, auraptene, angelicin, bergamottin, imperatorin and isopimpinellin) induced hepatic GST activities, whereas the linear furanocoumarins possessed the greatest abilities to induce hepatic P450 activities, in particular P450 2B and 3A. In both cases, this corresponded to an increase in protein expression of the enzymes. Induction of P4502B10, 3A11, and 2C9 by xenobiotics often is a result of activation of the pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Using a pregnane X receptor reporter system, our results demonstrated that isopimpinellin activated both PXR and its human ortholog SXR by recruiting coactivator SRC-1 in transfected cells. In CAR transfection assays, isopimpinellin counteracted the inhibitory effect of androstanol on full-length mCAR, a Gal4-mCAR ligand-binding domain fusion, and restored coactivator binding. Orally administered isopimpinellin induced hepatic mRNA expression of Cyp2b10, Cyp3a11, and GSTa in CAR(+/+) wild-type mice. In contrast, the induction of Cyp2b10 mRNA by isopimpinellin was attenuated in the CAR(-/-) mice, suggesting that isopimpinellin induces Cyp2b10 via the CAR receptor. Overall, the current data indicate that naturally occurring coumarins have

  19. Plastid Localization of the Key Carotenoid Enzyme Phytoene Synthase Is Altered by Isozyme, Allelic Variation, and Activity[W

    PubMed Central

    Shumskaya, Maria; Bradbury, Louis M.T.; Monaco, Regina R.; Wurtzel, Eleanore T.

    2012-01-01

    Plant carotenoids have unique physiological roles related to specific plastid suborganellar locations. Carotenoid metabolic engineering could enhance plant adaptation to climate change and improve food security and nutritional value. However, lack of fundamental knowledge on carotenoid pathway localization limits targeted engineering. Phytoene synthase (PSY), a major rate-controlling carotenoid enzyme, is represented by multiple isozymes residing at unknown plastid sites. In maize (Zea mays), the three isozymes were transiently expressed and found either in plastoglobuli or in stroma and thylakoid membranes. PSY1, with one to two residue modifications of naturally occurring functional variants, exhibited altered localization, associated with distorted plastid shape and formation of a fibril phenotype. Mutating the active site of the enzyme reversed this phenotype. Discovery of differential PSY locations, linked with activity and isozyme type, advances the engineering potential for modifying carotenoid biosynthesis. PMID:23023170

  20. Molecular architecture of DesI: a key enzyme in the biosynthesis of desosamine.

    PubMed

    Burgie, E Sethe; Holden, Hazel M

    2007-08-07

    Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found, for example, in such macrolide antibiotics as erthyromycin, azithromycin, and clarithromycin. The efficacies of these macrolide antibiotics are markedly reduced in the absence of desosamine. In the bacterium Streptomyces venezuelae, six enzymes are required for the production of dTDP-desosamine. The focus of this X-ray crystallographic analysis is the third enzyme in the pathway, a PLP-dependent aminotransferase referred to as DesI. The structure of DesI was solved in complex with its product, dTDP-4-amino-4,6-dideoxyglucose, to a nominal resolution of 2.1 A. Each subunit of the dimeric enzyme contains 12 alpha-helices and 14 beta-strands. Three cis-peptides are observed in each subunit, Phe 330, Pro 332, and Pro 339. The two active sites of the enzyme are located in clefts at the subunit/subunit interface. Electron density corresponding to the bound product clearly demonstrates a covalent bond between the amino group of the product and C-4' of the PLP cofactor. Interestingly, there are no hydrogen-bonding interactions between the protein and the dideoxyglucosyl group of the product (within 3.2 A). The only other sugar-modifying aminotransferase whose structure is known in the presence of product is PseC from Helicobacter pylori. This enzyme, as opposed to DesI, catalyzes amino transfer to the axial position of the sugar. A superposition of the two active sites for these proteins reveals that the major differences in ligand binding occur in the orientations of the deoxyglucosyl and phosphoryl groups. Indeed, the nearly 180 degrees difference in hexose orientation explains the equatorial versus axial amino transfer exhibited by DesI and PseC, respectively.

  1. Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis

    PubMed Central

    Chen, Sheng-Chia; Huang, Chi-Hung; Lai, Shu-Jung; Yang, Chia Shin; Hsiao, Tzu-Hung; Lin, Ching-Heng; Fu, Pin-Kuei; Ko, Tzu-Ping; Chen, Yeh

    2016-01-01

    The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, and it is feed-back inhibited by the downstream product CMP-Neu5Ac. Here the complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme. PMID:26980148

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

    PubMed

    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.

  3. Reaction phenotyping: advances in the experimental strategies used to characterize the contribution of drug-metabolizing enzymes.

    PubMed

    Zientek, Michael A; Youdim, Kuresh

    2015-01-01

    During the process of drug discovery, the pharmaceutical industry is faced with numerous challenges. One challenge is the successful prediction of the major routes of human clearance of new medications. For compounds cleared by metabolism, accurate predictions help provide an early risk assessment of their potential to exhibit significant interpatient differences in pharmacokinetics via routes of metabolism catalyzed by functionally polymorphic enzymes and/or clinically significant metabolic drug-drug interactions. This review details the most recent and emerging in vitro strategies used by drug metabolism and pharmacokinetic scientists to better determine rates and routes of metabolic clearance and how to translate these parameters to estimate the amount these routes contribute to overall clearance, commonly referred to as fraction metabolized. The enzymes covered in this review include cytochrome P450s together with other enzymatic pathways whose involvement in metabolic clearance has become increasingly important as efforts to mitigate cytochrome P450 clearance are successful. Advances in the prediction of the fraction metabolized include newly developed methods to differentiate CYP3A4 from the polymorphic enzyme CYP3A5, scaling tools for UDP-glucuronosyltranferase, and estimation of fraction metabolized for substrates of aldehyde oxidase.

  4. Bottom-up Metabolic Reconstruction of Arabidopsis and Its Application to Determining the Metabolic Costs of Enzyme Production[W

    PubMed Central

    Arnold, Anne; Nikoloski, Zoran

    2014-01-01

    Large-scale modeling of plant metabolism provides the possibility to compare and contrast different cellular and environmental scenarios with the ultimate aim of identifying the components underlying the respective plant behavior. The existing models of Arabidopsis (Arabidopsis thaliana) are top-down assembled, whereby the starting point is the annotated genome, in particular, the metabolic genes. Hence, dead-end metabolites and blocked reactions can arise that are subsequently addressed by using gap-filling algorithms in combination with species-unspecific genes. Here, we present a bottom-up-assembled, large-scale model that relies solely on Arabidopsis-specific annotations and results in the inclusion of only manually curated reactions. While the existing models are largely condition unspecific by employing a single biomass reaction, we provide three biomass compositions that pertain to realistic and frequently examined scenarios: carbon-limiting, nitrogen-limiting, and optimal growth conditions. The comparative analysis indicates that the proposed Arabidopsis core model exhibits comparable efficiency in carbon utilization and flexibility to the existing network alternatives. Moreover, the model is utilized to quantify the energy demand of amino acid and enzyme de novo synthesis in photoautotrophic growth conditions. Illustrated by the case of the most abundant protein in the world, Rubisco, we determine its synthesis cost in terms of ATP requirements. This, in turn, allows us to explore the tradeoff between protein synthesis and growth in Arabidopsis. Altogether, the model provides a solid basis for completely species-specific integration of high-throughput data, such as gene expression levels, and for condition-specific investigations of in silico metabolic engineering strategies. PMID:24808102

  5. Mycobacterium tuberculosis Glucosyl-3-Phosphoglycerate Synthase: Structure of a Key Enzyme in Methylglucose Lipopolysaccharide Biosynthesis

    PubMed Central

    Pereira, Pedro José Barbosa; Empadinhas, Nuno; Albuquerque, Luciana; Sá-Moura, Bebiana; da Costa, Milton S.; Macedo-Ribeiro, Sandra

    2008-01-01

    Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 Å, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme's preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental

  6. Posttranslational Peptide-Modification Enzymes in Action: Key Roles for Leaders and Glutamate.

    PubMed

    Montalbán-López, Manuel; Kuipers, Oscar P

    2016-03-17

    In this issue of Cell Chemical Biology, Ortega et al. (2016) determine the structure of another lantibiotic dehydratase with a tRNA(Glu)-dependent mechanism of modification. Moreover, they identify a common recognition motif involved in leader peptide binding in a number of different peptide-modification enzymes. These findings open up new mining possibilities and allow novel approaches in peptide engineering.

  7. Mechanisms for Regulation of the Activity of Key Enzymes in Developing Dormant and Germinated Bacterial Spores.

    DTIC Science & Technology

    1983-03-28

    Bacillus megaterium spores and characterized; it is a new member of the class of pyridine nucleotide dependent disulfide reductases.. The enzyme is...as noted, all of the studies utilized Bacillus megaterium . 1) Absence of cAMP from Clostridium perfringens. In collaboration with Dr. L.E. Sacks we...Disulfide Reductase from Bacillus megaterium Specific for Disulfides Containing Pantethine 4’,4". Diphosphate". J. Bacteriol. 153:475-481. 3. Swerdlow

  8. Targeted cellular metabolism for cancer chemotherapy with recombinant arginine-degrading enzymes

    PubMed Central

    Savaraj, Niramol; Feun, Lynn G.

    2010-01-01

    It has been shown that a subset of human cancers, notably, melanoma and hepatocellular carcinoma (HCC) are auxotrophic for arginine (Arg), because they do not express argininosuccinate synthetase (ASS), the rate-limiting enzyme for the biosynthesis of arginine from citrulline. These ASS-negative cancer cells require Arg from extracellular sources for survival. When they are exposed to recombinant Arg-degrading enzymes, e.g. arginine deiminase (ADI) or arginase, they die because of Arg starvation; whereas normal cells which express ASS are able to survive. A pegylated ADI (ADI-PEG20) has been developed for clinical trials for advanced melanoma and HCC; and favorable results have been obtained. ADI-PEG20 treatment induces autophagy in auxotrophic cancer cells leading to cell death. Clinical studies in melanoma patients show that re-expression of ASS is associated with ADI-PEG20 resistance. ADI-PEG20 treatment down-regulates the expression of HIF-1α but up-regulates c-Myc in culture melanoma cells. Induction of ASS by ADI-PEG20 involves positive regulators c-Myc and Sp4 and negative regulator HIF1α. Since both HIF-1α and c-Myc play important roles in cancer cell energy metabolism, together these results suggest that targeted cancer cell metabolism through modulation of HIF-1α and c-Myc expression may improve the efficacy of ADI-PEG20 in treating Arg auxotrophic tumors. PMID:21152246

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

    PubMed

    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.

  10. Cytochrome P4502C9: an enzyme of major importance in human drug metabolism

    PubMed Central

    Miners, John O; Birkett, Donald J

    1998-01-01

    Accumulating evidence indicates that CYP2C9 ranks amongst the most important drug metabolizing enzymes in humans. Substrates for CYP2C9 include fluoxetine, losartan, phenytoin, tolbutamide, torsemide, S-warfarin, and numerous NSAIDs. CYP2C9 activity in vivo is inducible by rifampicin. Evidence suggests that CYP2C9 substrates may also be induced variably by carbamazepine, ethanol and phenobarbitone. Apart from the mutual competitive inhibition which may occur between alternate substrates, numerous other drugs have been shown to inhibit CYP2C9 activity in vivo and/or in vitro. Clinically significant inhibition may occur with coadministration of amiodarone, fluconazole, phenylbutazone, sulphinpyrazone, sulphaphenazole and certain other sulphonamides. Polymorphisms in the coding region of the CYP2C9 gene produce variants at amino acid residues 144 (Arg144Cys) and 359 (Ile359Leu) of the CYP2C9 protein. Individuals homozygous for Leu359 have markedly diminished metabolic capacities for most CYP2C9 substrates, although the frequency of this allele is relatively low. Consistent with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates. Individualisation of dose is essential for those CYP2C9 substrates with a narrow therapeutic index. PMID:9663807

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

    PubMed Central

    Saruwatari, Junji; Ishitsu, Takateru; Nakagawa, Kazuko

    2010-01-01

    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. PMID:27713373

  12. Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula.

    PubMed

    Estelmann, Sebastian; Hügler, Michael; Eisenreich, Wolfgang; Werner, Katharina; Berg, Ivan A; Ramos-Vera, W Hugo; Say, Rafael F; Kockelkorn, Daniel; Gad'on, Nasser; Fuchs, Georg

    2011-03-01

    Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.

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

    PubMed Central

    Grimm, B

    1990-01-01

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

  14. Crystal structures of nitric oxide reductases provide key insights into functional conversion of respiratory enzymes.

    PubMed

    Tosha, Takehiko; Shiro, Yoshitsugu

    2013-03-01

    Respiration is an essential biological process to get bioenergy, ATP, for all kingdoms of life. Cytochrome c oxidase (COX) plays central role in aerobic respiration, catalyzing the reduction of O(2) coupled with pumping proton across the biological membrane. Nitric oxide reductase (NOR) involved in anaerobic nitrate respiration is suggested to be evolutionary related to COX and share the same progenitor with COX, on the basis of the amino acid sequence homology. Contrary to COX, NOR catalyzes the reduction of nitric oxide and shows no proton pumping ability. Thus, the respiratory enzyme acquires (or loses) proton pumping ability in addition to the conversion of the catalytic property along with the environmental change on earth. Recently, we solved the structures of two types of NORs, which provides novel insights into the functional conversion of the respiratory enzymes. In this review, we focus on the structural similarities and differences between COXs and NORs and discuss possible mechanism for the functional conversion of these enzymes during molecular evolution.

  15. Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei.

    PubMed

    Bautista, Vanesa; Esclapez, Julia; Pérez-Pomares, Francisco; Martínez-Espinosa, Rosa María; Camacho, Mónica; Bonete, María José

    2012-01-01

    A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.

  16. Muscle Transcriptional Profile Based on Muscle Fiber, Mitochondrial Respiratory Activity, and Metabolic Enzymes

    PubMed Central

    Liu, Xuan; Du, Yang; Trakooljul, Nares; Brand, Bodo; Muráni, Eduard; Krischek, Carsten; Wicke, Michael; Schwerin, Manfred; Wimmers, Klaus; Ponsuksili, Siriluck

    2015-01-01

    Skeletal muscle is a highly metabolically active tissue that both stores and consumes energy. Important biological pathways that affect energy metabolism and metabolic fiber type in muscle cells may be identified through transcriptomic profiling of the muscle, especially ante mortem. Here, gene expression was investigated in malignant hyperthermia syndrome (MHS)-negative Duroc and Pietrian (PiNN) pigs significantly differing for the muscle fiber types slow-twitch-oxidative fiber (STO) and fast-twitch-oxidative fiber (FTO) as well as mitochondrial activity (succinate-dependent state 3 respiration rate). Longissimus muscle samples were obtained 24 h before slaughter and profiled using cDNA microarrays. Differential gene expression between Duroc and PiNN muscle samples were associated with protein ubiquitination, stem cell pluripotency, amyloid processing, and 3-phosphoinositide biosynthesis and degradation pathways. In addition, weighted gene co-expression network analysis within both breeds identified several co-expression modules that were associated with the proportion of different fiber types, mitochondrial respiratory activity, and ATP metabolism. In particular, Duroc results revealed strong correlations between mitochondrion-associated co-expression modules and STO (r = 0.78), fast-twitch glycolytic fiber (r = -0.98), complex I (r=0.72) and COX activity (r = 0.86). Other pathways in the protein-kinase-activity enriched module were positively correlated with STO (r=0.93), while negatively correlated with FTO (r = -0.72). In contrast to PiNN, co-expression modules enriched in macromolecule catabolic process, actin cytoskeleton, and transcription activator activity were associated with fiber types, mitochondrial respiratory activity, and metabolic enzyme activities. Our results highlight the importance of mitochondria for the oxidative capacity of porcine muscle and for breed-dependent molecular pathways in muscle cell fibers. PMID:26681915

  17. Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

    PubMed Central

    Esser, Dominik; Rauch, Bernadette

    2014-01-01

    SUMMARY The metabolism of Archaea, the third domain of life, resembles in its complexity those of Bacteria and lower Eukarya. However, this metabolic complexity in Archaea is accompanied by the absence of many “classical” pathways, particularly in central carbohydrate metabolism. Instead, Archaea are characterized by the presence of unique, modified variants of classical pathways such as the Embden-Meyerhof-Parnas (EMP) pathway and the Entner-Doudoroff (ED) pathway. The pentose phosphate pathway is only partly present (if at all), and pentose degradation also significantly differs from that known for bacterial model organisms. These modifications are accompanied by the invention of “new,” unusual enzymes which cause fundamental consequences for the underlying regulatory principles, and classical allosteric regulation sites well established in Bacteria and Eukarya are lost. The aim of this review is to present the current understanding of central carbohydrate metabolic pathways and their regulation in Archaea. In order to give an overview of their complexity, pathway modifications are discussed with respect to unusual archaeal biocatalysts, their structural and mechanistic characteristics, and their regulatory properties in comparison to their classic counterparts from Bacteria and Eukarya. Furthermore, an overview focusing on hexose metabolic, i.e., glycolytic as well as gluconeogenic, pathways identified in archaeal model organisms is given. Their energy gain is discussed, and new insights into different levels of regulation that have been observed so far, including the transcript and protein levels (e.g., gene regulation, known transcription regulators, and posttranslational modification via reversible protein phosphorylation), are presented. PMID:24600042

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

    PubMed

    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

  19. Cytochrome P450 Drug Metabolizing Enzymes in Roma Population Samples: Systematic Review of the Literature.

    PubMed

    Szalai, Renata; Hadzsiev, Kinga; Melegh, Bela

    2016-01-01

    The cytochrome P450 drug metabolizing enzymes are highly polymorphic and show inter-individual differences in variability in drug response, which varies widely also with ethnicity. This study aims to summarize the available data on genetic polymorphisms associated with cytochrome enzymes conducted on Roma populations. Our goal was to compare the frequency of the variant alleles, genotypes and predicted phenotypes with corresponding rates from other populations. We carried out a systematic review including the papers published on the pharmacogenetically relevant variants of cytochrome P450 genes related to Roma population. The study was performed using several articles, websites and databases, including PubMed, Ensembl, dbSNP, HapMap and 1000 Genomes Project. This review attempts to summarize and discuss our current knowledge about the frequency distribution of the ever investigated 20 allelic variants of 9 cytochrome genes (CYP1A2, CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP2C8, CYP2D6, CYP3A5, CYP4F2) in Roma DNA samples and compare them with other populations. Differences between Roma and Hungarian samples are reported for 7 variant genotypes. CYP2C9 *2/*3 and CYP2C19 *2/*2 genotypes showed more than 3-fold differences. Additional differences are displayed for allele frequency of 7 variants (rs762551, rs3745274, rs1058930, rs1065852, rs3892097, rs1057910 and rs4244285) in Roma population samples. The interethnic variability in clinically relevant genetic polymorphisms of drug metabolizing enzymes, which may explain distinct drug response, highlights the need to allow for the ancestry of participants in pharmacogenetic studies.

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

    PubMed

    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.

  1. Overview of chitin metabolism enzymes in Manduca sexta: Identification, domain organization, phylogenetic analysis and gene expression.

    PubMed

    Tetreau, Guillaume; Cao, Xiaolong; Chen, Yun-Ru; Muthukrishnan, Subbaratnam; Jiang, Haobo; Blissard, Gary W; Kanost, Michael R; Wang, Ping

    2015-07-01

    Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27444012

  3. Human DHRS7, promising enzyme in metabolism of steroids and retinoids?

    PubMed

    Štambergová, Hana; Zemanová, Lucie; Lundová, Tereza; Malčeková, Beata; Skarka, Adam; Šafr, Miroslav; Wsól, Vladimír

    2016-01-01

    The metabolism of steroids and retinoids has been studied in detail for a long time, as these compounds are involved in a broad spectrum of physiological processes. Many enzymes participating in the conversion of such compounds are members of the short-chain dehydrogenase/reductase (SDR) superfamily. Despite great effort, there still remain a number of poorly characterized SDR proteins. According to various bioinformatics predictions, many of these proteins may play a role in the metabolism of steroids and retinoids. Dehydrogenase/reductase (SDR family) member 7 (DHRS7) is one such protein. In a previous study, we determined DHRS7 to be an integral membrane protein of the endoplasmic reticulum facing the lumen which has shown at least in vitro NADPH-dependent reducing activity toward several eobiotics and xenobiotics bearing a carbonyl moiety. In the present paper pure DHRS7 was used for a more detailed study of both substrate screening and an analysis of kinetics parameters of the physiologically important substrates androstene-3,17-dione, cortisone and all-trans-retinal. Expression patterns of DHRS7 at the mRNA as well as protein level were determined in a panel of various human tissue samples, a procedure that has enabled the first estimation of the possible biological function of this enzyme. DHRS7 is expressed in tissues such as prostate, adrenal glands, liver or intestine, where its activity could be well exploited. Preliminary indications show that DHRS7 exhibits dual substrate specificity recognizing not only steroids but also retinoids as potential substrates and could be important in the metabolism of these signalling molecules.

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

    PubMed

    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.

  5. Dorsomedial hindbrain catecholamine regulation of hypothalamic astrocyte glycogen metabolic enzyme protein expression: Impact of estradiol.

    PubMed

    Tamrakar, P; Shrestha, P K; Briski, K P

    2015-04-30

    The brain astrocyte glycogen reservoir is a vital energy reserve and, in the cerebral cortex, subject among other factors to noradrenergic control. The ovarian steroid estradiol potently stimulates nerve cell aerobic respiration, but its role in glial glycogen metabolism during energy homeostasis or mismatched substrate supply/demand is unclear. This study examined the premise that estradiol regulates hypothalamic astrocyte glycogen metabolic enzyme protein expression during normo- and hypoglycemia in vivo through dorsomedial hindbrain catecholamine (CA)-dependent mechanisms. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial hypothalamic (VMH), arcuate hypothalamic (ARH), and paraventricular hypothalamic (PVH) nuclei and the lateral hypothalamic area (LHA) of estradiol (E)- or oil (O)-implanted ovariectomized (OVX) rats after insulin or vehicle injection, and pooled within each site. Stimulation [VMH, LHA] or suppression [PVH, ARH] of basal glycogen synthase (GS) protein expression by E was reversed in the former three sites by caudal fourth ventricular pretreatment with the CA neurotoxin 6-hydroxydopamine (6-OHDA). E diminished glycogen phosphorylase (GP) protein profiles by CA-dependent [VMH, PVH] or -independent mechanisms [LHA]. Insulin-induced hypoglycemia (IIH) increased GS expression in the PVH in OVX+E, but reduced this protein in the PVH, ARH, and LHA in OVX+O. Moreover, IIH augmented GP expression in the VMH, LHA, and ARH in OVX+E and in the ARH in OVX+O, responses that normalized by 6-OHDA. Results demonstrate site-specific effects of E on astrocyte glycogen metabolic enzyme expression in the female rat hypothalamus, and identify locations where dorsomedial hindbrain CA input is required for such action. Evidence that E correspondingly increases and reduces basal GS and GP in the VMH and LHA, but augments the latter protein during IIH suggests that E regulates

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

    PubMed

    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.

  7. Correlation of Homocysteine Metabolic Enzymes Gene Polymorphism and Mild Cognitive Impairment in the Xinjiang Uygur Population

    PubMed Central

    Luo, Mei; Ji, Huihui; Zhou, Xiaohui; Liang, Jie; Zou, Ting

    2015-01-01

    Background The aim of this study was to investigate the genetic polymorphisms in the homocysteine (HCY) metabolic enzymes in the Xinjiang Uygur population who have mild cognitive impairment (MCI). Material/Methods Based on the epidemiological investigation, 129 cases of diagnosed Uygur MCI patients and a matched control group with 131 cases were enrolled for analyzing the association between the polymorphisms in the HCY metabolism related genes (C677T, A1298C, and G1968A polymorphisms in MTHFR, as well as the A2756G polymorphism in MS) and MCI by using the SNaPshot method. We then determined the homocysteine level in patients. Results In Xinjiang Uygur subjects, the A1298C polymorphisms in MTHFR and the A2756G polymorphisms in the MS gene in the MCI group were different from those in the control group. However, the C677T and G1968A polymorphisms in the MTHFR gene in MCI patients were not different from those in the control group. Multivariate logistic regression showed that, in addition to the well-known risk factors, such as low education level, high cholesterol level, high level of low-density lipoprotein, and high homocysteine levels, the A>G mutation in the MS gene at the rs1805087 locus was another independent risk factor for MCI in the Uyghur MCI population. The risk of MCI in G allele carriers was 2.265 times higher than that in matched control individuals (95% CI: 1.205~4.256, P<0.05). Conclusions The genetic polymorphism of HCY metabolizing enzymes is correlated to the occurrence of MCI in the Xinjiang Uygur population. The A2756G polymorphism in the MS gene could be an independent risk factor for MCI in the Xinjiang Uygur population. PMID:25625218

  8. Correlating Structure and Function of Drug-Metabolizing Enzymes: Progress and Ongoing Challenges

    PubMed Central

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

    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. PMID:24130370

  9. Sink filling, inulin metabolizing enzymes and carbohydrate status in field grown chicory (Cichorium intybus L.).

    PubMed

    van Arkel, Jeroen; Vergauwen, Rudy; Sévenier, Robert; Hakkert, Johanna C; van Laere, André; Bouwmeester, Harro J; Koops, Andries J; van der Meer, Ingrid M

    2012-10-15

    Inulin is a fructose-based polymer that is isolated from chicory (Cichorium intybus L.) taproots. The degree of polymerization (DP) determines its application and hence the value of the crop. The DP is highly dependent on the field conditions and harvest time. Therefore, the present study was carried out with the objective to understand the regulation of inulin metabolism and the process that determines the chain length and inulin yield throughout the whole growing season. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. The following characteristics were determined in taproots: concentrations of glucose, fructose and sucrose, the inulin mean polymer length (mDP), yield, gene expression and activity of enzymes involved in inulin metabolism. Inulin synthesis, catalyzed by sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) (1-SST) and fructan:fructan 1-fructosyltransferase (EC 2.4.1.100) (1-FFT), started at the onset of taproot development. Inulin yield as a function of time followed a sigmoid curve reaching a maximum in November. Inulin reached a maximum mDP of about 15 in September, than gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three different phases in the growing season and analyzed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST that starts in June is not regulated by day length and temperature. From mid-September onwards, the mean degree of polymerization (mDP) decreased gradually although inulin yield still increased. The decrease in mDP combined with increased yield results from fructan exohydrolase activity, induced by low temperature, and the back transfer activity of 1-FFT. Overall, this study provides background information on how to improve

  10. Reactive Intermediates Produced from Metabolism of the Vanilloid Ring of Capsaicinoids by P450 Enzymes

    PubMed Central

    Reilly, Christopher A.; Henion, Fred; Bugni, Tim S.; Ethirajan, Manivannan; Stockmann, Chris; Pramanik, Kartick C.; Srivastava, Sanjay K.; Yost, Garold S.

    2012-01-01

    This study characterized electrophilic and radical products derived from metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and β-mercaptoethanol conjugates (a.k.a., adducts), derived from trapping of quinone methide and quinone intermediates of capsaicin, its analogue nonivamide, and O-demethylated and aromatic hydroxylated metabolites thereof, were produced by human liver microsomes and individual recombinant human P450 enzymes. Conjugates derived from concomitant dehydrogenation of the alkyl terminus of capsaicin, were also characterized. Modifications to the 4-OH substituent of the vanilloid ring of capsaicinoids largely prevented the formation of electrophilic intermediates, consistent with the proposed structures and mechanisms of formation for the various conjugates. 5,5’-Dicapsaicin, presumably arising from bi-molecular coupling of free radical intermediates, was also characterized. Finally, the analysis of hepatic glutathione conjugates and urinary N-acetylcysteine conjugates from mice dosed with capsaicin confirmed the formation of glutathione conjugates of O-demethylated, quinone methide, and 5-OH-capsaicin in vivo. These data demonstrated that capsaicin and structurally similar analogues are converted to reactive intermediates by certain P450 enzymes, which may partially explain conflicting reports related to the cytotoxic, pro-carcinogenic, and chemoprotective effects of capsaicinoids in different cells and/or organ systems. PMID:23088752

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

    PubMed

    Li, C B; Li, J; Zhou, G H; Lametsch, R; Ertbjerg, P; Brüggemann, D A; Huang, H G; Karlsson, A H; Hviid, M; Lundström, K

    2012-05-01

    The objective of this study was to investigate the response of sarcoplasmic proteins in bovine LM to low-voltage electrical stimulation (ES; 80 V, 35 s) after dressing and its contribution to meat tenderization at an early postmortem time. Proteome analysis showed that ES resulted in decreased (P < 0.05) phosphorylation of creatine kinase M chain, fructose bisphosphate aldolase C-A, β-enolase, and pyruvate kinase at 3 h postmortem. Zymography indicated an earlier (P < 0.05) activation of μ-calpain in ES muscles. Free lysosomal cathepsin B and L activity increased faster (P < 0.05) in ES muscles up to 24 h. Immunohistochemistry and transmission electron microscopy further indicated that lysosomal enzymes were released at an early postmortem time. Electrical stimulation also induced ultrastructural disruption of sarcomeres. In addition, ES accelerated (P < 0.05) the depletion of ATP, creatine phosphate, and glycogen, as well as a pH decline and the more preferred pH/temperature decline mode. Finally, ES accelerated meat tenderization, resulting in lesser (P < 0.05) shear force values than the control over the testing time. A possible relationship was suggested between a change in the phosphorylation of energy metabolic enzymes and the postmortem tenderization of beef. Our results suggested the possible importance of the activation of μ-calpain, phosphorylation of sarcoplasmic proteins, and release of lysosomal enzymes for ES-induced tenderization of beef muscle.

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

    PubMed

    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.

  13. Altered expression of fatty acid–metabolizing enzymes in aromatase-deficient mice

    PubMed Central

    Nemoto, Yoshihisa; Toda, Katsumi; Ono, Masafumi; Fujikawa-Adachi, Kiyomi; Saibara, Toshiji; Onishi, Saburo; Enzan, Hideaki; Okada, Teruhiko; Shizuta, Yutaka

    2000-01-01

    Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid β-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid β-oxidation. In vitro assays of fatty acid β-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid β-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17β-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid β-oxidation and in maintaining hepatic lipid homeostasis. PMID:10862797

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

    PubMed

    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.

  15. The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles

    PubMed Central

    Frej, Anna D.; Clark, Jonathan; Le Roy, Caroline I.; Lilla, Sergio; Thomason, Peter A.; Otto, Grant P.; Churchill, Grant; Insall, Robert H.; Claus, Sandrine P.; Hawkins, Phillip; Stephens, Len

    2016-01-01

    Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1− mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism. PMID:26951199

  16. Lead (Pb)-inhibited radicle emergence in Brassica campestris involves alterations in starch-metabolizing enzymes.

    PubMed

    Singh, Harminder Pal; Kaur, Gurpreet; Batish, Daizy R; Kohli, Ravinder K

    2011-12-01

    Lead (Pb) is a toxic heavy metal released into the natural environment and known to cause oxidative damage and alter antioxidant mechanism in plants. However, not much is known about the interference of Pb with the biochemical processes and carbohydrate metabolism during seed germination. We, therefore, investigated the effect of Pb (50-500 μM) upon biochemical alterations in germinating seeds (at 24-h stage) of Brassica campestris L. Pb treatment significantly enhanced protein and carbohydrate contents that increased by ~43% and 200%, respectively, at 500-μM Pb over control. In contrast, the activities of starch/carbohydrate-metabolizing enzymes--α-amylases, β-amylases, acid invertases, and acid phosphatases--decreased by ~54%, 60%, 74%, and 52%, respectively, over control. Activities of peroxidases and polyphenol oxidases, involved in stress acclimation, however, increased by ~1.2- to 3.9-folds and 0.4- to 1.4-folds upon 50-500-μM Pb treatment. Pb enhanced oxidizing ability by 10 to 16.7 times over control suggesting interference with emerging root's oxidizing capacity. The study concludes that Pb exposure inhibits radicle emergence from B. campestris by interfering with the biochemical processes linked to protein and starch metabolism.

  17. NaCl stress impact on the key enzymes in glycolysis from Lactobacillus bulgaricus during freeze-drying.

    PubMed

    Li, Chun; Sun, Jinwei; Qi, Xiaoxi; Liu, Libo

    2015-01-01

    The viability of Lactobacillus bulgaricus in freeze-drying is of significant commercial interest to dairy industries. In the study, L.bulgaricus demonstrated a significantly improved (p < 0.05) survival rate during freeze-drying when subjected to a pre-stressed period under the conditions of 2% (w/v) NaCl for 2 h in the late growth phase. The main energy source for the life activity of lactic acid bacteria is related to the glycolytic pathway. To investigate the phenomenon of this stress-related viability improvement in L. bulgaricus, the activities and corresponding genes of key enzymes in glycolysis during 2% NaCl stress were studied. NaCl stress significantly enhanced (p < 0.05) glucose utilization. The activities of glycolytic enzymes (phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) decreased during freeze-drying, and NaCl stress were found to improve activities of these enzymes before and after freeze-drying. However, a transcriptional analysis of the corresponding genes suggested that the effect of NaCl stress on the expression of the pfk2 gene was not obvious. The increased survival of freeze-dried cells of L. bulgaricus under NaCl stress might be due to changes in only the activity or translation level of these enzymes in different environmental conditions but have no relation to their mRNA transcription level.

  18. NaCl stress impact on the key enzymes in glycolysis from Lactobacillus bulgaricus during freeze-drying

    PubMed Central

    Li, Chun; Sun, Jinwei; Qi, Xiaoxi; Liu, Libo

    2015-01-01

    Abstract The viability of Lactobacillus bulgaricus in freeze-drying is of significant commercial interest to dairy industries. In the study, L.bulgaricus demonstrated a significantly improved (p < 0.05) survival rate during freeze-drying when subjected to a pre-stressed period under the conditions of 2% (w/v) NaCl for 2 h in the late growth phase. The main energy source for the life activity of lactic acid bacteria is related to the glycolytic pathway. To investigate the phenomenon of this stress-related viability improvement in L. bulgaricus, the activities and corresponding genes of key enzymes in glycolysis during 2% NaCl stress were studied. NaCl stress significantly enhanced (p < 0.05) glucose utilization. The activities of glycolytic enzymes (phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) decreased during freeze-drying, and NaCl stress were found to improve activities of these enzymes before and after freeze-drying. However, a transcriptional analysis of the corresponding genes suggested that the effect of NaCl stress on the expression of the pfk2 gene was not obvious. The increased survival of freeze-dried cells of L. bulgaricus under NaCl stress might be due to changes in only the activity or translation level of these enzymes in different environmental conditions but have no relation to their mRNA transcription level. PMID:26691481

  19. Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

    PubMed Central

    Moraes, Caroline da Silva; Diaz-Albiter, Hector M.; Faria, Maiara do Valle; Sant'Anna, Maurício R. V.; Dillon, Rod J.; Genta, Fernando A.

    2014-01-01

    The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes. PMID:25140153

  20. The histone demethylase enzyme KDM3A is a key estrogen receptor regulator in breast cancer

    PubMed Central

    Wade, Mark A.; Jones, Dominic; Wilson, Laura; Stockley, Jacqueline; Coffey, Kelly; Robson, Craig N.; Gaughan, Luke

    2015-01-01

    Endocrine therapy has successfully been used to treat estrogen receptor (ER)-positive breast cancer, but this invariably fails with cancers becoming refractory to treatment. Emerging evidence has suggested that fluctuations in ER co-regulatory protein expression may facilitate resistance to therapy and be involved in breast cancer progression. To date, a small number of enzymes that control methylation status of histones have been identified as co-regulators of ER signalling. We have identified the histone H3 lysine 9 mono- and di-methyl demethylase enzyme KDM3A as a positive regulator of ER activity. Here, we demonstrate that depletion of KDM3A by RNAi abrogates the recruitment of the ER to cis-regulatory elements within target gene promoters, thereby inhibiting estrogen-induced gene expression changes. Global gene expression analysis of KDM3A-depleted cells identified gene clusters associated with cell growth. Consistent with this, we show that knockdown of KDM3A reduces ER-positive cell proliferation and demonstrate that KDM3A is required for growth in a model of endocrine therapy-resistant disease. Crucially, we show that KDM3A catalytic activity is required for both ER-target gene expression and cell growth, demonstrating that developing compounds which target demethylase enzymatic activity may be efficacious in treating both ER-positive and endocrine therapy-resistant disease. PMID:25488809

  1. The histone demethylase enzyme KDM3A is a key estrogen receptor regulator in breast cancer.

    PubMed

    Wade, Mark A; Jones, Dominic; Wilson, Laura; Stockley, Jacqueline; Coffey, Kelly; Robson, Craig N; Gaughan, Luke

    2015-01-01

    Endocrine therapy has successfully been used to treat estrogen receptor (ER)-positive breast cancer, but this invariably fails with cancers becoming refractory to treatment. Emerging evidence has suggested that fluctuations in ER co-regulatory protein expression may facilitate resistance to therapy and be involved in breast cancer progression. To date, a small number of enzymes that control methylation status of histones have been identified as co-regulators of ER signalling. We have identified the histone H3 lysine 9 mono- and di-methyl demethylase enzyme KDM3A as a positive regulator of ER activity. Here, we demonstrate that depletion of KDM3A by RNAi abrogates the recruitment of the ER to cis-regulatory elements within target gene promoters, thereby inhibiting estrogen-induced gene expression changes. Global gene expression analysis of KDM3A-depleted cells identified gene clusters associated with cell growth. Consistent with this, we show that knockdown of KDM3A reduces ER-positive cell proliferation and demonstrate that KDM3A is required for growth in a model of endocrine therapy-resistant disease. Crucially, we show that KDM3A catalytic activity is required for both ER-target gene expression and cell growth, demonstrating that developing compounds which target demethylase enzymatic activity may be efficacious in treating both ER-positive and endocrine therapy-resistant disease.

  2. Garlic oil attenuated nitrosodiethylamine-induced hepatocarcinogenesis by modulating the metabolic activation and detoxification enzymes.

    PubMed

    Zhang, Cui-Li; Zeng, Tao; Zhao, Xiu-Lan; Xie, Ke-Qin

    2013-01-01

    Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes.

  3. Garlic Oil Attenuated Nitrosodiethylamine-Induced Hepatocarcinogenesis by Modulating the Metabolic Activation and Detoxification Enzymes

    PubMed Central

    Zhang, Cui-Li; Zeng, Tao; Zhao, Xiu-Lan; Xie, Ke-Qin

    2013-01-01

    Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes. PMID:23494807

  4. Variation in the Activity of Some Enzymes of Photorespiratory Metabolism in C4 Grasses

    PubMed Central

    UENO, OSAMU; YOSHIMURA, YASUYUKI; SENTOKU, NAOKI

    2005-01-01

    • Background and Aims Photorespiration occurs in C4 plants, although rates are small compared with C3 plants. The amount of glycine decarboxylase in the bundle sheath (BS) varies among C4 grasses and is positively correlated with the granal index (ratio of the length of appressed thylakoid membranes to the total length of all thylakoid membranes) of the BS chloroplasts: C4 grasses with high granal index contained more glycine decarboxylase per unit leaf area than those with low granal index, probably reflecting the differences in O2 production from photosystem II and the potential photorespiratory capacity. Thus, it is hypothesized that the activities of peroxisomal enzymes involved in photorespiration are also correlated with the granal development. • Methods The granal development in BS chloroplasts was investigated and activities of the photorespiratory enzymes assayed in 28 C4 grasses and seven C3 grasses. • Key Results The NADP–malic enzyme grasses were divided into two groups: one with low granal index and the other with relatively high granal index in the BS chloroplasts. Both the NAD–malic enzyme and phosphoenolpyruvate carboxykinase grasses had high granal index in the BS chloroplasts. No statistically significant differences were found in activity of hydroxypyruvate reductase between the C3 and C4 grasses, or between the C4 subtypes. The activity of glycolate oxidase and catalase were smaller in the C4 grasses than in the C3 grasses. Among the C4 subtypes, glycolate oxidase activities were significantly smaller in the NADP–malic enzyme grasses with low granal index in the BS chloroplasts, compared with in the C4 grasses with substantial grana in the BS chloroplasts. • Conclusions There is interspecies variation in glycolate oxidase activity associated with the granal development in the BS chloroplasts and the O2 production from photosystem II, which suggests different potential photorespiration capacities among C4 grasses. PMID:16100226

  5. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid W

    SciTech Connect

    Messing, S.; Gabelli, S; Echeverria, I; Vogel, J; Guan, J; Tan, B; Klee, H; McCarty, D; Amzela, M

    2010-01-01

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  6. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid

    SciTech Connect

    Messing, Simon A.J.; Gabelli, Sandra B.; Echeverria, Ignacia; Vogel, Jonathan T.; Guan, Jiahn Chou; Tan, Bao Cai; Klee, Harry J.; McCarty, Donald R.; Amzel, L. Mario

    2011-09-06

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  7. Purification and characterization of rice alpha-glucosidase, a key enzyme for alcohol fermentation of rice polish.

    PubMed

    Iwata, Hiroshi; Suzuki, Toshiaki; Aramaki, Isao

    2003-01-01

    Alpha-glucosidase, a key enzyme for nuka-sake brewing, was purified from Oryza sativa cv. Yamadanishiki, which is widely used for sake brewing. The molecular weight of the purified enzyme was 95 kDa. The optimum pH and temperature were 4.5 and 55 degrees C, respectively. The substrate specificity differed from that of Oryza sativa cv. Shinsetsu, which is a variety of rice consumed as a cereal. The extraction of alpha-glucosidase from the rice was stimulated by lactic acid, which suggests that lactic acid plays an important role not only in preventing bacterial contamination, but also in stimulating the parallel fermentation that occurs in nuka-sake brewing.

  8. Quercetin-metabolizing CYP6AS enzymes of the pollinator Apis mellifera (Hymenoptera: Apidae).

    PubMed

    Mao, Wenfu; Rupasinghe, Sanjeewa G; Johnson, Reed M; Zangerl, Arthur R; Schuler, Mary A; Berenbaum, May R

    2009-12-01

    Although the honey bee (Apis mellifera) genome contains far fewer cytochrome P450 genes associated with xenobiotic metabolism than other insect genomes sequenced to date, the CYP6AS subfamily, apparently unique to hymenopterans, has undergone an expansion relative to the genome of the jewel wasp (Nasonia vitripennis). The relative dominance of this family in the honey bee genome is suggestive of a role in processing phytochemicals encountered by honey bees in their relatively unusual diet of honey (comprising concentrated processed nectar of many plant species) and bee bread (a mixture of honey and pollen from many plant species). In this study, quercetin was initially suggested as a shared substrate for CYP6AS1, CYP6AS3, and CYP6AS4, by its presence in honey, extracts of which induce transcription of these three genes, and by in silico substrate predictions based on a molecular model of CYP6AS3. Biochemical assays with heterologously expressed CYP6AS1, CYP6AS3, CYP6AS4 and CYP6AS10 enzymes subsequently confirmed their activity toward this substrate. CYP6AS1, CYP6AS3, CYP6AS4 and CYP6AS10 metabolize quercetin at rates of 0.5+/-0.1, 0.5+/-0.1, 0.2+/-0.1, and 0.2+/-0.1 pmol quercetin/ pmol P450/min, respectively. Substrate dockings and sequence alignments revealed that the positively charged amino acids His107 and Lys217 and the carbonyl group of the backbone between Leu302 and Ala303 are essential for quercetin orientation in the CYP6AS3 catalytic site and its efficient metabolism. Multiple replacements in the catalytic site of CYP6AS4 and CYP6AS10 and repositioning of the quercetin molecule likely account for the lower metabolic activities of CYP6AS4 and CYP6AS10 compared to CYP6AS1 and CYP6AS3.

  9. Maize Homologs of Hydroxycinnamoyltransferase, a Key Enzyme in Lignin Biosynthesis, Bind the Nucleotide Binding Leucine-Rich Repeat Rp1 Proteins to Modulate the Defense Response1

    PubMed Central

    Wang, Guan-Feng; He, Yijian; Strauch, Renee; Olukolu, Bode A.; Nielsen, Dahlia; Li, Xu; Balint-Kurti, Peter J.

    2015-01-01

    In plants, most disease resistance genes encode nucleotide binding Leu-rich repeat (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) upon pathogen recognition. The maize (Zea mays) NLR protein Rp1-D21 derives from an intragenic recombination between two NLRs, Rp1-D and Rp1-dp2, and confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified a single-nucleotide polymorphism locus highly associated with variation in the severity of Rp1-D21-induced HR. Two maize genes encoding hydroxycinnamoyltransferase (HCT; a key enzyme involved in lignin biosynthesis) homologs, termed HCT1806 and HCT4918, were adjacent to this single-nucleotide polymorphism. Here, we show that both HCT1806 and HCT4918 physically interact with and suppress the HR conferred by Rp1-D21 but not other autoactive NLRs when transiently coexpressed in Nicotiana benthamiana. Other maize HCT homologs are unable to confer the same level of suppression on Rp1-D21-induced HR. The metabolic activity of HCT1806 and HCT4918 is unlikely to be necessary for their role in suppressing HR. We show that the lignin pathway is activated by Rp1-D21 at both the transcriptional and metabolic levels. We derive a model to explain the roles of HCT1806 and HCT4918 in Rp1-mediated disease resistance. PMID:26373661

  10. Turmeric (Curcuma longa L.) volatile oil inhibits key enzymes linked to type 2 diabetes.

    PubMed

    Lekshmi, P C; Arimboor, Ranjith; Indulekha, P S; Menon, A Nirmala

    2012-11-01

    Anti-diabetic capacity of Curcuma longa volatile oil in terms of its ability to inhibit glucosidase activities was evaluated. Turmeric volatile oils inhibited glucosidase enzymes more effectively than the reference standard drug acarbose. Drying of rhizomes was found to enhance α-glucosidase (IC₅₀ = 1.32-0.38 μg/ml) and α-amylase (IC₅₀ = 64.7-34.3 μg/ml) inhibitory capacities of volatile oils. Ar-Turmerone, the major volatile component in the rhizome also showed potent α-glucosidase (IC₅₀ = 0.28 μg) and α-amylase (IC₅₀ = 24.5 μg) inhibition.

  11. Yucasin is a potent inhibitor of YUCCA, a key enzyme in auxin biosynthesis.

    PubMed

    Nishimura, Takeshi; Hayashi, Ken-Ichiro; Suzuki, Hiromi; Gyohda, Atsuko; Takaoka, Chihiro; Sakaguchi, Yusuke; Matsumoto, Sachiko; Kasahara, Hiroyuki; Sakai, Tatsuya; Kato, Jun-Ichi; Kamiya, Yuji; Koshiba, Tomokazu

    2014-02-01

    Indole-3-acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole-3-pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC-expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1-His suggested that yucasin strongly inhibited YUC1-His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over-expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss-of-function mutant of TAA1, sav3-2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l-kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin-treated sav3-2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.

  12. Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation.

    PubMed

    Mizutani, Masaharu; Nakanishi, Hidemitsu; Ema, Jun-ichi; Ma, Seung-Jin; Noguchi, Etsuko; Inohara-Ochiai, Misa; Fukuchi-Mizutani, Masako; Nakao, Masahiro; Sakata, Kanzo

    2002-12-01

    A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.

  13. UFGT: The Key Enzyme Associated with the Petals Variegation in Japanese Apricot

    PubMed Central

    Wu, Xinxin; Gong, Qinghua; Ni, Xiaopeng; Zhou, Yong; Gao, Zhihong

    2017-01-01

    Japanese apricot (Prunus mume Sieb.et Zucc.) is an important ornamental plant in China. One of the traits of petals color variegation is attractive, but its formation mechanism is unclear. In our study, RNA-seq technology was employed to characterize the transcriptome response to the mutation of “Fuban Tiaozhi” associated with petals variegation in Japanese apricot. As a result, 4,579,040 (white-flowered, WF) and 7,269,883 (red-flowered, RF) reads were mapped to P. persica genes, while 5,006,676 (WF) and 7,907,436 (RF) were mapped to P. persica genomes. There were 960 differentially expressed genes (DEGs) identified. Gene ontology analysis showed that these genes involved in 37 functional groups including 19 biological processes, 10 cellular components and eight molecular functions. Pathway enrichment annotation demonstrated that highly ranked genes were associated with flavonoid biosynthesis, anthocyanin biosynthesis, anthocyanins transports, plant hormone signal transduction, and transcriptional factors. The expression patterns part of them were validated by qRT-PCR. We found that UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) gene showed differential expression pattern. The UFGT enzyme activities in RF had a significantly higher than that of WF and lower in the initial stage and increased when the red appeared in the petals, which is identical to the accumulation of anthocyanins. And we also validated the SNPs, leading to the nonsynonymous mutations, in the UFGT by Sanger sequencing which may affect the enzyme activity. In summary, our results provide molecular candidates for better understanding the mechanisms of the variegation in Japanese Apricot. PMID:28223989

  14. Rooting depth: a key trait connecting water and carbon metabolism of trees

    NASA Astrophysics Data System (ADS)

    Savi, Tadeja; Dal Borgo, Anna; Casolo, Valentino; Bressan, Alice; Stenni, Barbara; Zini, Luca; Bertoncin, Paolo; Nardini, Andrea

    2015-04-01

    Drought episodes accompanied by heat waves are thought to be the main cause of increasing rates of tree decline and mortality in several biomes with consequent ecological/economical consequences. Three possible and not mutually exclusive mechanisms have been proposed to be the drivers of this phenomenon: hydraulic failure caused by massive xylem cavitation and leading to strong reduction of root-to-leaf water transport, carbon starvation caused by prolonged stomatal closure and leading to impairment of primary and secondary metabolism, and finally attacks of biotic agents. The different mechanisms have been reported to have different relevance in the different species. We analyzed the seasonal changes of water relations, xylem sap isotopic composition, and concentration of non-structural carbohydrates in four different woody species co-occurring in the same habitat during a summer drought. Analysis of rain and deep soil water isotopic composition were also performed. Different species showed differential access to deep water sources which influences the gas exchanges and the concentration of non structural carbohydrates (NSC) during the dry season. Species with access to deeper water maintained higher NSC content and were also able to better preserve the integrity of the water transport pathway. On the basis of our results, we propose that rooting depth is a key trait connecting water and carbon plant metabolism, thus mediating the likelihood of hydraulic failure vs carbon starvation in trees subjected to global warming.

  15. Target Organ Metabolism, Toxicity, and Mechanisms of Trichloroethylene and Perchloroethylene: Key Similarities, Differences, and Data Gaps.

    PubMed

    Cichocki, Joseph A; Guyton, Kathryn Z; Guha, Neela; Chiu, Weihsueh A; Rusyn, Ivan; Lash, Lawrence H

    2016-10-01

    Trichloroethylene (TCE) and perchloroethylene or tetrachloroethylene (PCE) are high-production volume chemicals with numerous industrial applications. As a consequence of their widespread use, these chemicals are ubiquitous environmental contaminants to which the general population is commonly exposed. It is widely assumed that TCE and PCE are toxicologically similar; both are simple olefins with three (TCE) or four (PCE) chlorines. Nonetheless, despite decades of research on the adverse health effects of TCE or PCE, few studies have directly compared these two toxicants. Although the metabolic pathways are qualitatively similar, quantitative differences in the flux and yield of metabolites exist. Recent human health assessments have uncovered some overlap in target organs that are affected by exposure to TCE or PCE, and divergent species- and sex-specificity with regard to cancer and noncancer hazards. The objective of this minireview is to highlight key similarities, differences, and data gaps in target organ metabolism and mechanism of toxicity. The main anticipated outcome of this review is to encourage research to 1) directly compare the responses to TCE and PCE using more sensitive biochemical techniques and robust statistical comparisons; 2) more closely examine interindividual variability in the relationship between toxicokinetics and toxicodynamics for TCE and PCE; 3) elucidate the effect of coexposure to these two toxicants; and 4) explore new mechanisms for target organ toxicity associated with TCE and/or PCE exposure.

  16. Polyhalogenated biphenyls and phenobarbital: evaluation as inducers of drug metabolizing enzymes in the sheepshead, Archosargus probatocephalus.

    PubMed

    James, M O; Little, P J

    1981-08-01

    Several doses of Aroclor 1254 (polychlorinated biphenyl (PCB) mixture), Firemaster FF1 (polybrominated biphenyl (PBB) mixture), 2,2',4,4',5,5'-hexabromobiphenyl (HBB), 3,3',4,4',5,5'-hexachlorobiphenyl (HCB) and phenobarbital (PB) were administered to the marine fish sheepshead (Archosargus probatocephalus). The PCB and PBB mixtures caused induction of hepatic microsomal benzo[a]pyrene hydroxylase (AHH), 7-ethoxycoumarin O-deethylase (7-EC) and 7-ethoxyresorufin O-deethylase (ERF) activities, but not benzphetamine N-demethylase (BND), epoxide hydrolase (EH) or glutathione S-transferase (GSH-T) activities. This induction pattern is typical of that caused by polycyclic aromatic hydrocarbons (PAH) in fish and mammals or by tetrachlorodibenzo-p-dioxin (TCDD) in mammals. The extent of induction of AHH-activity and cytochrome P-450 content was higher when experiments were carried out in summer (water temperature 25 +/- 4 degrees C) than in winter (water temperature 11 +/- 3 degrees C). Firemaster FF1 (15 mg/kg) induced fish for at least 56 days in both summer and winter at which time the liver concentrations of PBB were in the ppm range. PCB concentrations in the ppm range have been found in fish from polluted lakes and seas, thus we may expect that environmental exposure to PCB is sufficient to induce hepatic mixed function oxidase (MFO) activities. The PCB isomer 3,3'4,4'5,5'-HCB, which induces the same spectrum of hepatic drug-metabolizing activities as TCDD and PAH in rats, had a broadly similar effect in the sheepshead. Another purified isomer, 2,2',4,4',5,5'-HBB, which induces the same enzymes as PB in rats, had no effect on drug-metabolizing activities in sheepshead. PB was also without effect on sheepshead hepatic drug-metabolizing enzymes, although a typical narcotic effect was produced in PB-treated sheepshead. Our studies provide further evidence that drug-metabolizing activities in fish liver are readily induced by chemicals like TCDD or PAH, but we fail to

  17. Optimal response of key enzymes and uncoupling protein to cold in BAT depends on local T/sub 3/ generation

    SciTech Connect

    Bianco, A.C.; Silva, J.E.

    1987-09-01

    The authors have examined the activity of three lipogenic enzymes (malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), and acetyl coenzyme A (CoA) carboxylase), the activity of the mitochondrial FAD-dependent ..cap alpha..-glycerolphosphate dehydrogenase (..cap alpha..-GPD), and the mitochondrial concentration of uncoupling protein (UCP) in brown adipose tissue (BAT) of euthyroid and hypothyroid rats, both at room temperature and in response to acute cold stress. These enzymes and UCP are important for the thermogenic response of BAT in adaptation to cold. The basal level of the lipogenic enzymes was normal or slightly elevated in hypothyroid rats maintained at 23/sup 0/C, but the levels of ..cap alpha..-GPD and UCP were markedly reduced. Forty-eight hours at 4/sup 0/C resulted in an increase in the activity of G-6-PD, acetyl-CoA carboxylase, and ..cap alpha..-GPD and in the concentration of UCP both in euthyroid and hypothyroid animals, but the levels reached were invariably less in hypothyroid animals, indicating that thyroid hormone is necessary for a full metabolic response of BAT under maximal demands. Of all variables measured, the most affected was UCP followed by ..cap alpha..-GDP. Dose-response relationship analysis of the UCP response to T/sub 3/ indicated that the normalization of the response to cold requires saturation of the nuclear T/sub 3/ receptors. They concluded, therefore, that the activation of the BAT 5'-deiodinase induced by cold exposure is essential to provide the high levels of nuclear T/sub 3/ required for the full expression of BAT thermogenic potential.

  18. Complete Proteomic-Based Enzyme Reaction and Inhibition Kinetics Reveal How Monolignol Biosynthetic Enzyme Families Affect Metabolic Flux and Lignin in Populus trichocarpa[W

    PubMed Central

    Wang, Jack P.; Naik, Punith P.; Chen, Hsi-Chuan; Shi, Rui; Lin, Chien-Yuan; Liu, Jie; Shuford, Christopher M.; Li, Quanzi; Sun, Ying-Hsuan; Tunlaya-Anukit, Sermsawat; Williams, Cranos M.; Muddiman, David C.; Ducoste, Joel J.; Sederoff, Ronald R.; Chiang, Vincent L.

    2014-01-01

    We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants. PMID:24619611

  19. Optimizing the distribution of resources between enzymes of carbon metabolism can dramatically increase photosynthetic rate: a numerical simulation using an evolutionary algorithm.

    PubMed

    Zhu, Xin-Guang; de Sturler, Eric; Long, Stephen P

    2007-10-01

    The distribution of resources between enzymes of photosynthetic carbon metabolism might be assumed to have been optimized by natural selection. However, natural selection for survival and fecundity does not necessarily select for maximal photosynthetic productivity. Further, the concentration of a key substrate, atmospheric CO(2), has changed more over the past 100 years than the past 25 million years, with the likelihood that natural selection has had inadequate time to reoptimize resource partitioning for this change. Could photosynthetic rate be increased by altered partitioning of resources among the enzymes of carbon metabolism? This question is addressed using an "evolutionary" algorithm to progressively search for multiple alterations in partitioning that increase photosynthetic rate. To do this, we extended existing metabolic models of C(3) photosynthesis by including the photorespiratory pathway (PCOP) and metabolism to starch and sucrose to develop a complete dynamic model of photosynthetic carbon metabolism. The model consists of linked differential equations, each representing the change of concentration of one metabolite. Initial concentrations of metabolites and maximal activities of enzymes were extracted from the literature. The dynamics of CO(2) fixation and metabolite concentrations were realistically simulated by numerical integration, such that the model could mimic well-established physiological phenomena. For example, a realistic steady-state rate of CO(2) uptake was attained and then reattained after perturbing O(2) concentration. Using an evolutionary algorithm, partitioning of a fixed total amount of protein-nitrogen between enzymes was allowed to vary. The individual with the higher light-saturated photosynthetic rate was selected and used to seed the next generation. After 1,500 generations, photosynthesis was increased substantially. This suggests that the "typical" partitioning in C(3) leaves might be suboptimal for maximizing the light

  20. Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium.

    PubMed

    Osanai, Takashi; Shirai, Tomokazu; Iijima, Hiroko; Nakaya, Yuka; Okamoto, Mami; Kondo, Akihiko; Hirai, Masami Y

    2015-01-01

    Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.

  1. Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium

    PubMed Central

    Osanai, Takashi; Shirai, Tomokazu; Iijima, Hiroko; Nakaya, Yuka; Okamoto, Mami; Kondo, Akihiko; Hirai, Masami Y.

    2015-01-01

    Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique. PMID:26500619

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Structural basis of heroin and cocaine metabolism by a promiscuous human drug-processing enzyme.

    PubMed

    Bencharit, Sompop; Morton, Christopher L; Xue, Yu; Potter, Philip M; Redinbo, Matthew R

    2003-05-01

    We present the first crystal structures of a human protein bound to analogs of cocaine and heroin. Human carboxylesterase 1 (hCE1) is a broad-spectrum bioscavenger that catalyzes the hydrolysis of heroin and cocaine, and the detoxification of organophosphate chemical weapons, such as sarin, soman and tabun. Crystal structures of the hCE1 glycoprotein in complex with the cocaine analog homatropine and the heroin analog naloxone provide explicit details about narcotic metabolism in humans. The hCE1 active site contains both specific and promiscuous compartments, which enable the enzyme to act on structurally distinct chemicals. A selective surface ligand-binding site regulates the trimer-hexamer equilibrium of hCE1 and allows each hCE1 monomer to bind two narcotic molecules simultaneously. The bioscavenger properties of hCE1 can likely be used to treat both narcotic overdose and chemical weapon exposure.

  4. Epigenetic Silencing of the Key Antioxidant Enzyme Catalase in Karyotypically Abnormal Human Pluripotent Stem Cells

    PubMed Central

    Konki, Mikko; Pasumarthy, Kalyan; Malonzo, Maia; Sainio, Annele; Valensisi, Cristina; Söderström, Mirva; Emani, Maheswara Reddy; Stubb, Aki; Närvä, Elisa; Ghimire, Bishwa; Laiho, Asta; Järveläinen, Hannu; Lahesmaa, Riitta; Lähdesmäki, Harri; Hawkins, R. David; Lund, Riikka J.

    2016-01-01

    Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells, however, the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase, a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues. PMID:26911679

  5. Mono-hydroxy methoxychlor alters levels of key sex steroids and steroidogenic enzymes in cultured mouse antral follicles

    SciTech Connect

    Craig, Zelieann R.; Leslie, Traci C.; Hatfield, Kimberly P.; Gupta, Rupesh K.; Flaws, Jodi A.

    2010-12-01

    Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by decreasing antral follicle numbers and increasing follicular death. MXC is metabolized in the body to mono-hydroxy MXC (mono-OH). Little is known about the effects of mono-OH on the ovary. Thus, this work tested the hypothesis that mono-OH exposure decreases production of 17{beta}-estradiol (E{sub 2}) by cultured mouse antral follicles. Antral follicles were isolated from CD-1 mice (age 35-39 days) and exposed to dimethylsulfoxide (DMSO), or mono-OH (0.1-10 {mu}g/mL) for 96 h. Media and follicles were collected for analysis of sex steroid levels and mRNA expression, respectively. Mono-OH treatment (10 {mu}g/mL) decreased E{sub 2} (DMSO: 3009.72 {+-} 744.99 ng/mL; mono-OH 0.1 {mu}g/mL: 1679.66 {+-} 461.99 ng/mL; 1 {mu}g/mL: 1752.72 {+-} 532.41 ng/mL; 10 {mu}g/mL: 45.89 {+-} 33.83 ng/mL), testosterone (DMSO: 15.43 {+-} 2.86 ng/mL; mono-OH 0.1 {mu}g/mL: 17.17 {+-} 4.71 ng/mL; 1 {mu}g/mL: 13.64 {+-} 3.53 ng/mL; 10 {mu}g/mL: 1.29 {+-} 0.23 ng/mL), androstenedione (DMSO: 1.92 {+-} 0.34 ng/mL; mono-OH 0.1 {mu}g/mL: 1.49 {+-} 0.43 ng/mL; 1 {mu}g/mL: 0.64 {+-} 0.31 ng/mL; 10 {mu}g/mL: 0.12 {+-} 0.06 ng/mL) and progesterone (DMSO: 24.11 {+-} 4.21 ng/mL; mono-OH 0.1 {mu}g/mL: 26.77 {+-} 4.41 ng/mL; 1 {mu}g/mL: 20.90 {+-} 3.75 ng/mL; 10 {mu}g/mL: 9.44 {+-} 2.97 ng/mL) levels. Mono-OH did not alter expression of Star, Hsd3b1, Hsd17b1 and Cyp1b1, but it did reduce levels of Cyp11a1, Cyp17a1 and Cyp19a1 mRNA. Collectively, these data suggest that mono-OH significantly decreases levels of key sex steroid hormones and the expression of enzymes required for steroidogenesis.

  6. FireMaster BP-6: fractionation, metabolic and enzyme induction studies.

    PubMed

    Safe, S; Kohli, J; Crawford, A

    1978-04-01

    FireMaster BP-6 is a commercial polybrominated biphenyl (PBB) preparation containing a complex mixture of isomers with the major component being identified as 2,2',4,4',5,5'-hexabromobiphenyl. Column chromatographic techniques have been developed in which the crude FireMaster is separated into three fractions, F1, F2, and F3, in increasing order of polarity. F1 consists of highly purified 2,2',4,4',5,5'-hexabromobiphenyl (94%) whereas F2-F3 contain less of this isomer and correspondingly more of the other bromobiphenyl components. Previously we have shown that crude FireMaster BP-6 is metabolized in mammals to give hydroxylated degradation products and the metabolism of F1, not unexpectedly, gives comparable results. It is well known that PBBs are effective inducers of diverse microsomal enzymes including including the aryl hydrocarbon hydroxylase (AHH) system. The effects of FireMaster BP-6 and F1-F3 as AHH inducers have been investigated by using the following approach: the substrates used to monitor AHH activity are model halogenated aromatic compounds; the levels of metabolites and metabolite conjugates formed have been quantitated for control and induced enzymes; the levels of macromolecular adducts have also been quantitated for the inducers. This approach thus not only measures the rate of increase of detoxification products (metabolites and metabolite conjugates) but also monitors the macromolecule adduct formation which represents a toxification route. The effects of the PBBs as AHH inducers will be discussed in terms of the above approach.

  7. [Involvement of microRNA in the induction of drug-metabolizing enzymes].

    PubMed

    Shizu, Ryota; Numazawa, Satoshi; Yoshida, Takemi

    2012-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs of about 20 nucleotides in length and participate in the post-transcriptional regulation of gene expression. Accumulating evidence indicates that miRNA binds to 3'-UTR of its target mRNAs and thereby destabilizes the transcripts or suppresses the translation. It is expected that miRNAs could have diverse functions and therefore play a role in the gene expression caused by the drug treatment, which have yet to be determined. Demonstration of the participation of specific miRNA in the drug-mediated gene expression would make it a biomarker for the toxicological assessment and help an understanding of molecular machinery of the drug-drug interaction. Under these backgrounds, we investigated the change of miRNAs in the liver of mice treated with phenobarbital, a typical inducer for drug-metabolizing enzymes, and demonstrate the participation of miRNAs in the phenobarbital-regulated gene expression. We investigated the relationship between phenobarbital-mediated changes in miRNA and mRNA by using Agilent miRNA microarray and DNA microarray, followed by real time RT-PCR. From these experiments, it was suggested that the phenobarbital-induced changes in cyp2c29 and mrp3 are regulated by miR-30a and miR-29b, respectively. In addition, we obtained evidence that indicates a phenobarbital-mediated decrease in miR-122, a highly abundant liver-specific miRNA, leads to the activation of the transcription factor CAR and thereby induces drug-metabolizing enzymes.

  8. The key nickel enzyme of methanogenesis catalyses the anaerobic oxidation of methane.

    PubMed

    Scheller, Silvan; Goenrich, Meike; Boecher, Reinhard; Thauer, Rudolf K; Jaun, Bernhard

    2010-06-03

    Large amounts (estimates range from 70 Tg per year to 300 Tg per year) of the potent greenhouse gas methane are oxidized to carbon dioxide in marine sediments by communities of methanotrophic archaea and sulphate-reducing bacteria, and thus are prevented from escaping into the atmosphere. Indirect evidence indicates that the anaerobic oxidation of methane might proceed as the reverse of archaeal methanogenesis from carbon dioxide with the nickel-containing methyl-coenzyme M reductase (MCR) as the methane-activating enzyme. However, experiments showing that MCR can catalyse the endergonic back reaction have been lacking. Here we report that purified MCR from Methanothermobacter marburgensis converts methane into methyl-coenzyme M under equilibrium conditions with apparent V(max) (maximum rate) and K(m) (Michaelis constant) values consistent with the observed in vivo kinetics of the anaerobic oxidation of methane with sulphate. This result supports the hypothesis of 'reverse methanogenesis' and is paramount to understanding the still-unknown mechanism of the last step of methanogenesis. The ability of MCR to cleave the particularly strong C-H bond of methane without the involvement of highly reactive oxygen-derived intermediates is directly relevant to catalytic C-H activation, currently an area of great interest in chemistry.

  9. Correlations among antral follicular echotexture, apoptosis and expression of key steroidogenic enzymes in sheep

    PubMed Central

    VANDUZER, Taylor; DUGGAVATHI, Raj; MURAWSKI, Maciej; ZIEBA, Dorota A.; SROKA, Patrycja; BARTLEWSKI, Pawel M.

    2014-01-01

    Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3β-hydroxysteroid dehydrogenase, 3β-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3β-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays. PMID:25109269

  10. Lysophosphatidylcholine Acyltransferase 3 Is the Key Enzyme for Incorporating Arachidonic Acid into Glycerophospholipids during Adipocyte Differentiation

    PubMed Central

    Eto, Miki; Shindou, Hideo; Koeberle, Andreas; Harayama, Takeshi; Yanagida, Keisuke; Shimizu, Takao

    2012-01-01

    Cellular membranes contain glycerophospholipids, which have important structural and functional roles in cells. Glycerophospholipids are first formed in the de novo pathway (Kennedy pathway) and are matured in the remodeling pathway (Lands’ cycle). Recently, lysophospholipid acyltransferases functioning in Lands’ cycle were identified and characterized. Several enzymes involved in glycerophospholipid biosynthesis have been reported to have important roles in adipocytes. However, the role of Lands’ cycle in adipogenesis has not yet been reported. Using C3H10T1/2, a cell line capable of differentiating to adipocyte-like cells in vitro, changes of lysophospholipid acyltransferase activities were investigated. Lysophosphatidylcholine acyltransferase (LPCAT), lysophosphatidylethanolamine acyltransferase (LPEAT) and lysophosphatidylserine acyltransferase (LPSAT) activities were enhanced, especially with 18:2-CoA and 20:4-CoA as donors. Correspondingly, mRNA expression of LPCAT3, which possesses LPCAT, LPEAT and LPSAT activities with high specificity for 18:2- and 20:4-CoA, was upregulated during adipogenesis. Analysis of acyl-chain compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) showed a change in their profiles between preadipocytes and adipocytes, including an increase in the percentage of arachidonic acid-containing phospholipids. These changes are consistent with the activities of LPCAT3. Therefore, it is possible that enhanced phospholipid remodeling by LPCAT3 may be associated with adipocyte differentiation. PMID:23208369

  11. Protective effect of p-methoxycinnamic acid, an active phenolic acid against 1,2-dimethylhydrazine-induced colon carcinogenesis: modulating biotransforming bacterial enzymes and xenobiotic metabolizing enzymes.

    PubMed

    Gunasekaran, Sivagami; Venkatachalam, Karthikkumar; Jeyavel, Kabalimoorthy; Namasivayam, Nalini

    2014-09-01

    Objective of the study is to evaluate the modifying potential of p-methoxycinnamic acid (p-MCA), an active rice bran phenolic acid on biotransforming bacterial enzymes and xenobiotic metabolizing enzymes in 1,2-dimethylhydrazine-induced rat colon carcinogenesis. 48 male albino wistar rats were divided into six groups. Group1 (control) received modified pellet diet and 0.1 % carboxymethylcellulose; group2 received modified pellet diet along with p-MCA (80 mg/kg b.wt. p.o.) everyday for 16 weeks; groups 3-6 received 1,2-dimethylhydrazine (DMH) (20 mg/kg b.wt.) subcutaneous injection once a week for the first 4 weeks, while groups 4-6 received p-MCA at three different doses of 20, 40 and 80 mg/kg b.wt. p.o. everyday for 16 weeks. A significant increase in carcinogen-activating enzymes (cytochrome P450, cytochrome b5, cytochrome P4502E1, NADH-cytochrome-b5-reductase and NADPH-cytochrome-P450 reductase) with concomitant decrease in phaseII enzymes, DT-Diaphorase, glutathione S-transferase, UDP-glucuronyl-transferase and gamma glutamyltransferase were observed in group3 compared to control. DMH treatment significantly increased the activities of feacal and colonic bacterial enzymes (β-glucosidase, β-galactosidase, β-glucuronidase, nitroreductase, sulphatase and mucinase). p-MCA supplementation (40 mg/kg b.wt) to carcinogen exposed rats inhibited these enzymes, which were near those of control rats. The formation of dysplastic aberrant crypt foci in the colon and the histopathological observations of the liver also supports our biochemical findings. p-MCA (40 mg/kg b.wt.) offers remarkable modulating efficacy of biotransforming bacterial and xenobiotic metabolizing enzymes in colon carcinogenesis.

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

    PubMed

    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.

  13. Inhibition of Key Digestive Enzymes Related to Diabetes and Hyperlipidemia and Protection of Liver-Kidney Functions by Trigonelline in Diabetic Rats

    PubMed Central

    Hamden, Khaled; Mnafgui, Kais; Amri, Zahra; Aloulou, Ahmed; Elfeki, Abdelfattah

    2013-01-01

    Diabetes is a serious health problem and a source of risk for numerous severe complications such as obesity and hypertension. Treatment of diabetes and its related diseases can be achieved by inhibiting key digestive enzymes related to starch and lipid digestion. The findings revealed that the administration of trigonelline to surviving diabetic rats helped to protect the pancreas β-cells from death and damage. Additionally, the supplement of trigonelline to surviving diabetic rats significantly decreased intestinal α-amylase and maltase by 36 and 52%, respectively, which led to a significant decrease in the blood glucose rate by 46%. Moreover, the administration of trigonelline to surviving diabetic rats potentially inhibited key enzymes of lipid metabolism and absorption such as lipase activity in the small intestine by 56%, which led to a notable decrease in serum triglyceride (TG) and total cholesterol (TC) rates and an increase in the HDL cholesterol level. This treatment also improved glucose, maltase, starch, and lipid oral tolerance. Trigonelline was also observed to protect the liver-kidney functions efficiently, which was evidenced by the significant decrease in the serum aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transpeptidase (GGT), and lactate dehydrogenase (LDH) activities and creatinine, albumin, and urea rates. The histological analysis of the pancreas, liver, and kidney tissues further established the positive effect of trigonelline. Overall, the findings presented in this study demonstrate that the administration of trigonelline to diabetic rats can make it a potentially strong candidate for industrial application as a pharmacological agent for the treatment of hyperglycemia, hyperlipidemia, and liver-kidney dysfunctions. PMID:23641341

  14. Targeting of ECM molecules and their metabolizing enzymes and receptors for the treatment of CNS diseases.

    PubMed

    Berezin, Vladimir; Walmod, Peter S; Filippov, Mikhail; Dityatev, Alexander

    2014-01-01

    Extracellular matrix (ECM) molecules, their receptors at the cell surface, and cell adhesion molecules (CAMs) involved in cell-cell or cell-ECM interactions are implicated in processes related to major diseases of the central nervous system including Alzheimer's disease (AD), epilepsy, schizophrenia, addiction, multiple sclerosis, Parkinson's disease, and cancer. There are multiple strategies for targeting the ECM molecules and their metabolizing enzymes and receptors with antibodies, peptides, glycosaminoglycans, and other natural and synthetic compounds. ECM-targeting treatments include chondroitinase ABC, heparin/heparan sulfate-mimicking oligosaccharides, ECM cross-linking antibodies, and drugs stimulating expression of ECM molecules. The amount or activity of ECM-degrading enzymes like matrix metalloproteinases can be modulated indirectly via the regulation of endogenous inhibitors like TIMPs and RECK or at the transcriptional and translational levels using, e.g., histone deacetylase inhibitors, synthetic inhibitors like Periostat, microRNA-interfering drugs like AC1MMYR2, and natural compounds like flavonoids, epigallocatechin-3-gallate, anacardic acid, and erythropoietin. Among drugs targeting the major ECM receptors, integrins, are the anticancer peptide cilengitide and anti-integrin antibodies, which have a potential for treatment of stroke, multiple sclerosis, and AD. The latter can be also potentially treated with modulators of CAMs, such as peptide mimetics derived from L1-CAM and NCAM1.

  15. Polymorphisms in drug-metabolizing enzymes: What is their clinical relevance and why do they exist?

    SciTech Connect

    Nebert, D.W.

    1997-02-01

    The beautiful report by Sachse in this issue of the journal represents the culmination of 2 decades of increasingly exciting work on the {open_quotes}debrisoquine oxidation polymorphism,{close_quotes} one of dozens of pharmacogenetic or ecogenetic polymorphisms that have been shown to have an important impact on innumerable clinical diseases. Pharmacogenetics is the study of the hereditary basis of the differences in responses to drugs. Ecogenetics is the broader field of interindividual differences in response to all environmental chemical and physical agents (e.g., heavy metals, insecticides, compounds formed during combustion, and UV radiation). It is now clear that each of us has his or her own {open_quotes}individual fingerprint{close_quotes} of unique alleles encoding the so-called drug-metabolizing enzymes (DMEs) and the receptors that regulate these enzymes. In this invited editorial, I first introduce the current thinking in the field of DME (and DME-receptor) research and how DMEs have evolved from animal-plant interactions. I then describe the debrisoquine oxidation polymorphism, as well as two other relevant DME polymorphisms; show the relationship between these polymorphisms and human disease; provide examples of synergistic effects caused by the combination of two DME polymorphisms; and discuss the ethical considerations of such research. Last, I speculate on why these allelic frequencies of the DME genes might exist in human populations in the first place. 35 refs.

  16. Polymorphisms in carcinogen metabolism enzymes, fish intake, and risk of prostate cancer.

    PubMed

    Catsburg, Chelsea; Joshi, Amit D; Corral, Román; Lewinger, Juan Pablo; Koo, Jocelyn; John, Esther M; Ingles, Sue A; Stern, Mariana C

    2012-07-01

    Cooking fish at high temperature can produce potent carcinogens such as heterocyclic amines and polycyclic aromatic hydrocarbons. The effects of these carcinogens may undergo modification by the enzymes responsible for their detoxification and/or activation. In this study, we investigated genetic polymorphisms in nine carcinogen metabolism enzymes and their modifying effects on the association between white or dark fish consumption and prostate cancer (PCA) risk. We genotyped 497 localized and 936 advanced PCA cases and 760 controls from the California Collaborative Case-Control Study of Prostate Cancer. Three polymorphisms, EPHX1 Tyr113His, CYP1B1 Leu432Val and GSTT1 null/present, were associated with localized PCA risk. The PTGS2 765 G/C polymorphism modified the association between white fish consumption and advanced PCA risk (interaction P 5 0.002), with high white fish consumption being positively associated with risk only among carriers of the C allele. This effect modification by PTGS2 genotype was stronger when restricted to consumption of well-done white fish (interaction P 5 0.021). These findings support the hypotheses that changes in white fish brought upon by high-temperature cooking methods, such as carcinogen accumulation and/or fatty acid composition changes, may contribute to prostate carcinogenesis. However, the gene-diet interactions should be interpreted with caution given the limited sample size. Thus, our findings require further validation with additional studies.

  17. Toxicology in the Fast Lane: Application of High-Throughput Bioassays to Detect Modulation of Key Enzymes and Receptors

    PubMed Central

    Morisseau, Christophe; Merzlikin, Oleg; Lin, Amy; He, Guochun; Feng, Wei; Padilla, Isela; Denison, Michael S.; Pessah, Isaac N.; Hammock, Bruce D.

    2009-01-01

    Background Legislation at state, federal, and international levels is requiring rapid evaluation of the toxicity of numerous chemicals. Whole-animal toxicologic studies cannot yield the necessary throughput in a cost-effective fashion, leading to a critical need for a faster and more cost-effective toxicologic evaluation of xenobiotics. Objectives We tested whether mechanistically based screening assays can rapidly provide information on the potential for compounds to affect key enzymes and receptor targets, thus identifying those compounds requiring further in-depth analysis. Methods A library of 176 synthetic chemicals was prepared and examined in a high-throughput screening (HTS) manner using nine enzyme-based and five receptor-based bioassays. Results All the assays have high Z′ values, indicating good discrimination among compounds in a reliable fashion, and thus are suitable for HTS assays. On average, three positive hits were obtained per assay. Although we identified compounds that were previously shown to inhibit a particular enzyme class or receptor, we surprisingly discovered that triclosan, a microbiocide present in personal care products, inhibits carboxylesterases and that dichlone, a fungicide, strongly inhibits the ryanodine receptors. Conclusions Considering the need to rapidly screen tens of thousands of anthropogenic compounds, our study shows the feasibility of using combined HTS assays as a novel approach toward obtaining toxicologic data on numerous biological end points. The HTS assay approach is very useful to quickly identify potentially hazardous compounds and to prioritize them for further in-depth studies. PMID:20049205

  18. The Structure of the Human RNase H2 Complex Defines Key Interaction Interfaces Relevant to Enzyme Function and Human Disease*

    PubMed Central

    Reijns, Martin A. M.; Bubeck, Doryen; Gibson, Lucien C. D.; Graham, Stephen C.; Baillie, George S.; Jones, E. Yvonne; Jackson, Andrew P.

    2011-01-01

    Ribonuclease H2 (RNase H2) is the major nuclear enzyme involved in the degradation of RNA/DNA hybrids and removal of ribonucleotides misincorporated in genomic DNA. Mutations in each of the three RNase H2 subunits have been implicated in a human auto-inflammatory disorder, Aicardi-Goutières Syndrome (AGS). To understand how mutations impact on RNase H2 function we determined the crystal structure of the human heterotrimer. In doing so, we correct several key regions of the previously reported murine RNase H2 atomic model and provide biochemical validation for our structural model. Our results provide new insights into how the subunits are arranged to form an enzymatically active complex. In particular, we establish that the RNASEH2A C terminus is a eukaryotic adaptation for binding the two accessory subunits, with residues within it required for enzymatic activity. This C-terminal extension interacts with the RNASEH2C C terminus and both are necessary to form a stable, enzymatically active heterotrimer. Disease mutations cluster at this interface between all three subunits, destabilizing the complex and/or impairing enzyme activity. Altogether, we locate 25 out of 29 residues mutated in AGS patients, establishing a firm basis for future investigations into disease pathogenesis and function of the RNase H2 enzyme. PMID:21177854

  19. Folate cycle enzyme MTHFD1L confers metabolic advantages in hepatocellular carcinoma.

    PubMed

    Lee, Derek; Xu, Iris Ming-Jing; Chiu, David Kung-Chun; Lai, Robin Kit-Ho; Tse, Aki Pui-Wah; Lan Li, Lynna; Law, Cheuk-Ting; Tsang, Felice Ho-Ching; Wei, Larry Lai; Chan, Cerise Yuen-Ki; Wong, Chun-Ming; Ng, Irene Oi-Lin; Wong, Carmen Chak-Lui

    2017-04-10

    Cancer cells preferentially utilize glucose and glutamine, which provide macromolecules and antioxidants that sustain rapid cell division. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of these nutrients. The folate cycle, through transfer of a carbon unit between tetrahydrofolate and its derivatives in the cytoplasmic and mitochondrial compartments, produces other metabolites that are essential for cell growth, including nucleotides, methionine, and the antioxidant NADPH. Here, using hepatocellular carcinoma (HCC) as a cancer model, we have observed a reduction in growth rate upon withdrawal of folate. We found that an enzyme in the folate cycle, methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), plays an essential role in support of cancer growth. We determined that MTHFD1L is transcriptionally activated by NRF2, a master regulator of redox homeostasis. Our observations further suggest that MTHFD1L contributes to the production and accumulation of NADPH to levels that are sufficient to combat oxidative stress in cancer cells. The elevation of oxidative stress through MTHFD1L knockdown or the use of methotrexate, an antifolate drug, sensitizes cancer cells to sorafenib, a targeted therapy for HCC. Taken together, our study identifies MTHFD1L in the folate cycle as an important metabolic pathway in cancer cells with the potential for therapeutic targeting.

  20. Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR☆

    PubMed Central

    Tolson, Antonia H.; Wang, Hongbing

    2010-01-01

    Drug-metabolizing enzymes (DMEs) and transporters play pivotal roles in the disposition and detoxification of numerous foreign and endogenous chemicals. To accommodate chemical challenges, the expression of many DMEs and transporters is up-regulated by a group of ligand-activated transcription factors namely nuclear receptors (NRs). The importance of NRs in xenobiotic metabolism and clearance is best exemplified by the most promiscuous xenobiotic receptors: pregnane X receptor (PXR, NR1I2) and constitutive androstane/activated receptor (CAR, NR1I3). Together, these two receptors govern the inductive expression of a largely overlapping array of target genes encoding phase I and II DMEs, and drug transporters. Moreover, PXR and CAR also represent two distinctive mechanisms of NR activation, whereby CAR demonstrates both constitutive and ligand-independent activation. In this review, recent advances in our understanding of PXR and CAR as xenosensors are discussed with emphasis placed on the differences rather than similarities of these two xenobiotic receptors in ligand recognition and target gene regulation. PMID:20727377

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

    PubMed Central

    Andriotis, Vasilios M. E.; Rejzek, Martin; Rugen, Michael D.; Svensson, Birte; Smith, Alison M.; Field, Robert A.

    2016-01-01

    Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively 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. PMID:26862201

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

    PubMed

    Andriotis, Vasilios M E; Rejzek, Martin; Rugen, Michael D; Svensson, Birte; Smith, Alison M; Field, Robert A

    2016-02-01

    Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively 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.

  3. Two chitin metabolic enzyme genes from Hyriopsis cumingii: cloning, characterization, and potential functions.

    PubMed

    Wang, G-L; Xu, B; Bai, Z-Y; Li, J-L

    2012-12-19

    Chitin, the second most important natural polymer in the world, and its N-deacetylated derivative chitosan are found in a wide variety of organisms. These versatile biopolymers are associated with a broad range of biological functions. This article is the first to report the potential functions of 2 chitin metabolic enzyme genes from Hyriopsis cumingii. A chitinase-3 gene (Chi-3) and a chitin deacetylase gene (Cda) were cloned from H. cumingii and characterized. Semi-quantitative reverse transcription polymerase chain reaction analysis revealed that the Cda gene was expressed in blood, mantle, liver, stomach, kidney, intestine, gill, and foot, whereas Chi-3 was also expressed in those tissues but not in blood. The tissue-specific expression of H. cumingii Chi-3 indicated that other Chi genes may be involved in the H. cumingii immune system. Real-time quantitative polymerase chain reaction analysis showed that the expression of Chi-3 was significantly (P < 0.05) upregulated 12 h after shell damage, suggesting that Chi-3 might hydrolyze superfluous chitin after shell recovery and play a role in shell formation. Conversely, Cda expression did not change significantly (P > 0.05) to maintain a certain degree of acetylation in chitin/chitosan. This study enriches the basic research on chitin metabolic genes and lays foundations for further research of shell regeneration in mussels.

  4. Alteration of Fatty-Acid-Metabolizing Enzymes Affects Mitochondrial Form and Function in Hereditary Spastic Paraplegia

    PubMed Central

    Tesson, Christelle; Nawara, Magdalena; Salih, Mustafa A.M.; Rossignol, Rodrigue; Zaki, Maha S.; Al Balwi, Mohammed; Schule, Rebecca; Mignot, Cyril; Obre, Emilie; Bouhouche, Ahmed; Santorelli, Filippo M.; Durand, Christelle M.; Oteyza, Andrés Caballero; El-Hachimi, Khalid H.; Al Drees, Abdulmajeed; Bouslam, Naima; Lamari, Foudil; Elmalik, Salah A.; Kabiraj, Mohammad M.; Seidahmed, Mohammed Z.; Esteves, Typhaine; Gaussen, Marion; Monin, Marie-Lorraine; Gyapay, Gabor; Lechner, Doris; Gonzalez, Michael; Depienne, Christel; Mochel, Fanny; Lavie, Julie; Schols, Ludger; Lacombe, Didier; Yahyaoui, Mohamed; Al Abdulkareem, Ibrahim; Zuchner, Stephan; Yamashita, Atsushi; Benomar, Ali; Goizet, Cyril; Durr, Alexandra; Gleeson, Joseph G.; Darios, Frederic; Brice, Alexis; Stevanin, Giovanni

    2012-01-01

    Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function. PMID:23176821

  5. Rice Debranching Enzyme Isoamylase3 Facilitates Starch Metabolism and Affects Plastid Morphogenesis

    PubMed Central

    Yun, Min-Soo; Umemoto, Takayuki; Kawagoe, Yasushi

    2011-01-01

    Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on β-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3–green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice. PMID:21551159

  6. Oxidative metabolism in guinea pig ventricular myocytes protected from proteolytic enzyme activity.

    PubMed

    Bailey, L E; Carlos, H; Amian, A; Moon, K E

    1987-07-01

    Surface structures on guinea pig ventricular myocytes were protected from proteolytic enzyme activity with 100 KIU.ml-2 aprotinin during mechanical disaggregation. Intact myocytes, approximately 7.5 X 10(6) cells.g-1 ventricular wet weight, were separated from debris and damaged cells using Cytodex I tissue culture supports. Cellular ultrastructure did not differ from that observed in intact tissue. Neither spontaneous contractions nor contracture were ever observed in these myocytes in calcium concentrations of 10 mmol.litre-1. Dinitrophenol (0.2 mmol. litre-1) uncoupled respiration in the myocytes but only after the sarcolemma had been disrupted with Triton X100. The adenosine diphosphate to oxygen ratio of mitochondria isolated from the myocytes was 2.4(0.2) and the respiratory control index 2.6(0.3). Calcium (1.8 mmol.litre-1) increased oxygen uptake in the presence of 10 mmol.litre-1 pyruvate or 11 mmol.litre-1 glucose but not 17 mmol. litre-1 succinate. Succinate dependent oxygen consumption was greater than pyruvate dependent oxygen consumption (1090.0(190.0) and 40.1(0.8) nl.min-1.mg-1 protein respectively). The Crabtree effect was present. Oxidative metabolism was normal in cells stored at 10 degrees C for seven days but deteriorated rapidly thereafter. The results indicate that myocytes disaggregated by this procedure retain many of the morphological and metabolic characteristics of intact cardiac muscle cells and are relatively homogeneous with respect to calcium tolerance and metabolic function.

  7. Vasomotor symptom prevalence is associated with polymorphisms in sex steroid-metabolizing enzymes and receptors.

    PubMed

    Crandall, Carolyn J; Crawford, Sybil L; Gold, Ellen B

    2006-09-01

    The relation of single nucleotide polymorphisms (SNPs) of genes involved in estrogen function to vasomotor symptoms (VMS) has been inadequately explored. We evaluated SNPs in sex steroid-metabolizing genes and estrogen receptors (ERs) for their association with VMS (hot flashes, night sweats, and/or cold sweats) reported by women who were premenopausal or in early perimenopause at baseline. The study population was drawn from participants in the Study of Women's Health Across the Nation (SWAN). African American, Caucasian, Chinese, and Japanese women, 42 to 52 years of age at baseline, who were enrolled in the longitudinal, community-based cohort of SWAN provided questionnaire, interview, weight and height measurements, and serum samples through the sixth annual visit. SNPs associated with the sex steroid hormone pathway were genotyped and available for 1,538 participants. These SNPs were associated with reporting VMS > or =6 days compared with <6 days in the past 2 weeks using race/ethnicity-specific repeated measures logistic regression models. Participants were on average 46 years old at baseline. The prevalence of VMS reporting increased in all racial/ethnic groups from baseline to the sixth annual follow-up visit. After adjustment for covariates, several SNPs encoding genes responsible for estrogen metabolism and ERs were associated with decreased odds of reporting VMS, including the CYP1B1 rs1056836 GC genotype in African American women; 17HSD rs615942 TG, 17HSD rs592389 TG, and 17HSD rs2830 AG genotypes in Caucasian women; and the CYP1A1 rs2606345 AC genotype in Chinese women. We identified race/ethnicity-specific associations between VMS reporting and specific polymorphisms for sex steroid-metabolizing enzymes and sex steroid receptors. Clarification of the mechanisms of the associations and confirmation in other populations is warranted.

  8. Xenobiotic metabolizing enzyme activities in cells used for testing skin sensitization in vitro.

    PubMed

    Fabian, E; Vogel, D; Blatz, V; Ramirez, T; Kolle, S; Eltze, T; van Ravenzwaay, B; Oesch, F; Landsiedel, R

    2013-09-01

    For ethical and regulatory reasons, in vitro tests for scoring potential toxicities of cosmetics are essential. A test strategy for investigating potential skin sensitization using two human keratinocytic and two human dendritic cell lines has been developed (Mehling et al. Arch Toxicol 86:1273–1295, 2012). Since prohaptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (XME) activities in these cell lines is of high interest. In this study, XME activity assays, monitoring metabolite or cofactor, showed the following: all three passages of keratinocytic (KeratinoSens® and LuSens) and dendritic (U937 und THP-1) cells displayed N-acetyltransferase 1 (NAT1) activities (about 6–60 nmol/min/mg S9-protein for acetylation of para-aminobenzoic acid). This is relevant since reactive species of many cosmetics are metabolically controlled by cutaneous NAT1. Esterase activities of about 1–4 nmol fluorescein diacetate/min/mg S9-protein were observed in all passages of investigated keratinocytic and about 1 nmol fluorescein diacetate/min/mg S9-protein in dendritic cell lines. This is also of practical relevance since many esters and amides are detoxified and others activated by cutaneous esterases. In both keratinocytic cell lines, activities of aldehyde dehydrogenase (ALDH) were observed (5–17 nmol product/min/mg cytosolic protein). ALDH is relevant for the detoxication of reactive aldehydes. Activities of several other XME were below detection, namely the investigated cytochrome P450-dependent alkylresorufin O-dealkylases 7-ethylresorufin O-deethylase, 7-benzylresorufin O-debenzylase and 7-pentylresorufin O-depentylase (while NADPH cytochrome c reductase activities were much above the limit of quantification), the flavin-containing monooxygenase, the alcohol dehydrogenase as well as the UDP glucuronosyl transferase activities.

  9. Cigarette Smoking, Genetic Variants in Carcinogen-metabolizing Enzymes, and Colorectal Cancer Risk

    PubMed Central

    Cleary, Sean P.; Cotterchio, Michelle; Shi, Ellen; Gallinger, Steven; Harper, Patricia

    2010-01-01

    The risk of colorectal cancer associated with smoking is unclear and may be influenced by genetic variation in enzymes that metabolize cigarette carcinogens. The authors examined the colorectal cancer risk associated with smoking and 26 variants in carcinogen metabolism genes in 1,174 colorectal cancer cases and 1,293 population-based controls recruited in Canada by the Ontario Familial Colorectal Cancer Registry from 1997 to 2001. Adjusted odds ratios were calculated by multivariable logistic regression. Smoking for >27 years was associated with a statistically significant increased colorectal cancer risk (adjusted odds ratio (AOR) = 1.25, 95% confidence interval (CI): 1.02, 1.53) in all subjects. Colorectal cancer risk associated with smoking was higher in males for smoking status, duration, and intensity. The CYP1A1-3801-CC (AOR = 0.47, 95% CI: 0.23, 0.94) and CYP2C9-430-CT (AOR = 0.82, 95% CI: 0.68, 0.99) genotypes were associated with decreased risk, and the GSTM1-K173N-CG (AOR = 1.99, 95% CI: 1.21, 3.25) genotype was associated with an increased risk of colorectal cancer. Statistical interactions between smoking and genetic variants were assessed by comparing logistic regression models with and without a multiplicative interaction term. Significant interactions were observed between smoking status and SULT1A1-638 (P = 0.02), NAT2-857 (P = 0.01), and CYP1B1-4390 (P = 0.04) variants and between smoking duration and NAT1-1088 (P = 0.02), SULT1A1-638 (P = 0.04), and NAT1-acetylator (P = 0.03) status. These findings support the hypothesis that prolonged cigarette smoking is associated with increased risk of colorectal cancer and that this risk may be modified by variation in carcinogen metabolism genes. PMID:20937634

  10. Temporal repeatability of metabolic rate and the effect of organ mass and enzyme activity on metabolism in European eel (Anguilla anguilla).

    PubMed

    Boldsen, Martin Maagaard; Norin, Tommy; Malte, Hans

    2013-05-01

    Intraspecific variation in metabolic rate of fish can be pronounced and have been linked to various fitness-related behavioural and physiological traits, but the underlying causes for this variation have received far less attention than the consequences of it. In the present study we investigated whether European eels (Anguilla anguilla) displayed temporal repeatability of body-mass-corrected (residual) metabolic rate over a two-month period and if variations in organ mass and enzyme activity between individual fish could be the cause for the observed variation in metabolic rate. Both standard metabolic rate (SMR; Pearson's r=0.743) and routine metabolic rate (RMR; r=0.496) were repeatable over the two-month period. Repeatability of RMR is an interesting finding as it indicates that the level of spontaneous activity in respirometer-confined fish is not random. Cumulative organ mass (liver, heart, spleen and intestine; mean 1.6% total body mass) was found to explain 38% of the variation in SMR (r=0.613) with the liver (one of the metabolically most active organs) being the driver for the correlation between organ mass and metabolic rate. No relationships were found for either liver citrate synthase or cytochrome oxidase activity and metabolic rate in the European eels. Reasons for, and contributions to, the observed variation in metabolic rate are discussed.

  11. Activity of xenobiotic-metabolizing enzymes in the liver of rats with multi-vitamin deficiency.

    PubMed

    Tutelyan, Victor A; Kravchenko, Lidia V; Aksenov, Ilya V; Trusov, Nikita V; Guseva, Galina V; Kodentsova, Vera M; Vrzhesinskaya, Oksana A; Beketova, Nina A

    2013-01-01

    The purpose of the study was to determine how multi-vitamin deficiency affects xenobiotic-metabolizing enzyme (XME) activities in the rat liver. Vitamin levels and XME activities were studied in the livers of male Wistar rats who were fed for 4 weeks with semi-synthetic diets containing either adequate (100 % of recommended vitamin intake) levels of vitamins (control), or decreased vitamin levels (50 % or 20 % of recommended vitamin intake). The study results have shown that moderate vitamin deficiency (50 %) leads to a decrease of vitamin A levels only, and to a slight increase, as compared with the control, in the following enzyme activities: methoxyresorufin O-dealkylase (MROD) activity of CYP1 A2 - by 34 % (p < 0.05), UDP-glucuronosyl transferase - by 26 % (p < 0.05), and quinone reductase - by 55 % (p < 0.05). Profound vitamin deficiency (20 %) led to a decrease of vitamins A, E, B1, B2, and C, and enzyme activities in the liver: MROD - to 78 % of the control level (p < 0.05), 4-nitrophenol hydroxylase - to 74 % (p < 0.05), heme oxygenase-1 - to 83 % (p < 0.05), and quinone reductase - to 60 % (p < 0.05). At the same time, the UDP-glucuronosyl transferase activity and ethoxyresorufin O-dealkylase activity of CYP1A1, pentoxyresorufin O-dealkylase activity of CYP2B1/2 and 6β-testosterone hydroxylase, as well as the total activity of glutathione transferase did not differ from the control levels. The study has demonstrated that profound multi-vitamin deficiency is associated with a decrease in the expression of CYP1A2 and CYP3A1 mRNAs to 62 % and 79 %, respectively. These data indicated that a short-term but profound multi-vitamin deficiency in rats leads to a decrease in the activities and expression of the some XME that play an important role in detoxification of xenobiotics and metabolism of drugs and antioxidant protection.

  12. Identification of metabolic pathways involved in the biotransformation of tolperisone by human microsomal enzymes.

    PubMed

    Dalmadi, Balázs; Leibinger, János; Szeberényi, Szabolcs; Borbás, Tímea; Farkas, Sándor; Szombathelyi, Zsolt; Tihanyi, Károly

    2003-05-01

    The in vitro metabolism of tolperisone, 1-(4-methyl-phenyl)-2-methyl-3-(1-piperidino)-1-propanone-hydrochloride, a centrally acting muscle relaxant, was examined in human liver microsomes (HLM) and recombinant enzymes. Liquid chromatography-mass spectrometry measurements revealed methyl-hydroxylation (metabolite at m/z 261; M1) as the main metabolic route in HLM, however, metabolites of two mass units greater than the parent compound and the hydroxy-metabolite were also detected (m/z 247 and m/z 263, respectively). The latter was identified as carbonyl-reduced M1, the former was assumed to be the carbonyl-reduced parent compound. Isoform-specific cytochrome P450 (P450) inhibitors, inhibitory antibodies, and experiments with recombinant P450s pointed to CYP2D6 as the prominent enzyme in tolperisone metabolism. CYP2C19, CYP2B6, and CYP1A2 are also involved to a smaller extent. Hydroxymethyl-tolperisone formation was mediated by CYP2D6, CYP2C19, CYP1A2, but not by CYP2B6. Tolperisone competitively inhibited dextromethorphan O-demethylation and bufuralol hydroxylation (K(i) = 17 and 30 microM, respectively). Tolperisone inhibited methyl p-tolyl sulfide oxidation (K(i) = 1200 microM) in recombinant flavin-containing monooxygenase 3 (FMO3) and resulted in a 3-fold (p < 0.01) higher turnover number using rFMO3 than that of control microsomes. Experiments using nonspecific P450 inhibitors-SKF-525A, 1-aminobenzotriazole, 1-benzylimidazole, and anti-NADPH-P450-reductase antibodies-resulted in 61, 47, 49, and 43% inhibition of intrinsic clearance in HLM, respectively, whereas hydroxymethyl-metabolite formation was inhibited completely by nonspecific chemical inhibitors and by 80% with antibodies. Therefore, it was concluded that tolperisone undergoes P450-dependent and P450-independent microsomal biotransformations to the same extent. On the basis of metabolites formed and indirect evidences of inhibition studies, a considerable involvement of a microsomal reductase is

  13. Vascularization, High-Volume Solution Flow, and Localized Roles for Enzymes of Sucrose Metabolism during Tumorigenesis by Agrobacterium tumefaciens1

    PubMed Central

    Wächter, Rebecca; Langhans, Markus; Aloni, Roni; Götz, Simone; Weilmünster, Anke; Koops, Ariane; Temguia, Leopoldine; Mistrik, Igor; Pavlovkin, Jan; Rascher, Uwe; Schwalm, Katja; Koch, Karen E.; Ullrich, Cornelia I.

    2003-01-01

    Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens. Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host. Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment. CO2 release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks. However, tumor water loss contributed little to water flow to host shoots. Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance. Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors. Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization. Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase. Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport. Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment. PMID:14526106

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

    PubMed Central

    Arriarán, Sofía; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio

    2015-01-01

    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. PMID:26587356

  15. β-d-Glucosidase as "key enzyme" for sorghum cyanogenic glucoside (dhurrin) removal and beer bioflavouring.

    PubMed

    Tokpohozin, Sedjro Emile; Fischer, Susann; Sacher, Bertram; Becker, Thomas

    2016-11-01

    Sorghum malt used during African beer processing contains a high level of cyanogenic glucoside (dhurrin), up to 1375 ppm. In traditional sorghum malting and mashing, dhurrin is not sufficiently hydrolyzed due to uncontrolled germination and a high gelatinization temperature. The cyanide content of traditional African beers (11 ppm) is higher than the minimum dose (1 ppm) required to form carcinogenic ethyl carbamate during alcoholic fermentation. In the detoxification process, aryl-β-d-glucosidase (dhurrinase) is the "key component". For significant dhurrin hydrolysis during mashing, optimizing dhurrinase synthesis during malting is a good solution to reduce dhurrin completely to below the harmful dose in the sorghum wort. Lactic acid bacteria which exhibit aryl-β-d-glucosidase prior to alcoholic fermentation may help to reduce ethyl carbamate content in alcoholic beverages. Moreover, some specific β-d-glucosidases have a dual property, being able to cleave and synthesize glucosides bonds and thereby generating good precursors for beer bioflavouring.

  16. Transcription of genes coding for metabolic key functions in Nitrosomonas europaea during aerobic and anaerobic growth.

    PubMed

    Beyer, Sonja; Gilch, Stefan; Meyer, Ortwin; Schmidt, Ingo

    2009-01-01

    Nitrosomonas europaea can grow under conditions of chemolithoautotrophic aerobic (oxygen as oxidant) as well as anaerobic [nitrogen dioxide (NO(2)) as oxidant] nitrification or chemoorganotrophic anaerobic pyruvate-dependent denitrification. In this study, the adaptation of the transcription (mRNA synthesis/concentration) of N. europaea to aerobic and anaerobic growth conditions was evaluated and the transcription of genes coding for metabolic key functions was analyzed: nitrogen and energy metabolism (amoA, hao, rh1, nirK, norB, nsc, aceE, ldhA, ppc, gltA, odhA, coxA), carbon dioxide fixation (cbbL), gluconeogenesis (ppsA), cell growth (ftsZ), and oxidative stress (sodB). During aerobic ammonia oxidation the specific activities of ammonia oxidation, nitrite reduction, and the growth rates correlated with the transcription level of the corresponding genes amoA/hao, nirK/norB/nsc, and cbbL/ftsZ. In anaerobically ammonia-oxidizing cells of N. europaea, the cellular mRNA concentrations of amoA, hao, rh1,coxA, cbbL, ftsZ, and sodB were reduced compared with aerobically nitrifying cells, but the mRNA levels of nirK, norB, and nsc were significantly increased. During anaerobic pyruvate-dependent denitrification, the mRNA abundance of nirK, norB, nsc, aceE, gltA, and odhA was increased, while the concentrations of amoA,hao, rh1, coxAcbbL, ftsZ, and sodB were significantly reduced. Temperature, pH value, and NH(4)(+), O(2), NO, and NO(2) concentrations had comparatively small effects on the transcription of the studied genes.

  17. Adaptive Control Model Reveals Systematic Feedback and Key Molecules in Metabolic Pathway Regulation

    PubMed Central

    Moffitt, Richard A.; Merrill, Alfred H.; Wang, May D.

    2011-01-01

    Abstract Robust behavior in metabolic pathways resembles stabilized performance in systems under autonomous control. This suggests we can apply control theory to study existing regulation in these cellular networks. Here, we use model-reference adaptive control (MRAC) to investigate the dynamics of de novo sphingolipid synthesis regulation in a combined theoretical and experimental case study. The effects of serine palmitoyltransferase over-expression on this pathway are studied in vitro using human embryonic kidney cells. We report two key results from comparing numerical simulations with observed data. First, MRAC simulations of pathway dynamics are comparable to simulations from a standard model using mass action kinetics. The root-sum-square (RSS) between data and simulations in both cases differ by less than 5%. Second, MRAC simulations suggest systematic pathway regulation in terms of adaptive feedback from individual molecules. In response to increased metabolite levels available for de novo sphingolipid synthesis, feedback from molecules along the main artery of the pathway is regulated more frequently and with greater amplitude than from other molecules along the branches. These biological insights are consistent with current knowledge while being new that they may guide future research in sphingolipid biology. In summary, we report a novel approach to study regulation in cellular networks by applying control theory in the context of robust metabolic pathways. We do this to uncover potential insight into the dynamics of regulation and the reverse engineering of cellular networks for systems biology. This new modeling approach and the implementation routines designed for this case study may be extended to other systems. Supplementary Material is available at www.liebertonline.com/cmb. PMID:21314456

  18. Acetate and other Volatile Fatty Acids - Key Intermediates in marine sediment metabolism - Thermodynamic and kinetic implications

    NASA Astrophysics Data System (ADS)

    Glombitza, C.; Jaussi, M.; Røy, H.; Jørgensen, B. B.

    2014-12-01

    Volatile fatty acids (VFAs) play important roles as key intermediates in the anaerobic metabolism of subsurface microbial communities. Usually they are present in marine sediment pore water in low concentrations as a result of balanced production and consumption, both occurring in the same sediment zone. Thus their low concentrations represent a steady state condition regulated by either thermodynamics or kinetics. We have developed a novel analytical approach for the parallel measurement of several VFAs directly from marine pore water without any sample pretreatment by the use of a 2-dimensional ion chromatography coupled to mass spectrometry. In a first study we analyzed acetate, formate, and propionate in pore water from sediment cores retrieved from 5 different stations within and offshore of the Godhåbsfjord (Greenland). The sediment cores represent different sedimentological conditions, ranging from a typical marine sedimentation site to a glacier/freshwater dominated site. In addition to VFA concentrations, we measured sulfate concentrations, sulfate reduction rates, and cell abundances. We calculated the Gibbs free energy (ΔG) available for sulfate reduction (SR), as well as the VFA turnover times by the in-situ SR rates. The turnover time for acetate by SR ranged from several hours to days in the top cm of sediment and increased to several hundred years at the bottom of the SR zone. From the associated cell abundances we calculated that the VFA turnover times were significantly longer than the diffusion times of the VFA between individual cells. This shows that VFA consumption in the SR zone, and concomitantly the observed pore water concentrations, are not constrained by diffusion. DG values for SR using acetate were >36 kJ/mol which is significantly above the lower limit for anaerobic microbial energy metabolism. It thus remains unclear what controls the VFA concentrations in the sediment.

  19. Expression profiles of key transcription factors involved in lipid metabolism in Beijing-You chickens.

    PubMed

    Fu, R Q; Liu, R R; Zhao, G P; Zheng, M Q; Chen, J L; Wen, J

    2014-03-01

    Intramuscular fat (IMF) is a crucial factor for the meat quality of chickens. With the aim of studying the molecular mechanisms underlying IMF deposition in chickens, the expression profiles of five candidate transcription factors involved in lipid metabolism in several tissues were examined in Beijing-You (BJY) chickens at five ages (0, 4, 8, 14 and 20 wk). Results showed that accumulation of IMF in breast (IMFbr), thigh (IMFth) and abdominal fat weight increased significantly (P<0.01) after 8 wk. Accumulation of both IMFbr and IMFth from 8 to 14 wk exceeded that from 14 to 20 wk; IMFth was 4-7 times of IMFbr. As for the expression profiles of key transcription factors: 1) expression of C/EBPα and PPARγ in abdominal fat was significantly higher than that in breast and thigh muscles at all ages. The expression of C/EBPα was positively correlated with PPARγ in both breast and thigh muscles, which indicated that both C/EBPα and PPARγ promoted fat deposition and might act through a unified pathway; 2) the expression of SREBP-1 in 0, 4, and 8 wk in thigh muscle was significantly higher than that in breast; 3) expression of C/EBPβ at 4 and 8 wk was significantly higher than that at 14 and 20 wk; and it was positively correlated with IMFth and IMFbr from 0 to 8 wk; 4) expression of PPARα in breast and thigh muscles was significantly higher than that in abdominal fat. Taken together, all five transcription factors studied play roles in lipid metabolism in chickens with C/EBPα and PPARγ being important effectors.

  20. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin-cadmium induced diabetic nephrotoxic rats.

    PubMed

    Kandasamy, Neelamegam; Ashokkumar, Natarajan

    2014-09-01

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)-cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine, blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ-Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ-Cd induced diabetic nephrotoxic rats.

  1. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin–cadmium induced diabetic nephrotoxic rats

    SciTech Connect

    Kandasamy, Neelamegam; Ashokkumar, Natarajan

    2014-09-01

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)–cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine, blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ–Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ–Cd induced diabetic nephrotoxic rats. - Highlights: • Diabetic rats are more susceptible to cadmium nephrotoxicity. • Cadmium plays as a cumulative

  2. Simultaneous modulation of transport and metabolism of acyclovir prodrugs across rabbit cornea: An approach involving enzyme inhibitors.

    PubMed

    Katragadda, Suresh; Talluri, Ravi S; Mitra, Ashim K

    2006-08-31

    The aim of this study is to identify the class of enzymes responsible for the hydrolysis of amino acid and dipeptide prodrugs of acyclovir (ACV) and to modulate transport and metabolism of amino acid and dipeptide prodrugs of acyclovir by enzyme inhibitors across rabbit cornea. l-Valine ester of acyclovir, valacyclovir (VACV) and l-glycine-valine ester of acyclovir, gly-val-acyclovir (GVACV) were used as model compounds. Hydrolysis studies of VACV and GVACV in corneal homogenate were conducted in presence of various enzyme inhibitors. IC(50) values were determined for the enzyme inhibitors. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Complete inhibition of VACV hydrolysis was observed in the presence of Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl-fluoride) and PCMB (p-chloromercuribenzoic acid). Similar trend was also observed with GVACV in the presence of bestatin. IC(50) values of PCMB and bestatin for VACV and GVACV were found to be 3.81+/-0.94 and 0.34+/-0.08muM respectively. Eserine, tetraethyl pyrophosphate (TEPP) and diisopropyl fluorophosphate (DFP) also produced significant inhibition of VACV hydrolysis. Transport of VACV and GVACV across cornea showed decreased metabolic rate and modulation of transport in presence of PCMB and bestain respectively. The principle enzyme classes responsible for the hydrolysis of VACV and GVACV were carboxylesterases and aminopeptidases respectively. Enzyme inhibitors modulated the transport and metabolism of prodrugs simultaneously even though their affinity towards prodrugs was distinct. In conclusion, utility of enzyme inhibitors to modulate transport and metabolism of prodrugs appears to be promising strategy for enhancing drug transport across cornea.

  3. TM6SF2 and MAC30, new enzyme homologs in sterol metabolism and common metabolic disease

    PubMed Central

    Sanchez-Pulido, Luis; Ponting, Chris P.

    2014-01-01

    Carriers of the Glu167Lys coding variant in the TM6SF2 gene have recently been identified as being more susceptible to non-alcoholic fatty liver disease (NAFLD), yet exhibit lower levels of circulating lipids and hence are protected against cardiovascular disease. Despite the physiological importance of these observations, the molecular function of TM6SF2 remains unknown, and no sequence similarity with functionally characterized proteins has been identified. In order to trace its evolutionary history and to identify functional domains, we embarked on a computational protein sequence analysis of TM6SF2. We identified a new domain, the EXPERA domain, which is conserved among TM6SF, MAC30/TMEM97 and EBP (D8, D7 sterol isomerase) protein families. EBP mutations are the cause of chondrodysplasia punctata 2 X-linked dominant (CDPX2), also known as Conradi-Hünermann-Happle syndrome, a defective cholesterol biosynthesis disorder. Our analysis of evolutionary conservation among EXPERA domain-containing families and the previously suggested catalytic mechanism for the EBP enzyme, indicate that TM6SF and MAC30/TMEM97 families are both highly likely to possess, as for the EBP family, catalytic activity as sterol isomerases. This unexpected prediction of enzymatic functions for TM6SF and MAC30/TMEM97 is important because it now permits detailed experiments to investigate the function of these key proteins in various human pathologies, from cardiovascular disease to cancer. PMID:25566323

  4. Chemical characterization, antioxidant and inhibitory effects of some marine sponges against carbohydrate metabolizing enzymes

    PubMed Central

    2012-01-01

    Background More than 15,000 marine products have been described up to now; Sponges are champion producers, concerning the diversity of products that have been found. Most bioactive compounds from sponges were classified into anti-inflammatory, antitumor, immuno- or neurosurpressive, antiviral, antimalarial, antibiotic, or antifouling. Evaluation of in vitro inhibitory effects of different extracts from four marine sponges versus some antioxidants indices and carbohydrate hydrolyzing enzymes concerned with diabetes mellitus was studied. The chemical characterizations for the extracts of the predominating sponges; SP1 and SP3 were discussed. Methods All chemicals served in the biological study were of analytical grade and purchased from Sigma, Merck and Aldrich. All kits were the products of Biosystems (Spain), Sigma Chemical Company (USA), Biodiagnostic (Egypt). Carbohydrate metabolizing enzymes; α-amylase, α-glucosidase, and β-galactosidase (EC3.2.1.1, EC3.2.1.20, and EC3.2.1.23, respectively) were obtained from Sigma Chemical Company (USA). Results Four marine sponges; Smenospongia (SP1), Callyspongia (SP2), Niphates (SP3), and Stylissa (SP4), were collected from the Red Sea at Egyptian coasts, and taxonomically characterized. The sponges' extracts exhibited diverse inhibitory effects on oxidative stress indices and carbohydrate hydrolyzing enzymes in linear relationships to some extent with concentration of inhibitors (dose dependant). The extracts of sponges (3, 1, and 2) showed, respectively, potent-reducing power. Purification and Chemical characterization of sponge 1 using NMR and mass spectroscopy, recognized the existence of di-isobutyl phthalate (1), di-n-butyl phthalate (2), linoleic acid (3), β-sitosterol (4), and cholesterol (5). Sponge 3 produced bis-[2-ethyl]-hexyl-phthylester (6) and triglyceride fatty acid ester (7). Conclusion Marine sponges are promising sources for delivering of bioactive compounds. Four marine sponges, collected from

  5. Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism*

    PubMed Central

    Daniel, Bastian; Pavkov-Keller, Tea; Steiner, Barbara; Dordic, Andela; Gutmann, Alexander; Nidetzky, Bernd; Sensen, Christoph W.; van der Graaff, Eric; Wallner, Silvia; Gruber, Karl; Macheroux, Peter

    2015-01-01

    Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family. PMID:26037923

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

    NASA Technical Reports Server (NTRS)

    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.

  7. Characterization of the Impact of Life Stage on Xenobiotic Metabolizing Enzyme Expression and Gene -Chemical Interactions in the Liver

    EPA Science Inventory

    Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). We have carried out a comprehensive analysis of the mRNA expression of XMETs thro...

  8. Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  9. Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  10. Relationships between environmental organochlorine contaminant residues, plasma corticosterone concentrations, and intermediary metabolic enzyme activities in Great Lakes herring gull embryos.

    PubMed Central

    Lorenzen, A; Moon, T W; Kennedy, S W; Glen, G A

    1999-01-01

    Experiments were conducted to survey and detect differences in plasma corticosterone concentrations and intermediary metabolic enzyme activities in herring gull (Larus argentatus) embryos environmentally exposed to organochlorine contaminants in ovo. Unincubated fertile herring gull eggs were collected from an Atlantic coast control site and various Great Lakes sites in 1997 and artificially incubated in the laboratory. Liver and/or kidney tissues from approximately half of the late-stage embryos were analyzed for the activities of various intermediary metabolic enzymes known to be regulated, at least in part, by corticosteroids. Basal plasma corticosterone concentrations were determined for the remaining embryos. Yolk sacs were collected from each embryo and a subset was analyzed for organochlorine contaminants. Regression analysis of individual yolk sac organochlorine residue concentrations, or 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs), with individual basal plasma corticosterone concentrations indicated statistically significant inverse relationships for polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDDs/PCDFs), total polychlorinated biphenyls (PCBs), non-ortho PCBs, and TEQs. Similarly, inverse relationships were observed for the activities of two intermediary metabolic enzymes (phosphoenolpyruvate carboxykinase and malic enzyme) when regressed against PCDDs/PCDFs. Overall, these data suggest that current levels of organochlorine contamination may be affecting the hypothalamo-pituitary-adrenal axis and associated intermediary metabolic pathways in environmentally exposed herring gull embryos in the Great Lakes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:10064546

  11. Comparative genomic, phylogenetic, and functional investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated (Glenn and Bacon, 2009; Glenn et al., 2010). The NAT1 gene of Gibberella moniliformis was the...

  12. Somatostatin Modulates Insulin-Degrading-Enzyme Metabolism: Implications for the Regulation of Microglia Activity in AD

    PubMed Central

    Tundo, Grazia; Ciaccio, Chiara; Sbardella, Diego; Boraso, Mariaserena; Viviani, Barbara; Coletta, Massimiliano; Marini, Stefano

    2012-01-01

    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. PMID:22509294

  13. Potato steroidal glycoalkaloid levels and the expression of key isoprenoid metabolic genes.

    PubMed

    Krits, Pinchas; Fogelman, Edna; Ginzberg, Idit

    2007-12-01

    The potato steroidal glycoalkaloids (SGA) are toxic secondary metabolites, and their total content in tubers should not exceed 20 mg/100 g fresh weight. The two major SGA in cultivated potato (Solanum tuberosum) are alpha-chaconine and alpha-solanine. SGA biosynthetic genes and the genetic factors that control their expression have not yet been determined. In the present study, potato genotypes exhibiting different levels of SGA content showed an association between high SGA levels in their leaves and tubers and high expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (hmg1) and squalene synthase 1 (pss1), genes of the mevalonic/isoprenoid pathway. Transcripts of other key enzymes of branches of the isoprenoid pathway, vetispiradiene/sesquiterpene synthase (pvs1) and sterol C24-methyltransferase type1 (smt1), were undetectable or exhibited stable expression regardless of SGA content, respectively, suggesting facilitated precursor flow to the SGA biosynthetic branch. The transcript ratio of solanidine glucosyltransferase (sgt2) to solanidine galactosyltransferase (sgt1) was correlated to the documented chaconine-to-solanine ratio in the tested genotypes. Significantly higher expression of hmg1, pss1, smt1, sgt1 and sgt2 was monitored in the tuber phelloderm than in the parenchyma of the tuber's flesh, targeting the former as the main SGA-producing tissue in the tuber, in agreement with the known high SGA content in the layers directly under the tuber skin.

  14. Sex- and age-dependent gene expression in human liver: An implication for drug-metabolizing enzymes.

    PubMed

    Uno, Yasuhiro; Takata, Ryo; Kito, Go; Yamazaki, Hiroshi; Nakagawa, Kazuko; Nakamura, Yusuke; Kamataki, Tetsuya; Katagiri, Toyomasa

    2017-02-01

    Sex and age differences in hepatic expression of drug-metabolizing enzyme genes could cause variations in drug metabolism, but has not been fully elucidated, especially in Asian population. In this study, the global expression of human hepatic genes was analyzed by microarrays in 40 Japanese subjects (27 males and 13 females). Thirty-five sex-biased genes were identified (P < 0.005). Whereas, 60 age-biased genes in two age groups, <60 years and ≥70 years (P < 0.001), were identified in males. By Gene Ontology analysis, the sex-biased genes were related to protein catabolism and modification, while the age-biased genes were related to transcription regulation and cell death. Quantitative polymerase chain reaction confirmed the female-biased expression of drug-metabolizing enzyme genes BChE, CYP4X1, and SULT1E1 (≥1.5-fold, P < 0.05). Further analysis of drug-metabolizing enzyme genes indicated that expression of CYP2A6 and CYP3A4 in females in the ≥70 age group was less than in the <60 age group (≥1.5-fold, P < 0.05), and this trend was also observed for PXR expression in males (≥1.5-fold, P < 0.05). The results presented provide important insights into hepatic physiology and function, especially drug metabolism, with respect to sex and age.

  15. Leaf responses of micropropagated apple plants to water stress: nonstructural carbohydrate composition and regulatory role of metabolic enzymes.

    PubMed

    Li, Tian H; Li, Shao H

    2005-04-01

    We examined changes in nonstructural carbohydrate biosynthesis and activities of related enzymes in leaves of micropropagated apple plants (Malus domestica Borkh. cv. 'NaganoFuji') in response to water stress, with particular emphasis on the enzymes associated with sorbitol, sucrose and starch metabolism. Water stress resulted in the accumulation of photosynthates in leaves, mainly sorbitol, sucrose, glucose and fructose, accompanied by a reduction in starch concentration. Correlation and path analysis indicated that water stress affected the partitioning of newly fixed carbon among terminal products. In response to water stress, ADP-glucose-pyrophosphorylase (ADPGPPase) activity decreased, becoming a critical and limiting step in shifting partitioning of photosynthetically fixed carbon. Amylase and ADPGPPase affected sucrose and sorbitol metabolism, mainly by regulating substrate supply; however, competition for limited substrate had a greater effect on the biosynthesis of sorbitol than of sucrose. Starch metabolism was also strictly regulated by ADPGPPase and amylase, whereas other related enzymes were downstream of the pathway for synthesis and degradation of carbohydrates and thus had relatively little effect on starch metabolism. Sorbitol dehydrogenase and sucrose phosphate synthase were critical regulators of sorbitol and sucrose metabolism, respectively.

  16. A pangenomic analysis of the Nannochloropsis organellar genomes reveals novel genetic variations in key metabolic genes

    PubMed Central

    2014-01-01

    Background Microalgae in the genus Nannochloropsis are photosynthetic marine Eustigmatophytes of significant interest to the bioenergy and aquaculture sectors due to their ability to efficiently accumulate biomass and lipids for utilization in renewable transportation fuels, aquaculture feed, and other useful bioproducts. To better understand the genetic complement that drives the metabolic processes of these organisms, we present the assembly and comparative pangenomic analysis of the chloroplast and mitochondrial genomes from Nannochloropsis salina CCMP1776. Results The chloroplast and mitochondrial genomes of N. salina are 98.4% and 97% identical to their counterparts in Nannochloropsis gaditana. Comparison of the Nannochloropsis pangenome to other algae within and outside of the same phyla revealed regions of significant genetic divergence in key genes that encode proteins needed for regulation of branched chain amino synthesis (acetohydroxyacid synthase), carbon fixation (RuBisCO activase), energy conservation (ATP synthase), protein synthesis and homeostasis (Clp protease, ribosome). Conclusions Many organellar gene modifications in Nannochloropsis are unique and deviate from conserved orthologs found across the tree of life. Implementation of secondary and tertiary structure prediction was crucial to functionally characterize many proteins and therefore should be implemented in automated annotation pipelines. The exceptional similarity of the N. salina and N. gaditana organellar genomes suggests that N. gaditana be reclassified as a strain of N. salina. PMID:24646409

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

    PubMed

    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.

  18. Phenotypic knockouts of selected metabolic pathways by targeting enzymes with camel-derived nanobodies (V(HH)s).

    PubMed

    Jiménez, José I; Fraile, Sofía; Zafra, Olga; de Lorenzo, Víctor

    2015-07-01

    Surveying the dynamics of metabolic networks of Gram-negative bacteria often requires the conditional shutdown of enzymatic activities once the corresponding proteins have been produced. We show that given biochemical functions can be entirely suppressed in vivo with camel antibodies (VHHs, nanobodies) that target active sites of cognate enzymes expressed in the cytoplasm. As a proof of principle, we raised VHHs against 2,5-dihydroxypyridine dioxygenase (NicX) of Pseudomonas putida, involved in nicotinic acid metabolism. Once fused to a thioredoxin domain, the corresponding nanobodies inhibited the enzyme both in Escherichia coli and in P. putida cells, which then accumulated the metabolic substrate of NicX. VHHs were further engineered to track the antigen in vivo by C-terminal fusion to a fluorescent protein. Conditional expression of the resulting VHHs allows simultaneously to track and target proteins of interest and enables the design of transient phenotypes without mutating the genetic complement of the bacteria under study.

  19. Cadmium effect on microsomal drug-metabolizing enzyme activity in rat livers with respect to differences in age and sex

    SciTech Connect

    Ando, M.

    1982-04-01

    The effect of cadmium on the hepatic microsomal drug-metabolizing enzyme system was investigated. Cadmium chloride caused the conversion of cytochrome P-450 to P-420 in rat liver microsomes. The destruction of cytochrome P-450 by cadmium caused the reduction of microsomal drug-metabolizing enzyme activity and prolonged the pentobarbital sleeping time. There is a sex-related difference in the ability of cadmium to inhibit the hepatic drug metabolism in rats: male rats are more sensitive to cadmium than females. The effective period when cadmium prolonged their sleep depended upon the age of rats; older rats were more sensitive to cadmium than younger ones. The maximum increase of sleeping time depended upon the dose level of cadium, and the rate constant of the equations seems to depend upon the age of the animals.

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

    PubMed

    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.

  1. Temperature during embryonic development has persistent effects on metabolic enzymes in the muscle of zebrafish.

    PubMed

    Schnurr, Meghan E; Yin, Yi; Scott, Graham R

    2014-04-15

    Global warming is intensifying interest in the physiological consequences of temperature change in ectotherms, but we still have a relatively poor understanding of the effects of temperature on early life stages. This study determined how embryonic temperature (TE) affects development and the activity of metabolic enzymes in the swimming muscle of zebrafish. Embryos developed successfully to hatching (survival ≥ 88%) from 22 to 32°C, but suffered sharp increases in mortality outside of this range. Embryos that were incubated until hatching at a control TE (27°C) or near the extremes for successful development (22 or 32°C) were next raised to adulthood under control conditions at 27°C. Growth trajectories after hatching were altered in the 22°C and 32°C TE groups compared with 27°C TE controls, but growth slowed after 3 months of age in all groups. Maximal enzyme activities of cytochrome c oxidase (COX), citrate synthase (CS), hydroxyacyl-coA dehydrogenase (HOAD), pyruvate kinase (PK) and lactate dehydrogenase (LDH) were measured across a range of assay temperatures (22, 27, 32 and 36°C) in adults from each TE group that were acclimated to 27 or 32°C. Substrate affinities (Km) were also determined for COX and LDH. In adult fish acclimated to 27°C, COX and PK activities were higher in 22°C and 32°C TE groups than in 27°C TE controls, and the temperature optimum for COX activity was higher in the 32°C TE group than in the 22°C TE group. Warm acclimation reduced COX, CS and/or PK activities in the 22 and 32°C TE groups, possibly to compensate for thermal effects on molecular activity. This response did not occur in the 27°C TE controls, which instead increased LDH and HOAD activities. Warm acclimation also increased thermal sensitivity (Q10) of HOAD to cool temperatures across all TE groups. We conclude that the temperature experienced during early development can have a persistent impact on energy metabolism pathways and acclimation capacity in

  2. Can chronic maternal drug therapy alter the nursing infant's hepatic drug metabolizing enzyme pattern?

    PubMed

    Toddywalla, V S; Patel, S B; Betrabet, S S; Kulkarni, R D; Kombo, I; Saxena, B N

    1995-10-01

    This study was carried out to investigate whether minute quantities of maternal drugs ingested over an extended period of time by a breast-feeding infant can alter the activity pattern of the infant's hepatic drug metabolizing enzyme (HDME). The HDME activity patterns of 12 breast-fed infants whose mothers were not on drug therapy were compared with those of 11 infants whose mothers had been taking 30 micrograms levo-norgesterel daily for 90 to 195 days (oral contraceptives group) and of 10 infants whose mothers had been taking ethambutol and isoniazid daily since pregnancy (tuberculosis group). As 6 beta hydroxycortisol in urine is considered to be a good and acceptable reflector of HDME activity, it was estimated from the infants' urine using enzyme-linked immunosorbent assay (ELISA) technique. A comparison of the patterns between 90 days of age and 195 days of age of the infants in the control group and the two study groups indicated an increase from 36.6 ng/mL to 58.4 ng/mL at 195 days in the control group. An initial decrease from 36.6 ng/mL to 26.2 ng/mL was noted with commencement of maternal levo-norgesterel therapy, followed by a slow and steady rise to 47.8 ng/mL at 195 days of age, with a shift in the peak from 120 to 135 days of infants age in the oral contraceptive group. A suppressed pattern with decreased levels of 6 beta hydroxycortisol ranging from 19.3 ng/mL to 26.5 ng/mL at 195 days was found in the tuberculosis group. The data were analyzed by two-way analysis of variance (ANOVA) coupled with Duncan's Multiple range test. Both treatment group showed significant differences from the control group at the 0.050 level. The HDME plays an important role in determining the final outcome of any drug in humans, as it controls the metabolism of drugs. Hence, alterations in its activity caused by the transfer of maternal drugs over a prolonged period of time could pose a serious problem to nurslings when they require drugs for their own benefit.

  3. In vitro and cellular effects of 4-pyridone-3-carboxamide riboside on enzymes of nucleotide metabolism.

    PubMed

    Slominska, Ewa M; Borkowski, Tomasz; Rybakowska, Iwona; Abramowicz-Glinka, Magdalena; Orlewska, Czesława; Smolenski, Ryszard T

    2014-01-01

    4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that has recently been identified as a substrate for intracellular phosphorylation to form nucleotide derivatives. Low level of 4PYR is normally present in human plasma, but 4PYR massively accumulates in patients with renal failure. This study aimed to evaluate effects of 4PYR and its monophosphate derivative (4PYMP) on several enzymes of nucleotide metabolism in homogenates and intact cells. Activities of adenosine monophosphate deaminase (AMPD), adenosine deaminase, ecto-5'-nucleotidase (e5NT), adenine phosphoribosyltransferase (APRT), hypoxanthine/guanine phosphoribosyltransferase, purine nucleoside phosphorylase, and S-adenosylhomocysteine hydrolase (SAHH) were evaluated in erythrocyte lysates, rat heart homogenates, and in the intact rat cardiomyocytes by high performance liquid chromatography-based assays. 4PYMP caused significant inhibition of AMPD in both erythrocyte lysate and heart homogenate with 50% inhibitory concentration (IC50) of 74 and 55 μM, respectively. Inhibition of e5NT in heart homogenates was also noted with IC50 of 63 μM. 4PYMP slightly inhibited APRT and 4PYR caused moderate activation of SAHH. No effects on other enzymes studied were noted. Inhibition of AMPD by 4PYMP in homogenates was confirmed in the intact cell experiments with isolated cardiomyocytes that were allowed to accumulate 4PYMP by incubation with 4PYR. We conclude that among pathways studied, most important is the effect of 4PYMP on AMPD and that such effect could be one of the consequences of elevated plasma 4PYR concentration.

  4. [Effects of waterlogging on the growth and energy-metabolic enzyme activities of different tree species].

    PubMed

    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.

  5. Effect of deuterium oxide on neutrophil oxidative metabolism, phagocytosis, and lysosomal enzyme release

    SciTech Connect

    Tsan, M.F.; Turkall, R.M.

    1982-12-01

    We have previously shown that deuterium oxide (D/sub 2/O) enhances the oxidation of methionine, a myeloperoxidase (MPO) -mediated reaction, by human neutrophils during phagocytosis. However, D/sub 2/O has no effect on the oxidation of methionine by the purified MPO-H/sub 2/O/sub 2/-Cl- system. To explain this observation, we studied the effect of D/sub 2/O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D/sub 2/O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D/sub 2/O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D/sub 2/O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the