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Sample records for key metabolic enzyme

  1. Yeast RNase III as a Key Processing Enzyme in Small Nucleolar RNAs Metabolism

    E-print Network

    Chanfreau, Guillaume

    Yeast RNase III as a Key Processing Enzyme in Small Nucleolar RNAs Metabolism Guillaume Chanfreau searched for yeast snoRNAs which are affected by the depletion of the yeast ortholog of bacterial RNase III, Rnt1. In a yeast strain inactivated for RNT1, almost half of the snoRNAs tested are depleted

  2. Lactate Dehydrogenase Is the Key Enzyme for Pneumococcal Pyruvate Metabolism and Pneumococcal Survival in Blood

    PubMed Central

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

    2014-01-01

    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. PMID:25245810

  3. 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.

  4. 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

  5. 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

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

    PubMed

    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

    2014-07-01

    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

  7. 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}.

  8. Inhibitory Potential of Turbinaria ornata against Key Metabolic Enzymes Linked to Diabetes

    PubMed Central

    Unnikrishnan, P. S.; Suthindhiran, K.; Jayasri, M. A.

    2014-01-01

    One of the therapeutic approaches in treating diabetes is to reduce postprandial hyperglycemia by inhibiting major carbohydrate hydrolyzing enzymes. In the present study, crude extracts of marine seaweed, Turbinaria ornata, were tested for their antidiabetic potential using enzyme inhibitory assays (?-amylase, ?-glucosidase, and dipeptidyl peptidase-IV). Among the tested extracts, methanol and acetone extracts showed significant inhibitory effects on ?-amylase (IC50 250.9??g/mL), ?-glucosidase (535.6??g/mL), and dipeptidyl peptidase-4 (55.2??g/mL), respectively. Free radical scavenging activity of these extracts was analyzed using DPPH assay (65%). Extracts were tested for in vitro toxicity using DNA fragmentation assay, haemolytic assay, and MTT assay. None of the extracts showed toxicity in tested models. Furthermore, GC-MS analysis of lead extracts showed the presence of major compounds, hentriacontane, z, z-6, 28-heptatriactontadien-2-one, 8-heptadecene, and 1-heptacosanol. Our findings suggest that Turbinaria ornata can be used as a potential source for further in vivo studies in controlling hyperglycemia. PMID:25050371

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

    PubMed Central

    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. PMID:24451681

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

    PubMed

    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. PMID:24451681

  11. Lysine acetylation is a common post-translational modification of key metabolic pathway enzymes of the anaerobe Porphyromonas gingivalis.

    PubMed

    Butler, Catherine A; Veith, Paul D; Nieto, Matthew F; Dashper, Stuart G; Reynolds, Eric C

    2015-10-14

    Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a keystone pathogen in the development of the bacterial-associated inflammatory oral disease chronic periodontitis. Although post-translational modifications (PTMs) of proteins are commonly found to modify protein function in eukaryotes and prokaryotes, PTMs such as lysine acetylation have not been examined in P. gingivalis. Lysine acetylation is the addition of an acetyl group to a lysine which removes this amino acid's positive charge and can induce changes in a protein's secondary structure and reactivity. A proteomics based approach combining immune-affinity enrichment with high sensitivity Orbitrap mass spectrometry identified 130 lysine acetylated peptides from 92 P. gingivalis proteins. The majority of these peptides (71) were attributed to 45 proteins with predicted metabolic activity; these proteins could be mapped to several P. gingivalis metabolic pathways where enzymes catalysing sequential reactions within the same pathway were often found acetylated. In particular, the catabolic pathways of complex anaerobic fermentation of amino acids to produce energy had 12 enzymes lysine acetylated. The results suggest that lysine acetylation may be an important mechanism in metabolic regulation in P. gingivalis, which is vital for P. gingivalis survival and adaptation of its metabolism throughout infection. Statement of significance. Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The ability of the pathogen to induce dysbiosis and disease is related to an array of specific virulence factors and metabolic regulation that enables the bacterium to proliferate in an inflamed periodontal pocket. The mechanisms P. gingivalis uses to adapt to a changing and hostile environment are poorly understood and here we show, for the first time, that enzymes of critical metabolic pathways for energy production in this bacterium were acetylated on certain lysine residues. These enzymes were often found catalysing sequential reactions within the same catabolic pathway. The results suggest that lysine acetylation is an important mechanism of metabolic regulation in P. gingivalis vital for its adaptation and proliferation to produce disease. PMID:26341301

  12. Polyphenols rich fraction from Geoffroea decorticans fruits flour affects key enzymes involved in metabolic syndrome, oxidative stress and inflammatory process.

    PubMed

    Costamagna, M S; Zampini, I C; Alberto, M R; Cuello, S; Torres, S; Pérez, J; Quispe, C; Schmeda-Hirschmann, G; Isla, M I

    2016-01-01

    Geoffroea decorticans (chañar), is widely distributed throughout Northwestern Argentina. Its fruit is consumed as flour, arrope or hydroalcoholic beverage. The chañar fruits flour was obtained and 39 phenolic compounds were tentatively identified by HPLC-MS/MS(n). The compounds comprised caffeic acid glycosides, simple phenolics (protocatechuic acid and vanillic acid), a glycoside of vanillic acid, p-coumaric acid and its phenethyl ester as well as free and glycosylated flavonoids. The polyphenols enriched extract with and without gastroduodenal digestion inhibited enzymes associated with metabolic syndrome, including ?-amylase, ?-glucosidase, lipase and hydroxyl methyl glutaryl CoA reductase. The polyphenolic extract exhibited antioxidant activity by different mechanisms and inhibited the pro-inflammatory enzymes (ciclooxygenase, lipoxygenase and phospholipase A2). The polyphenolic extract did not showed mutagenic effect by Ames test against Salmonella typhimurium TA98 and TA100 strains. These findings add evidence that chañar fruit flour may be considered a functional food with preventive properties against diseases associated with oxidative stress, inflammatory mediators and metabolic syndrome. PMID:26212988

  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 ?-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. PMID:25636882

  14. 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. PMID:26002973

  15. Chicken Cytochrome P450 1A5 Is the Key Enzyme for Metabolizing T-2 Toxin to 3?OH-T-2

    PubMed Central

    Shang, Shufeng; Jiang, Jun; Deng, Yiqun

    2013-01-01

    The transmission of T-2 toxin and its metabolites into the edible tissues of poultry has potential effects on human health. We report that T-2 toxin significantly induces CYP1A4 and CYP1A5 expression in chicken embryonic hepatocyte cells. The enzyme activity assays of CYP1A4 and CYP1A5 heterologously expressed in HeLa cells indicate that only CYP1A5 metabolizes T-2 to 3?OH-T-2 by the 3?-hydroxylation of isovaleryl groups. In vitro enzyme assays of recombinant CYP1A5 expressed in DH5? further confirm that CYP1A5 can convert T-2 into TC-1 (3?OH-T-2). Therefore, CYP1A5 is critical for the metabolism of trichothecene mycotoxin in chickens. PMID:23702848

  16. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device...

  17. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification . A drug metabolizing enzyme genotyping system is a device...

  18. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification . A drug metabolizing enzyme genotyping system is a device...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification . A drug metabolizing enzyme genotyping system is a device...

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification . A drug metabolizing enzyme genotyping system is a device...

  1. Enzyme clustering can induce metabolic channeling

    NASA Astrophysics Data System (ADS)

    Castellana, Michele

    2015-03-01

    Direct channeling of intermediates via a physical tunnel between enzyme active sites is an established mechanism to improve metabolic efficiency. In this talk, I will present a theoretical model that demonstrates that coclustering multiple enzymes into proximity can yield the full efficiency benefits of direct channeling. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with the spacing between coclusters in yeast and mammalian cells. The model also predicts that enzyme agglomerates can regulate steady-state flux division at metabolic branch points: we experimentally test this prediction for a fundamental branch point in Escherichia coli, and the results confirm that enzyme colocalization within an agglomerate can accelerate the processing of a shared intermediate by one branch. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation.

  2. 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

  3. Histone Acetylation Enzymes Coordinate Metabolism and Gene Expression.

    PubMed

    Shen, Yuan; Wei, Wei; Zhou, Dao-Xiu

    2015-10-01

    Histone lysine acetylation is well known for being important in the epigenetic regulation of gene expression in eukaryotic cells. Recent studies have uncovered a plethora of acetylated proteins involved in important metabolic pathways, such as photosynthesis and respiration in plants. Enzymes involved in histone acetylation and deacetylation are being identified as regulators of acetylation of metabolic enzymes. Importantly, key metabolites, such as acetyl-CoA and NAD(+), are involved in protein acetylation and deacetylation processes, and their cellular levels may regulate the activity of histone acetyltransferases (HAT) and deacetylases (HDAC). Further research is required to determine whether and how HATs and HDACs sense cellular metabolite signals to control gene expression and metabolic enzyme activity through lysine acetylation and deacetylation. PMID:26440431

  4. Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux.

    PubMed

    Wang, Qijun; Zhang, Yakun; Yang, Chen; Xiong, Hui; Lin, Yan; Yao, Jun; Li, Hong; Xie, Lu; Zhao, Wei; Yao, Yufeng; Ning, Zhi-Bin; Zeng, Rong; Xiong, Yue; Guan, Kun-Liang; Zhao, Shimin; Zhao, Guo-Ping

    2010-02-19

    Lysine acetylation regulates many eukaryotic cellular processes, but its function in prokaryotes is largely unknown. We demonstrated that central metabolism enzymes in Salmonella were acetylated extensively and differentially in response to different carbon sources, concomitantly with changes in cell growth and metabolic flux. The relative activities of key enzymes controlling the direction of glycolysis versus gluconeogenesis and the branching between citrate cycle and glyoxylate bypass were all regulated by acetylation. This modulation is mainly controlled by a pair of lysine acetyltransferase and deacetylase, whose expressions are coordinated with growth status. Reversible acetylation of metabolic enzymes ensure that cells respond environmental changes via promptly sensing cellular energy status and flexibly altering reaction rates or directions. It represents a metabolic regulatory mechanism conserved from bacteria to mammals. PMID:20167787

  5. 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.

  6. 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... 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. Composition of native Australian herbs polyphenolic-rich fractions and in vitro inhibitory activities against key enzymes relevant to metabolic syndrome.

    PubMed

    Sakulnarmrat, Karunrat; Konczak, Izabela

    2012-09-15

    Polyphenolic-rich fractions obtained from three native Australian herbs: Tasmannia pepper leaf, anise myrtle and lemon myrtle were characterised with regards to their composition, antioxidant capacities and inhibitory activities against ?-glucosidase, pancreatic lipase and angiotensin I-converting enzyme, using in vitro models. Ellagic acid and derivatives were the dominant compounds of anise myrtle and lemon myrtle fractions, accompanied by flavonoids (catechin, myricetin, hesperetin, and quercetin). Tasmannia pepper leaf fraction comprised chlorogenic acid and quercetin derivatives, exhibited the highest oxygen radical absorbance capacity and effectively inhibited ?-glucosidase (IC(50): 0.83 mg/ml) and pancreatic lipase (IC(50): 0.60 mg/ml). Anise myrtle and lemon myrtle fractions had pronounced ?-glucosidase-inhibitory activities (IC(50): 0.30 and 0.13 mg/ml, respectively) and were less effective against lipase. Enzyme-inhibitory activities showed various levels of correlation with the levels of total phenolics and antioxidant capacities, indicating a specificity of individual phenolic compounds present in the isolated fractions to complex with proteins. PMID:23107721

  12. Isolation and structural characterization of 2R, 3R taxifolin 3-O-rhamnoside from ethyl acetate extract of Hydnocarpus alpina and its hypoglycemic effect by attenuating hepatic key enzymes of glucose metabolism in streptozotocin-induced diabetic rats.

    PubMed

    Balamurugan, Rangachari; Vendan, Subramanian Ezhil; Aravinthan, Adithan; Kim, Jong-Hoon

    2015-04-01

    Hydnocarpus alpina Wt. (Flacourtiaceae) (H. alpina) is a large tree traditionally used to treat leprosy; it also posses antidiabetic property. The present study was undertaken to isolate, characterize and to evaluate the antidiabetic effect of 2R, 3R taxifolin 3-O-rhamnoside. (rhamnoside) and its impact on carbohydrate metabolic key enzymes in control and streptozotocin (STZ)-induced diabetic rats. Diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (STZ) (40 mg/kg). Oral administration of rhamnoside for 21 days significantly reduced food intake, calorie intake, blood glucose and glycosylated hemoglobin levels, and improved plasma insulin levels. Administration of rhamnoside showed significant increase in the body weight, body composition (Lean body weight (LBW) and retro body fat), glycolytic hexokinase, glucose-6-phophate dehydrogenase and pyruvate kinase levels where as significant decrease was observed in the levels of glucose-6-phosphatase fructose-1, 6-bisphosphatase and lactate dehydrogenase in diabetic treated rats. Further, administration of rhamnoside significantly improved the glycogen content, glycogen synthase and glycogen phosphorylase, suggesting the antihyperglycemic potential of rhamnoside in diabetic rats. The results obtained were compared with glibenclamide a standard hypoglycaemic drug. Immunohistopathological study of pancreas revealed increased number of ?-cells and insulin granules in diabetes-induced rats after treatment with rhamnoside for 21 days. Furthermore, Co-administration of rhamnoside (50 mg/kg) with nifedipine (13.6 mg/kg), a Ca(2+)ion channel blocker, or nicorandil (6.8 mg/kg), an ATP-sensitive K(+) ion channel opener, reveals the insulin secretion property of rhamnoside via a K(+)-ATP channels dependent pathway in diabetic rats. In conclusion, rhamnoside normalized blood glucose, glycosylated hemoglobin, key hepatic enzymes and glycogen content by increasing insulin secretion via K(+)-ATP channels dependent signaling pathway. The results suggest that the rhamnoside from H. alpina could be used as a therapeutic agent to treat diabetes mellitus. PMID:25698613

  13. Modulating effects of hesperidin on key carbohydrate-metabolizing enzymes, lipid profile, and membrane-bound adenosine triphosphatases against 7,12-dimethylbenz(a)anthracene-induced breast carcinogenesis.

    PubMed

    Nandakumar, N; Rengarajan, T; Balamurugan, A; Balasubramanian, M P

    2014-05-01

    The aim of this study was to document the effect of hesperidin on the key enzyme activities of carbohydrate metabolism, lipid profile, and membrane-bound adenosine triphosphatases (ATPases) during 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast carcinogenesis. Hesperidin has been reported to have multiple biological properties. Breast cancer was induced by single dose of DMBA (20 mg/kg body weight (bw)). The results revealed that there was a significant increase in the activities of hexokinase, phosphoglucoisomerase, and aldolase and a concomitant decrease in the activities of glucose-6-phosphatase and fructose-1,6-diphosphatase in cancer-induced animals. The activities of ATPases were found to be decreased both in erythrocyte membrane and in the liver of mammary cancer-bearing animals. The lipid profiles such as total cholesterol, free cholesterol, phospholipids, triglycerides, and free fatty acids significantly increased and in contrast the ester cholesterol in plasma was found to be decreased, whereas it was found to be elevated in the liver of cancer-bearing groups. The altered levels of the above-mentioned biochemical parameters in cancer-bearing animals were significantly ameliorated by the administration of hesperidin at the dosage of 30 mg/kg bw for 45 days. The histopathological analysis of breast and liver tissues were well supported the modulatory property of hesperidin, and this might be associated with normalizing the gluconeogenesis process, stabilization of cell membranes, and modulation of lipid biosynthesis. PMID:23690228

  14. 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

  15. Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

    PubMed Central

    Shi, Xueyan; Dick, Ryan A.; Ford, Kevin A.; Casida, John E.

    2009-01-01

    Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19 and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19 and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than that of TMX. Imidacloprid (IMI), CLO and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI and 4-hydroxy-thiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid and CLO metabolism in vivo in mice, elevating the brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites. PMID:19391582

  16. 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. PMID:25433633

  17. Controls on the Temperature Sensitivity of Soil Enzymes: A Key Driver of In Situ Enzyme

    E-print Network

    Allison, Steven D.

    few studies. In theory, the temperature sensitivity of enzyme activities can be described from first the activation energy of biochemical reactions. There are two aspects to the temperature sensitivity of enzymesChapter 13 Controls on the Temperature Sensitivity of Soil Enzymes: A Key Driver of In Situ Enzyme

  18. 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-treatment. Not all recovered completely by the final time point tested: with 6 Gy exposure, metallothionein expression was 132-fold more than control at the 4 hr time point, and fell at each later time point (11-fold at 24 hrs, and 8-fold at 7 days). In contrast, there were other genes whose expression was altered and remained relatively constant through the 7 day period we tested. One examples is Cyp17a1, which showed a 4-fold elevation at 4 hrs after exposure and remained constant for 7 days after the last treatment. Spaceflight samples evaluated with similar methods and comparisons will be made between the radiation-treated groups and the spaceflight samples. CONCLUSION It seems likely that radiation exposure triggers homeostatic mechanisms, which could include alterations of gene expression. Better understanding of these pathways could aid in optimizing medications doses given to crewmembers who require treatment and eventually, to development of new countermeasures to ameliorate or prevent radiation-induced damage to cells and tissues.

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

    PubMed Central

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

    2015-01-01

    In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial cellular fate changes, cell differentiation, or malignant transformation are now known to involve ‘metabolic remodeling’ with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation (‘moonlighting’) and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme–RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future. PMID:26520658

  20. Enzymes of ?,?-Trehalose Metabolism in Soybean Nodules 1

    PubMed Central

    Salminen, Seppo O.; Streeter, John G.

    1986-01-01

    Metabolism of trehalose, ?,d-glucopyranosyl-?,d-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max [L.] Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6-phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6-phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and ?-glucose-1-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6-phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase. PMID:16664852

  1. 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

  2. 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.

  3. Bifidobacterial Enzymes Involved in the Metabolism of Human Milk Oligosaccharides123

    PubMed Central

    Kitaoka, Motomitsu

    2012-01-01

    Intestinal colonization of bifidobacteria is important for the health of infants. Human milk oligosaccharides (HMO) have been identified as growth factors for bifidobacteria. Recently, a bifidobacterial enzymatic system to metabolize HMO was identified. 1,3-?-Galactosyl-N-acetylhexosamine phosphorylase (GLNBP, EC 2.4.1.211), which catalyzes the reversible phosphorolysis of galacto-N-biose (GNB) (Gal?1?3GalNAc)] and lacto-N-biose I (LNB) (Gal?1?3GlcNAc), is a key enzyme to explain the metabolism of HMO. Infant-type bifidobacteria possess the intracellular pathway to specifically metabolize GNB and LNB (GNB/LNB pathway). Bifidobacterium bifidum possesses extracellular enzymes to liberate LNB from HMO. However, Bifidobacterium longum subsp. infantis imports intact HMO to be hydrolyzed by intracellular enzymes. Bifidobacterial enzymes related to the metabolism of HMO are useful tools for preparing compounds related to HMO. For instance, LNB and GNB were produced from sucrose and GlcNAc/GalNAc in 1 pot using 4 bifidobacterial enzymes, including GLNBP. LNB is expected to be a selective bifidus factor for infant-type strains. PMID:22585921

  4. 2006 Nature Publishing Group A trehalose metabolic enzyme controls

    E-print Network

    Jackson, David

    © 2006 Nature Publishing Group A trehalose metabolic enzyme controls inflorescence architecture Inflorescence branching is a major yield trait in crop plants controlled by the developmental fate of axillary (inflorescences) and affect crop yield by influencing seed number or harvesting ability2,3 . Several growth

  5. Unraveling the toxicity mechanisms of the herbicide diclofop-methyl in rice: modulation of the activity of key enzymes involved in citrate metabolism and induction of cell membrane anion channels.

    PubMed

    Ding, Haiyan; Lu, Haiping; Lavoie, Michel; Xie, Jun; Li, Yali; Lv, Xiaolu; Fu, Zhengwei; Qian, Haifeng

    2014-11-01

    Residual soil concentrations of the herbicide diclofop-methyl (DM) can be toxic to other nontarget plant species, but the toxicity mechanisms at play are not fully understood. In the present study, we analyzed the toxic effect of DM on root growth and metabolism in the rice species Oryza sativa. The results show that a 48-h exposure to a trace level (5 ?g/L) of DM inhibits rice root growth by almost 70%. A 48-h exposure to 5 ?g/L DM also leads to an ?2.5-fold increase in citrate synthase (CS) activity (and CS gene transcription) and an ?2-fold decrease in the citrate lyase gene transcripts, which lead to an increase in the intracellular concentration of citrate and in citrate exudation rate. Addition of a specific inhibitor of cell membrane anion channel, anthracene-9-carboxylic acid, decreased citrate release in the culture, suggesting that DM-induced citrate loss from the cells is mediated by a specific membrane-bound channel protein. This study brings new insights into the key biochemical mechanisms leading to DM toxicity in rice. PMID:25307187

  6. 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

  7. Engineering of Metabolic Pathways by Artificial Enzyme Channels.

    PubMed

    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

  8. Enzymes of Starch Metabolism in the Developing Rice Grain 1

    PubMed Central

    Baun, Lyda C.; Palmiano, Evelyn P.; Perez, Consuelo M.; Juliano, Bjenvenido O.

    1970-01-01

    The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism—?-amylase, ?-amylase, phosphorylase, Q-enzyme, R-enzyme, and starch synthetase —were assayed in dehulled developing rice grains (Oryzasativa L., variety IR8). Phosphorylase, Q-enzyme, and R-enzyme had peak activities 10 days after flowering, whereas ?- and ?-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule. Images PMID:16657480

  9. Enzyme Regulation& Catalysis in Carbon Fixation Metabolism

    SciTech Connect

    Henry M. Miziorko

    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. Potential activities of androgen metabolizing enzymes in human prostate.

    PubMed

    Krieg, M; Weisser, H; Tunn, S

    1995-06-01

    The entire androgen metabolism of the human prostate is an integral part of the DHT mediated cellular processes, which eventually give rise to the androgen responsiveness of the prostate. Therefore, the potential activities of various androgen metabolizing enzymes were studied. Moreover, the impact of aging on the androgen metabolism and the inhibition of 5 alpha-reductase by finasteride were studied. In epithelium (E) and stroma (S) of normal (NPR) and hyperplastic human prostate (BPH), for each enzyme being involved in the conversion either of testosterone via DHT, 3 alpha- and 3 beta-diol to the C19O3-triols or from testosterone to androstenedione and vice versa, the amount (Vmax) and Michaelis constant (Km) were determined by Lineweaver-Burk plots. Furthermore, Vmax/Km quotients were calculated, which served as an index for the potential enzyme activity. 17 enzymes showed a mean Vmax/Km > or = 0.10. The top four were the 5 alpha-reductases in E and S of NPR and BPH. Among those, the highest activity was found in E of NPR (1.6 +/- 0.2). Moreover, in E a significant age-dependent decrease of 5 alpha-reductase activity occurred, whereas in stroma rather constant activities were found over the whole age range. Similar age-dependent alterations were found for the cellular DHT levels. Finally, the finasteride inhibition of 5 alpha-reductase (IC50;nM) was stronger in E (35 +/- 17) than in S (126 +/- 15). In conclusion, 5 alpha-reductase is: (a) the outstanding androgen metabolizing enzyme in NPR and BPH; (b) dictating the DHT enrichment in the prostate; (c) under the impact of aging; and (d) preferentially inhibited by finasteride in E. PMID:7542902

  11. 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.

  12. 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.

  13. Role of Sphingolipids and Metabolizing Enzymes in Hematological Malignancies

    PubMed Central

    Kitatani, Kazuyuki; Taniguchi, Makoto; Okazaki, Toshiro

    2015-01-01

    Sphingolipids such as ceramide, sphingosine-1-phosphate and sphingomyelin have been emerging as bioactive lipids since ceramide was reported to play a role in human leukemia HL-60 cell differentiation and death. Recently, it is well-known that ceramide acts as an inducer of cell death, that sphingomyelin works as a regulator for microdomain function of the cell membrane, and that sphingosine-1-phosphate plays a role in cell survival/proliferation. The lipids are metabolized by the specific enzymes, and each metabolite could be again returned to the original form by the reverse action of the different enzyme or after a long journey of many metabolizing/synthesizing pathways. In addition, the metabolites may serve as reciprocal bio-modulators like the rheostat between ceramide and sphingosine-1-phosphate. Therefore, the change of lipid amount in the cells, the subcellular localization and the downstream signal in a specific subcellular organelle should be clarified to understand the pathobiological significance of sphingolipids when extracellular stimulation induces a diverse of cell functions such as cell death, proliferation and migration. In this review, we focus on how sphingolipids and their metabolizing enzymes cooperatively exert their function in proliferation, migration, autophagy and death of hematopoetic cells, and discuss the way developing a novel therapeutic device through the regulation of sphingolipids for effectively inhibiting cell proliferation and inducing cell death in hematological malignancies such as leukemia, malignant lymphoma and multiple myeloma. PMID:25997737

  14. 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

  15. Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids

    PubMed Central

    Romanov, Vassily I.; Hernández-Lucas, Ismael; Martínez-Romero, Esperanza

    1994-01-01

    We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids. PMID:16349319

  16. 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

  17. Gene expression analysis of membrane transporters and drug-metabolizing enzymes in the lung of healthy and COPD subjects

    PubMed Central

    Berg, Tove; Hegelund Myrbäck, Tove; Olsson, Marita; Seidegård, Janeric; Werkström, Viktoria; Zhou, Xiao-Hong; Grunewald, Johan; Gustavsson, Lena; Nord, Magnus

    2014-01-01

    This study describes for the first time the expression levels of genes encoding membrane transporters and drug-metabolizing enzymes in the lungs of ex-smoking patients with chronic obstructive pulmonary disease (COPD). Membrane transporters and drug-metabolizing enzymes are key determinants of drug uptake, metabolism, and elimination for systemically administered as well as inhaled drugs, with consequent influence on clinical efficacy and patient safety. In this study, while no difference in gene expression was found between healthy and COPD subjects, we identified a significant regional difference in mRNA expression of both membrane transporters and drug-metabolizing enzymes between central and peripheral tissue in both healthy and COPD subjects. The majority of the differentially expressed genes were higher expressed in the central airways such as the transporters SLC2A1 (GLUT1), SLC28A3 (CNT3), and SLC22A4 (OCTN1) and the drug-metabolizing enzymes GSTZ1, GSTO2, and CYP2F1. Together, this increased knowledge of local pharmacokinetics in diseased and normal lung may improve modeling of clinical outcomes of new chemical entities intended for inhalation therapy delivered to COPD patients. In addition, based on the similarities between COPD and healthy subjects regarding gene expression of membrane transporters and drug-metabolizing enzymes, our results suggest that clinical pharmacological studies in healthy volunteers could be a valid model of COPD patients regarding drug disposition of inhaled drugs in terms of drug metabolism and drug transporters. PMID:25505599

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

    PubMed Central

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

    2015-01-01

    In this study, we investigated the enzymes catalyzing the phase?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. PMID:26404338

  19. 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

  20. Altered drug metabolism during pregnancy: Hormonal regulation of drug-metabolizing enzymes

    PubMed Central

    Jeong, Hyunyoung

    2013-01-01

    Importance of the field Medication use during pregnancy is prevalent, but pharmacokinetic information of most drugs used during pregnancy is lacking in spite of known effects of pregnancy on drug disposition. Accurate pharmacokinetic information is essential for optimal drug therapy in mother and fetus. Thus, understanding how pregnancy influences drug disposition is important for better prediction of pharmacokinetic changes of drugs in pregnant women. Areas covered in this review Pregnancy is known to affect hepatic drug metabolism, but the underlying mechanisms remain unknown. Physiological changes accompanying pregnancy are likely responsible for the reported alteration in drug metabolism during pregnancy. These include elevated concentrations of various hormones such as estrogen, progesterone, placental growth hormones and prolactin. This review covers how these hormones influence expression of drug-metabolizing enzymes, thus potentially responsible for altered drug metabolism during pregnancy. What the reader will gain The reader will gain a greater understanding of the altered drug metabolism in pregnant women and the regulatory effects of pregnancy hormones on expression of drug-metabolizing enzymes. Take home message In-depth studies in hormonal regulatory mechanisms as well as confirmatory studies in pregnant women are warranted for systematic understanding and prediction of the changes in hepatic drug metabolism during pregnancy. PMID:20367533

  1. Evolution of Domain Architectures and Catalytic Functions of Enzymes in Metabolic Systems

    E-print Network

    Yeang, Chen-Hsiang

    Evolution of Domain Architectures and Catalytic Functions of Enzymes in Metabolic Systems Summit architectures and catalytic functions of enzymes constitute the centerpieces of a metabolic network. These types. In contrast, prokaryotic enzymes become more versatile by catalyzing multiple reactions with similar chemical

  2. METABOLIC ENZYME ACTIVITIES OF BENTHIC ZOARCIDS OFF THE COAST OF CALIFORNIA

    E-print Network

    Qiu, Bo

    METABOLIC ENZYME ACTIVITIES OF BENTHIC ZOARCIDS OFF THE COAST OF CALIFORNIA A THESIS SUBMITTED, therefore, a great model for this research. The rates of four enzymes in twelve species of zoarcids were examined as a biochemical proxy for metabolic activity. Enzyme assays were performed on two anaerobic

  3. 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

  4. The effect of polymorphic metabolism enzymes on serum phenytoin level.

    PubMed

    Ozkaynakci, Aydan; Gulcebi, Medine Idrizoglu; Ergeç, Deniz; Ulucan, Korkut; Uzan, Mustafa; Ozkara, Cigdem; Guney, Ilter; Onat, Filiz Yilmaz

    2015-03-01

    Phenytoin has a widespread use in epilepsy treatment and is mainly metabolized by hepatic cytochrome P450 enzymes (CYP). We have investigated CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3 allelic variants in a Turkish population of patients on phenytoin therapy. Patients on phenytoin therapy (n = 102) for the prevention of epileptic seizures were included. Polymorphic alleles were analyzed by restriction fragment length polymorphism method. Serum concentrations of phenytoin were measured by fluorescence polarization immune assay method. The most frequent genotype was detected for CYP2C9 wild-type alleles (78.43 %), whereas CYP2C19*2/*2 (5.88 %) was the least frequent genotype group. According to the classification made with both enzyme polymorphisms, CYP2C9*1/*1-CYP2C19*1/*1 (G1: 41.17 %) genotype group was the most frequent whereas CYP2C9*1/*2-CYP2C19*1/*3 (G7: 0.98 %) was the least frequent one. The highest mean phenytoin level (27.95 ± 1.85 µg/ml) was detected in the G8 genotype group (CYP2C9*1/*3-CYP2C19*2/*3) and the G1 genotype group showed the lowest mean phenytoin level (7.43 ± 0.73 µg/ml). The mean serum concentration of phenytoin of the polymorphic patients with epilepsy was higher than that for the wild-type alleles both in the monotherapy and polytherapy patients. These results show the importance of the genetic polymorphism analysis of the main metabolizing enzyme groups of phenytoin for the dose adjustment. PMID:25311916

  5. Hormonal Regulation of Hepatic Drug Metabolizing Enzyme Activity During Adolescence

    PubMed Central

    Kennedy, M.J.

    2009-01-01

    Activities of drug metabolizing enzymes (DME) are known to change throughout the course of physical and sexual maturation with the greatest variability noted during infancy and adolescence. The mechanisms responsible for developmental regulation of DME are currently unknown. However, the hormonal changes of puberty/adolescence provide a theoretical framework for understanding biochemical regulation of DME activity during growth and maturation. Important information regarding potential influences of growth and sex hormones can also be extrapolated from studies evaluating changes in activities of DMEs occurring as a consequence of physiologic, pathologic and/or pharmacologic hormonal fluctuations. Collectively, current data support the hypothesis that isoform-specific alterations in DME activity during adolescence are mediated via sex and/or growth hormones. Characterization of the underlying biochemical alterations responsible for developmental changes in DME activity will require additional studies in which relationships between DME and important hormonal axes are evaluated during the course of pubertal development. PMID:18971926

  6. Ammonium Metabolism Enzymes Aid Helicobacter pylori Acid Resistance

    PubMed Central

    Miller, Erica F.

    2014-01-01

    The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH3 that is immediately protonated to form NH4+. This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg2+ at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium. PMID:24936052

  7. Ammonium metabolism enzymes aid Helicobacter pylori acid resistance.

    PubMed

    Miller, Erica F; Maier, Robert J

    2014-09-01

    The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH3 that is immediately protonated to form NH4 (+). This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg(2+) at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium. PMID:24936052

  8. 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.

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

    E-print Network

    Montgomerie, Bob

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

  10. 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

  11. 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

    2015-12-01

    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. PMID:26524672

  12. Comparison of the Small Molecule Metabolic Enzymes of Escherichia coli and Saccharomyces cerevisiae

    E-print Network

    Gough, Julian

    Comparison of the Small Molecule Metabolic Enzymes of Escherichia coli and Saccharomyces cerevisiae pathways in Escherichia coli and Saccharomyces cerevisiae (yeast) shows that 271 enzymes are common to both organisms. These common enzymes involve 384 gene products in E. coli and 390 in yeast, which are between one

  13. Reference: Bid. Bull. 187: 84-98. (August, 1994) Oxygen Consumption Rates and Metabolic Enzyme

    E-print Network

    Thuesen, Erik V.

    Reference: Bid. Bull. 187: 84-98. (August, 1994) Oxygen Consumption Rates and Metabolic Enzyme in Krebs cycle enzyme activities. LDH activities, on the other hand, in- creased with increasing wet weight (Childress et al., 1989). In addition, we recently measured the respiratory rates and enzyme activities

  14. Characterization of xenobiotic metabolizing enzymes in bovine small intestinal mucosa.

    PubMed

    Virkel, G; Carletti, M; Cantiello, M; Della Donna, L; Gardini, G; Girolami, F; Nebbia, C

    2010-06-01

    The intestinal mucosa plays a capital role in dictating the bioavailability of a large array of orally ingested drugs and toxicants. The activity and the expression of several xenobiotic metabolizing enzymes were measured in subcellular fractions from the duodenal mucosa of male veal calves and beef cattle displaying a functional rumen but differing in both age (about 8 months vs. 18 to 24 months) and dietary regimens (i.e., milk replacer plus hay and straw vs. corn and concentrated meal). Intestinal microsomes showed cytochrome P450 (CYP) 2B, 2C- and 3A-mediated activities and the presence of the corresponding immunorelated proteins, but no proof of CYP1A expression and/or functions could be provided. Intestinal microsomes were also active in performing reactions typically mediated by carboxylesterases (indophenylacetate hydrolysis), flavin-containing monooxygenases (methimazole S-oxidation), and uridindiphosphoglucuronyltransferases (1-naphthol glucuronidation), respectively. Cytosolic fractions displayed the glutathione S-transferase (GST)-dependent conjugation of 1-chloro-2,4-dinitrobenzene; besides, the GST-mediated conjugation of ethacrinic acid (GSTpi) or cumene hydroperoxide (GSTalpha) was matched by the presence of the corresponding immunorelated proteins. Conversely, despite the lack of measurable activity with 3,4-dichloronitrobenzene, a protein cross reacting with anti-rat GSTmu antibodies could be clearly detected. Although, as detected by densitometry, CYPs and GST isoenzymes tended to be more expressed in beef cattle than in veal calf preparations, there was a general poor correlation with the rate of the in vitro metabolism of the selected diagnostic probes. PMID:20557447

  15. 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...

  16. 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

  17. Enzymes of carbohydrate metabolism in the developing rice grain.

    PubMed

    Perez, C M; Perdon, A A; Resurreccion, A P; Villareal, R M; Juliano, B O

    1975-11-01

    The levels of reducing and nonreducing sugars, starch, soluble protein, and selected enzymes involved in the metabolism of sucrose, glucose-1-P, and glucose nucleotides were assayed in dehulled developing rice grains (Oryza sativa L. line IR1541-76-3) during the first 3 weeks after flowering. The level of reducing sugars in the grain was highest 5 to 6 days after flowering. The level of nonreducing sugars and the rate of starch accumulation were maximum 11 to 12 days after flowering, when the level of soluble protein was also the highest. The activities of bound and free invertase, sucrose-UDP and sucrose-ADP glucosyltransferases, hexokinase, phosphoglucomutase, nucleoside diphosphokinase, and UDP-glucose and ADP-glucose pyrophosphorylases were high throughout starch deposition, and were maximum, except for nucleoside diphosphokinase which did not increase in activity, between 8 and 18 days after flowering. Soluble primed phosphorylase and ADP glucose-alpha-glucosyltransferase (starch synthetase) were both present during starch accumulation. Phosphorylase activity was at least 2-fold that of soluble starch synthetase but the synthetase followed more closely the rate of starch accumulation in the grain. The activity of starch synthetase bound to the starch granule also increased progressively with increased starch content of the grain. PMID:16659348

  18. Enzymes of Carbohydrate Metabolism in the Developing Rice Grain

    PubMed Central

    Perez, Consuelo M.; Perdon, Alicia A.; Resurreccion, Adoracion P.; Villareal, Ruth M.; Juliano, Bienvenido O.

    1975-01-01

    The levels of reducing and nonreducing sugars, starch, soluble protein, and selected enzymes involved in the metabolism of sucrose, glucose-1-P, and glucose nucleotides were assayed in dehulled developing rice grains (Oryza sativa L. line IR1541-76-3) during the first 3 weeks after flowering. The level of reducing sugars in the grain was highest 5 to 6 days after flowering. The level of nonreducing sugars and the rate of starch accumulation were maximum 11 to 12 days after flowering, when the level of soluble protein was also the highest. The activities of bound and free invertase, sucrose-UDP and sucrose-ADP glucosyltransferases, hexokinase, phosphoglucomutase, nucleoside diphosphokinase, and UDP-glucose and ADP-glucose pyrophosphorylases were high throughout starch deposition, and were maximum, except for nucleoside diphosphokinase which did not increase in activity, between 8 and 18 days after flowering. Soluble primed phosphorylase and ADP glucose-?-glucosyltransferase (starch synthetase) were both present during starch accumulation. Phosphorylase activity was at least 2-fold that of soluble starch synthetase but the synthetase followed more closely the rate of starch accumulation in the grain. The activity of starch synthetase bound to the starch granule also increased progressively with increased starch content of the grain. PMID:16659348

  19. 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, cofactors and vitamins. Conclusions The ECemble method is able to hierarchically assign high quality enzyme annotations to genomic and metagenomic data. This study demonstrated the real application of ECemble to understand the indispensable role played by microbe-encoded enzymes in the healthy functioning of human metabolic systems. PMID:26099921

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

    PubMed Central

    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. PMID:26132489

  1. 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. PMID:23545442

  2. 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) ?1 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 MAT?1, including accumulation of nuclear tetramers. H35 cells that overexpress tagged-MAT?1 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-MAT?1 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-MAT?1 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 redox-dependent mechanisms in the control of MAT?1 subcellular distribution. Antioxid. Redox Signal. 20, 2541–2554. PMID:24124652

  3. 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 ...

  4. 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)

  5. 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. PMID:26362128

  6. 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

    2014-03-01

    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. PMID:24564900

  7. 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...

  8. 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

  9. Health-related effects of genetic variations of alcohol-metabolizing enzymes in African Americans.

    PubMed

    Scott, Denise M; Taylor, Robert E

    2007-01-01

    Alcohol metabolism involves two key enzymes-alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). There are several types of ADH and ALDH, each of which may exist in several variants (i.e., isoforms) that differ in their ability to break down alcohol and its toxic metabolite acetaldehyde. The isoforms are encoded by different gene variants (i.e., alleles) whose distribution among ethnic groups differs. One variant of ADH is ADH1B, which is encoded by several alleles. An allele called ADH1 B*3 is unique to people of African descent and certain Native American tribes. This allele is associated with more rapid breakdown of alcohol, leading to a transient accumulation of acetaldehyde. African Americans carrying this allele are less likely to have a family history of alcoholism and experience a less rewarding subjective response to alcohol. Moreover, children of mothers with this allele are less vulnerable to alcohol-related birth defects. The enzyme ALDH1 also is encoded by several alleles. Two of these alleles that are found in African Americans-ALDH1 A1 *2 and ALDH1A1 *3--may be associated with a reduced risk of alcoholism. PMID:17718396

  10. 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 prediction of in vivo metabolism, which is reflected by the diverse and comprehensive data sources and methods for metabolism prediction reviewed here. This review attempts to survey the range and scope of computational methods applied to metabolism prediction and also to compare and contrast their applicability and performance. PMID:22339582

  11. 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 those found in control soleus muscles. It would be interesting to know if this represents a transition stage, and whether with prolonged weightlessness most of the fibers would be transformed into a low glycogenolytic type.

  12. Water at Biological Phase Boundaries: Its Role in Interfacial Activation of Enzymes and Metabolic Pathways.

    PubMed

    Damodaran, Srinivasan

    2015-01-01

    Many life-sustaining activities in living cells occur at the membrane-water interface. The pertinent questions that we need to ask are, what are the evolutionary reasons in biology for choosing the membrane-water interface as the site for performing and/or controlling crucial biological reactions, and what is the key physical principle that is very singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this chapter, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes through manipulation of the thermodynamic activity of water in the lipid-water interfacial region. The hypothesis is based on the fact that the surface pressure of a lipid monolayer is a direct measure of the thermodynamic activity of water at the lipid-water interface. Accordingly, the surface pressure-dependent activation or inactivation of interfacial enzymes is directly related to changes in the thermodynamic activity of interfacial water. Extension of this argument suggests that cells may manipulate conformations (and activities) of membrane-bound enzymes by manipulating the (re)activity of interfacial water at various locations in the membrane by localized compression or expansion of the interface. In this respect, cells may use the membrane-bound hormone receptors, lipid phase transition, and local variations in membrane lipid composition as effectors of local compression and/or expansion of membrane, and thereby local water activity. Several experimental data in the literature will be reexamined in the light of this hypothesis. PMID:26438268

  13. 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

  14. Oral cancer cells may rewire alternative metabolic pathways to survive from siRNA silencing of metabolic enzymes

    PubMed Central

    2014-01-01

    Background Cancer cells may undergo metabolic adaptations that support their growth as well as drug resistance properties. The purpose of this study is to test if oral cancer cells can overcome the metabolic defects introduced by using small interfering RNA (siRNA) to knock down their expression of important metabolic enzymes. Methods UM1 and UM2 oral cancer cells were transfected with siRNA to transketolase (TKT) or siRNA to adenylate kinase (AK2), and Western blotting was used to confirm the knockdown. Cellular uptake of glucose and glutamine and production of lactate were compared between the cancer cells with either TKT or AK2 knockdown and those transfected with control siRNA. Statistical analysis was performed with student T-test. Results Despite the defect in the pentose phosphate pathway caused by siRNA knockdown of TKT, the survived UM1 or UM2 cells utilized more glucose and glutamine and secreted a significantly higher amount of lactate than the cells transferred with control siRNA. We also demonstrated that siRNA knockdown of AK2 constrained the proliferation of UM1 and UM2 cells but similarly led to an increased uptake of glucose/glutamine and production of lactate by the UM1 or UM2 cells survived from siRNA silencing of AK2. Conclusions Our results indicate that the metabolic defects introduced by siRNA silencing of metabolic enzymes TKT or AK2 may be compensated by alternative feedback metabolic mechanisms, suggesting that cancer cells may overcome single defective pathways through secondary metabolic network adaptations. The highly robust nature of oral cancer cell metabolism implies that a systematic medical approach targeting multiple metabolic pathways may be needed to accomplish the continued improvement of cancer treatment. PMID:24666435

  15. Organization of Enzyme Concentration across the Metabolic Network in Cancer Cells

    PubMed Central

    Madhukar, Neel S.; Warmoes, Marc O.; Locasale, Jason W.

    2015-01-01

    Rapid advances in mass spectrometry have allowed for estimates of absolute concentrations across entire proteomes, permitting the interrogation of many important biological questions. Here, we focus on a quantitative aspect of human cancer cell metabolism that has been limited by a paucity of available data on the abundance of metabolic enzymes. We integrate data from recent measurements of absolute protein concentration to analyze the statistics of protein abundance across the human metabolic network. At a global level, we find that the enzymes in glycolysis comprise approximately half of the total amount of metabolic proteins and can constitute up to 10% of the entire proteome. We then use this analysis to investigate several outstanding problems in cancer metabolism, including the diversion of glycolytic flux for biosynthesis, the relative contribution of nitrogen assimilating pathways, and the origin of cellular redox potential. We find many consistencies with current models, identify several inconsistencies, and find generalities that extend beyond current understanding. Together our results demonstrate that a relatively simple analysis of the abundance of metabolic enzymes was able to reveal many insights into the organization of the human cancer cell metabolic network. PMID:25621879

  16. De novo transcriptome characterization of Lilium 'Sorbonne' and key enzymes related to the flavonoid biosynthesis.

    PubMed

    Zhang, Ming-fang; Jiang, Ling-min; Zhang, Dong-mei; Jia, Gui-xia

    2015-02-01

    Lily is an important cut-flower and bulb crop in the commercial market. Here, transcriptome profiling of Lilium 'Sorbonne' was conducted through de novo sequencing based on Illumina platform. This research aims at revealing basic information and data that can be used for applied purposes especially the molecular regulatory information on flower color formation in lily. In total, 36,920,680 short reads which corresponded to 3.32 GB of total nucleotides, were produced through transcriptome sequencing. These reads were assembled into 39,636 Unigenes, of which 30,986 were annotated in Nr, Nt, Swiss-Prot, KEGG, COG, GO databases. Based on the three public protein databases, a total of 32,601 coding sequences were obtained. Meanwhile, 19,242 Unigenes were assigned to 128 KEGG pathways. Those with the greatest representation by unique sequences were for ''metabolic pathways'' (5,406 counts, 28.09 %). Our transcriptome revealed 156 Unigenes that encode key enzymes in the flavonoid biosynthesis pathway including CHS, CHI, F3H, FLS, DFR, etc. MISA software identified 2,762 simple sequence repeats, from which 1,975 primers pairs were designed. Over 2,762 motifs were identified, of which the most frequent was AG/CT (659, 23.86 %), followed by A/T (615, 22.27 %) and CCG/CGG (416, 15.06 %). Based on the results, we believe that the color formation of the Lilium 'Sorbonne' flower was mainly controlled by the flavonoid biosynthesis pathway. Additionally, this research provides initial genetic resources that will be valuable to the lily community for other molecular biology research, and the SSRs will facilitate marker-assisted selection in lily breeding. PMID:25307066

  17. 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 . PMID:26542446

  18. Comparison of the Small Molecule Metabolic Enzymes of Escherichia coli and Saccharomyces cerevisiae

    PubMed Central

    Jardine, Oliver; Gough, Julian; Chothia, Cyrus; Teichmann, Sarah A.

    2002-01-01

    The comparison of the small molecule metabolism pathways in Escherichia coli and Saccharomyces cerevisiae (yeast) shows that 271 enzymes are common to both organisms. These common enzymes involve 384 gene products in E. coli and 390 in yeast, which are between one half and two thirds of the gene products of small molecule metabolism in E. coli and yeast, respectively. The arrangement and family membership of the domains that form all or part of 374 E. coli sequences and 343 yeast sequences was determined. Of these, 70% consist entirely of homologous domains, and 20% have homologous domains linked to other domains that are unique to E. coli, yeast, or both. Over two thirds of the enzymes common to the two organisms have sequence identities between 30% and 50%. The remaining groups include 13 clear cases of nonorthologous displacement. Our calculations show that at most one half to two thirds of the gene products involved in small molecule metabolism are common to E. coli and yeast. We have shown that the common core of 271 enzymes has been largely conserved since the separation of prokaryotes and eukaryotes, including modifications for regulatory purposes, such as gene fusion and changes in the number of isozymes in one of the two organisms. Only one fifth of the common enzymes have nonhomologous domains between the two organisms. Around the common core very different extensions have been made to small molecule metabolism in the two organisms. [Online supplementary material available a http://www.genome.org.] PMID:12045145

  19. 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. PMID:26335727

  20. Structural studies of radical enzymes in bacterial central metabolism

    E-print Network

    Funk, Michael A. (Michael Andrew)

    2015-01-01

    Anaerobic bacteria play a crucial role in cycling of nutrients in diverse ecosystems, degradation of organic compounds, and as key members of the human gut microbiome. The absence of oxygen limits the chemistry that bacteria ...

  1. Novel TPP-riboswitch activators bypass metabolic enzyme dependency

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

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

  2. Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

    PubMed

    Sun, Junsong; Hopkins, Robert C; 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

  3. 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, in response to B-deficiency. In conclusion, respiration, organic acid (i.e., glycolysis and the TCA cycle) metabolism, the anapleurotic pathway and amino acid biosynthesis were upregulated in B-deficient leaves with excessive accumulation of carbohydrates to 'consume' the excessive carbon available, but downregulated in B-deficient roots with less accumulation of carbohydrates to maintain the net carbon balance. PMID:24957048

  4. 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. PMID:26512807

  5. The effect of phytic acid on the levels of blood glucose and some enzymes of carbohydrate and lipid metabolism.

    PubMed

    Dilworth, L L; Omoruyi, F O; Simon, O R; Morrison, E Y St A; Asemota, H N

    2005-03-01

    In this study, six groups of rats were fed as follows: Groups 1 and 2 were fed formulated diets supplemented with zinc or without zinc respectively. Groups 3 and 4 were fed formulated diets supplemented with zinc plus phytic acid extracted from sweet potato (Ipomea batatas) or commercial phytic acid respectively. Groups 5 and 6 were fed formulated diets supplemented with phytic acid extract from sweet potato or commercial phytic acid respectively. The animals were fed for three weeks and then sacrificed The activities of key enzymes of carbohydrate and lipid metabolism as well as transaminases in the liver were determined. Blood glucose level was also assessed. Phytic acid extract consumption from sweet potato and commercial phytic acid plus zinc supplement lowered blood glucose levels. There was no significant change in the activity of 6-phosphogluconate dehydrogenase among the groups. Similarly, phytic acid supplementation showed no significant decrease in the activity of pyruvate kinase compared to the group fed formulated diets. There was a significant increase in the activity of glucose-6-phosphate dehydrogenase in the groups fed phytic extract from sweet potato compared to the other groups. The activities of malic enzyme and ATP-citrate lyase in this study were not significantly altered among the groups. There is a lowering of blood glucose levels which is desirable for diabetics who consume sweet potato diets. The changes in some of the hepatic metabolic enzymes are geared towards compensating for the decreased glycolytic responses. PMID:15999878

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

    PubMed Central

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

    2014-01-01

    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. PMID:25034694

  7. Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

    PubMed Central

    Sapir, Amir; Tsur, Assaf; Koorman, Thijs; Ching, Kaitlin; Mishra, Prashant; Bardenheier, Annabelle; Podolsky, Lisa; Bening-Abu-Shach, Ulrike; Boxem, Mike; Chou, Tsui-Fen; Broday, Limor; Sternberg, Paul W.

    2014-01-01

    Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin–proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies. PMID:25187565

  8. Absolute quantitative profiling of the key metabolic pathways in slow and fast skeletal muscle.

    PubMed

    Rakus, Dariusz; Gizak, Agnieszka; Deshmukh, Atul; Wi?niewski, Jacek R

    2015-03-01

    Slow and fast skeletal muscles are composed of, respectively, mainly oxidative and glycolytic muscle fibers, which are the basic cellular motor units of the motility apparatus. They largely differ in excitability, contraction mechanism, and metabolism. Because of their pivotal role in body motion and homeostasis, the skeletal muscles have been extensively studied using biochemical and molecular biology approaches. Here we describe a simple analytical and computational approach to estimate titers of enzymes of basic metabolic pathways and proteins of the contractile machinery in the skeletal muscles. Proteomic analysis of mouse slow and fast muscles allowed estimation of the titers of enzymes involved in the carbohydrate, lipid, and energy metabolism. Notably, we observed that differences observed between the two muscle types occur simultaneously for all proteins involved in a specific process such as glycolysis, free fatty acid catabolism, Krebs cycle, or oxidative phosphorylation. These differences are in a good agreement with the well-established biochemical picture of the muscle types. We show a correlation between maximal activity and the enzyme titer, suggesting that change in enzyme concentration is a good proxy for its catalytic potential in vivo. As a consequence, proteomic profiling of enzyme titers can be used to monitor metabolic changes in cells. Additionally, quantitative data of structural proteins allowed studying muscle type specific cell architecture and its remodeling. The presented proteomic approach can be applied to study metabolism in any other tissue or cell line. PMID:25597705

  9. 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 (GSK3?) 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 GSK3? 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. PMID:25152463

  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. 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...

  12. Evolution of a Major Drug Metabolizing Enzyme Defect in the Domestic Cat and Other Felidae: Phylogenetic

    E-print Network

    Starks, Philip

    Evolution of a Major Drug Metabolizing Enzyme Defect in the Domestic Cat and Other Felidae, University of California, Berkeley, California, United States of America Abstract The domestic cat (Felis UGT1A6 mutations and explored the hypothesis that gene inactivation in cats was enabled by minimal

  13. 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...

  14. 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...

  15. 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...

  16. FDH: an Aldehyde Dehydrogenase Fusion Enzyme in Folate Metabolism

    PubMed Central

    Krupenko, Sergey A.

    2009-01-01

    FDH (10-formyltetrahydrofolate dehydrogenase, Aldh1L1, EC 1.5.1.6) converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate and CO2 in a NADP+-dependent reaction. It is a tetramer of four identical 902 amino acid residue subunits. The protein subunit is a product of a natural fusion of three unrelated genes and consists of three distinct domains. The N-terminal domain of FDH (residues 1–310) carries the folate binding site and shares sequence homology and structural topology with other enzymes utilizing 10-formyl-THF as a substrate. In vitro it functions as 10-formyl-THF hydrolase, and evidence indicate that this activity is a part of the overall FDH mechanism. The C-terminal domain of FDH (residues 400–902) originated from an aldehyde dehydrogenase-related gene and is capable of oxidation of short-chain aldehydes to corresponding acids. Similar to class 1 and 2 aldehyde dehydrogenases, this domain exists as a tetramer and defines the oligomeric structure of the full-length enzyme. The two catalytic domains are connected by an intermediate linker (residues 311–399), which is a structural and functional homolog of carrier proteins possessing a 4?-phosphopantetheine prosthetic group. In the FDH mechanism, the intermediate linker domain transfers a formyl, covalently attached to the sulfhydryl group of the phosphopantetheine arm, from the N-terminal domain to the C-terminal domain. The overall FDH mechanism is a coupling of two sequential reactions, a hydrolase and a formyl dehydrogenase, bridged by a substrate transfer step. In this mechanism, one domain provides the folate binding site and a hydrolase catalytic center to remove the formyl group from the folate substrate, another provides a transfer vehicle between catalytic centers and the third one contributes the dehydrogenase machinery further oxidizing formyl to CO2. PMID:18848533

  17. 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.

  18. Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins.

    PubMed

    Yamaji, Toshiyuki; Hanada, Kentaro

    2015-02-01

    In recent decades, many sphingolipid enzymes, sphingolipid-metabolism regulators and sphingolipid transfer proteins have been isolated and characterized. This review will provide an overview of the intracellular localization and topology of sphingolipid enzymes in mammalian cells to highlight the locations where respective sphingolipid species are produced. Interestingly, three sphingolipids that reside or are synthesized in cytosolic leaflets of membranes (ceramide, glucosylceramide and ceramide-1-phosphate) all have cytosolic lipid transfer proteins (LTPs). These LTPs consist of ceramide transfer protein (CERT), four-phosphate adaptor protein 2 (FAPP2) and ceramide-1-phosphate transfer protein (CPTP), respectively. These LTPs execute functions that affect both the location and metabolism of the lipids they bind. Molecular details describing the mechanisms of regulation of LTPs continue to emerge and reveal a number of critical processes, including competing phosphorylation and dephosphorylation reactions and binding interactions with regulatory proteins and lipids that influence the transport, organelle distribution and metabolism of sphingolipids. PMID:25382749

  19. Pharmacogenetics of drug-metabolizing enzymes: implications for a safer and more effective drug therapy

    PubMed Central

    Ingelman-Sundberg, Magnus; Rodriguez-Antona, Cristina

    2005-01-01

    The majority of phase I- and phase II-dependent drug metabolism is carried out by polymorphic enzymes which can cause abolished, quantitatively or qualitatively decreased or enhanced drug metabolism. Several examples exist where subjects carrying certain alleles do not benefit from drug therapy due to ultrarapid metabolism caused by multiple genes or by induction of gene expression or, alternatively, suffer from adverse effects of the drug treatment due to the presence of defective alleles. It is likely that future predictive genotyping for such enzymes might benefit 15–25% of drug treatments, and thereby allow prevention of adverse drug reactions and causalities, and thus improve the health of a significant fraction of the patients. However, it will take time before this will be a reality within the clinic. We describe some important aspects in the field with emphasis on cytochrome P450 and discuss also polymorphic aspects of foetal expression of CYP3A5 and CYP3A7. PMID:16096104

  20. Odorant Metabolism Catalyzed by Olfactory Mucosal Enzymes Influences Peripheral Olfactory Responses in Rats

    PubMed Central

    Thiebaud, Nicolas; Veloso Da Silva, Stéphanie; Jakob, Ingrid; Sicard, Gilles; Chevalier, Joëlle; Ménétrier, Franck; Berdeaux, Olivier; Artur, Yves; Heydel, Jean-Marie; Le Bon, Anne-Marie

    2013-01-01

    A large set of xenobiotic-metabolizing enzymes (XMEs), such as the cytochrome P450 monooxygenases (CYPs), esterases and transferases, are highly expressed in mammalian olfactory mucosa (OM). These enzymes are known to catalyze the biotransformation of exogenous compounds to facilitate elimination. However, the functions of these enzymes in the olfactory epithelium are not clearly understood. In addition to protecting against inhaled toxic compounds, these enzymes could also metabolize odorant molecules, and thus modify their stimulating properties or inactivate them. In the present study, we investigated the in vitro biotransformation of odorant molecules in the rat OM and assessed the impact of this metabolism on peripheral olfactory responses. Rat OM was found to efficiently metabolize quinoline, coumarin and isoamyl acetate. Quinoline and coumarin are metabolized by CYPs whereas isoamyl acetate is hydrolyzed by carboxylesterases. Electro-olfactogram (EOG) recordings revealed that the hydroxylated metabolites derived from these odorants elicited lower olfactory response amplitudes than the parent molecules. We also observed that glucurono-conjugated derivatives induced no olfactory signal. Furthermore, we demonstrated that the local application of a CYP inhibitor on rat olfactory epithelium increased EOG responses elicited by quinoline and coumarin. Similarly, the application of a carboxylesterase inhibitor increased the EOG response elicited by isoamyl acetate. This increase in EOG amplitude provoked by XME inhibitors is likely due to enhanced olfactory sensory neuron activation in response to odorant accumulation. Taken together, these findings strongly suggest that biotransformation of odorant molecules by enzymes localized to the olfactory mucosa may change the odorant’s stimulating properties and may facilitate the clearance of odorants to avoid receptor saturation. PMID:23555703

  1. 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

  2. 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. PMID:25954206

  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

    In 2005 researchers at the Centre for Geobiology, University of Bergen, Norway, discovered two active vent fields at the southwestern Mohns Ridge in the Norwegian-Greenland Sea. The fields harbours both low-temperature iron deposits and high-temperature white smoker vents. Distinct microbial mats were abundantly present and located in close vicinity to the hydrothermal vent sites. Characteristics of the mat environment were steep physical and chemical gradients with temperatures ranging from 10°C in the top layer to 90°C at 10 cm bsf and high concentrations of hydrogen sulfide and methane. The work presented here focus on the In situ community activities, and is part of an integrated strategy combining metagenomics, metatranscriptomics and metaproteomics to in-depth characterise these newly discovered hydrothermal vent communities. Extracted proteins were separated via SDS-PAGE. Peptides extracted after In-gel tryptic digest was injected into an Ultimate 3000 nanoLC system connected to a linear quadropole ion trap-orbitrap (LTQ-Orbitrap XL) mass spectrometer equipped with a nanoelectrospray ion source. A custom database of open reading frames (ORFs) from the combined metatranscriptome and metagenome datasets was implemented and searched against using Mascot 2.2; the IRMa tool box [1] was used in peptide validation. Validated ORFs were subjected to a Blastp search against Refseq with an E-value cut-off of 0.001. A total of 1097 proteins with ? 2 peptides were identified of which 921 gave a hit against Refseq, containing 519 unique proteins. Key enzymes of the sulfur oxidation pathway (sox) were found, which were taxonomically affiliated to Epsilonproteobacteria. In addition, this group actively expressed hydrogenases and membrane proteins involved in aerobic and anaerobic respiratory chains. Enzymes of dissimilatory sulfate-reduction (APS-reductase, AprAB and DsrA2) were found with closest hit to members of the Deltaproteobacteria. These findings indicate an 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. Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of rosiglitazone

    PubMed Central

    Baldwin, S J; Clarke, S E; Chenery, R J

    1999-01-01

    Aims To identify the human cytochrome P450 enzyme(s) involved in the in vitro metabolism of rosiglitazone, a potential oral antidiabetic agent for the treatment of type 2 diabetes-mellitus. Method The specific P450 enzymes involved in the metabolism of rosiglitazone were determined by a combination of three approaches; multiple regression analysis of the rates of metabolism of rosiglitazone in human liver microsomes against selective P450 substrates, the effect of selective chemical inhibitors on rosiglitazone metabolism and the capability of expressed P450 enzymes to mediate the major metabolic routes of rosiglitazone metabolism. Result The major products of metabolism following incubation of rosiglitazone with human liver microsomes were para-hydroxy and N-desmethyl rosiglitazone. The rate of formation varied over 38-fold in the 47 human livers investigated and correlated with paclitaxel 6?-hydroxylation (P<0.001). Formation of these metabolites was inhibited significantly (>50%) by 13-cis retinoic acid, a CYP2C8 inhibitor, but not by furafylline, quinidine or ketoconazole. In addition, both metabolites were produced by microsomes derived from a cell line transfected with human CYP2C8 cDNA. There was some evidence for CYP2C9 playing a minor role in the metabolism of rosiglitazone. Sulphaphenazole caused limited inhibition (<30%) of both pathways in human liver microsomes and microsomes from cells transfected with CYP2C9 cDNA were able to mediate the metabolism of rosiglitazone, in particular the N-demethylation pathway, albeit at a much slower rate than CYP2C8. Rosiglitazone caused moderate inhibition of paclitaxel 6?-hydroxylase activity (CYP2C8; IC50=18 ?m), weak inhibition of tolbutamide hydroxylase activity (CYP2C9; IC50 =50 ?m), but caused no marked inhibition of the other cytochrome P450 activities investigated (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, 3A and 4A). Conclusion CYP2C8 is primarily responsible for the hydroxylation and N-demethylation of rosiglitazone in human liver; with minor contributions from CYP2C9. PMID:10510156

  5. Vitamin C Stimulates Epidermal Ceramide Production by Regulating Its Metabolic Enzymes.

    PubMed

    Kim, Kun Pyo; Shin, Kyong-Oh; Park, Kyungho; Yun, Hye Jeong; Mann, Shivtaj; Lee, Yong Moon; Cho, Yunhi

    2015-11-01

    Ceramide is the most abundant lipid in the epidermis and plays a critical role in maintaining epidermal barrier function. Overall ceramide content in keratinocyte increases in parallel with differentiation, which is initiated by supplementation of calcium and/or vitamin C. However, the role of metabolic enzymes responsible for ceramide generation in response to vitamin C is still unclear. Here, we investigated whether vitamin C alters epidermal ceramide content by regulating the expression and/or activity of its metabolic enzymes. When human keratinocytes were grown in 1.2 mM calcium with vitamin C (50 ?g/ml) for 11 days, bulk ceramide content significantly increased in conjunction with terminal differentiation of keratinocytes as compared to vehicle controls (1.2 mM calcium alone). Synthesis of the ceramide fractions was enhanced by increased de novo ceramide synthesis pathway via serine palmitoyltransferase and ceramide synthase activations. Moreover, sphingosine-1-phosphate (S1P) hydrolysis pathway by action of S1P phosphatase was also stimulated by vitamin C supplementation, contributing, in part, to enhanced ceramide production. However, activity of sphingomyelinase, a hydrolase enzyme that converts sphingomyelin to ceramide, remained unaltered. Taken together, we demonstrate that vitamin C stimulates ceramide production in keratinocytes by modulating ceramide metabolic-related enzymes, and as a result, could improve overall epidermal barrier function. PMID:26535077

  6. Vitamin C Stimulates Epidermal Ceramide Production by Regulating Its Metabolic Enzymes

    PubMed Central

    Kim, Kun Pyo; Shin, Kyong-Oh; Park, Kyungho; Yun, Hye Jeong; Mann, Shivtaj; Lee, Yong Moon; Cho, Yunhi

    2015-01-01

    Ceramide is the most abundant lipid in the epidermis and plays a critical role in maintaining epidermal barrier function. Overall ceramide content in keratinocyte increases in parallel with differentiation, which is initiated by supplementation of calcium and/or vitamin C. However, the role of metabolic enzymes responsible for ceramide generation in response to vitamin C is still unclear. Here, we investigated whether vitamin C alters epidermal ceramide content by regulating the expression and/or activity of its metabolic enzymes. When human keratinocytes were grown in 1.2 mM calcium with vitamin C (50 ?g/ml) for 11 days, bulk ceramide content significantly increased in conjunction with terminal differentiation of keratinocytes as compared to vehicle controls (1.2 mM calcium alone). Synthesis of the ceramide fractions was enhanced by increased de novo ceramide synthesis pathway via serine palmitoyltransferase and ceramide synthase activations. Moreover, sphingosine-1-phosphate (S1P) hydrolysis pathway by action of S1P phosphatase was also stimulated by vitamin C supplementation, contributing, in part, to enhanced ceramide production. However, activity of sphingomyelinase, a hydrolase enzyme that converts sphingomyelin to ceramide, remained unaltered. Taken together, we demonstrate that vitamin C stimulates ceramide production in keratinocytes by modulating ceramide metabolic-related enzymes, and as a result, could improve overall epidermal barrier function. PMID:26535077

  7. The role of carbonyl reducing enzymes in oxcarbazepine in vitro metabolism in man.

    PubMed

    Malátková, Petra; Havlíková, Lucie; Wsól, Vladimír

    2014-09-01

    Oxcarbazepine, a second generation antiepileptic drug belonging to the family of dibenz[b,f]azepines, is subjected to a rapid and extensive biotransformation. Oxcarbazepine demonstrates a low potential for drug interactions because its biotransformation is mainly mediated by the reduction pathway instead of oxidative pathways, which are very susceptible to drug interactions. The reductive metabolism of oxcarbazepine yields a 10-monohydroxy derivative (10,11-dihydro-10-hydroxy-carbazepine), which is responsible for the pharmacological activity. The identity and localization of enzymes participating in the reduction of oxcarbazepine in response to this active metabolite have remained unknown until now. Thus, we investigated the reductive metabolism of oxcarbazepine in human liver subcellular fractions and using recombinant carbonyl reducing enzymes. The reduction of oxcarbazepine was shown to occur largely in the liver cytosol rather than liver microsomes. Furthermore, the activity and stereospecificity of cytosolic carbonyl reducing enzymes toward oxcarbazepine were assessed. Of the eight tested enzymes, six reductases were identified to contribute to the reduction of oxcarbazepine. The highest activities were demonstrated by AKR1C1, AKR1C2, AKR1C3, and AKR1C4. The contribution of CBR1 and CBR3 to the reduction of oxcarbazepine was also significant, although their role in oxcarbazepine metabolism in vivo is unclear. PMID:25063510

  8. Genetic polymorphisms of metabolic enzymes and the pharmacokinetics of indapamide in Taiwanese subjects.

    PubMed

    Wang, Teng-Hsu; Hsiong, Cheng-Huei; Ho, Hsin-Tien; Shih, Tung-Yuan; Yen, San-Jan; Wang, Hui-Hung; Wu, Jer-Yuarn; Kuo, Benjamin Pei-Chung; Chen, Yuan-Tsong; Ho, Shung-Tai; Hu, Oliver Yoa-Pu

    2014-03-01

    To understand the genetic makeup and impact on pharmacokinetics (PK) in the Taiwanese population, we analyzed the pharmacogenetic (PG) profile and demonstrated its effects on enzyme metabolism using indapamide as an example. A multiplex mass spectrometry method was used to examine the single nucleotide polymorphism (SNP) profile of eight major phases I and II metabolic enzymes in 1,038 Taiwanese subjects. A PG/PK study was conducted in 24 healthy subjects to investigate the possible effects of 28 SNPs on drug biotransformation. Among the genetic profile analyzed, eight SNPs from CYP2A6, CYP2C19, CYP2D6, CYP2E1, CYP3A5, and UGT2B7 showed higher variant frequencies than those previously reported in Caucasians or Africans. For instance, we observed 14.7% frequency of the SNP rs5031016 (I471T) from CYP2A6 in Taiwanese, whereas 0% variation was reported in Caucasians and Africans. The PG/PK study of indapamide demonstrated that the polymorphic SNPs CYP2C9 rs4918758 and CYP2C19 rs4244285 appeared to confer lowered enzyme activity, as indicated by increased C max (25%???64%), increased area under the plasma level-time curves (30~76%), increased area under the time infinity (43%???80%), and lower apparent clearance values than PK for wild-type indapamide. Our results reinforce the biochemical support of CYP2C19 in indapamide metabolism and identify a possible new participating enzyme CYP2C9. The PG/PK approach contributed toward understanding the genetic makeup of different ethnic groups and associations of enzymes in drug metabolism. It could be used to identify two genetic markers that enable to differentiate subjects with varied PK outcomes of indapamide. PMID:24357089

  9. Liver enzymes and metabolic syndrome: a large-scale case-control study.

    PubMed

    Zhang, Lu; Ma, Xiangyu; Jiang, Zhi; Zhang, Kejun; Zhang, Mengxuan; Li, Yafei; Zhao, Xiaolan; Xiong, Hongyan

    2015-09-29

    Previous studies suggested that elevated liver enzymes could be used as potential novel biomarkers of Metabolic syndrome (MetS) and its clinical outcomes, although the results were inconsistent and the conclusions were underpowered. A case-control study with 6,268 MetS subjects and 6,330 frequency-matched healthy controls was conducted to systematically evaluated levels of four liver enzymes (ALT, AST, GGT and ALP), both in overall populations and in subjects with normal liver enzymes, with MetS risk using both quartiles and continuous unit of liver enzymes. We found significant associations were detected for all above analyses. Compared with quartile 1 (Q1), other quartiles have significant higher MetS risk, with ORs ranging from 1.15 to 18.15. The highest effected was detected for GGT, for which the OR value for the highest versus lowest quartile was 18.15 (95% CI: 15.7-20.9). Mutual adjustment proved the independence of the relations for all four liver enzymes. Sensitivity analyses didn't materially changed the trend. To the best of our knowledge, this study should be the largest, which aimed at evaluating the association between liver enzymes measures and MetS risk. The results can better support that liver enzyme levels could be used as clinical predictors of MetS. PMID:26449189

  10. 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.

  11. Effects of bisphenol A on key enzymes in cellular respiration of soybean seedling roots.

    PubMed

    Nie, Lijun; Wang, Lihong; Wang, Qingqing; Wang, Shengman; Zhou, Qing; Huang, Xiaohua

    2015-10-01

    The environmental endocrine disrupter bisphenol A (BPA) is ubiquitous in the environment, with potential toxic effects on plants. Previous studies have found a significant effect of BPA on levels of mineral nutrients in plant roots, but the underlying mechanism remains unknown. To determine how BPA influences root mineral nutrients, the effects of BPA (1.5?mg?L(-1) , 3.0?mg?L(-1) , 6.0?mg?L(-1) , 12.0?mg?L(-1) , 24.0?mg?L(-1) , 48.0?mg?L(-1) , and 96.0?mg?L(-1) ) on activities of critical respiratory enzymes (hexokinase, phosphofructokinase, pyruvate kinase, isocitrate dehydrogenase, and cytochrome c oxidase) were investigated in soybean seedling roots. After BPA exposure for 7 d, the low concentrations of BPA increased the activities of critical respiratory enzymes in roots, whereas opposite effects were observed in roots exposed to high concentrations of BPA, and the inhibitory effect was greater for higher BPA concentrations. In addition, evident morphological anomalies and decreases in root lengths and volumes were induced by high concentrations of BPA. Following withdrawal of BPA exposure after 7 d, the activities of respiratory enzymes and visible signs of toxicity recovered, and the extent of recovery depended on the type of enzyme and the BPA concentration. Furthermore, correlation analysis showed that the disturbance by BPA to activities of respiratory enzymes, which led to interference in the energy metabolism in roots, might be an effect mechanism of BPA on mineral element accumulation in plant roots. PMID:26010676

  12. The spectrum of enzymes involved in activation of 2-aminoanthracene varies with the metabolic system applied.

    PubMed

    Veres, Zsuzsa; Török, Géza; Tóth, Eva; Vereczkey, László; Jemnitz, Katalin

    2005-09-01

    The aim of this study was to estimate the involvement of cytochrome P450s (CYPs) in the metabolic activation of 2-aminoanthracene (2AA) by use of metabolic systems such as liver S9 or hepatocytes from untreated and beta-naphthoflavone (BNF)- or phenobarbital (PB)-treated rats. Metabolic activation was determined in the Salmonella reverse mutation assay (Ames test). Unexpectedly, both enzyme inducers, BNF and PB, significantly decreased the mutagenicity of 2AA activated by S9 fractions. 2AA mutagenicity was detected in the presence of cytochrome P450 inhibitors such as alpha-naphthoflavone (ANF), clotrimazole and N-benzylimidazole to study the contribution of CYP isoenzymes to the activation process. ANF significantly decreased the activation of 2AA by S9 from untreated rats. In contrast, ANF significantly increased the metabolic activation of 2AA by S9 from BNF- and PB-treated rats. The enhanced mutagenicity was not altered by co-incubation with clotrimazole and ANF. Pre-incubation of 2AA in the presence of N-benzylimidazole significantly increased the activation of 2AA by S9 from BNF- and PB-treated rats, which suggests that CYPs play minor role in 2AA metabolic activation by rat liver S9 fractions. In contrast with the results described above, BNF treatment of rats significantly enhanced the activation of 2AA by hepatocytes. ANF attenuated the extent of this activation suggesting that different enzymes play a major role in the activation processes in these metabolic systems. Our results indicate that identification of mutagenic hazard by use of the Ames test may depend on the metabolic system applied. PMID:16006184

  13. Dual function of MIPS1 as a metabolic enzyme and transcriptional regulator.

    PubMed

    Latrasse, David; Jégu, Teddy; Meng, Pin-Hong; Mazubert, Christelle; Hudik, Elodie; Delarue, Marianne; Charon, Céline; Crespi, Martin; Hirt, Heribert; Raynaud, Cécile; Bergounioux, Catherine; Benhamed, Moussa

    2013-03-01

    Because regulation of its activity is instrumental either to support cell proliferation and growth or to promote cell death, the universal myo-inositol phosphate synthase (MIPS), responsible for myo-inositol biosynthesis, is a critical enzyme of primary metabolism. Surprisingly, we found this enzyme to be imported in the nucleus and to interact with the histone methyltransferases ATXR5 and ATXR6, raising the question of whether MIPS1 has a function in transcriptional regulation. Here, we demonstrate that MIPS1 binds directly to its promoter to stimulate its own expression by locally inhibiting the spreading of ATXR5/6-dependent heterochromatin marks coming from a transposable element. Furthermore, on activation of pathogen response, MIPS1 expression is reduced epigenetically, providing evidence for a complex regulatory mechanism acting at the transcriptional level. Thus, in plants, MIPS1 appears to have evolved as a protein that connects cellular metabolism, pathogen response and chromatin remodeling. PMID:23341037

  14. 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. PMID:21666928

  15. 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. PMID:25799954

  16. 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, 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

  17. 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

  18. Stem sugar accumulation in sweet sorghum - activity and expression of sucrose metabolizing enzymes and sucrose transporters.

    PubMed

    Qazi, Hilal Ahmad; Paranjpe, Sharayu; Bhargava, Sujata

    2012-04-15

    Sugar metabolism was studied in sweet sorghum (SSV74) that is known to store sugars in the mature internodes and which is reported to give grain yields twice that of a grain sorghum variety (SPV1616). Comparison of sugar accumulation in these two varieties was carried out at three stages of growth and in the upper and lower internodes. In spite of large differences in the level of sugar accumulation, osmolarity of the sap did not vary as significantly in the two varieties. Significant contribution of variety, stage and internode position was seen for the variation observed in sugar content. Though the activities of sugar metabolizing enzymes namely sucrose synthase (in the synthesis and cleavage directions), sucrose phosphate synthase and invertase (cytoplasmic and vacuolar) also varied in a stage- and internode-specific manner in the two varieties, these enzymes did not contribute significantly to the variation observed in sugar content. Transcriptional expression of one sucrose synthase (SUC1), two sucrose phosphate synthase (SPS2 and SPS3) and a vacuolar invertase (INV3) gene were lower in sweet sorghum as compared to grain sorghum. Sweet sorghum also showed lower expression of two sucrose transporters (SUT1 and SUT4), which correlated to higher sugar accumulation in this variety. Differential expression of the sugar metabolizing enzymes and sucrose transporters in sweet and grain sorghum suggest a role for signaling molecules and transcription factors in regulating sugar accumulation observed in the mature internodes of sweet sorghum, which needs to be investigated. PMID:22325624

  19. [Nitrogen enzymes and energy metabolism from dogfish liver and in a Catrex preparation].

    PubMed

    Kvesitadze, G I; Kokonashvili, G N; Sadunishvili, T A

    1993-01-01

    The activity of enzymes of nitrogen and energy metabolisms from dogfish liver and a commercial preparation Catrex manufactured in the Scientific-Industrial Association "Adaptogen" (Tbilisi) was studied. The liver homogenate contains active glutamate dehydrogenase (GD), malate dehydrogenase (MD) and lactate dehydrogenase (LD) catalysing in vitro the reaction in both directions, as well as active glutamine synthetase, aspartate transaminase and alanine transaminase. These enzymes are also present in Catrex, but their activities are less. After 10-day storage of the liver homogenate and the Catrex preparation the enzymes slightly inactivated. Two isozymes of MD and four isozymes of LD were detected in the liver homogenate by polyacrylamide gel electrophoresis. In Catrex the two MD isozymes and only three LD isozymes were found. PMID:8097312

  20. Entropy is key to the formation of pentacyclic terpenoids by enzyme-catalyzed polycyclization.

    PubMed

    Syrén, Per-Olof; Hammer, Stephan C; Claasen, Birgit; Hauer, Bernhard

    2014-05-01

    Polycyclizations constitute a cornerstone of chemistry and biology. Multicyclic scaffolds are generated by terpene cyclase enzymes in nature through a carbocationic polycyclization cascade of a prefolded polyisoprene backbone, for which electrostatic stabilization of transient carbocationic species is believed to drive catalysis. Computational studies and site-directed mutagenesis were used to assess the contribution of entropy to the polycyclization cascade catalyzed by the triterpene cyclase from A. acidocaldarius. Our results show that entropy contributes significantly to the rate enhancement through the release of water molecules through specific channels. A single rational point mutation that results in the disruption of one of these water channels decreased the entropic contribution to catalysis by 60?kcal?mol(-1) . This work demonstrates that entropy is the key to enzyme-catalyzed polycyclizations, which are highly relevant in biology since 90?% of all natural products contain a cyclic subunit. PMID:24711227

  1. 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 ...

  2. 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

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

    PubMed

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

    2015-01-01

    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

  4. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation.

    PubMed

    Zanger, Ulrich M; Schwab, Matthias

    2013-04-01

    Cytochromes P450 (CYP) are a major source of variability in drug pharmacokinetics and response. Of 57 putatively functional human CYPs only about a dozen enzymes, belonging to the CYP1, 2, and 3 families, are responsible for the biotransformation of most foreign substances including 70-80% of all drugs in clinical use. The highest expressed forms in liver are CYPs 3A4, 2C9, 2C8, 2E1, and 1A2, while 2A6, 2D6, 2B6, 2C19 and 3A5 are less abundant and CYPs 2J2, 1A1, and 1B1 are mainly expressed extrahepatically. Expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, regulation by cytokines, hormones and during disease states, as well as sex, age, and others. Multiallelic genetic polymorphisms, which strongly depend on ethnicity, play a major role for the function of CYPs 2D6, 2C19, 2C9, 2B6, 3A5 and 2A6, and lead to distinct pharmacogenetic phenotypes termed as poor, intermediate, extensive, and ultrarapid metabolizers. For these CYPs, the evidence for clinical significance regarding adverse drug reactions (ADRs), drug efficacy and dose requirement is rapidly growing. Polymorphisms in CYPs 1A1, 1A2, 2C8, 2E1, 2J2, and 3A4 are generally less predictive, but new data on CYP3A4 show that predictive variants exist and that additional variants in regulatory genes or in NADPH:cytochrome P450 oxidoreductase (POR) can have an influence. Here we review the recent progress on drug metabolism activity profiles, interindividual variability and regulation of expression, and the functional and clinical impact of genetic variation in drug metabolizing P450s. PMID:23333322

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

    PubMed Central

    Michalak, Krzysztof Piotr; Ma?kowska-K?dziora, Agnieszka; Sobolewski, Bogus?aw; Wo?niak, 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

  6. Evolution of Domain Architectures and Catalytic Functions of Enzymes in Metabolic Systems

    PubMed Central

    Suen, Summit; Lu, Henry Horng-Shing; Yeang, Chen-Hsiang

    2012-01-01

    Domain architectures and catalytic functions of enzymes constitute the centerpieces of a metabolic network. These types of information are formulated as a two-layered network consisting of domains, proteins, and reactions—a domain–protein–reaction (DPR) network. We propose an algorithm to reconstruct the evolutionary history of DPR networks across multiple species and categorize the mechanisms of metabolic systems evolution in terms of network changes. The reconstructed history reveals distinct patterns of evolutionary mechanisms between prokaryotic and eukaryotic networks. Although the evolutionary mechanisms in early ancestors of prokaryotes and eukaryotes are quite similar, more novel and duplicated domain compositions with identical catalytic functions arise along the eukaryotic lineage. In contrast, prokaryotic enzymes become more versatile by catalyzing multiple reactions with similar chemical operations. Moreover, different metabolic pathways are enriched with distinct network evolution mechanisms. For instance, although the pathways of steroid biosynthesis, protein kinases, and glycosaminoglycan biosynthesis all constitute prominent features of animal-specific physiology, their evolution of domain architectures and catalytic functions follows distinct patterns. Steroid biosynthesis is enriched with reaction creations but retains a relatively conserved repertoire of domain compositions and proteins. Protein kinases retain conserved reactions but possess many novel domains and proteins. In contrast, glycosaminoglycan biosynthesis has high rates of reaction/protein creations and domain recruitments. Finally, we elicit and validate two general principles underlying the evolution of DPR networks: 1) duplicated enzyme proteins possess similar catalytic functions and 2) the majority of novel domains arise to catalyze novel reactions. These results shed new lights on the evolution of metabolic systems. PMID:22936075

  7. Estrogen Exposure, Metabolism, and Enzyme Variants in a Model for Breast Cancer Risk Prediction

    PubMed Central

    Parl, Fritz F.; Egan, Kathleen M.; Li, Chun; Crooke, Philip S.

    2009-01-01

    Estrogen is a well-known risk factor for breast cancer. Current models of breast cancer risk prediction are based on cumulative estrogen exposure but do not directly reflect mammary estrogen metabolism or address genetic variability between women in exposure to carcinogenic estrogen metabolites. We are proposing a mathematical model that forecasts breast cancer risk for a woman based on three factors: (1) estimated estrogen exposure, (2) kinetic analysis of the oxidative estrogen metabolism pathway in the breast, and (3) enzyme genotypes responsible for inherited differences in the production of carcinogenic metabolites. The model incorporates the main components of mammary estrogen metabolism, i.e. the conversion of 17?-estradiol (E2) by the phase I and II enzymes cytochrome P450 (CYP) 1A1 and 1B1, catechol-O-methyltransferase (COMT), and glutathione S-transferase P1 (GSTP1) into reactive metabolites, including catechol estrogens and estrogen quinones, such as E2-3,4-Q which can damage DNA. Each of the four genes is genotyped and the SNP data used to derive the haplotype configuration for each subject. The model then utilizes the kinetic and genotypic data to calculate the amount of E2-3,4-Q carcinogen as ultimate risk factor for each woman. The proposed model extends existing models by combining the traditional “phenotypic” measures of estrogen exposure with genotypic data associated with the metabolic fate of E2 as determined by critical phase I and II enzymes. Instead of providing a general risk estimate our model would predict the risk for each individual woman based on her age, reproductive experiences as well as her genotypic profile. PMID:19718449

  8. 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

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

    SciTech Connect

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

    2012-01-20

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

  10. 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

  11. 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. PMID:25644215

  12. 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

  13. Effect of acute and repeated chlordimeform treatment on rat hepatic microsomal drug metabolizing enzymes.

    PubMed

    Budris, D M; Yim, G K; Carlson, G P; Schnell, R C

    1983-08-01

    The effect of chlordimeform (CDM) treatment on the hepatic microsomal drug metabolizing enzymes was examined in male and female rats following either acute or repeated treatment. After acute administration of chlordimeform (100 mg/kg, i.p., 1 hour before killing) differential effects were observed in various parameters of the hepatic microsomal mixed function oxidase system with significant decreases in ethylmorphine metabolism, cytochrome P-450 content, NADPH cytochrome c reductase, and in the spectral binding of hexobarbital and aniline while no changes were found in the metabolism of aniline or p-nitroanisole. Durations of zoxazolamine-induced paralysis and pentobarbital-induced hypnosis were increased significantly after acute CDM administration. Following repeated administration of CDM (75 mg/kg, i.p., for 4 days) to adult male rats, a decrease was observed in zoxazolamine-induced paralysis time while pentobarbital-induced hypnosis was not altered. Metabolism studies using isolated hepatic microsomal fractions showed a decreased rate of biotransformation of ethylmorphine and aniline while the activity of p-nitroanisole O-demethylase was not changed. No differences were found in cytochrome P-450 levels whereas microsomal spectral binding of hexobarbital was reduced while that of aniline was not affected. Following acute or repeated administration of CDM to adult female rats, decreases in the hepatic microsomal metabolism of aniline, but not ethylmorphine or p-nitroanisole, were observed. Addition of CDM to microsomal suspensions yielded a Type I binding curve. PMID:6623550

  14. Flux Analysis Uncovers Key Role of Functional Redundancy in Formaldehyde Metabolism

    PubMed Central

    2005-01-01

    Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and 14C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop. PMID:15660163

  15. In silico study of binding motifs in squalene synthase enzyme of secondary metabolic pathway of solanaceae [corrected].

    PubMed

    Sanchita; Singh, Garima; Sharma, Ashok

    2014-11-01

    Solanaceae is an important family with several plants of medicinal importance. These medicinal plants have distinctive pathways for secondary metabolite biosynthesis. In most of the plants, two important compounds, dimethylallyl diphosphate and isopentenyl diphosphate, synthesize isoprenoid or terpenoids. Squalene synthase (SQS) is a key enzyme of the biosynthesis of isoprenoid (farnesyl pyrophosphate (FPP) ? squalene). Withania somnifera (ashwagandha), an important medicinal plant of family solanaceae produces withanolides. Withanolides are secondary metabolites synthesized through isoprenoid pathway. In this study, 13 SQS protein sequences from the plants of solanacae family and Arabidopsis thaliana were analyzed. The conserved domains in corresponding sequences were searched. The multiple sequence alignment of conserved domains revealed the important motifs and identified the residue substitution in each motif. Our result further indicated that residue substitution in motifs might not lead to functional variation, although it may affect the binding affinity of Mg(++), FPP and NAD(P)H. In addition, the homology modelling of SQS enzyme of W. somnifera was done for the prediction of three-dimensional structure. Molecular docking study of considered substrates with WsSQS was performed and the docked structure were analyzed further. The docked structures showed binding affinity for motif 2 of WsSQS. Our analysis revealed that 29 residues of motif 2 might be important for catalytic/functional activity of SQS enzyme of W. somnifera. This study may provide an understanding of metabolic pathways responsible for the production of secondary metabolites. The motifs may play a key role in regulating the pathway towards enhanced production of metabolites. PMID:25106523

  16. 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

  17. Metabolism of halogenated alkanes by cytochrome P450 enzymes. Aerobic oxidation versus anaerobic reduction.

    PubMed

    Ji, Li; Zhang, Jing; Liu, Weiping; de Visser, Sam P

    2014-04-01

    The cytochromes P450 are a large class of heme-containing enzymes that catalyze a broad range of chemical reactions in biosystems, mainly through oxygen-atom transfer to substrates. A relatively unknown reaction catalyzed by the P450s, but very important for human health, is the activation of halogenated substrates, which may lead to toxicity problems. However, its catalytic mechanism is currently unknown and, therefore, we performed a detailed computational study. To gain insight into the metabolism of halogenated compounds by P450 enzymes, we have investigated the oxidative and reductive P450-mediated activation of tetra- and trichloromethane as halogenated models with density functional theory (DFT) methods. We propose an oxidative halosylation mechanism for CCl4 under aerobic conditions by Compound?I of P450, which follows the typical Groves-type rebound mechanism. By contrast, the metabolism of CHCl3 occurs preferentially via an initial hydrogen-atom abstraction rather than halosylation. Kinetic isotope effect studies should, therefore, be able to distinguish the mechanistic pathways of CCl4 versus CHCl3 . We find a novel mechanism that is different from the well accepted P450 substrate activation mechanisms reported previously. Moreover, the studies highlight the substrate specific activation pathways by P450 enzymes leading to different products. These reactivity differences are rationalized using Marcus theory equations, which reproduce experimental product distributions. PMID:24501011

  18. Flubendazole metabolism and biotransformation enzymes activities in healthy sheep and sheep with haemonchosis.

    PubMed

    Bártíková, H; Krízová, V; Lamka, J; Kubícek, V; Skálová, L; Szotáková, B

    2010-02-01

    The aim of this project was to study the influence of haemonchosis, a common parasitic infection of small ruminants caused by Haemonchus contortus, on the activity of biotransformation enzymes and on in vitro flubendazole (FLU) biotransformation in liver and small intestine of lambs (Ovis aries). Twelve lambs were divided into three groups: non-infected animals, animals orally infected with larvae of H. contortus ISE strain for 7 weeks and for 11 weeks. At the end of the experiment, hepatic and intestinal subcellular fractions were prepared and used for assays of biotransformation enzymes activities and FLU metabolism testing. The activities of hepatic cytochromes P450, flavine monooxygenases and carbonyl-reducing enzymes were decreased in infected animals. UDP-glucuronosyl transferase activity was significantly lower (by 35%) in 11 weeks infected animals than that in control animals. When in vitro metabolism of FLU was compared in control and infected animals, significantly lower velocity of FLU reduction was found in infected animals. Slower FLU reduction may be beneficial for the haemonchosis treatment using FLU, because FLU will remain longer in the organism and could cause longer contact of parasites with FLU. PMID:20444026

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

    PubMed

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

    2014-01-01

    The present study aimed to investigate in vitro biological activities of extract of Eugenia punicifolia leaves (EEP), emphasizing the inhibitory activity of enzymes related to metabolic syndrome and its antioxidant effects. The antioxidant activity was analyzed by free radicals scavengers in vitro assays: DPPH·, ABTS(·+), O2(·?), and NO· and a cell-based assay. EEP were tested in inhibitory colorimetric assays using ?-amylase, ?-glucosidase, xanthine oxidase, and pancreatic lipase enzymes. The EEP exhibited activity in ABTS(·+), DPPH·, and O2(·?) scavenger (IC50 = 10.5 ± 1.2, 28.84 ± 0.54, and 38.12 ± 2.6 ?g/mL), respectively. EEP did not show cytotoxic effects, and it showed antioxidant activity in cells in a concentration-dependent manner. EEP exhibited inhibition of ?-amylase, ?-glucosidase, and xanthine oxidase activities in vitro assays (IC50 = 122.8 ± 6.3; 2.9 ± 0.1; 23.5 ± 2.6), respectively; however, EEP did not inhibit the lipase activity. The findings supported that extract of E. punicifolia leaves is a natural antioxidant and inhibitor of enzymes, such as ?-amylase, ?-glucosidase, and xanthine oxidase, which can result in a reduction in the carbohydrate absorption rate and decrease of risks factors of cardiovascular disease, thereby providing a novel dietary opportunity for the prevention of metabolic syndrome. PMID:24078187

  20. 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.

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

    PubMed

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

    2014-11-19

    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

  2. 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

  3. Spatial modulation of key pathway enzymes by DNA-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by Bacillus subtilis.

    PubMed

    Liu, Yanfeng; Zhu, Yanqiu; Ma, Wenlong; Shin, Hyun-dong; Li, Jianghua; Liu, Long; Du, Guocheng; Chen, Jian

    2014-07-01

    Previously we constructed a Bacillus subtilis strain for efficient production of N-acetylglucosamine (GlcNAc) by engineering of GlcNAc synthetic and catabolic pathways. However, the further improvement of GlcNAc titer is limited by the intrinsic inefficiency of GlcNAc synthetic pathway and undesirable cellular properties including sporulation and high maintenance metabolism. In this work, we further improved GlcNAc titer through spatial modulation of key pathway enzymes and by blocking sporulation and decreasing maintenance metabolism. Specifically, a DNA-guided scaffold system was firstly used to modulate the activities of glucosamine-6-phosphate synthase and GlcNAc-6-phosphate N-acetyltransferase, increasing the GlcNAc titer from 1.83g/L to 4.55g/L in a shake flask. Next, sporulation was blocked by respectively deleting spo0A (gene encoding the initiation regulon of sporulation) and sigE (gene encoding RNA polymerase sporulation-specific sigma factor). Deletion of sigE more effectively blocked sporulation without altering cell growth or GlcNAc production. The respiration chain was then engineered to decrease the maintenance metabolism of recombinant B. subtilis by deleting cydB and cydC, genes encoding cytochrome bd ubiquinol oxidase (subunit II) and ATP-binding protein for the expression of cytochrome bd, respectively. The respiration-engineered B. subtilis produced 6.15g/L GlcNAc in a shake flask and 20.58g/L GlcNAc in a 3-L fed-batch bioreactor. To the best of our knowledge, this report is the first to describe the modulation of pathway enzymes via a DNA-guided scaffold system in B. subtilis. The combination of spatial modulation of key pathway enzymes and optimization of cellular properties may be used to develop B. subtilis as a well-organized cell factory for the production of the other industrially useful chemicals. PMID:24815549

  4. 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.

  5. Effects of sexually dimorphic growth hormone secretory patterns on arachidonic acid metabolizing enzymes in rodent heart.

    PubMed

    Zhang, Furong; Yu, Xuming; He, Chunyan; Ouyang, Xiufang; Wu, Jinhua; Li, Jie; Zhang, Junjie; Duan, Xuejiao; Wan, Yu; Yue, Jiang

    2015-12-15

    The arachidonic acid (AA) metabolizing enzymes are the potential therapeutic targets of cardiovascular diseases (CVDs). As sex differences have been shown in the risk and outcome of CVDs, we investigated the regulation of heart AA metabolizing enzymes (COXs, LOXs, and CYPs) by sex-dependent growth hormone (GH) secretory patterns. The pulsatile (masculine) GH secretion at a physiological concentration decreased CYP1A1 and CYP2J3 mRNA levels more efficiently in the H9c2 cells compared with the constant (feminine) GH secretion; however, CYP1B1 mRNA levels were higher following the pulsatile GH secretion. Sex differences in CYP1A1, CYP1B1, and CYP2J11 mRNA levels were observed in both the wild-type and GHR deficient mice. No sex differences in the mRNA levels of COXs, LOXs, or CYP2E1 were observed in the wild-type mice. The constant GH infusion induced heart CYP1A1 and CYP2J11, and decreased CYP1B1 in the male C57/B6 mice constantly infused with GH (0.4?g/h, 7days). The activity of rat Cyp2j3 promoter was inhibited by the STAT5B protein, but was activated by C/EBP? (CEBPA). Compared with the constant GH administration, the levels of the nuclear phosphorylated STAT5B protein and its binding to the rat Cyp2j3 promoter were higher following the pulsatile GH administration. The constant GH infusion decreased the binding of the nuclear phosphorylated STAT5B protein to the mouse Cyp2j11 promoter. The data suggest the sexually dimorphic transcription of heart AA metabolizing enzymes, which might alter the risk and outcome of CVDs. GHR-STAT5B signal transduction pathway may be involved in the sex difference in heart CYP2J levels. PMID:26493931

  6. 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.

  7. 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.

  8. The effects of benzopyrene and safrole on biphenyl 2-hydroxylase and other drug-metabolizing enzymes.

    PubMed Central

    McPherson, F J; Bridges, J W; Parke, D V

    1976-01-01

    A study was made of the nature and specificity of the increase in biphenyl 2-hydroxylase activity after preincubation of liver microsomal preparations with various carcinogens in vitro. This enhancement of enzyme activity in vitro was investigated in mouse, hamster and rat, and although the rat appears to be atypical in the variation of the pattern of 2- and 4-hydroxylation with age, similar enhancements were detectable in each species examined. An increase in biphenyl 2-hydroxylase activity was apparent 2h after intraperitoneal administration of safrole or benzopyrene to mature Wistar albino rats and appeared to be similar in nature to that observed after preincubation of liver microsomal preparations with the same chemical in vitro. Investigation of other drug-metabolizing enzyme systems suggests that the enhancing effects of carcinogens in vitro are specific for biphenyl 2-hydroxylase. No correlation between the enhancement of biphenyl 2-hydroxylase and inhibtion of biphenyl 4-hydroxylase was apparent. PMID:821473

  9. Effect of Schistosoma mansoni infection and treatment on drug metabolizing enzymes.

    PubMed

    Ebeid, F A; Seif el-Din, S H; Ezzat, A R

    2000-09-01

    The effect of Schistosoma mansoni infection on drug-metabolizing enzymes was investigated before and after treatment of S. mansoni-infected male albino mice with the antibilharzial drug praziquantel (CAS 55268-74-1). The drug was given in a total dose of 1000 mg/kg, and was administered at 500 mg/kg of body weight for two consecutive days each of 500 mg/kg of body weight. The drug was given 49 days after infection with 80 Egyptian strains of S. mansoni cercariae/mouse. The hepatic content of cytochrome P-450, cytochrome b-5 and NADPH cytochrome P-450 reductase were determined at 4, 8, 24, 72 h, one and two weeks after the second dose of treatment. The enzymes were determined in the microsomal fraction after homogenization and ultracentrifugation at 105,000 x g. The results indicate a marked decrease of most enzymes in the infected groups compared to normal controls. Two weeks after treatment there was an improvement of the level of most enzymes towards the normal values. The levels of liver function tests were also improved. Concerning the oogram pattern, there was a rapid change even after 8 h after the second dose of treatment. It was concluded that the antibilharzial drug could be considered as a safe drug with a rapid onset of action. PMID:11050708

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

    PubMed

    Barker, Brandon E; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R; Xi, Hongwei; Locasale, Jason W; Gu, Zhenglong

    2015-12-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with high-throughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability and improve our understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present an algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation, the ability to handle large enzyme complex rules that may incorporate multiple isoforms, and either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB. PMID:26381164

  11. 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 diverse activities in terms of inducing various xenobiotic metabolizing enzymes based on their chemical structure.

  12. Ontogeny of hepatic enzymes involved in serine- and folate-dependent one-carbon metabolism in rabbits.

    PubMed

    Thompson, H R; Jones, G M; Narkewicz, M R

    2001-05-01

    Serine occupies a central position in folate-dependent, one-carbon metabolism through 5,10-methylenetetrahydrofolate (MTHF) and 5-formyltetrahydrofolate (FTHF). We characterized the ontogeny of the specific activity of key enzymes involved in serine, 5,10-MTHF, and 5-FTHF metabolism: methenyltetrahydrofolate synthetase (MTHFS), MTHF reductase (MTHFR), the glycine cleavage system (GCS), methionine synthase (MS), and serine hydroxymethyltransferase (SHMT) in rabbit liver, placenta, brain, and kidney. In liver, MTHFS activity is low in the fetus (0.36 +/- 0.07 nmol. min(-1). mg protein(-1)), peaks at 3 wk (1.48 +/- 0.50 nmol. min(-1). mg protein(-1)), and then decreases to adult levels (1.13 +/- 0.32 nmol. min(-1). mg protein(-1)). MTHFR activity is highest early in gestation (24.9 +/- 2.4 nmol. h(-1). mg protein(-1)) and declines rapidly by birth (4.7 +/- 1.3 nmol. h(-1). mg protein(-1)). MS is highest during fetal life and declines after birth. Cytosolic SHMT activity does not vary during development, but mitochondrial SHMT peaks at 23 days. GCS activity is high in the fetus and the neonate, declining after weaning. In placenta and brain, all activities are low throughout gestation. Cytosolic and mitochondrial SHMT activities are low in kidney and rise after weaning, whereas MTHFS is low throughout development. These data suggest that the liver is the primary site of activity for these enzymes. Throughout development, there are multiple potential sources for production of 5,10-MTHF, but early in gestation high MTHFR activity and low MTHFS activity could reduce 5,10-MTHF availability. PMID:11292595

  13. Intron features of key functional genes mediating nitrogen metabolism in marine phytoplankton

    E-print Network

    Ward, Bess

    Intron features of key functional genes mediating nitrogen metabolism in marine phytoplankton on the evolutionary history of genes and organisms. The number and frequency of introns vary widely in the small in sequences of functional genes of phytoplankton, both in published genomes and in sequences obtained from

  14. Identification of metabolic pathways and enzyme systems involved in the in vitro human hepatic metabolism of dronedarone, a potent new oral antiarrhythmic drug

    PubMed Central

    Klieber, Sylvie; Arabeyre-Fabre, Catherine; Moliner, Patricia; Marti, Eric; Mandray, Martine; Ngo, Robert; Ollier, Céline; Brun, Priscilla; Fabre, Gérard

    2014-01-01

    The in vitro metabolism of dronedarone and its major metabolites has been studied in human liver microsomes and cryopreserved hepatocytes in primary culture through the use of specific or total cytochrome P450 (CYP) and monoamine oxidase (MAO) inhibitors. The identification of the main metabolites and enzymes participating in their metabolism was also elucidated by using rhCYP, rhMAO, flavin monooxygenases (rhFMO) and UDP-glucuronosyltransferases (rhUGT) and liquid chromatography/tandem mass spectrometry (LC/MS-MS) analysis. Dronedarone was extensively metabolized in human hepatocytes with a metabolic clearance being almost completely inhibited (98 ± 2%) by 1-aminobenzotriazole. Ketoconazole also inhibited dronedarone metabolism by 89 ± 7%, demonstrating the crucial role of CYP3A in its metabolism. CYP3A isoforms mostly contributed to N-debutylation while hydroxylation on the butyl-benzofuran moiety was catalyzed by CYP2D6. However, hydroxylation on the dibutylamine moiety did not appear to be CYP-dependent. N-debutyl-dronedarone was less rapidly metabolized than dronedarone, the major metabolic pathway being catalyzed by MAO-A to form propanoic acid-dronedarone and phenol-dronedarone. Propanoic acid-dronedarone was metabolized at a similar rate to that of N-debutyl-dronedarone and was predominantly hydroxylated by CYP2C8 and CYP1A1. Phenol-dronedarone was extensively glucuronidated while C-dealkyl-dronedarone was metabolized at a slow rate. The evaluation of the systemic clearance of each metabolic process together with the identification of both the major metabolites and predominant enzyme systems and isoforms involved in the formation and subsequent metabolism of these metabolites has enhanced the overall understanding of metabolism of dronedarone in humans. PMID:25505590

  15. Normal liver enzymes are correlated with severity of metabolic syndrome in a large population based cohort

    PubMed Central

    Kälsch, Julia; Bechmann, Lars P.; Heider, Dominik; Best, Jan; Manka, Paul; Kälsch, Hagen; Sowa, Jan-Peter; Moebus, Susanne; Slomiany, Uta; Jöckel, Karl-Heinz; Erbel, Raimund; Gerken, Guido; Canbay, Ali

    2015-01-01

    Key features of the metabolic syndrome are insulin resistance and diabetes. The liver as central metabolic organ is not only affected by the metabolic syndrome as non-alcoholic fatty liver disease (NAFLD), but may contribute to insulin resistance and metabolic alterations. We aimed to identify potential associations between liver injury markers and diabetes in the population-based Heinz Nixdorf RECALL Study. Demographic and laboratory data were analyzed in participants (n?=?4814, age 45 to 75y). ALT and AST values were significantly higher in males than in females. Mean BMI was 27.9?kg/m2 and type-2-diabetes (known and unkown) was present in 656 participants (13.7%). Adiponectin and vitamin D both correlated inversely with BMI. ALT, AST, and GGT correlated with BMI, CRP and HbA1c and inversely correlated with adiponectin levels. Logistic regression models using HbA1c and adiponectin or HbA1c and BMI were able to predict diabetes with high accuracy. Transaminase levels within normal ranges were closely associated with the BMI and diabetes risk. Transaminase levels and adiponectin were inversely associated. Re-assessment of current normal range limits should be considered, to provide a more exact indicator for chronic metabolic liver injury, in particular to reflect the situation in diabetic or obese individuals. PMID:26269425

  16. Regulation of Key Enzymes of Sucrose Biosynthesis in Soybean Leaves 1

    PubMed Central

    Cheikh, Nordine; Brenner, Mark L.

    1992-01-01

    An important part in the understanding of the regulation of carbon partitioning within the leaf is to investigate the endogenous variations of parameters related to carbon metabolism. This study of diurnal changes in the activities of sucrose-synthesizing enzymes and levels of nonstructural carbohydrates in intact leaves of field-grown soybean plants (Glycine max [L.]) showed pronounced diurnal fluctuations in sucrose phosphate synthase (SPS) activity. However, there was no distinct diurnal change in the activity of fructose-1,6-bisphosphatase (F1,6BPase). SPS activity in leaves from plants grown in controlled environments presented two peaks during the light period. In contrast to field-grown plants, F1,6BPase activity in leaves from growth chamber-grown plants manifested one peak during the first half of the light period. In plants grown under both conditions, sucrose and starch accumulation rates were highest during early hours of the light period. By the end of the dark period, most of the starch was depleted. A pattern of diurnal fluctuations of abscisic acid (ABA) levels in leaves was also observed under all growing conditions. Either imposition of water stress or exogenous applications of ABA inhibited F1,6BPase activity. However, SPS-extractable activity increased following water deficit but did not change in response to ABA treatment. Gibberellin application to intact soybean leaves increased levels of both starch and sucrose. Both gibberellic acid (10?6m) and gibberellins 4 and 7 (10?5m) increased the activity of SPS but had an inconsistent effect on F1,6BPase. Correlation studies between the activities of SPS and F1,6BPase suggest that these two enzymes are coordinated in their function, but the factors that regulate them may be distinct because they respond differently to certain environmental and physiological changes. PMID:16653110

  17. Chromium Picolinate Does Not Improve Key Features of Metabolic Syndrome in Obese Nondiabetic Adults

    PubMed Central

    Iqbal, Nayyar; Cardillo, Serena; Volger, Sheri; Bloedon, LeAnne T.; Anderson, Richard A.; Boston, Raymond

    2009-01-01

    Abstract Background The use of chromium-containing dietary supplements is widespread among patients with type 2 diabetes. Chromium's effects in patients at high risk for developing diabetes, especially those with metabolic syndrome, is unknown. The objective of this study was to determine the effects of chromium picolinate (CrPic) on glucose metabolism in patients with metabolic syndrome. Method A double-blind, placebo-controlled, randomized trial was conducted at a U.S. academic medical center. Sixty three patients with National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III)-defined metabolic syndrome were included. The primary end point was a change in the insulin sensitivity index derived from a frequently sampled intravenous glucose tolerance test. Prespecified secondary end points included changes in other measurements of glucose metabolism, oxidative stress, fasting serum lipids, and high sensitivity C-reactive protein. Results After 16 weeks of CrPic treatment, there was no significant change in insulin sensitivity index between groups (P?=?0.14). However, CrPic increased acute insulin response to glucose (P?=?0.02). CrPic had no significant effect on other measures of glucose metabolism, body weight, serum lipids, or measures of inflammation and oxidative stress. Conclusion CrPic at 1000 ?g/day does not improve key features of the metabolic syndrome in obese nondiabetic patients. PMID:19422140

  18. 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

  19. Dose-related effect of aflatoxin B1 on liver drug metabolizing enzymes in rabbit.

    PubMed

    Guerre, P; Eeckhoutte, C; Larrieu, G; Burgat, V; Galtier, P

    1996-04-15

    The effects of chronic administration of aflatoxin B1 (AFB1) on liver drug metabolism enzymes were measured in New Zealand rabbits divided into three groups of 5 animals, each receiving over 5 days either arabic gum or AFB1 in arabic gum at a daily oral dose of 0.05 or 0.10 mg/kg. These treatments did not lead to any lethality in any of the treated groups, but the body weight gain was altered. Biochemical exploration of plasma components revealed a dose-dependent hepatotoxicity characterized by cytolysis and cholestasis. At 0.10 mg/kd/day of AFB1, significant decreases were observed in total liver microsomal cytochrome P450, several P450-dependent monooxygenase activities, all individual P450 isoenzymes levels analysed by Western-blotting and glutathione S-transferase activities. By contrast, at 0.05 mg/kg/day of AFB1, even though total cytochrome P450 was decreased by 30%, only P450 1A1 and 3A6 isoenzymes, and aniline hydroxylation, pentoxyresorufin O-depentylation, aminopyrine, erythromycin, ethylmorphine and dimethylnitrosamine N-demethylations were affected. In the same animal group, the only glutathione S-transferase accepting CDNB (1-chloro-2,4-dinitrobenzene) as substrate was decreased by 22%. UDP-glucuronyltransferase accepting p-nitrophenol as substrate was increased in both groups of animals (33-62%). The mechanisms that could contribute to the observed changes in drug metabolizing enzymes are discussed. PMID:8644116

  20. 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

  1. Metabolism of Oral Turinabol by Human Steroid Hormone-Synthesizing Cytochrome P450 Enzymes.

    PubMed

    Schiffer, Lina; Brixius-Anderko, Simone; Hannemann, Frank; Zapp, Josef; Neunzig, Jens; Thevis, Mario; Bernhardt, Rita

    2016-02-01

    The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco- and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17?-hydroxy-17?-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 µM and 5.4 µM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min(-1) mM(-1) for CYP11A1, 741 min(-1) mM(-1) for CYP11B1, and 3338 min(-1) mM(-1) for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli-based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11?-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11?,18-diOH-OT and 11?-OH-OT-18-al, which rearranges to its tautomeric form 11?,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography-tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies. PMID:26658226

  2. Thermoanaerobacter spp. control ethanol pathway via transcriptional regulation and versatility of key enzymes.

    PubMed

    Pei, Jianjun; Zhou, Qing; Jiang, Yu; Le, Yilin; Li, Huazhong; Shao, Weilan; Wiegel, Juergen

    2010-09-01

    Ethanologenic Thermoanaerobacter species produce ethanol from lignocellulose derived substrates at temperatures above 70 degrees C. In the final steps of ethanol formation, two bifunctional acetaldehyde/alcohol dehydrogenases, AdhB and AdhE, and an alcohol dehydrogenase, AdhA, catalyze redox reactions between acetyl-CoA and ethanol via an acetaldehyde intermediate. DNA cloning and analysis revealed that the dehydrogenase genes and their transcriptional regulatory regions were highly conserved in these species. As determined by real-time PCR, the transcription of adhE was activated by ethanol, while adhB was transcribed without ethanol; however, all of their transcription was reduced at higher ethanol concentrations. Under imitating physiological conditions, AdhE played a crucial role in ethanol formation, and AdhB favored ethanol consumption when ethanol concentration was high e.g. 1%. Thus, the ethanol titer of fermentation is controlled via transcriptional regulation and the properties of specific enzymes in Thermoanaerobacter. These results provide evidence for an ethanol balance model and offer the possibility to raise the ethanol titer by metabolic engineering. PMID:20547239

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

    PubMed

    Štambergová, Hana; Zemanová, Lucie; Lundová, Tereza; Mal?eková, 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. PMID:26466768

  4. 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 glycogen content and turnover in these structures during glucose sufficiency and shortage. PMID:25701713

  5. 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

  6. 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

  7. 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 ?-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

  8. 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

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

    2015-12-01

    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

  9. Mesaconyl-Coenzyme A Hydratase, a New Enzyme of Two Central Carbon Metabolic Pathways in Bacteria?

    PubMed Central

    Zarzycki, Jan; Schlichting, Ansgar; Strychalsky, Nina; Müller, Michael; Alber, Birgit E.; Fuchs, Georg

    2008-01-01

    The coenzyme A (CoA)-activated C5-dicarboxylic acids mesaconyl-CoA and ?-methylmalyl-CoA play roles in two as yet not completely resolved central carbon metabolic pathways in bacteria. First, these compounds are intermediates in the 3-hydroxypropionate cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium. Second, mesaconyl-CoA and ?-methylmalyl-CoA are intermediates in the ethylmalonyl-CoA pathway for acetate assimilation in various bacteria, e.g., in Rhodobacter sphaeroides, Methylobacterium extorquens, and Streptomyces species. In both cases, mesaconyl-CoA hydratase was postulated to catalyze the interconversion of mesaconyl-CoA and ?-methylmalyl-CoA. The putative genes coding for this enzyme in C. aurantiacus and R. sphaeroides were cloned and heterologously expressed in Escherichia coli, and the proteins were purified and studied. The recombinant homodimeric 80-kDa proteins catalyzed the reversible dehydration of erythro-?-methylmalyl-CoA to mesaconyl-CoA with rates of 1,300 ?mol min?1 mg protein?1. Genes coding for similar enzymes with two (R)-enoyl-CoA hydratase domains are present in the genomes of Roseiflexus, Methylobacterium, Hyphomonas, Rhodospirillum, Xanthobacter, Caulobacter, Magnetospirillum, Jannaschia, Sagittula, Parvibaculum, Stappia, Oceanicola, Loktanella, Silicibacter, Roseobacter, Roseovarius, Dinoroseobacter, Sulfitobacter, Paracoccus, and Ralstonia species. A similar yet distinct class of enzymes containing only one hydratase domain was found in various other bacteria, such as Streptomyces species. The role of this widely distributed new enzyme is discussed. PMID:18065535

  10. Evaluation of the possible role of five enzymes in the metabolism of IBA in mustard aphid, Lipaphis erysimi (Kalt.).

    PubMed

    Rup, P J; Sohal, S K; Kaur, G; Pal, S

    2004-04-01

    The enzymatic activity of five enzymes viz. Glutathione S-transferases, Esterases, NADH dehydrogenase, NADH oxidase and Glutathione reductase were assessed under the influence of Indole butyric acid (IBA) (400 ppm) in the nymphs (48-52h old) of mustard aphid, Lipaphis erysimi fed on radish plants treated for 13, 25 and 37h. The activity of Glutathione S-transferases, Esterases and NADH dehydrogenase increased compared to that found in the control of the same age group of nymphs and it was concluded that these enzymes might be involved in the metabolism of IBA. The other two enzymes, NADH oxidase and Glutathione reductase showed no significant increase in their activity compared to that in the control of the same age group. It was hypothesized that the latter enzymes do not play any significant role in the metabolism of IBA. PMID:15529874

  11. Changes in nitrogen metabolism and antioxidant enzyme activities of maize tassel in black soils region of northeast China

    PubMed Central

    Xu, Hongwen; Lu, Yan; Xie, Zhiming; Song, Fengbin

    2014-01-01

    Two varieties of maize (Zea mays L.) grown in fields in black soils of northeast China were tested to study the dynamic changes of nitrogen metabolism and antioxidant enzyme activity in tassels of maize. Results showed that antioxidant enzyme activity in tassels of maize increased first and then decreased with the growing of maize, and reached peak value at shedding period. Pattern of proline was consistent with antioxidant enzyme activity, showing that osmotic adjustment could protect many enzymes, which are important for cell metabolism. Continuous reduction of soluble protein content along with the growing of maize was observed in the study, which indicated that quantitative material and energy were provided for pollen formation. Besides, another major cause was that a large proportion of nitrogen was used for the composition of structural protein. Nitrate nitrogen concentrations of tassels were more variable than ammonium nitrogen, which showed that nitrate nitrogen was the favored nitrogen source for maize. PMID:25324855

  12. The Subcellular Compartmentalization of Arginine Metabolizing Enzymes and Their Role in Endothelial Dysfunction

    PubMed Central

    Chen, Feng; Lucas, Rudolf; Fulton, David

    2013-01-01

    The endothelial production of nitric oxide (NO) mediates endothelium-dependent vasorelaxation and restrains vascular inflammation, smooth muscle cell proliferation, and platelet aggregation. Impaired production of NO is a hallmark of endothelial dysfunction and promotes the development of cardiovascular disease. In endothelial cells, NO is generated by endothelial nitric oxide synthase (eNOS) through the conversion of its substrate, l-arginine to l-citrulline. Reduced access to l-arginine has been proposed as a major mechanism underlying reduced eNOS activity and NO production in cardiovascular disease. The arginases (Arg1 and Arg2) metabolize l-arginine to generate l-ornithine and urea and increased expression of arginase has been proposed as a mechanism of reduced eNOS activity secondary to the depletion of l-arginine. Indeed, supplemental l-arginine and suppression of arginase activity has been shown to improve endothelium-dependent relaxation and ameliorate cardiovascular disease. However, this simple relationship is complicated by observations that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. Accordingly, the subcellular compartmentalization of intracellular l-arginine into poorly interchangeable pools has been proposed to allow for the local depletion of pools or pockets of l-arginine. In agreement with this, there is considerable evidence supporting the importance of the subcellular localization of l-arginine metabolizing enzymes. In endothelial cells in vitro and in vivo, eNOS is found in discrete intracellular locations and the capacity to generate NO is heavily influenced by its localization inside the cell. Arg1 and Arg2 also reside in different subcellular environments and are thought to differentially influence endothelial function. The plasma membrane solute transporter, CAT-1 and the arginine recycling enzyme, arginosuccinate lyase, co-localize with eNOS and facilitate NO release. Herein, we highlight the importance of the subcellular location of eNOS and arginine transporting and metabolizing enzymes to NO release and cardiovascular disease. PMID:23847624

  13. 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

  14. 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

  15. Gene Expression Variability in Human Hepatic Drug Metabolizing Enzymes and Transporters

    PubMed Central

    Yang, Lun; Price, Elvin T.; Chang, Ching-Wei; Li, Yan; Huang, Ying; Guo, Li-Wu; Guo, Yongli; Kaput, Jim; Shi, Leming; Ning, Baitang

    2013-01-01

    Interindividual variability in the expression of drug-metabolizing enzymes and transporters (DMETs) in human liver may contribute to interindividual differences in drug efficacy and adverse reactions. Published studies that analyzed variability in the expression of DMET genes were limited by sample sizes and the number of genes profiled. We systematically analyzed the expression of 374 DMETs from a microarray data set consisting of gene expression profiles derived from 427 human liver samples. The standard deviation of interindividual expression for DMET genes was much higher than that for non-DMET genes. The 20 DMET genes with the largest variability in the expression provided examples of the interindividual variation. Gene expression data were also analyzed using network analysis methods, which delineates the similarities of biological functionalities and regulation mechanisms for these highly variable DMET genes. Expression variability of human hepatic DMET genes may affect drug-gene interactions and disease susceptibility, with concomitant clinical implications. PMID:23637747

  16. 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.

  17. Non-Viral Gene Transfer as a Tool for Studying Transcription Regulation of Xenobiotic Metabolizing Enzymes

    PubMed Central

    Bonamassa, Barbara; Liu, Dexi

    2010-01-01

    Numerous xenobiotic metabolizing enzymes are regulated by nuclear receptors at transcriptional level. The challenge we currently face is to understand how a given nuclear receptor interacts with its xenobiotics, migrates into nucleus, binds to the xenobiotic response element of a target gene, and regulates transcription. Toward this end, new methods have been developed to introduce the nuclear receptor gene into appropriate cells and study its activity in activating reporter gene expression under the control of a promoter containing xenobiotic response elements. The goal of this review is to critically examine the gene transfer methods currently available. We concentrate on the gene transfer mechanism, advantages and limitations of each method when employed for nuclear receptor-mediated gene regulation studies. It is our hope that the information provided highlights the importance of gene transfer in studying the mechanisms by which our body eliminates the potentially harmful substances and maintains the homeostasis. PMID:20713102

  18. 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

  19. 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 (?-glucosidase, ?-galactosidase, ?-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. PMID:24908112

  20. Impact of a community-based diabetes self-management program on key metabolic parameters

    PubMed Central

    Johnson, Courtney; Ruisinger, Janelle F.; Bates, Jessica; Barnes., Brian J.

    2014-01-01

    Objective: Characterize the impact of a pharmacist-led diabetes self-management program on three key metabolic parameters: glycosylated hemoglobin (HbA1c), low-density lipoprotein cholesterol (LDL-C), and mean arterial blood pressure (MAP) among employee health program participants. Methods: A self-insured company in the Kansas City metropolitan area began offering a pharmacist-led diabetes self-management program to eligible company employees and their dependents in 2008. A retrospective pre-post analysis was conducted to determine if the program affected key metabolic parameters in participants by determining mean change after one year of participation. Results: Among 183 program participants, 65 participants met inclusion criteria. All three key metabolic parameters were significantly reduced from baseline to one year of program participation: HbA1c decreased from 8.1 % to 7.3% (p=0.007); LDL-C decreased from 108.3 mg/dL to 96.4 mg/dL (p=0.009); and MAP decreased from 96.1 to 92.3 mm Hg (p=0.005). Conclusions: The pharmacist-led diabetes self-management program demonstrated significant reductions in HbA1c, LDL-C, and MAP from baseline to one year of program participation. Improvements were statistically significant and clinically relevant for each parameter. Previous studies indicate these reductions may cause reduced overall healthcare costs. PMID:25580174

  1. Novel role of TLR4 in NAFLD development: Modulation of metabolic enzymes expression.

    PubMed

    Ferreira, Darkiane Fernandes; Fiamoncini, Jarlei; Prist, Iryna Hirata; Ariga, Suely Kubo; de Souza, Heraldo Possolo; de Lima, Thais Martins

    2015-10-01

    The rise in the prevalence of obesity and metabolic syndrome turned NAFLD as the most common cause of chronic liver diseases worldwide. Although the role of toll like receptors, especially TLR4, as activators of inflammatory pathways in liver diseases is well established, our goal was to investigate if TLR4 activation could modulate metabolic lipid pathways and alter the onset of NAFLD. We used LDL receptor-deficient mice (LDLrKO) fed with an atherogenic diet as a model. The role of TLR4 activation was evaluated by crossing LDLrKO mice with the TLR4 knockout mice. Animals were fed for 12weeks with high-fat high-cholesterol diet (HFD) containing 18% saturated fat and 1.25% cholesterol. TLR4/LDLr KO mice presented lower triacylglyceride (TAG) plasma levels when compared to LDLrKO, despite the type of diet ingested. HFD induced TAG and cholesterol accumulation in the liver of all mice genotypes studied, but TLR4/LDLr KO presented lower TAG accumulation than LDLrKO mice. Gene expression of TAG synthesis enzymes (ApoB100, MTTP, GPAT1 and GPAT4) was not differentially altered in TLR4/LDLr KO and LDLrKO mice. On the other hand, TLR4 deficiency enhanced the expression of several enzymes involved in the oxidation of fatty acids, as follows: ACOX, CPT-1, MTPa, MTBb, PBE and 3-ketoacyl-CoA thiolase. Acyl-carnitine plasma profile showed an increase in C0 and C2 concentration in TLR4/LDLr KO group, corroborating the hypothesis of increased fat oxidation. Our results indicate that TLR4 may have an important role in the onset of steatosis, once its depletion enhances fatty acid oxidation in the liver of mice, preventing triglyceride accumulation. PMID:26172853

  2. 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. PMID:24399236

  3. YCF1-Mediated Cadmium Resistance in Yeast Is Dependent on Copper Metabolism and Antioxidant Enzymes

    PubMed Central

    Wei, Wenzhong; Smith, Nathan; Wu, Xiaobin; Kim, Heejeong; Seravalli, Javier; Khalimonchuk, Oleh

    2014-01-01

    Abstract Aims: Acquisition and detoxification of metal ions are vital biological processes. Given the requirement of metallochaperones in cellular copper distribution and metallation of cuproproteins, this study investigates whether the metallochaperones also deliver metal ions for transporters functioning in metal detoxification. Results: Resistance to excess cadmium and copper of the yeast Saccharomyces cerevisiae, which is conferred by PCA1 and CaCRP1 metal efflux P-type ATPases, respectively, does not rely on known metallochaperones, Atx1p, Ccs1p, and Cox17p. Copper deficiency induced by the expression of CaCRP1 encoding a copper exporter occurs in the absence of Atx1p. Intriguingly, CCS1 encoding the copper chaperone for superoxide dismutase 1 (Sod1p) is necessary for cadmium resistance that is mediated by Ycf1p, a vacuolar cadmium sequestration transporter. This is attributed to Ccs1p's role in the maturation of Sod1p rather than its direct interaction with Ycf1p for cadmium transfer. Functional defect in Ycf1p associated with the absence of Sod1p as well as another antioxidant enzyme Glr1p is rescued by anaerobic growth or substitutions of specific cysteine residues of Ycf1p to alanine or serine. This further supports oxidative inactivation of Ycf1p in the absence of Ccs1p, Sod1p, or Glr1p. Innovation: These results provide new insights into the mechanisms of metal metabolism, interaction among metal ions, and the roles for antioxidant systems in metal detoxification. Conclusion: Copper metabolism and antioxidant enzymes maintain the function of Ycf1p for cadmium defense. Antioxid. Redox Signal. 21, 1475–1489. PMID:24444374

  4. Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells.

    PubMed

    Ek, Monica; Söderdahl, Therese; Küppers-Munther, Barbara; Edsbagge, Josefina; Andersson, Tommy B; Björquist, Petter; Cotgreave, Ian; Jernström, Bengt; Ingelman-Sundberg, Magnus; Johansson, Inger

    2007-08-01

    Human embryonic stem cells (hESC) offer a potential unlimited source for functional human hepatocytes, since they can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing several hepatic markers. Such cells could be used for, e.g. studies of drug metabolism and hepatotoxicity, which however would require a significant expression of drug metabolising enzymes. Thus, we have investigated the expression of cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs), drug transporters, transcription factors and other liver specific genes in hepatocyte-like cells derived from hESC using a simple direct differentiation protocol. The mRNA and protein expression of several important CYPs were determined using low density arrays, real time PCR and Western blotting. Significant CYP expression on the mRNA level was detected in hepatocyte-like cells derived from one out of two different hESC lines tested, which was much higher than in undifferentiated hESC and generally higher than in HepG2 cells. CYP1A2, CYP3A4/7 and low levels of CYP1A1 and CYP2C8/9/19 protein were detected in both lines. The mRNAs for a variety of CYPs and liver specific factors were shown to be inducible in both cell lines, and this was reflected in induced levels of CYP1A2 and CYP3A4/7 protein. This first report on expression of all major CYPs in hepatocyte-like cells derived from hESC represents an important step towards functional hepatocytes, but efforts to further differentiate the cells using optimized protocols are needed before they exhibit similar levels of drug metabolizing enzymes as primary human hepatocytes and liver. PMID:17568565

  5. 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

  6. Involvement of glutathione and glutathione metabolizing enzymes in human colorectal cancer cell lines and tissues.

    PubMed

    Kim, Areum Daseul; Zhang, Rui; Han, Xia; Kang, Kyoung Ah; Piao, Mei Jing; Maeng, Young Hee; Chang, Weon Young; Hyun, Jin Won

    2015-09-01

    Reduced glutathione (GSH) is an abundant tripeptide present in the majority of cell types. GSH is highly reactive and is often conjugated to other molecules, via its sulfhydryl moiety. GSH is synthesized from glutamic acid, cysteine, and glycine via two sequential ATP?consuming steps, which are catalyzed by glutamate cysteine ligase (GCL) and GSH synthetase (GSS). However, the role of GSH in cancer remains to be elucidated. The present study aimed to determine the levels of GSH and GSH synthetic enzymes in human colorectal cancer. The mRNA and protein expression levels of GSH, the catalytic subunit of GCL (GCLC) and GSS were significantly higher in the following five colon cancer cell lines: Caco?2, SNU?407, SNU?1033, HCT?116, and HT?29, as compared with the normal colon cell line, FHC. Similarly, in 9 out of 15 patients with colon cancer, GSH expression levels were higher in tumor tissue, as compared with adjacent normal tissue. In addition, the protein expression levels of GCLC and GSS were higher in the tumor tissue of 8 out of 15, and 10 out of 15 patients with colon cancer respectively, as compared with adjacent normal tissue. Immunohistochemical analyses confirmed that GCLC and GSS were expressed at higher levels in colon cancer tissue, as compared with normal mucosa. Since GSH and GSH metabolizing enzymes are present at elevated levels in colonic tumors, they may serve as clinically useful biomarkers of colon cancer, and/or targets for anti-colon cancer drugs. PMID:26059756

  7. Effects of 4-tertiary butyl catechol on glutathione-metabolizing enzymes in vivo and in vitro.

    PubMed

    Kawashima, T; Yonemoto, K; Gellin, G A; Epstein, W L; Fukuyama, K

    1984-01-01

    4-Tertiary butyl catechol (TBC) causes depigmentation in humans and animals and stimulates formation of pheomelanosomes. In this study, we investigated the effects of noncytotoxic doses of TBC on glutathione S-transferase (GST) activity in the skin of Uscd strain mice and B16 murine melanoma cells in culture, in relation to changes in activities of glutathione reductase (GR) and gamma-glutamyl transpeptidase (GGT) reported to be involved in pheomelanogenesis. Occurrence of pheomelanosomes in skin melanocytes was demonstrated by electron microscopy and reduction (25%) of eumelanin content in melanoma cells was shown by spectrophotometry. Topical application of 1 M TBC-DMSO-acetone solution on the ear skin elevated GST activity about 27%, and activities of GGT and GR to 35% and 19%, respectively, within 1 week. Melanoma cells cultured in 10(-4) M TBC-containing medium for 2 h showed no changes in GST and GGT activities, but 12% increase of GR activity during the first 12 h. Activities of all 3 enzymes was elevated (11-17%) 24 h later. The elevation detected by 48 h was 25% for GST, 26% for GGT, and 14% for GR. The findings were interpreted to show that depigmentation produced by the antioxidant results from stimulated pheomelanogenesis through activation of glutathione-metabolizing enzymes and suppressed oxidation of eumelanin intermediates. PMID:6140289

  8. Polymorphisms in Genes of Tricarboxylic Acid Cycle Key Enzymes Are Associated with Early Recurrence of Hepatocellular Carcinoma

    PubMed Central

    Zhou, Xingchun; Chen, Yibing; An, Jiaze; Yu, Xiaohe; Zhang, Huiqing; Yang, Hushan; Xing, Jinliang

    2015-01-01

    Alterations of activity and expression in tricarboxylic acid (TCA) cycle key enzymes have been indicated in several malignancies, including hepatocellular carcinoma (HCC). They play an important role in the progression of cancer. However, the impact of single nucleotide polymorphisms (SNPs) in genes encoding these key enzymes on the recurrence of HCC has not been investigated. In this study, we genotyped 17 SNPs in genes encoding TCA cycle key enzymes and analyzed their association with recurrence-free survival (RFS) in a cohort of 492 Chinese HCC patients by Cox proportional hazard model and survival tree analysis. We identified 7 SNPs in SDHC, SDHD, FH, and IDH2 genes to be significantly associated with the RFS of HCC patients. Moreover, all these SNPs were associated with the early recurrence (within 2 years after surgery) risk of diseases. Cumulative effect analysis showed that these SNPs exhibited a dose-dependent effect on the overall and early recurrence. Further stratified analysis suggested that number of risk genotypes modified the protective effect on HCC recurrence conferred by transcatheter arterial chemoembolization treatment. Finally, the survival tree analysis revealed that SNP rs10789859 in SDHD gene was the primary factor contributing to HCC recurrence in our population. To the best of our knowledge, we for the first time observed the association between SNPs in genes encoding TCA cycle key enzymes and HCC recurrence risk. Further observational and functional studies are needed to validate our findings and generalize its clinical usage. PMID:25894340

  9. Effect of inhibitors of arachidonic acid metabolism on efflux of intracellular enzymes from skeletal muscle following experimental damage.

    PubMed

    Jackson, M J; Wagenmakers, A J; Edwards, R H

    1987-01-15

    The role of arachidonic acid metabolism in the efflux of intracellular enzymes from damaged skeletal muscle has been examined in vitro using inhibitors of cyclo-oxygenase and lipoxygenase enzymes. Damage to skeletal muscle induced by either calcium ionophore A23187 (25 microM) or dinitrophenol (1 mM) caused an increase in the efflux of prostaglandins E2 and F2 alpha together with a large efflux of intracellular creatine kinase. Use of a cyclo-oxygenase inhibitor completely prevented the efflux of prostaglandins, but had no effect on creatine kinase efflux. However, several agents having the ability to inhibit lipoxygenase enzymes dramatically reduced creatine kinase efflux following damage. These data suggest that a product or products of lipoxygenase enzymes may be mediators of the changes in plasma membrane integrity which permit efflux of intracellular enzymes as a consequence of skeletal muscle damage. PMID:3109374

  10. 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

  11. 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. PMID:23958860

  12. The effects of T-2 toxin exposure on liver drug metabolizing enzymes in rabbit.

    PubMed

    Guerre, P; Eeckhoutte, C; Burgat, V; Galtier, P

    2000-12-01

    High doses of T-2 toxin are known to decrease protein synthesis and mono-oxygenase activities in rat liver. The purpose of this study was to investigate whether exposure at a low dose could alter the normal metabolism of the xenobiotic by the liver. Three doses of T-2 toxin, dissolved in olive oil, were orally and daily administered to New Zealand white rabbits for five days. At 0.5 mg/kg, three of the five animals died, whereas only a weak decrease in body weight gain and moderate signs of toxicity occurred in rabbits receiving 0.25 mg/kg/day, and the body weight increased without signs of toxicity at 0.1 mg/kg/day. At 0.25 mg/kg/day, total liver microsomal P450 content, and the activities of aminopyrine and benzphetamine N-demethylases, pentoxyresorufin O-depentylase, glutathione S-transferases accepting 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, were decreased. By contrast, ethylmorphine and erythromycin N-demethylases, ethoxyresorufin and methoxyresorufin O-dealkylases, aniline hydroxylase, and UDP-glucuronyltransferase accepting p-nitrophenol as substrate, were unaffected. The expression of P450 1A1, 1A2, 2A1, and 2B4, but not P450 2C3 and 3A6, were also decreased, whereas microsomal conjugated dienes, fluorescent substances, and malondialdehyde contents were increased. At 0.1 mg/kg/day, neither significant effects on drug metabolizing enzymes nor microsomal oxidative damages were obtained. Taken together, these results suggest that a short exposure time to the mycotoxin would not be associated with significant changes in the normal metabolism of xenobiotics by the liver. PMID:11271836

  13. Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism.

    PubMed

    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

    2015-07-24

    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

  14. 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

  15. 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...

  16. 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

  17. Crystallization and preliminary X-ray analysis of Thermoactinomyces vulgaris R-47 maltooligosaccharide-metabolizing enzyme homologous to glucoamylase

    SciTech Connect

    Ichikawa, Kazuhiro; Tonozuka, Takashi; Mizuno, Masahiro; Tanabe, Yoshihiro; Kamitori, Shigehiro; Nishikawa, Atsushi; Sakano, Yoshiyuki

    2005-03-01

    A maltooligosaccharide-metabolizing enzyme from T. vulgaris R-47 (TGA) homologous to glucoamylase degrades maltooligosaccharides more efficiently than starch, unlike fungal glucoamylases. TGA was crystallized and the state of the protein in solution was analyzed by gel-filtration chromatography.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. 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...

  20. 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...

  1. 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...

  2. Expression level, cellular compartment and metabolic network position all influence the average selective constraint on mammalian enzymes

    PubMed Central

    2011-01-01

    Background A gene's position in regulatory, protein interaction or metabolic networks can be predictive of the strength of purifying selection acting on it, but these relationships are neither universal nor invariably strong. Following work in bacteria, fungi and invertebrate animals, we explore the relationship between selective constraint and metabolic function in mammals. Results We measure the association between selective constraint, estimated by the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions, and several, primarily metabolic, measures of gene function. We find significant differences between the selective constraints acting on enzyme-coding genes from different cellular compartments, with the nucleus showing higher constraint than genes from either the cytoplasm or the mitochondria. Among metabolic genes, the centrality of an enzyme in the metabolic network is significantly correlated with Ka/Ks. In contrast to yeasts, gene expression magnitude does not appear to be the primary predictor of selective constraint in these organisms. Conclusions Our results imply that the relationship between selective constraint and enzyme centrality is complex: the strength of selective constraint acting on mammalian genes is quite variable and does not appear to exclusively follow patterns seen in other organisms. PMID:21470417

  3. 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. PMID:25528545

  4. 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

  5. Short Communication: Expression of Transporters and Metabolizing Enzymes in the Female Lower Genital Tract: Implications for Microbicide Research

    PubMed Central

    Zhou, Tian; Hu, Minlu; Cost, Marilyn; Poloyac, Samuel

    2013-01-01

    Abstract Topical vaginal microbicides have been considered a promising option for preventing the male-to-female sexual transmission of HIV; however, clinical trials to date have not clearly demonstrated robust and reproducible effectiveness results. While multiple approaches may help enhance product effectiveness observed in clinical trials, increasing the drug exposure in lower genital tract tissues is a compelling option, given the difficulty in achieving sufficient drug exposure and positive correlation between tissue exposure and microbicide efficacy. Since many microbicide drug candidates are substrates of transporters and/or metabolizing enzymes, there is emerging interest in improving microbicide exposure and efficacy through local modulation of transporters and enzymes in the female lower genital tract. However, no systematic information on transporter/enzyme expression is available for ectocervical and vaginal tissues of premenopausal women, the genital sites most relevant to microbicide drug delivery. The current study utilized reverse transcriptase polymerase chain reaction (RT-PCR) to examine the mRNA expression profile of 22 transporters and 19 metabolizing enzymes in premenopausal normal human ectocervix and vagina. Efflux and uptake transporters important for antiretroviral drugs, such as P-gp, BCRP, OCT2, and ENT1, were found to be moderately or highly expressed in the lower genital tract as compared to liver. Among the metabolizing enzymes examined, most CYP isoforms were not detected while a number of UGTs such as UGT1A1 were highly expressed. Moderate to high expression of select transporters and enzymes was also observed in mouse cervix and vagina. The implications of this information on microbicide research is also discussed, including microbicide pharmacokinetics, the utilization of the mouse model in microbicide screening, as well as the in vivo functional studies of cervicovaginal transporters and enzymes. PMID:23607746

  6. Short communication: expression of transporters and metabolizing enzymes in the female lower genital tract: implications for microbicide research.

    PubMed

    Zhou, Tian; Hu, Minlu; Cost, Marilyn; Poloyac, Samuel; Rohan, Lisa

    2013-11-01

    Topical vaginal microbicides have been considered a promising option for preventing the male-to-female sexual transmission of HIV; however, clinical trials to date have not clearly demonstrated robust and reproducible effectiveness results. While multiple approaches may help enhance product effectiveness observed in clinical trials, increasing the drug exposure in lower genital tract tissues is a compelling option, given the difficulty in achieving sufficient drug exposure and positive correlation between tissue exposure and microbicide efficacy. Since many microbicide drug candidates are substrates of transporters and/or metabolizing enzymes, there is emerging interest in improving microbicide exposure and efficacy through local modulation of transporters and enzymes in the female lower genital tract. However, no systematic information on transporter/enzyme expression is available for ectocervical and vaginal tissues of premenopausal women, the genital sites most relevant to microbicide drug delivery. The current study utilized reverse transcriptase polymerase chain reaction (RT-PCR) to examine the mRNA expression profile of 22 transporters and 19 metabolizing enzymes in premenopausal normal human ectocervix and vagina. Efflux and uptake transporters important for antiretroviral drugs, such as P-gp, BCRP, OCT2, and ENT1, were found to be moderately or highly expressed in the lower genital tract as compared to liver. Among the metabolizing enzymes examined, most CYP isoforms were not detected while a number of UGTs such as UGT1A1 were highly expressed. Moderate to high expression of select transporters and enzymes was also observed in mouse cervix and vagina. The implications of this information on microbicide research is also discussed, including microbicide pharmacokinetics, the utilization of the mouse model in microbicide screening, as well as the in vivo functional studies of cervicovaginal transporters and enzymes. PMID:23607746

  7. 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 (?) 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.

  8. Structure and function of human cytochromes P450 enzymes: Xenobiotic metabolism by CYP2A and steroid biosynthesis by CYP17A1

    E-print Network

    DeVore, Natasha M.

    2011-05-31

    of xenobiotic compounds, including drugs. Several isoforms of cytochromes P450 of both types are targets for the prevention or treatment of cancers. The xenobiotic metabolizing enzyme CYP2A13 and the steroid synthesis enzyme CYP17A1 are two such enzymes...

  9. 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 presence or absence of latex particles or PMA. Superoxide and H/sub 2/O/sub 2/ production by neutrophils in the presence of latex particles or PMA were also stimulated by D/sub 2/O. In contrast, D/sub 2/O inhibited the ingestion of latex particles. D/sub 2/O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D/sub 2/O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D/sub 2/O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.

  10. Meat intake, heterocyclic amine exposure, and metabolizing enzyme polymorphisms in relation to colorectal polyp risk

    PubMed Central

    Shin, Aesun; Shrubsole, Martha J.; Rice, Jeffrey M.; Cai, Qiuyin; Doll, Mark A.; Long, Jirong; Smalley, Walter E.; Shyr, Yu; Sinha, Rashmi; Ness, Reid M.; Hein, David W.; Zheng, Wei

    2008-01-01

    Most colorectal cancers arise from adenomatous polyps or certain hyperplastic polyps. Only a few studies have investigated potential genetic modifiers of the associations between meat intake and polyp risk, and results are inconsistent. Using data from the Tennessee Colorectal Polyp Study (TCPS), a large colonoscopy-based study including 1,002 polyp cases (557 adenoma only, 250 hyperplastic polyp only, 195 both polyps) and 1,493 polyp-free patients, we evaluated the association of colorectal polyp risk with carcinogen exposure from meat and genetic polymorphisms in enzymes involved in heterocyclic amine (HCA) metabolism including, N-acetyltransferase 1 (NAT1) and 2 (NAT2), cytochrome P450 1A2 (CYP1A2), and aryl hydrocarbon receptor (AhR). Intake levels of meats by preparation methods, doneness preferences, and other lifestyle factors were obtained. Fourteen SNPs in the AhR, CYP1A2, NAT1 and NAT2 genes were evaluated. No clear association was found for any polymorphisms with polyp risk. However, apparent interactions were found for intake of meat and HCAs with AhR, NAT1, and NAT2 genotypes, and the interactions were statistically significant for the group with both adenomatous and hyperplastic polyps. Dose-response relationships with meat or HCA intake were found only among those with the AhR GA/AA (rs2066853) genotype, NAT1 rapid, or NAT2 rapid/intermediate acetylators, but not among those with other genotypes of these genes. This dose-response relationship was more evident among those with both AhR GA/AA and the NAT1 rapid acetylator than those without this genotype combination. These results provide strong evidence for a modifying effect of metabolizing genes on the association of meat intake and HCA exposure with colorectal polyp risk. PMID:18268115

  11. 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.

  12. [Effects of exogenous NO3- on cherry root function and enzyme activities related to nitrogen metabolism under hypoxia stress].

    PubMed

    Feng, Li-guo; Sheng, Li-xi; Shu, Huai-rui

    2010-12-01

    A water culture experiment with controlled dissolved oxygen concentration was conducted to explore the effects of exogenous NO3- on the root function and enzyme activities related to nitrogen metabolism of cherry (Prunun cerasus x P. canescens) seedlings under hypoxia stress. Comparing with the control (7.5 mmol NO3- x L(-1)), treatments 15 and 22.5 mmol NO3- x L(-1) made the materials for plant metabolism abundant, ensured the synthesis of enzyme proteins, increased root activity, maintained root respiration, improved the activities of enzymes related to nitrogen metabolism, such as nitrate reductase (NR), glutamine synthethase (GS), and glutamate dehydrogenase (NADH-GDH) in roots, and thereby, supplied enough energy for root respiration and NAD+ to glycolytic pathway, ensured electron transfer, and avoid ammonium toxicity under hypoxia stress. As a result, the injury of hypoxia stress to cherry plant was alleviated. Applying NO3- at the concentration of 22.5 mmol x L(-1) was more advisable. However, NO3- deficiency (0 mmol x L(-1)) showed opposite results. The above results suggested that applying exogenous NO3- to growth medium could regulate cherry root function and nitrogen metabolism, and antagonize the damage of hypoxia stress on cherry roots. PMID:21443020

  13. Metabolic Syndrome and Serum Liver Enzymes Level at Patients with Type 2 Diabetes Mellitus

    PubMed Central

    Music, Miralem; Dervisevic, Amela; Pepic, Esad; Lepara, Orhan; Fajkic, Almir; Ascic-Buturovic, Belma; Tuna, Enes

    2015-01-01

    Objectives: The aim of this study was to evaluate liver function in patients with type 2 diabetes mellitus (T2DM) with and without metabolic syndrome (MS) by determining serum levels of gamma glutamyltransferase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). We also investigated correlation between levels of liver enzymes and some components of MS in both groups of patients. Methods: This cross-sectional study included 96 patients (age 47–83 years) with T2DM. All patients were divided according to the criteria of the National Cholesterol Education Program (NCEP) in two groups: 50 patients with T2 DM and MS (T2DM-MS) and 46 patients with T2DM without MS (T2DM-Non MS). The analysis included blood pressure monitoring and laboratory tests: fasting blood glucose (FBG), total lipoprotein cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), fibrinogen and liver enzymes: GGT, ALT and AST. T2DM-MS group included patients which had FBG ? 6,1 mmol/L, TG ? 1,7 mmol/L and blood pressure ? 130/85 mm Hg. Results: T2DM-MS patients had significant higher values of systolic blood pressure, diastolic blood pressure and medium arterial pressure compared to T2DM-Non MS patients. Serum levels of TC, TG, LDL-C, VLDL-C and FBG were significantly higher in the T2DM-MS group compared to the T2DM-Non MS group. Serum fibrinogen level and GGT level were significantly higher in patients with T2DM-MS compared to the serum fibrinogen level and GGT level in T2DM-Non MS patients. Mean serum AST and ALT level were higher, but not significantly, in patients with T2DM and MS compared to the patients with T2DM without MS. Significant negative correlations were observed between TC and AST (r= -0,28, p<0,05), as well as between TC and ALT level (r= -0,29, p<0,05) in T2DM-MS group of patients. Conclusion: These results suggest that patients with T2DM and MS have markedly elevated liver enzymes. T2DM and MS probably play a role in increasing the risk of liver injury. PMID:26543313

  14. Stereoselective Formation and Metabolism of 4-Hydroxy-Retinoic Acid Enantiomers by Cytochrome P450 Enzymes*

    PubMed Central

    Shimshoni, Jakob A.; Roberts, Arthur G.; Scian, Michele; Topletz, Ariel R.; Blankert, Sean A.; Halpert, James R.; Nelson, Wendel L.; Isoherranen, Nina

    2012-01-01

    All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the ?-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids. PMID:23071109

  15. 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.

  16. Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering.

    PubMed

    Suflita, Matthew; Fu, Li; He, Wenqin; Koffas, Mattheos; Linhardt, Robert J

    2015-09-01

    Glycosaminoglycans are linear anionic polysaccharides that exhibit a number of important biological and pharmacological activities. The two most prominent members of this class of polysaccharides are heparin/heparan sulfate and the chondroitin sulfates (including dermatan sulfate). These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of most animal cells. The chemical synthesis of these glycosaminoglycans is precluded by their structural complexity. Today, we depend on food animal tissues for their isolation and commercial production. Ton quantities of these glycosaminoglycans are used annually as pharmaceuticals and nutraceuticals. The variability of animal-sourced glycosaminoglycans, their inherent impurities, the limited availability of source tissues, the poor control of these source materials, and their manufacturing processes suggest a need for new approaches for their production. Over the past decade, there have been major efforts in the biotechnological production of these glycosaminoglycans. This mini-review focuses on the use of recombinant enzymes and metabolic engineering for the production of heparin and chondroitin sulfates. PMID:26219501

  17. Protein Acetylation Microarray Reveals NuA4 Controls Key Metabolic Target Regulating Gluconeogenesis

    PubMed Central

    Lin, Yu-yi; Lu, Jin-ying; Zhang, Junmei; Walter, Wendy; Dang, Weiwei; Wan, Jun; Tao, Sheng-Ce; Qian, Jiang; Zhao, Yingming; Boeke, Jef D.; Berger, Shelley L.; Zhu, Heng

    2009-01-01

    SUMMARY Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys 19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys 514 was crucial for enzymatic activity and the ability of yeast cells to grow on non-fermentable carbon sources. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production was dependent on TIP60, the human homolog of ESA1. Our results demonstrate a novel regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme. PMID:19303850

  18. Effect of consumption of milk from goats fed Senecio jacobaea on hepatic drug metabolizing enzyme activities in rats.

    PubMed

    Miranda, C L; Cheeke, P R; Goeger, D E; Buhler, D R

    1981-08-01

    Milk from lactating goats fed tansy ragwort (Senecio jacobaea) was evaluated for its ability to influence hepatic drug metabolism. The milk after being freeze-dried was fed to male rats for 1 week ad lib. A significant reduction in the activities of hepatic aminopyrine N-demethylase and aryl hydrocarbon hydroxylase was obtained. There was no significant change in the activities of microsomal epoxide hydrolase and cytosolic glutathione S-transferase. The data suggest that consumption of milk from goats fed Senecio jacobaea produces a selective alteration of the activities of hepatic drug-metabolizing enzymes. PMID:7302964

  19. Maize Homologs of Hydroxycinnamoyltransferase, a Key Enzyme in Lignin Biosynthesis, Bind the Nucleotide Binding Leucine-Rich Repeat Rp1 Proteins to Modulate the Defense Response.

    PubMed

    Wang, Guan-Feng; He, Yijian; Strauch, Renee; Olukolu, Bode A; Nielsen, Dahlia; Li, Xu; Balint-Kurti, Peter J

    2015-11-01

    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

  20. Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism1[W][OPEN

    PubMed Central

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

    2014-01-01

    To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum ‘Moneymaker’) plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels. PMID:24474652

  1. Effect of Graded Nrf2 Activation on Phase-I and -II Drug Metabolizing Enzymes and Transporters in Mouse Liver

    PubMed Central

    Wu, Kai Connie; Cui, Julia Yue; Klaassen, Curtis D.

    2012-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes in response to oxidative/electrophilic stress. Kelch-like ECH associating protein 1 (Keap1) sequesters Nrf2 in the cytosol. The purpose of this study was to investigate the role of Nrf2 in regulating the mRNA of genes encoding drug metabolizing enzymes and xenobiotic transporters. Microarray analysis was performed in livers of Nrf2-null, wild-type, Keap1-knockdown mice with increased Nrf2 activation, and Keap1-hepatocyte knockout mice with maximum Nrf2 activation. In general, Nrf2 did not have a marked effect on uptake transporters, but the mRNAs of organic anion transporting polypeptide 1a1, sodium taurocholate cotransporting polypeptide, and organic anion transporter 2 were decreased with Nrf2 activation. The effect of Nrf2 on cytochrome P450 (Cyp) genes was minimal, with only Cyp2a5, Cyp2c50, Cyp2c54, and Cyp2g1 increased, and Cyp2u1 decreased with enhanced Nrf2 activation. However, Nrf2 increased mRNA of many other phase-I enzymes, such as aldo-keto reductases, carbonyl reductases, and aldehyde dehydrogenase 1. Many genes involved in phase-II drug metabolism were induced by Nrf2, including glutathione S-transferases, UDP- glucuronosyltransferases, and UDP-glucuronic acid synthesis enzymes. Efflux transporters, such as multidrug resistance-associated proteins, breast cancer resistant protein, as well as ATP-binding cassette g5 and g8 were induced by Nrf2. In conclusion, Nrf2 markedly alters hepatic mRNA of a large number of drug metabolizing enzymes and xenobiotic transporters, and thus Nrf2 plays a central role in xenobiotic metabolism and detoxification. PMID:22808024

  2. Cytochromes P450: Inhibition of CYP2A Enzymes Involved in Xenobiotic Metabolism and Generation of CYP26 Enzymes Involved in Retinoic Acid Metabolism

    E-print Network

    Stephens, Eva Susanne

    2012-08-31

    of retinoic acid (RA) catabolism in the body. In humans, the CYP26 family of enzymes consists of three isoforms: CYP26A1, CYP26B1, and CYP26C1. As a result of RA's endogenous role in regulating cellular growth and differentiation, geometric isomers of RA...

  3. Metabolism of monoterpenes: oxidation of isopiperitenol to isopiperitenone, and subsequent isomerization to piperitenone by soluble enzyme preparations from peppermint (Mentha piperita) leaves

    SciTech Connect

    Kjonaas, R.B.; Venkatachalam, K.V.; Croteau, R.

    1985-04-01

    Soluble enzyme extracts from peppermint leaves, when treated with polystyrene resin to remove endogenous monoterpenes and assayed with unlabeled substrates coupled with capillary gas-liquid chromatographic/mass spectrometric detection methods, were shown to oxidize isopiperitenol to isopiperitenone, and to isomerize isopiperitenone to piperitenone. The enzymes responsible for the monoterpenol dehydrogenation and the subsequent allylic isomerization were separated and partially purified by chromatography on Sephadex G-150, and were shown to have molecular weights of approximately 66,000 and 54,000, respectively. The general properties of the NAD-dependent dehydrogenase were examined, and specificity studies indicated that a double bond adjacent to the carbinol carbon was a required structural feature of the monoterpenol substrate. General properties of the isomerase were also determined, and it was demonstrated that the double bond migration catalyzed by this enzyme involved an intramolecular 1,3-hydrogen transfer. These enzymatic transformations represent two key steps in the metabolic pathway for the conversion of the initially formed cyclic olefin, (+/-)-limonene, to (-)-menthol and related monoterpenes characteristic of peppermint. Some stereochemical features of these reactions, and of the overall biogenetic scheme, are described. 39 references, 5 figures.

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

    PubMed Central

    Karagianni, Eleni P.; Kontomina, Evanthia; Davis, Britton; Kotseli, Barbara; Tsirka, Theodora; Garefalaki, Vasiliki; Sim, Edith; Glenn, Anthony E.; Boukouvala, Sotiria

    2015-01-01

    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 in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism. PMID:26245863

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

    PubMed

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

    2004-11-01

    Although it is well established that cellular transformation with tumor virus leads to changes on glucose metabolism, the effects of cell infection by non-transforming virus are far to be completely elucidated. In this study, we report the first evidence that cultured Vero cells infected with the alphavirus Mayaro show several alterations on glucose metabolism. Infected cells presented a two fold increase on glucose consumption, accompanied by an increment in lactate production. This increase in glycolytic flux was also demonstrated by a significant increase on the activity of 6-phosphofructo 1-kinase, one of the regulatory enzymes of glycolysis. Analysis of the kinetic parameters revealed that the regulation of 6-phosphofructo 1-kinase is altered in infected cells, presenting an increase in Vmax along with a decrease in Km for fructose-6-phosphate. Another fact contributing to an increase in enzyme activity was the decrease in ATP levels observed in infected cells. Additionally, the levels of fructose 2,6-bisphosphate, a potent activator of this enzyme, was significantly reduced in infected cells. These observations suggest that the increase in PFK activity may be a compensatory cellular response to the viral-induced metabolic alterations that could lead to an impairment of the glycolytic flux and energy production. PMID:15646042

  6. The role of human cytochrome P450 enzymes in the metabolism of anticancer agents: implications for drug interactions.

    PubMed Central

    Kivistö, K T; Kroemer, H K; Eichelbaum, M

    1995-01-01

    1. Little information is available about the pharmacokinetic interactions of anticancer drugs in man. However, clinically significant drug interactions do occur in cancer chemotherapy, and it is likely that important interactions have not been recognized. 2. Specific cytochrome P450 (CYP) enzymes have been recently shown to be involved in the metabolism of several essential anticancer agents. In particular, enzymes of the CYP3A subfamily play a role in the metabolism of many anticancer drugs, including epipodophyllotoxins, ifosphamide, tamoxifen, taxol and vinca alkaloids. CYP3A4 has been shown to catalyse the activation of the prodrug ifosphamide, raising the possibility that ifosphamide could be activated in tumour tissues containing this enzyme. 3. As examples of recently found, clinically significant interactions, cyclosporin considerably increases plasma doxorubicin and etoposide concentrations. Although cyclosporin and calcium channel blockers may influence the pharmacokinetics of certain anticancer agents by inhibiting their CYP3A mediated metabolism, it is more likely that these P-glycoprotein inhibitors inhibit P-glycoprotein mediated drug elimination. 4. Appropriate caution should be exercised when combining P-glycoprotein inhibitors and potential CYP3A inhibitors with cancer chemotherapy. PMID:8703657

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

    PubMed

    Karagianni, Eleni P; Kontomina, Evanthia; Davis, Britton; Kotseli, Barbara; Tsirka, Theodora; Garefalaki, Vasiliki; Sim, Edith; Glenn, Anthony E; Boukouvala, Sotiria

    2015-01-01

    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 in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism. PMID:26245863

  8. Effects of tin-protoporphyrin administration on hepatic xenobiotic metabolizing enzymes in the juvenile rat

    SciTech Connect

    Stout, D.L.; Becker, F.F.

    1988-01-01

    The heme analogue tin-protoporphyrin IX (SnP) is a potent inhibitor of microsomal heme oxygenase. Administration of SnP to neonatal rats can prevent hyperbilirubinemia by blocking the postnatal increase of heme oxygenase activity. Apparently innocuous at therapeutic doses, it is of potential clinical value for chemoprevention of neonatal jaundice. We found that when 50-g male Sprague-Dawley rats were treated daily with 50 mumol of SnP/kg sc for 6 days, hepatic microsomal cytochromes b5 and P-450 were significantly diminished. Cytochrome P-450 reductase, two P-450-dependent monooxygenases, aminopyrine demethylase and benzo(a)pyrene hydroxylase, and catalase, a peroxisomal hemoprotein, were also significantly diminished. These results suggested that SnP might significantly affect the metabolism of other xenobiotics. This possibility was confirmed by the finding that hexobarbital-induced sleep lasted 4 times longer in SnP-treated rats than in controls. Inhibition of protein synthesis by SnP was ruled out as the cause of hemoprotein loss when administration of (/sup 3/H)leucine to SnP-treated and control rats demonstrated that proteins of the microsomal, cytosolic, and plasma membrane fractions of the livers from both groups incorporated similar levels of leucine. When /sup 55/FeCl/sub 3/ and (2-/sup 14/C)glycine were administered to measure heme synthesis, heme extract from the livers of SnP-treated rats contained 4 times more label from iron and glycine than did heme from control livers. Despite the apparent increased rate of heme synthesis in SnP-treated rats, each of the three cell fractions demonstrated a significant loss of heme but contained sizable amounts of SnP. These findings suggest that SnP causes a decrease of functional hemoprotein and partial loss of enzymic activity by displacing intracellular heme.

  9. Effects of Arachis hypogaea nutshell extract on lipid metabolic enzymes and obesity parameters.

    PubMed

    Moreno, Diego A; Ilic, Nebojsa; Poulev, Alexander; Raskin, Ilya

    2006-05-01

    The aim of the present study was to assess the effects of peanut (Arachis hypogaea L.) shell extracts (PSE) on lipases and to evaluate its potential development for the treatment of obesity. The peanut shells were extracted in 95% ethanol, and the extracts were screened for inhibitory effects on pancreatic lipase (PL) and lipoprotein lipase (LPL) activities as well as on lipolysis of 3T3-L1 adipocytes. We also examined in vivo whether PSE could prevent the body weight gain induced by feeding a high-fat diet to male Wistar rats for 12 weeks. PSE inhibits a number of lipases, including PL, LPL and, possibly, hormone sensitive lipase (HSL). PSE-treated Wistar rats showed increased fecal lipid excretion respect to the control group. Body weight and body weight gain, and liver size, were significantly lower in rats fed the high-fat diet with 1% of PSE (w:w diet) than in those fed the high-fat diet alone. The rats treated with PSE showed reduced triacylglycerol content in the liver, as well as the serum glucose and insulin. The inhibitory activity of PSE on the lipid metabolic enzymes and the increase in fecal fat excretion suggests that PSE might be useful as a treatment to reduce the dietary fat absorption. The observed reduction in intracellular lipolytic activity of cultured 3T3-L1 adipocytes may reduce the levels of circulating free fatty acids. The observed effects are likely induced by more than one bioactive component of PSE. The PSE actions may, at least in part, be attributed to the inhibition of fat absorption in the digestive tract and the reduction of the adipocyte lipolysis. PMID:16337240

  10. ?-Viniferin and resveratrol induced alteration in the activities of some energy metabolism related enzymes in the cestode parasite Raillietina echinobothrida.

    PubMed

    Roy, Bishnupada; Giri, Bikash Ranjan

    2016-02-01

    ?-Viniferin (AVF) and its monomer resveratrol (RESV) are natural phytostilbenes produced by several plants in response to injury or under the influence of pathogens such as bacteria or fungi. Our earlier studies have revealed that both the compounds exert anthelmintic activity through alterations of cestode tegument and its associated enzymes. The present study investigates the effects of these phytochemicals on some energy metabolism related enzymes in the fowl tapeworm, Raillietina echinobothrida. The phytostilbenes AVF, RESV and the reference drug praziquantel (PZQ) were tested against some selected enzymes i.e., phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) of R. echinobothrida. Exposure of the tapeworm to AVF, RESV and PZQ causes reduction in activity of PEPCK to the extent of 40.57/41.96, 24.58/23.75 and 41.11/13.47%, respectively, and LDH up to 48.95/16.25, 38.31/38.42 and 45.67/41.87%, respectively, at the time of paralysis. Whereas activity of MDH decreased by 34.22/37.7, 39.1/35.24 and 28.83/19.26%, respectively. Decrease in activities of LDH and MDH was also visible through histochemical observations. The results suggest that both the phytochemicals interfere with the energy transducing pathways by inhibiting the studied energy metabolism related enzymes of the parasite. PMID:26603215

  11. [Investigation of metabolic kinetics and reaction phenotyping of ligustrazin by using liver microsomes and recombinant human enzymes].

    PubMed

    Tan, Yan; Zhuang, Xiao-Mei; Shen, Guo-Lin; Li, Hua; Gao, Yue

    2014-03-01

    The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions. PMID:24961110

  12. Mitochondrial dysfunction in obesity: potential benefit and mechanism of Co-enzyme Q10 supplementation in metabolic syndrome

    PubMed Central

    2014-01-01

    Co-enzyme Q10 (Co-Q10) is an essential component of the mitochondrial electron transport chain. Most cells are sensitive to co-enzyme Q10 (Co-Q10) deficiency. This deficiency has been implicated in several clinical disorders such as heart failure, hypertension, Parkinson’s disease and obesity. The lipid lowering drug statin inhibits conversion of HMG-CoA to mevalonate and lowers plasma Co-Q10 concentrations. However, supplementation with Co-Q10 improves the pathophysiological condition of statin therapy. Recent evidence suggests that Co-Q10 supplementation may be useful for the treatment of obesity, oxidative stress and the inflammatory process in metabolic syndrome. The anti-inflammatory response and lipid metabolizing effect of Co-Q10 is probably mediated by transcriptional regulation of inflammation and lipid metabolism. This paper reviews the evidence showing beneficial role of Co-Q10 supplementation and its potential mechanism of action on contributing factors of metabolic and cardiovascular complications. PMID:24932457

  13. In vitro metabolism of ?7 neuronal nicotinic receptor agonist AZD0328 and enzyme identification for its N-oxide metabolite.

    PubMed

    Zhou, Diansong; Zhang, Minli; Ye, Xiaomei; Gu, Chungang; Piser, Timothy M; Lanoue, Bernard A; Schock, Sara A; Cheng, Yi-Fang; Grimm, Scott W

    2011-03-01

    1. AZD0328 was pharmacologically characterized as a ?7 neuronal nicotinic receptor agonist intended for treatment of Alzheimer's disease. In vitro AZD0328 cross species metabolite profile and enzyme identification for its N-oxide metabolite were evaluated in this study. 2. AZD0328 was very stable in the human hepatocyte incubation, whereas extensively metabolized in rat, dog and guinea pig hepatocyte incubations. The N-oxidation metabolite (M6) was the only metabolite detected in human hepatocyte incubations, and it also appeared to be the major in vitro metabolic pathway in a number of preclinical species. In addition, N-glucuronide metabolite of AZD0328 was observed in human liver microsomes. 3. Other metabolic pathways in the preclinical species include hydroxylation in azabicyclo octane or furopyridine part of the molecule. Pyridine N-methylation of AZD0328 (M2) was identified as a dog specific metabolite, not observed in human or other preclinical species. 4. Multiple enzymes including CYP2D6, CYP3A4/5, FMO1 and FMO3 catalyzed AZD0328 metabolism. The potential for AZD0328 to be inhibited clinically by co-administered drugs or genetic polymorphism is relative low. PMID:21226652

  14. Anaerobic Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica—a Result of Promiscuous Enzymes and Regulators??

    PubMed Central

    Ding, Bin; Schmeling, Sirko; Fuchs, Georg

    2008-01-01

    The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO2 as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism. PMID:18156265

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    2011-01-01

    Background Cytochrome P450 enzymes (P450s) have been implicated in insecticide resistance. Anopheles minumus mosquito P450 isoforms CYP6AA3 and CYP6P7 are capable of metabolizing pyrethroid insecticides, however CYP6P8 lacks activity against this class of compounds. Findings Homology models of the three An. minimus P450 enzymes were constructed using the multiple template alignment method. The predicted enzyme model structures were compared and used for molecular docking with insecticides and compared with results of in vitro enzymatic assays. The three model structures comprise common P450 folds but differences in geometry of their active-site cavities and substrate access channels are prominent. The CYP6AA3 model has a large active site allowing it to accommodate multiple conformations of pyrethroids. The predicted CYP6P7 active site is more constrained and less accessible to binding of pyrethroids. Moreover the predicted hydrophobic interface in the active-site cavities of CYP6AA3 and CYP6P7 may contribute to their substrate selectivity. The absence of CYP6P8 activity toward pyrethroids appears to be due to its small substrate access channel and the presence of R114 and R216 that may prevent access of pyrethroids to the enzyme heme center. Conclusions Differences in active site topologies among CYPAA3, CYP6P7, and CYP6P8 enzymes may impact substrate binding and selectivity. Information obtained using homology models has the potential to enhance the understanding of pyrethroid metabolism and detoxification mediated by P450 enzymes. PMID:21892968

  17. Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis.

    PubMed

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V; Mühlemann, Joëlle K; Bomati, Erin K; Bowman, Marianne E; Dudareva, Natalia; Dixon, Richard A; Noel, Joseph P; Wang, Xiaoqiang

    2014-09-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

  18. 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 ?-cells from death and damage. Additionally, the supplement of trigonelline to surviving diabetic rats significantly decreased intestinal ?-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

  19. Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells.

    PubMed

    Xu, Bo; Chen, Minjian; Ji, Xiaoli; Yao, Mengmeng; Mao, Zhilei; Zhou, Kun; Xia, Yankai; Han, Xiao; Tang, Wei

    2015-10-01

    Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1?, IL-1?, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment. PMID:26165742

  20. 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.

  1. 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.

  2. 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.

  3. Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity

    SciTech Connect

    Murray, A.J.S.; Blackwell, R.D.; Lea, P.J. )

    1989-09-01

    A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO{sub 2} fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O{sub 2}. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C)serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either ({sup 14}C)serine or {sup 14}CO{sub 2}, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied ({sup 14}C)serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolize a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.

  4. Metabolism of diosbulbin B in vitro and in vivo in rats: formation of reactive metabolites and human enzymes involved.

    PubMed

    Yang, Baohua; Liu, Wei; Chen, Kaixian; Wang, Zhengtao; Wang, Changhong

    2014-10-01

    Diosbulbin B (DB), a major constituent of the furano-norditerpenes in Dioscorea bulbifera Linn, exhibits potential antineoplasmic activity and hepatotoxicity. The metabolism and reactive metabolites of DB in vitro (with human and animal liver microsomes) and in vivo in rats were investigated. The human enzymes involved in DB metabolism were identified. DB was first catalyzed into reactive metabolites of 2-butene-1,4-dial derivatives dependent on NADPH and then trapped by Tris base or oxidized to hemiacetal lactones (M12 and M13) in microsomal incubations. Tris base was used as buffer constituent and as a trapping agent for aldehyde. Methoxylamine and glutathione (GSH) were also used as trapping agents. DB metabolism in vivo in rats after oral administration was consistent with that in vitro. The structures of M12 and M13, as well as mono-GSH conjugates of DB (M31), were confirmed by nuclear magnetic resonance spectroscopy of the chemically synthesized products. The bioactivation enzymes of DB were identified as CYP3A4/5, 2C9, and 2C19. CYP3A4 was found to be the primary enzyme using human recombinant cytochrome P450 enzymes, specific inhibitory studies, and a relative activity factor approach for pooled human liver microsomes. Michaelis-Menten constants K(m) and V(max) were determined by the formation of M31. The reactive metabolites may be related to the hepatotoxicity of DB. The gender difference in CYP3A expression in mice and rats contributed to the gender-related liver injury and pharmacokinetics in mice and rats, respectively. PMID:25053620

  5. Persistent Overexpression of Phosphoglycerate Mutase, a Glycolytic Enzyme, Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice

    PubMed Central

    Shioi, Tetsuo; Kato, Takao; Inuzuka, Yasutaka; Kawashima, Tsuneaki; Tamaki, Yodo; Kawamoto, Akira; Tanada, Yohei; Iwanaga, Yoshitaka; Narazaki, Michiko; Matsuda, Tetsuya; Adachi, Souichi; Soga, Tomoyoshi; Takemura, Genzou; Kondoh, Hiroshi; Kita, Toru; Kimura, Takeshi

    2013-01-01

    Background Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function. Methods and Results Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/?ATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload. Conclusions Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice. PMID:23951293

  6. Carbon metabolism of peach fruit after harvest: changes in enzymes involved in organic acid and sugar level modifications.

    PubMed

    Borsani, Julia; Budde, Claudio O; Porrini, Lucía; Lauxmann, Martin A; Lombardo, Verónica A; Murray, Ricardo; Andreo, Carlos S; Drincovich, María F; Lara, María V

    2009-01-01

    Peach (Prunus persica L. Batsch) is a climacteric fruit that ripens after harvest, prior to human consumption. Organic acids and soluble sugars contribute to the overall organoleptic quality of fresh peach; thus, the integrated study of the metabolic pathways controlling the levels of these compounds is of great relevance. Therefore, in this work, several metabolites and enzymes involved in carbon metabolism were analysed during the post-harvest ripening of peach fruit cv 'Dixiland'. Depending on the enzyme studied, activity, protein level by western blot, or transcript level by quantitative real time-PCR were analysed. Even though sorbitol did not accumulate at a high level in relation to sucrose at harvest, it was rapidly consumed once the fruit was separated from the tree. During the ripening process, sucrose degradation was accompanied by an increase of glucose and fructose. Specific transcripts encoding neutral invertases (NIs) were up-regulated or down-regulated, indicating differential functions for each putative NI isoform. Phosphoenolpyruvate carboxylase was markedly induced, and may participate as a glycolytic shunt, since the malate level did not increase during post-harvest ripening. The fermentative pathway was highly induced, with increases in both the acetaldehyde level and the enzymes involved in this process. In addition, proteins differentially expressed during the post-harvest ripening process were also analysed. Overall, the present study identified enzymes and pathways operating during the post-harvest ripening of peach fruit, which may contribute to further identification of varieties with altered levels of enzymes/metabolites or in the evaluation of post-harvest treatments to produce fruit of better organoleptic attributes. PMID:19264753

  7. 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.

  8. CelE, a Multidomain Cellulase from Clostridium cellulolyticum: a Key Enzyme in the Cellulosome?

    PubMed Central

    Gaudin, Christian; Belaich, Anne; Champ, Stéphanie; Belaich, Jean-Pierre

    2000-01-01

    CelE, one of the three major proteins of the cellulosome of Clostridium cellulolyticum, was characterized. The amino acid sequence of the protein deduced from celE DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active on p-nitrophenyl–cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from C. cellulolyticum and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome. PMID:10714996

  9. Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on TRP channels and endocannabinoid metabolic enzymes

    PubMed Central

    De Petrocellis, Luciano; Ligresti, Alessia; Moriello, Aniello Schiano; Allarà, Marco; Bisogno, Tiziana; Petrosino, Stefania; Stott, Colin G; Di Marzo, Vincenzo

    2011-01-01

    BACKGROUND AND PURPOSE Cannabidiol (CBD) and ?9-tetrahydrocannabinol (THC) interact with transient receptor potential (TRP) channels and enzymes of the endocannabinoid system. EXPERIMENTAL APPROACH The effects of 11 pure cannabinoids and botanical extracts [botanical drug substance (BDS)] from Cannabis varieties selected to contain a more abundant cannabinoid, on TRPV1, TRPV2, TRPM8, TRPA1, human recombinant diacylglycerol lipase ? (DAGL?), rat brain fatty acid amide hydrolase (FAAH), COS cell monoacylglycerol lipase (MAGL), human recombinant N-acylethanolamine acid amide hydrolase (NAAA) and anandamide cellular uptake (ACU) by RBL-2H3 cells, were studied using fluorescence-based calcium assays in transfected cells and radiolabelled substrate-based enzymatic assays. Cannabinol (CBN), cannabichromene (CBC), the acids (CBDA, CBGA, THCA) and propyl homologues (CBDV, CBGV, THCV) of CBD, cannabigerol (CBG) and THC, and tetrahydrocannabivarin acid (THCVA) were also tested. KEY RESULTS CBD, CBG, CBGV and THCV stimulated and desensitized human TRPV1. CBC, CBD and CBN were potent rat TRPA1 agonists and desensitizers, but THCV-BDS was the most potent compound at this target. CBG-BDS and THCV-BDS were the most potent rat TRPM8 antagonists. All non-acid cannabinoids, except CBC and CBN, potently activated and desensitized rat TRPV2. CBDV and all the acids inhibited DAGL?. Some BDS, but not the pure compounds, inhibited MAGL. CBD was the only compound to inhibit FAAH, whereas the BDS of CBC > CBG > CBGV inhibited NAAA. CBC = CBG > CBD inhibited ACU, as did the BDS of THCVA, CBGV, CBDA and THCA, but the latter extracts were more potent inhibitors. CONCLUSIONS AND IMPLICATIONS These results are relevant to the analgesic, anti-inflammatory and anti-cancer effects of cannabinoids and Cannabis extracts. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7 PMID:21175579

  10. The effect of exogenous calcium on mitochondria, respiratory metabolism enzymes and ion transport in cucumber roots under hypoxia.

    PubMed

    He, Lizhong; Li, Bin; Lu, Xiaomin; Yuan, Lingyun; Yang, Yanjuan; Yuan, Yinghui; Du, Jing; Guo, Shirong

    2015-01-01

    Hypoxia induces plant stress, particularly in cucumber plants under hydroponic culture. In plants, calcium is involved in stress signal transmission and growth. The ultimate goal of this study was to shed light on the mechanisms underlying the effects of exogenous calcium on the mitochondrial antioxidant system, the activity of respiratory metabolism enzymes, and ion transport in cucumber (Cucumis sativus L. cv. Jinchun No. 2) roots under hypoxic conditions. Our experiments revealed that exogenous calcium reduces the level of reactive oxygen species (ROS) and increases the activity of antioxidant enzymes in mitochondria under hypoxia. Exogenous calcium also enhances the accumulation of enzymes involved in glycolysis and the tricarboxylic acid (TCA) cycle. We utilized fluorescence and ultrastructural cytochemistry methods to observe that exogenous calcium increases the concentrations of Ca(2+) and K(+) in root cells by increasing the activity of plasma membrane (PM) H(+)-ATPase and tonoplast H(+)-ATPase and H(+)-PPase. Overall, our results suggest that hypoxic stress has an immediate and substantial effect on roots. Exogenous calcium improves metabolism and ion transport in cucumber roots, thereby increasing hypoxia tolerance in cucumber. PMID:26304855

  11. The effect of exogenous calcium on mitochondria, respiratory metabolism enzymes and ion transport in cucumber roots under hypoxia

    PubMed Central

    He, Lizhong; Li, Bin; Lu, Xiaomin; Yuan, Lingyun; Yang, Yanjuan; Yuan, Yinghui; Du, Jing; Guo, Shirong

    2015-01-01

    Hypoxia induces plant stress, particularly in cucumber plants under hydroponic culture. In plants, calcium is involved in stress signal transmission and growth. The ultimate goal of this study was to shed light on the mechanisms underlying the effects of exogenous calcium on the mitochondrial antioxidant system, the activity of respiratory metabolism enzymes, and ion transport in cucumber (Cucumis sativus L. cv. Jinchun No. 2) roots under hypoxic conditions. Our experiments revealed that exogenous calcium reduces the level of reactive oxygen species (ROS) and increases the activity of antioxidant enzymes in mitochondria under hypoxia. Exogenous calcium also enhances the accumulation of enzymes involved in glycolysis and the tricarboxylic acid (TCA) cycle. We utilized fluorescence and ultrastructural cytochemistry methods to observe that exogenous calcium increases the concentrations of Ca2+ and K+ in root cells by increasing the activity of plasma membrane (PM) H+-ATPase and tonoplast H+-ATPase and H+-PPase. Overall, our results suggest that hypoxic stress has an immediate and substantial effect on roots. Exogenous calcium improves metabolism and ion transport in cucumber roots, thereby increasing hypoxia tolerance in cucumber. PMID:26304855

  12. Fine quantitative trait loci mapping of carbon and nitrogen metabolism enzyme activities and seedling biomass in the intermated maize IBM mapping population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the genetic basis of nitrogen and carbon metabolism will accelerate development of plant varieties with high yield and improved nitrogen use efficiency. In this study, we measured the activities of ten enzymes from carbon and nitrogen metabolism and seedling/juvenile biomass in the mai...

  13. Novel Systems Modeling Methodology in Comparative Microbial Metabolomics: Identifying Key Enzymes and Metabolites Implicated in Autism Spectrum Disorders

    PubMed Central

    Heberling, Colin; Dhurjati, Prasad

    2015-01-01

    Autism spectrum disorders are a group of mental illnesses highly correlated with gastrointestinal dysfunction. Recent studies have shown that there may be one or more microbial “fingerprints” in terms of the composition characterizing individuals with autism, which could be used for diagnostic purposes. This paper proposes a computational approach whereby metagenomes characteristic of “healthy” and autistic individuals are artificially constructed via genomic information, analyzed for the enzymes coded within, and then these enzymes are compared in detail. This is a text mining application. A custom-designed online application was built and used for the comparative metabolomics study and made publically available. Several of the enzyme-catalyzing reactions involved with the amino acid glutamate were curiously missing from the “autism” microbiome and were coded within almost every organism included in the “control” microbiome. Interestingly, there exists a leading hypothesis regarding autism and glutamate involving a neurological excitation/inhibition imbalance; but the association with this study is unclear. The results included data on the transsulfuration and transmethylation pathways, involved with oxidative stress, also of importance to autism. The results from this study are in alignment with leading hypotheses in the field, which is impressive, considering the purely in silico nature of this study. The present study provides new insight into the complex metabolic interactions underlying autism, and this novel methodology has potential to be useful for developing new hypotheses. However, limitations include sparse genome data availability and conflicting literature experimental data. We believe our software tool and methodology has potential for having great utility as data become more available, comprehensive and reliable. PMID:25913376

  14. 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. PMID:24299123

  15. COMPARATIVE ENZYME INDUCTION AND LINDANE METABOLISM IN RATS PRE-TREATED WITH VARIOUS ORGANOCHLORINE PESTICIDES

    EPA Science Inventory

    The comparative effect of 7 days pre-treatment with one of seven organochlorine pesticides on the metabolism of lindane in vivo and on the metabolism of EPN, p-nitroanisole and methyl orange in vitro was investigated. Mirex was the most potent inducer of the oxidative hydrolysis ...

  16. GENE EXPRESSION PROFILING OF XENOBIOTIC METABOLIZING ENZYMES (XMES) IN THE AGING MALE FISHER RAT

    EPA Science Inventory

    Detoxification and elimination of xenobiotics is a major function of the liver and is important in maintaining the metabolic homeostasis of the organism. The degree to which aging affects hepatic metabolism is not known. The expression of XMEs, in part, determines the fate of the...

  17. Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes

    PubMed Central

    Yang, Junling; He, Minxia M; Niu, Wei; Wrighton, Steven A; Li, Li; Liu, Yang; Li, Chuan

    2012-01-01

    AIM The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin. METHODS The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4?-hydroxylation (S)-mephenytoin 4?-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1?-hydroxylation/testosterone 6?-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences. RESULTS The samples of the two ethnic groups were not significantly different in cytochrome-b5 concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A. CONCLUSIONS These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins. PMID:21815912

  18. [The metabolic fingerprint of the compatibility of Radix Aconite and Radix Paeoniae Alba and its effect on CYP450 enzymes].

    PubMed

    Bi, Yun-Feng; Zheng, Zhong; Pi, Zi-Feng; Liu, Zhi-Qiang; Song, Feng-Rui

    2014-12-01

    Using a UPLC-MS/MS (MRM) and cocktail probe substrates method, the metabolic fingerprint of the compatibility of Radix Aconite (RA) and Radix Paeoniae Alba (RPA) and its effect on CYP450 enzymes were investigated. These main CYP isoforms include CYP 1A2, CYP 2C, CYP 2E1, CYP 2D and CYP 3A. Compared with the inhibition effect of RA decoctions on CYP450 isoforms, their co-decoctions of RA and RPA with different proportions can decrease RA' inhibition on CYP3A, CYP2D, CYP2C and CYP1A2, but can not reduce RA' effect on CYP2E1. The metabolic fingerprints of RA decoction and co-decoctions with different proportions of RPA in CYP450 of rat liver were analyzed by UPLC-MS. Compared with the metabolic fingerprints of RA decoction, the intensity of diester-diterpenoid aconitum alkaloids decreased significantly, while the intensity of monoester-diterpenoid alkaloids significantly increased in the metabolic fingerprints of co-decoctions of RA and RPA. The results suggest that RA coadministration with RPA increased the degradation of toxic alkaloid and show the effect of toxicity reducing and efficacy enhancing. PMID:25920201

  19. Variation in sulfur and selenium accumulation is controlled by naturally occurring isoforms of the key sulfur assimilation enzyme ADENOSINE 5'-PHOSPHOSULFATE REDUCTASE2 across the Arabidopsis species range.

    PubMed

    Chao, Dai-Yin; Baraniecka, Patrycja; Danku, John; Koprivova, Anna; Lahner, Brett; Luo, Hongbing; Yakubova, Elena; Dilkes, Brian; Kopriva, Stanislav; Salt, David E

    2014-11-01

    Natural variation allows the investigation of both the fundamental functions of genes and their role in local adaptation. As one of the essential macronutrients, sulfur is vital for plant growth and development and also for crop yield and quality. Selenium and sulfur are assimilated by the same process, and although plants do not require selenium, plant-based selenium is an important source of this essential element for animals. Here, we report the use of linkage mapping in synthetic F2 populations and complementation to investigate the genetic architecture of variation in total leaf sulfur and selenium concentrations in a diverse set of Arabidopsis (Arabidopsis thaliana) accessions. We identify in accessions collected from Sweden and the Czech Republic two variants of the enzyme ADENOSINE 5'-PHOSPHOSULFATE REDUCTASE2 (APR2) with strongly diminished catalytic capacity. APR2 is a key enzyme in both sulfate and selenate reduction, and its reduced activity in the loss-of-function allele apr2-1 and the two Arabidopsis accessions Hodonín and Shahdara leads to a lowering of sulfur flux from sulfate into the reduced sulfur compounds, cysteine and glutathione, and into proteins, concomitant with an increase in the accumulation of sulfate in leaves. We conclude from our observation, and the previously identified weak allele of APR2 from the Shahdara accession collected in Tadjikistan, that the catalytic capacity of APR2 varies by 4 orders of magnitude across the Arabidopsis species range, driving significant differences in sulfur and selenium metabolism. The selective benefit, if any, of this large variation remains to be explored. PMID:25245030

  20. Relationship between pathological findings and enzymes of the energy metabolism in liver of rats infected by Trypanosoma evansi.

    PubMed

    Baldissera, Matheus D; Rech, Virginia C; Grings, Mateus; Kolling, Janaína; Da Silva, Aleksandro S; Gressler, Lucas T; Souza, Carine De F; Vaucher, Rodrigo A; Schwertz, Claiton I; Mendes, Ricardo E; Leipnitz, Guilhian; Wyse, Angela T S; Stefani, Lenita M; Monteiro, Silvia G

    2015-12-01

    The aim of this study was to investigate the activities of important enzymes involved in the energetic metabolism in the liver of rats experimentally infected by Trypanosoma evansi. Adenylate kinase (AK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) in liver homogenate, as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and clotting time in plasma were evaluated at 5 and 15days post-infection (PI). The activities of the respiratory chain complexes and of Na(+), K(+)-ATPase were also evaluated. This study demonstrates energetic metabolism impairment in rats infected by T. evansi. A reduced energy metabolism in the liver of rats infected by T. evansi was observed, demonstrated by AK decreased and PK increased activities at 5days PI, a mechanism known as energetic compensation. However, at 15days PI a decrease of AK and PK activities were observed. In addition, an increase in the activities of respiratory chain complexes II, II-III and IV in infected rats at 15days PI, and a decrease of Na(+), K(+)-ATPase activities in infected rats on days 5 and 15 PI were verified. In the plasma, we observed an increase in ALT and AST activities on days 5 and 15 PI, and increase in clotting time in infected rats. The changes caused by T. evansi infection on the activity of enzymes of hepatic energy metabolism can corroborate to elucidate the mechanisms that lead to liver injury and inflammatory infiltration verified in T. evansi infected rats. Therefore, these alterations are directly related to disease pathogenesis. PMID:26239575

  1. Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut.

    PubMed

    Araji, Soha; Grammer, Theresa A; Gertzen, Ross; Anderson, Stephen D; Mikulic-Petkovsek, Maja; Veberic, Robert; Phu, My L; Solar, Anita; Leslie, Charles A; Dandekar, Abhaya M; Escobar, Matthew A

    2014-03-01

    The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut. PMID:24449710

  2. Metabolic organization and effects of feeding on enzyme activities of the dogfish shark (Squalus acanthias) rectal gland.

    PubMed

    Walsh, Patrick J; Kajimura, Makiko; Mommsen, Thomas P; Wood, Chris M

    2006-08-01

    In order to investigate the metabolic poise of the elasmobranch rectal gland, we conducted two lines of experimentation. First, we examined the effects of feeding on plasma metabolites and enzyme activities from several metabolic pathways in several tissues of the dogfish shark, Squalus acanthias, after starvation and at 6, 20, 30 and 48 h post-feeding. We found a rapid and sustained ten-fold decrease in plasma beta-hydroxybutyrate at 6 h and beyond compared with starved dogfish, suggesting an upregulation in the use of this substrate, a decrease in production, or both. Plasma acetoacetate levels remain unchanged, whereas there was a slight and transient decrease in plasma glucose levels at 6 h. Several enzymes showed a large increase in activity post-feeding, including beta-hydroxybutyrate dehydrogenase in rectal gland and liver, and in rectal gland, isocitrate dehydrogenase, citrate synthase, lactate dehydrogenase, aspartate amino transferase, alanine amino transferase, glutamine synthetase and Na(+)/K(+) ATPase. Also notable in these enzyme measurements was the overall high level of activity in the rectal gland in general. For example, activity of the Krebs' TCA cycle enzyme citrate synthase (over 30 U g(-1)) was similar to activities in muscle from other species of highly active fish. Surprisingly, lactate dehydrogenase activity in the gland was also high (over 150 U g(-1)), suggesting either an ability to produce lactate anaerobically or use lactate as an aerobic fuel. Given these interesting observations, in the second aspect of the study we examined the ability of several metabolic substrates (alone and in combination) to support chloride secretion by the rectal gland. Among the substrates tested at physiological concentrations (glucose, beta-hydroxybutyrate, lactate, alanine, acetoacetate, and glutamate), only glucose could consistently maintain a viable preparation. Whereas beta-hydroxybutyrate could enhance gland activity when presented in combination with glucose, surprisingly it could not sustain chloride secretion when used as a lone substrate. Our results are discussed in the context of the in vivo role of the gland and mechanisms of possible upregulation of enzyme activities. PMID:16857877

  3. Inhibition of Key Digestive Enzymes by Cocoa Extracts 1 and Procyanidins

    PubMed Central

    Gu, Yeyi; Hurst, William J.; Stuart, David A.; Lambert, Joshua D.

    2011-01-01

    We determined the in vitro inhibitory effects of cocoa extracts and procyanidins against pancreatic ?-amylase (PA), pancreatic lipase (PL) and secreted phospholipase A2 (PLA2), and characterized the kinetics of such inhibition. Lavado, regular and Dutch-processed cocoa extracts as well as cocoa procyanidins (degree of polymerization (DP) = 2 to 10) were examined. Cocoa extracts and procyanidins dose-dependently inhibited PA, PL and PLA2. Lavado cocoa extract was the most potent inhibitor (IC50 = 8.5 – 47 ?g/mL). An inverse correlation between Log IC50 and DP (R2 > 0.93) was observed. Kinetic analysis suggested that regular cocoa extract, the pentamer and decamer inhibited PL activity in a mixed mode. The pentamer and decamer non-competitively inhibited PLA2 activity, whereas regular cocoa extract inhibited PLA2 competitively. Our study demonstrates that cocoa polyphenols can inhibit digestive enzymes in vitro, and may, in conjunction with a low calorie diet, play a role in body weight management. PMID:21495725

  4. Zinc biochemistry: from a single zinc enzyme to a key element of life.

    PubMed

    Maret, Wolfgang

    2013-01-01

    The nutritional essentiality of zinc for the growth of living organisms had been recognized long before zinc biochemistry began with the discovery of zinc in carbonic anhydrase in 1939. Painstaking analytical work then demonstrated the presence of zinc as a catalytic and structural cofactor in a few hundred enzymes. In the 1980s, the field again gained momentum with the new principle of "zinc finger" proteins, in which zinc has structural functions in domains that interact with other biomolecules. Advances in structural biology and a rapid increase in the availability of gene/protein databases now made it possible to predict zinc-binding sites from metal-binding motifs detected in sequences. This procedure resulted in the definition of zinc proteomes and the remarkable estimate that the human genome encodes ?3000 zinc proteins. More recent developments focus on the regulatory functions of zinc(II) ions in intra- and intercellular information transfer and have tantalizing implications for yet additional functions of zinc in signal transduction and cellular control. At least three dozen proteins homeostatically control the vesicular storage and subcellular distribution of zinc and the concentrations of zinc(II) ions. Novel principles emerge from quantitative investigations on how strongly zinc interacts with proteins and how it is buffered to control the remarkably low cellular and subcellular concentrations of free zinc(II) ions. It is fair to conclude that the impact of zinc for health and disease will be at least as far-reaching as that of iron. PMID:23319127

  5. 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

  6. The evolution of new enzyme function: lessons from xenobiotic metabolizing bacteria versus insecticide-resistant insects

    PubMed Central

    Russell, Robyn J; Scott, Colin; Jackson, Colin J; Pandey, Rinku; Pandey, Gunjan; Taylor, Matthew C; Coppin, Christopher W; Liu, Jian-Wei; Oakeshott, John G

    2011-01-01

    Here, we compare the evolutionary routes by which bacteria and insects have evolved enzymatic processes for the degradation of four classes of synthetic chemical insecticide. For insects, the selective advantage of such degradative activities is survival on exposure to the insecticide, whereas for the bacteria the advantage is simply a matter of access to additional sources of nutrients. Nevertheless, bacteria have evolved highly efficient enzymes from a wide variety of enzyme families, whereas insects have relied upon generalist esterase-, cytochrome P450- and glutathione-S-transferase-dependent detoxification systems. Moreover, the mutant insect enzymes are less efficient kinetically and less diverged in sequence from their putative ancestors than their bacterial counterparts. This presumably reflects several advantages that bacteria have over insects in the acquisition of new enzymatic functions, such as a broad biochemical repertoire from which new functions can be evolved, large population sizes, high effective mutation rates, very short generation times and access to genetic diversity through horizontal gene transfer. Both the insect and bacterial systems support recent theory proposing that new biochemical functions often evolve from ‘promiscuous’ activities in existing enzymes, with subsequent mutations then enhancing those activities. Study of the insect enzymes will help in resistance management, while the bacterial enzymes are potential bioremediants of insecticide residues in a range of contaminated environments. PMID:25567970

  7. Phase I to II cross-induction of xenobiotic metabolizing enzymes: A feedforward control mechanism for potential hormetic responses

    SciTech Connect

    Zhang Qiang Pi Jingbo; Woods, Courtney G.; Andersen, Melvin E.

    2009-06-15

    Hormetic responses to xenobiotic exposure likely occur as a result of overcompensation by the homeostatic control systems operating in biological organisms. However, the mechanisms underlying overcompensation that leads to hormesis are still unclear. A well-known homeostatic circuit in the cell is the gene induction network comprising phase I, II and III metabolizing enzymes, which are responsible for xenobiotic detoxification, and in many cases, bioactivation. By formulating a differential equation-based computational model, we investigated in this study whether hormesis can arise from the operation of this gene/enzyme network. The model consists of two feedback and one feedforward controls. With the phase I negative feedback control, xenobiotic X activates nuclear receptors to induce cytochrome P450 enzyme, which bioactivates X into a reactive metabolite X'. With the phase II negative feedback control, X' activates transcription factor Nrf2 to induce phase II enzymes such as glutathione S-transferase and glutamate cysteine ligase, etc., which participate in a set of reactions that lead to the metabolism of X' into a less toxic conjugate X''. The feedforward control involves phase I to II cross-induction, in which the parent chemical X can also induce phase II enzymes directly through the nuclear receptor and indirectly through transcriptionally upregulating Nrf2. As a result of the active feedforward control, a steady-state hormetic relationship readily arises between the concentrations of the reactive metabolite X' and the extracellular parent chemical X to which the cell is exposed. The shape of dose-response evolves over time from initially monotonically increasing to J-shaped at the final steady state-a temporal sequence consistent with adaptation-mediated hormesis. The magnitude of the hormetic response is enhanced by increases in the feedforward gain, but attenuated by increases in the bioactivation or phase II feedback loop gains. Our study suggests a possibly common mechanism for the hormetic responses observed with many mutagens/carcinogens whose activities require bioactivation by phase I enzymes. Feedforward control, often operating in combination with negative feedback regulation in a homeostatic system, may be a general control theme responsible for steady-state hormesis.

  8. Recruitment of co-metabolic enzymes for environmental detoxification of organohalides.

    PubMed Central

    Wackett, L P

    1995-01-01

    Polyhalogenated compounds are often environmentally persistent and toxic to mammals. Microorganisms that metabolize these compounds can detoxify contaminated environments. Different biochemical mechanisms are used to metabolize polyhalogenated compounds, but few naturally occurring bacteria have this capability. A recombinant bacterium was constructed to metabolize polyhalogenated compounds to nonhalogenated end products. Seven genes were expressed in Pseudomonas putida G786 to biosynthesize cytochrome P450CAM and toluene dioxygenase. Cytochrome P450CAM catalyzed reductive dechlorinated reactions and toluene dioxygenase catalyzed oxidative dechlorination. With pentachloroethane, reductive dechlorination yielded trichloroethylene, which was further oxidized to formate and glyoxylate. The sequential action of cytochrome P450CAM and toluene dioxygenase with polyhalogenated compounds constitutes a novel engineered metabolic pathway. PMID:8565909

  9. Metabolism at Evolutionary Optimal States

    PubMed Central

    Rabbers, Iraes; van Heerden, Johan H.; Nordholt, Niclas; Bachmann, Herwig; Teusink, Bas; Bruggeman, Frank J.

    2015-01-01

    Metabolism is generally required for cellular maintenance and for the generation of offspring under conditions that support growth. The rates, yields (efficiencies), adaptation time and robustness of metabolism are therefore key determinants of cellular fitness. For biotechnological applications and our understanding of the evolution of metabolism, it is necessary to figure out how the functional system properties of metabolism can be optimized, via adjustments of the kinetics and expression of enzymes, and by rewiring metabolism. The trade-offs that can occur during such optimizations then indicate fundamental limits to evolutionary innovations and bioengineering. In this paper, we review several theoretical and experimental findings about mechanisms for metabolic optimization. PMID:26042723

  10. Effect of geraniol, a plant derived monoterpene on lipids and lipid metabolizing enzymes in experimental hyperlipidemic hamsters.

    PubMed

    Jayachandran, Muthukumaran; Chandrasekaran, Balaji; Namasivayam, Nalini

    2015-01-01

    Hyperlipidemia is a major, modifiable risk factor for atherosclerosis and cardiovascular disease. In the present study, we have focused on the effect of different doses of geraniol (GOH) on the lipid profile and lipid metabolizing enzymes in atherogenic diet (AD) fed hamsters. Male Syrian hamsters were grouped into seven: group 1 were control animals; group 2 were animals fed GOH alone (200 mg/kg b.w); group 3 were animals fed AD (10 % coconut oil, 0.25 % cholesterol, and 0.25 % cholic acid); group 4 were animals fed AD + corn oil (2.5 ml/kg b.w); and groups 5, 6, and 7 were fed AD as in group 3 + different doses of GOH (50, 100, and 200 mg/kg b.w), respectively, for 12 weeks. At the end of the experimental period, animals were sacrificed by cervical dislocation and various assays were performed in the plasma and tissues. The AD hamsters showed marked changes in lipid profile and lipid metabolizing enzymes. However, supplementation with GOH counteracted the hyperlipidemia by inhibiting HMG CoA reductase and suppressing lipogenesis. The antihyperlipidemic efficacy of GOH was found to be effective at the dose of 100 mg/kg b.w. This study illustrates that GOH is effective in lowering the risk of hyperlipidemia in AD fed hamsters. PMID:25218494

  11. Studies on the role of six enzymes in the metabolism of kinetin in mustard aphid, Lipaphis erysimi (Kalt.).

    PubMed

    Rup, Pushpinder J; Sohal, S K; Kaur, H

    2006-07-01

    The activity of catalase, glutathione peroxidase, superoxide dismutase, O-demethylase, ATPase and succinate dehydrogenase, belonging to two main classes of detoxification enzymes (i.e. hydrolases and oxido-reductases), mostly involved in metabolism and degradation of xenobiotics in insects, were assessed under the influence of kinetin, a plant growth regulator (PGR). The nymphs (48-52 hr old) of Lipaphis erysimi (Kalt.) were permitted to feed on radish plant, Raphanus sativus L. treated with kinetin (400 ppm) for 13, 25 and 37 hr. It was found that the activity of catalase, glutathione peroxidase and superoxide dismutase increased significantly when compared with the control of the same age group, which indicated that these enzymes might be playing a significant role in the metabolism of kinetin in this insect. The activity of O-demethylase showed an increase up to 25 hr of the treatment but it decreased under prolonged treatment whereas the activity of succinate dehydrogenase fluctuated insignificantly. ATPase showed a decrease in the activity with the treatment suggesting kinetin's interference in synthesis of ATPase. PMID:17402253

  12. Effect of cobalt protoporphyrin on hepatic drug-metabolizing enzymes. Specificity for cytochrome P-450.

    PubMed

    Spaethe, S M; Jollow, D J

    1989-06-15

    Cobaltic protoporphyrin IX (cobalt protoporphyrin) is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. To examine the specificity of cobalt protoporphyrin for hepatic cytochrome P-450, cobalt protoporphyrin was administered to rats and hamsters, and its effects on cytochrome P-450-dependent and non-P-450-dependent phase I and phase II metabolism were determined. Cobalt protoporphyrin pretreatment depleted hepatic cytochrome P-450 in both species and lowered their Vmax values for the hepatic microsomal metabolism of ethylmorphine, aminopyrine, ethoxyresorufin and ethoxycoumarin, without change in their Km values. In the rat, cobalt protoporphyrin treatment lowered both the Vmax and Km for microsomal metabolism of aniline. In vivo hepatic cytochrome P-450-dependent metabolism, as measured by antipyrine clearance, was decreased in both species. UDP-Glucuronyltransferase, phenolsulfotransferase and glutathione-S-transferase were unaffected, as was hepatic glutathione. Modest effects of cobalt protoporphyrin were seen on the hepatic microsomal flavoprotein mixed-function oxidase (hamster only), cytochrome P-450 reductase, cytochrome b5 (rat only), UDPGA (rat only), and glycogen, and on blood glucose (rat). In in vivo studies with hamsters given a low dose of acetaminophen, cobalt protoporphyrin suppressed the apparent rate constants for the cytochrome P-450-dependent pathways of acetaminophen metabolism but had no effect on acetaminophen glucuronidation and sulfation. Polyacrylamide gel electrophoresis analysis indicated that cobalt protoporphyrin markedly reduced the levels of the cytochrome P-450 holoenzyme but did not alter either the content or profile of the cytochrome P-450 apoenzyme. collectively, the data indicate that cobalt protoporphyrin shows relatively high selectivity for the hepatic cytochrome P-450 system, and support the use of this compound as a tool for resolution of the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. PMID:2742604

  13. Key elements of plant-based diets associated with reduced risk of metabolic syndrome.

    PubMed

    Turner-McGrievy, Gabrielle; Harris, Metria

    2014-01-01

    Approximately 20 %-25 % of adults worldwide have metabolic syndrome. Vegetarian and vegan diets have demonstrated effectiveness in improving body weight, glycemic control, and cardiovascular risk factors, as compared with conventional therapeutic approaches, and are potentially useful in the prevention of metabolic syndrome. This article consists of two steps: (1) a review of the literature on studies examining vegetarian and vegan diets and metabolic syndrome and (2) a review of foods and nutrients that are protective against or associated with metabolic syndromes that may help to explain the beneficial effects of plant-based dietary approaches for metabolic syndrome. The present review found eight observational research studies, and no intervention studies, examining the association of plant-based dietary approaches with metabolic syndrome. These studies, conducted mostly in Asian populations, yielded varying results. The majority, however, found better metabolic risk factors and lowered risk of metabolic syndrome among individuals following plant-based diets, as compared with omnivores. Some dietary components that are lower in the diets of vegetarians, such as energy intake, saturated fat, heme iron, and red and processed meat, may influence metabolic syndrome risk. In addition, plant-based diets are higher in fruits, vegetables, and fiber, which are protective against the development of metabolic syndrome. PMID:25084991

  14. Enzymes of glucose and methanol metabolism in the actinomycete Amycolatopsis methanolica.

    PubMed Central

    Alves, A M; Euverink, G J; Hektor, H J; Hessels, G I; van der Vlag, J; Vrijbloed, J W; Hondmann, D; Visser, J; Dijkhuizen, L

    1994-01-01

    The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PPi-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PPi-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PPi-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P(i) and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively. PMID:7961441

  15. Genetic perturbation of key central metabolic genes extends lifespan in Drosophila and affects response to dietary restriction.

    PubMed

    Talbert, Matthew E; Barnett, Brittany; Hoff, Robert; Amella, Maria; Kuczynski, Kate; Lavington, Erik; Koury, Spencer; Brud, Evgeny; Eanes, Walter F

    2015-09-22

    There is a connection between nutrient inputs, energy-sensing pathways, lifespan variation and aging. Despite the role of metabolic enzymes in energy homeostasis and their metabolites as nutrient signals, little is known about how their gene expression impacts lifespan. In this report, we use P-element mutagenesis in Drosophila to study the effect on lifespan of reductions in expression of seven central metabolic enzymes, and contrast the effects on normal diet and dietary restriction. The major observation is that for five of seven genes, the reduction of gene expression extends lifespan on one or both diets. Two genes are involved in redox balance, and we observe that lower activity genotypes significantly extend lifespan. The hexokinases also show extension of lifespan with reduced gene activity. Since both affect the ATP/ADP ratio, this connects with the role of AMP-activated protein kinase as an energy sensor in regulating lifespan and mediating caloric restriction. These genes possess significant expression variation in natural populations, and our experimental genotypes span this level of natural activity variation. Our studies link the readout of energy state with the perturbation of the genes of central metabolism and demonstrate their effect on lifespan. PMID:26378219

  16. The metabolism of flubendazole and the activities of selected biotransformation enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics.

    PubMed

    Vok?ál, Ivan; Bártíková, Hana; Prchal, Lukáš; Stuchlíková, Lucie; Skálová, Lenka; Szotáková, Barbora; Lamka, Ji?í; Várady, Marián; Kubí?ek, Vladimír

    2012-09-01

    Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g. sheep and goat). The treatment of haemonchosis is complicated because of recurrent resistance of H. contortus to common anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug flubendazole (FLU) and the activities of selected biotransformation enzymes towards model xenobiotics in 4 different strains of H. contortus: the ISE strain (susceptible to common anthelmintics), ISE-S (resistant to ivermectin), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (resistant to all common anthelmintics). H. contortus adults were collected from the abomasums from experimentally infected lambs. The in vitro as well as ex vivo experiments were performed and analysed using HPLC with spectrofluorimetric and mass-spectrometric detection. In all H. contortus strains, 4 different FLU metabolites were detected: FLU with a reduced carbonyl group (FLU-R), glucose conjugate of FLU-R and 2 glucose conjugates of FLU. In the resistant strains, the ex vivo formation of all FLU metabolites was significantly higher than in the susceptible ISE strain. The multi-resistant WR strain formed approximately 5 times more conjugates of FLU than the susceptible ISE strain. The in vitro data also showed significant differences in FLU metabolism, in the activities of UDP-glucosyltransferase and several carbonyl-reducing enzymes between the susceptible and resistant H. contortus strains. The altered activities of certain detoxifying enzymes might protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites. PMID:22717022

  17. Natural variations in xenobiotic-metabolizing enzymes: developing tools for coral monitoring

    NASA Astrophysics Data System (ADS)

    Rougée, L. R. A.; Richmond, R. H.; Collier, A. C.

    2014-06-01

    The continued deterioration of coral reefs worldwide demonstrates the need to develop diagnostic tools for corals that go beyond general ecological monitoring and can identify specific stressors at sublethal levels. Cellular diagnostics present an approach to defining indicators (biomarkers) that have the potential to reflect the impact of stress at the cellular level, allowing for the detection of intracellular changes in corals prior to outright mortality. Detoxification enzymes, which may be readily induced or inhibited by environmental stressors, present such a set of indicators. However, in order to apply these diagnostic tools for the detection of stress, a detailed understanding of their normal, homeostatic levels within healthy corals must first be established. Herein, we present molecular and biochemical evidence for the expression and activity of major Phase I detoxification enzymes cytochrome P450 (CYP450), CYP2E1, and CYP450 reductase, as well as the Phase II enzymes UDP, glucuronosyltransferase (UGT), ?-glucuronidase, glutathione- S-transferase (GST), and arylsulfatase C (ASC) in the coral Pocillopora damicornis. Additionally, we characterized enzyme expression and activity variations over a reproductive cycle within a coral's life history to determine natural endogenous changes devoid of stress exposure. Significant changes in enzyme activity over the coral's natural lunar reproductive cycle were observed for CYP2E1 and CYP450 reductase as well as UGT and GST, while ?-glucuronidase and ASC did not fluctuate significantly. The data represent a baseline description of `health' for the expression and activity of these enzymes that can be used toward understanding the impact of environmental stressors on corals. Such knowledge can be applied to address causes of coral reef ecosystem decline and to monitor effectiveness of mitigation strategies. Achieving a better understanding of cause-and-effect relationships between putative stressors and biological responses in corals, and other marine invertebrates, can guide and evaluate mitigation and conservation approaches for marine ecosystem protection.

  18. Drug-metabolizing enzymes and praziquantel bioavailability in mice harboring Schistosoma mansoni isolates of different drug susceptibilities.

    PubMed

    Botros, Sanaa S; El-Din, Sayed H Seif; El-Lakkany, Naglaa M; Sabra, Abdel Nasser A; Ebeid, Fatma A

    2006-12-01

    The level of drug-metabolizing enzymes (cytochrome P450 [CYP450] and cytochrome b5 [cyt b5]) and the bioavailability of praziquantel (PZQ) were investigated in batches of mice infected with Schistosoma mansoni displaying either a decreased susceptibility to PZQ ("EE2" and "BANL"-isolates), or a normal susceptibility to the drug ("CD" isolate). Each batch was divided into 2 groups. The first group was further subdivided into 5 subgroups. Subgroups 1 to 4 were treated 7 wk postinfection (PI) with oral PZQ at 25, 50, 100, and 200 mg/kg for 5 consecutive days, whereas the fifth subgroup was administered the vehicle only as control. Animals were perfused 9 wk PI, and worms were counted to estimate PZQ ED50. CYP450 and cyt b5 were examined in hepatic microsomes of infected untreated mice and of infected mice treated with 25 and 200 mg/ kg PZQ. The second group was given PZQ 7 wk PI and was further subdivided into 11 subgroups, killed at 2, 5, 15, 30, 60, 90, 120, 150, 180, 240, and 360 min postdosing to study pharmacokinetic parameters of PZQ. Mice harboring S. mansoni isolates having higher PZQ ED50 (170.3 mg/kg for EE2 and 249.9 mg/kg for BANL vs. 82.96 mg/kg for CD) had higher levels of CYP450 and cyt b5, a PZQ Cmax decreased by 19-30% and area under the serum concentration-time curve0-6 hr decreased by 57-74%. Data suggest that S. mansoni isolates that are less sensitive to PZQ induce a lower inhibition of hepatic drug-metabolizing enzymes, with a consequently higher metabolic transformation of PZQ. PMID:17304818

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  20. Metabolic gene clusters encoding the enzymes of two branches of the 3-oxoadipate pathway in the pathogenic yeast Candida albicans.

    PubMed

    Gérecová, Gabriela; Nebohá?ová, Martina; Zeman, Igor; Pryszcz, Leszek P; Tomáška, ?ubomír; Gabaldón, Toni; Nosek, Jozef

    2015-05-01

    The pathogenic yeast Candida albicans utilizes hydroxyderivatives of benzene via the catechol and hydroxyhydroquinone branches of the 3-oxoadipate pathway. The genetic basis and evolutionary origin of this catabolic pathway in yeasts are unknown. In this study, we identified C. albicans genes encoding the enzymes involved in the degradation of hydroxybenzenes. We found that the genes coding for core components of the 3-oxoadipate pathway are arranged into two metabolic gene clusters. Our results demonstrate that C. albicans cells cultivated in media containing hydroxybenzene substrates highly induce the transcription of these genes as well as the corresponding enzymatic activities. We also found that C. albicans cells assimilating hydroxybenzenes cope with the oxidative stress by upregulation of cellular antioxidant systems such as alternative oxidase and catalase. Moreover, we investigated the evolution of the enzymes encoded by these clusters and found that most of them share a particularly sparse phylogenetic distribution among Saccharomycotina, which is likely to have been caused by extensive gene loss. We exploited this fact to find co-evolving proteins that are suitable candidates for the missing enzymes of the pathway. PMID:25743787

  1. Physiological approach to explain the ecological success of 'superclones' in aphids: interplay between detoxification enzymes, metabolism and fitness.

    PubMed

    Castañeda, Luis E; Figueroa, Christian C; Fuentes-Contreras, Eduardo; Niemeyer, Hermann M; Nespolo, Roberto F

    2010-09-01

    'Superclones' are predominant and time-persistent genotypes, exhibiting constant fitness across different environments. However, causes of this ecological success are still unknown. Therefore, we studied the physiological mechanisms that could explain this success, evaluating the effects of wheat chemical defences on detoxification enzymes [cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST), esterases (EST)], standard metabolic rate (SMR), and fitness-related traits [adult body mass and intrinsic rate of increase (r(m))] of two 'superclones' (Sa1 and Sa2) of the grain aphid, Sitobion avenae. Additionally, we compared 'superclones' with a less-frequent genotype (Sa46). Genotypes were reared on three wheat cultivars with different levels of hydroxamic acids (Hx; wheat chemical defences). Detoxification enzymes and SMR did not differ between wheat hosts. However, GST and EST were different between 'superclones' and Sa46, while Sa1 showed a higher SMR than Sa2 or Sa46 (p=0.03). Differences between genotypes were found for r(m), which was higher for Sa1 than for Sa2 or Sa46. For all cases, genotype-host interactions were non-significant, except for aphid body mass. In conclusion, 'superclones' exhibit a broad host range, flat energetic costs for non-induced detoxification enzymes, and low variation in their reproductive performance on different defended hosts. However, physiological specialization of 'superclones' that could explain their ecological success was not evident in this study. PMID:20223246

  2. Gene expression of transporters and phase I/II metabolic enzymes in murine small intestine during fasting

    PubMed Central

    van den Bosch, Heleen M; Bünger, Meike; de Groot, Philip J; van der Meijde, Jolanda; Hooiveld, Guido JEJ; Müller, Michael

    2007-01-01

    Background Fasting has dramatic effects on small intestinal transport function. However, little is known on expression of intestinal transport and phase I/II metabolism genes during fasting and the role the fatty acid-activated transcription factor PPAR? may play herein. We therefore investigated the effects of fasting on expression of these genes using Affymetrix GeneChip MOE430A arrays and quantitative RT-PCR. Results After 24 hours of fasting, expression levels of 33 of the 253 analyzed transporter and phase I/II metabolism genes were changed. Upregulated genes were involved in transport of energy-yielding molecules in processes such as glycogenolysis (G6pt1) and mitochondrial and peroxisomal oxidation of fatty acids (Cact, Mrs3/4, Fatp2, Cyp4a10, Cyp4b1). Other induced genes were responsible for the inactivation of the neurotransmitter serotonin (Sert, Sult1d1, Dtd, Papst2), formation of eicosanoids (Cyp2j6, Cyp4a10, Cyp4b1), or for secretion of cholesterol (Abca1 and Abcg8). Cyp3a11, typically known because of its drug metabolizing capacity, was also increased. Fasting had no pronounced effect on expression of phase II metabolic enzymes, except for glutathione S-transferases which were down-regulated. Time course studies revealed that some genes were acutely regulated, whereas expression of other genes was only affected after prolonged fasting. Finally, we identified 8 genes that were PPAR?-dependently upregulated upon fasting. Conclusion We have characterized the response to fasting on expression of transporters and phase I/II metabolic enzymes in murine small intestine. Differentially expressed genes are involved in a variety of processes, which functionally can be summarized as a) increased oxidation of fat and xenobiotics, b) increased cholesterol secretion, c) increased susceptibility to electrophilic stressors, and d) reduced intestinal motility. This knowledge increases our understanding of gut physiology, and may be of relevance for e.g. pre-surgery regimen of patients. PMID:17683626

  3. Consumption of poisonous plants (Senecio jacobaea, Symphytum officinale, Pteridium aquilinum, Hypericum perforatum) by rats: chronic toxicity, mineral metabolism, and hepatic drug-metabolizing enzymes.

    PubMed

    Garrett, B J; Cheeke, P R; Miranda, C L; Goeger, D E; Buhler, D R

    1982-02-01

    Effect of dietary tancy ragwort (Senecio jacobaea), comfrey (Symphytum officinale), bracken (Pteridium aquilinum) and alfalfa (Medicago sativa) on hepatic drug-metabolizing enzymes in rats were measured. Tansy ragwort and bracken increased (P less than 0.05) the activity of glutathione transferase and epoxide hydrolase. Comfrey and alfalfa increased (P less than 0.05) the activity of aminopyrine N-demethylase. Feeding bracken or St. John's wort (Hypericum perforatum) in conjunction with tansy ragwort did not influence chronic toxicity of tansy ragwort as assessed by rat survival time. Dietary tansy ragwort resulted in increased (P less than 0.05) hepatic copper levels; the other plants did not affect copper levels. The results do not suggest any major interaction in the toxicity of tansy ragwort with bracken or St. John's wort. PMID:7080084

  4. 6-Gingerol Protects against Nutritional Steatohepatitis by Regulating Key Genes Related to Inflammation and Lipid Metabolism

    PubMed Central

    Tzeng, Thing-Fong; Liou, Shorong-Shii; Chang, Chia Ju; Liu, I-Min

    2015-01-01

    Non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH), appears to be increasingly common worldwide. The aim of the study was to investigate the effects of 6-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone), a bioactive ingredient of plants belonging to the Zingiberaceae family, on experimental models of NASH. In HepG2 cells, 6-gingerol (100 ?mol/L) treatment inhibited free fatty acids mixture (0.33 mmol/L palmitate and 0.66 mmol/L oleate)-induced triglyceride and inflammatory marker accumulations. Male C57BL/6 mice were fed with a methionine and choline-deficient (MCD) diet to induce steatohepatitis. After four weeks of MCD diet feeding, the mice were dosed orally with 6-gingerol (25, 50 or 100 mg/kg/day) once daily for another four weeks. 6-Gingerol (100 mg/kg/day) attenuated liver steatosis and necro-inflammation in MCD diet-fed mice. The expressions of inflammatory cytokine genes, including those for monocyte chemoattractant protein-1, tumor necrosis factor-?, and interleukin-6, and nuclear transcription factor (NF-?B), which were increased in the livers of MCD diet-fed mice, were attenuated by 6-gingerol. 6-Gingerol possesses a repressive property on hepatic steatosis, which is associated with induction of peroxisome proliferator-activated receptor ?. Our study demonstrated the protective role of 6-gingerol in ameliorating nutritional steatohepatitis. The effect was mediated through regulating key genes related to lipid metabolism and inflammation. PMID:25658238

  5. 6-gingerol protects against nutritional steatohepatitis by regulating key genes related to inflammation and lipid metabolism.

    PubMed

    Tzeng, Thing-Fong; Liou, Shorong-Shii; Chang, Chia Ju; Liu, I-Min

    2015-01-01

    Non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH), appears to be increasingly common worldwide. The aim of the study was to investigate the effects of 6-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone), a bioactive ingredient of plants belonging to the Zingiberaceae family, on experimental models of NASH. In HepG2 cells, 6-gingerol (100 ?mol/L) treatment inhibited free fatty acids mixture (0.33 mmol/L palmitate and 0.66 mmol/L oleate)-induced triglyceride and inflammatory marker accumulations. Male C57BL/6 mice were fed with a methionine and choline-deficient (MCD) diet to induce steatohepatitis. After four weeks of MCD diet feeding, the mice were dosed orally with 6-gingerol (25, 50 or 100 mg/kg/day) once daily for another four weeks. 6-Gingerol (100 mg/kg/day) attenuated liver steatosis and necro-inflammation in MCD diet-fed mice. The expressions of inflammatory cytokine genes, including those for monocyte chemoattractant protein-1, tumor necrosis factor-?, and interleukin-6, and nuclear transcription factor (NF-?B), which were increased in the livers of MCD diet-fed mice, were attenuated by 6-gingerol. 6-Gingerol possesses a repressive property on hepatic steatosis, which is associated with induction of peroxisome proliferator-activated receptor ?. Our study demonstrated the protective role of 6-gingerol in ameliorating nutritional steatohepatitis. The effect was mediated through regulating key genes related to lipid metabolism and inflammation. PMID:25658238

  6. Enhancing enzyme stability and metabolic functional ability of ?-galactosidase through functionalized polymer nanofiber immobilization.

    PubMed

    Misson, Mailin; Jin, Bo; Chen, Binghui; Zhang, Hu

    2015-10-01

    A functionalized polystyrene nanofiber (PSNF) immobilized ?-galactosidase assembly (PSNF-Gal) was synthesized as a nanobiocatalyst aiming to enhance the biocatalyst stability and functional ability. The PSNF fabricated by electrospinning was functionalized through a chemical oxidation method for enzyme binding. The bioengineering performance of the enzyme carriers was further evaluated for bioconversion of lactose to galacto-oligosaccharides (GOS). The modified PSNF-Gal demonstrated distinguished performances to preserve the same activity as the free ?-galactosidase at the optimum pH of 7.0, and to enhance the enzyme stability of PSNF-Gal in an alkaline condition up to pH 10. The PSNF assembly demonstrated improved thermal stability from 37 to 60 °C. The nanobiocatalyst was able to retain 30 % of its initial activity after ninth operation cycles comparing to four cycles with the unmodified counterpart. In contrast with free ?-galactosidase, the modified PSNF-Gal enhanced the GOS yield from 14 to 28 %. These findings show the chemically modified PSNF-based nanobiocatalyst may be pertinent for various enzyme-catalysed bioprocessing applications. PMID:26104537

  7. Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery

    E-print Network

    Tong, Liang

    in ACC2 have continuous fatty acid oxidation and reduced body fat and body weight, val- idating; protein struc- ture and function; biotin-dependent carboxylases; enzyme catalysis and mechanism [6]. While life-style changes (less food intake and more physical activity) are an important

  8. Asparagus racemosus competitively inhibits in vitro the acetylcholine and monoamine metabolizing enzymes.

    PubMed

    Meena, Jairam; Ojha, Rakesh; Muruganandam, A V; Krishnamurthy, Sairam

    2011-09-26

    Asparagus racemosus (AR) has earlier been reported to possess antidepressant activity possibly mediated through the monoaminergic system, and nootropic and anti amnestic activities possibly through the cholinergic system. In the present study to further understand the mechanism of action, we evaluated the kinetics of acetyl (AChE) and butyryl (BuChE) cholinesterases, and monoamine oxidase (MAO-A and B) enzyme inhibitory activities of different fractions of AR. The results showed that methanolic extract of AR (MAR) significantly inhibited cholinesterase and MAO activities as compared to hexane (HAR) and chloroform (CAR) extracts of AR as evident from the IC(50) values. The kinetic analysis of enzyme inhibition of MAR shows that the V(max) does not change with different concentrations of MAR but the K(m) value increases. This indicates that MAR is a non-selective competitive inhibitor for both cholinesterase and monoamine oxidase enzymes. Evaluation of K(i) values show that MAR inhibited these enzymes less potently compared to the respective standard drugs. There seems to be a positive correlation between the saponin content and, cholinesterase and monoamine inhibitory activities as MAR had 62.20% of saponins, whereas HAR and CAR had no measurable saponin content. The non-selective competitive inhibitory activity on cholinesterase and monoamine oxidase enzymes can explain many reported neuropharmacological activities of AR. AR apart being used as a drug is also used as a food. As such AR may have potential drug-drug, drug-food and food-food interactions with drugs and foods sharing the cholinergic and monoaminergic pathways. PMID:21843599

  9. Thermophilic and thermoacidophilic metabolism genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOEpatents

    Thompson, Vicki S; Apel, William A; Reed, David W; Lee, Brady D; Thompson, David N; Roberto, Francisco F; Lacey, Jeffrey A

    2014-05-20

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering metabolism in a cell using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  10. Thermophilic and thermoacidophilic metabolism genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOEpatents

    Thompson, Vicki S.; Apel, William A.; Reed, David William; Lee, Brady D.; Thompson, David N.; Roberto, Francisco F.; Lacey, Jeffrey A.

    2015-12-29

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering metabolism in a cell using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  11. Crystal Structure of Baeyer-Villiger Monooxygenase MtmOIV, the Key Enzyme of the Mithramycin Biosynthetic Pathway†

    PubMed Central

    Beam, Miranda P.; Bosserman, Mary A.; Noinaj, Nicholas; Wehenkel, Marie; Rohr, Jürgen

    2009-01-01

    Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kD homo-dimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIV’s structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9Å. MtmOIV cleaves a C-C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments implicate several residues to participate in co-factor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis. PMID:19364090

  12. Chromium picolinate does not improve key features of metabolic syndrome in obese nondiabetic adults

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of chromium-containing dietary supplements is widespread among patients with type 2 diabetes as a means of improving glucose metabolism. However, chromium’s role as a potential therapy for patients at high risk for developing type 2 diabetes, specifically those with metabolic syndrome, is n...

  13. [Regulation of terpene metabolism]. [Mentha piperita, Mentha spicata

    SciTech Connect

    Croteau, R.

    1989-01-01

    Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

  14. Metabolism

    MedlinePLUS

    ... Metabolic Disorders Metabolism Basics Our bodies get the energy they need from food through metabolism, the chemical ... that convert the fuel from food into the energy needed to do everything from moving to thinking ...

  15. 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 1; Metabolic Enzymes of Individual Muscle Fibers

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

    Individual fibers of any given muscle vary widely in enzyme composition, a fact obscured when enzyme levels of whole muscle are measured. Therefore, the purpose of this part of the study was to assess the effects of microgravity and hind limb suspension on the enzyme patterns within a slow twitch muscle (soleus) and a fast twitch muscle (tibialis anterior).

  16. Stable Carbon Isotope Discrimination by Form IC Rubisco Enzymes of the Extremely Metabolically Versatile Rhodobacter sphaeroides and Ralstonia eutropha}

    NASA Astrophysics Data System (ADS)

    Thomas, P. J.; Boller, A. J.; Zhao, Z.; Tabita, F. R.; Cavanaugh, C. M.; Scott, K. M.

    2006-12-01

    Variations in the relative amounts of 12C and 13C in microbial biomass can be used to infer the pathway(s) autotrophs use to fix and assimilate dissolved inorganic carbon. Discrimination against 13C by the enzymes catalyzing autotrophic carbon fixation is a major factor dictating biomass stable carbon isotopic compositions (?13C = {[13C/12Csample/13C/12Cstandard] - 1} × 1000). Five different forms of RubisCO (IA, IB, IC, ID, and II) are utilized by algae and autotrophic bacteria reliant on the Calvin-Benson cycle for carbon fixation. To date, isotope discrimination has been measured for form IA, IB, and II RubisCOs, and their ? values (={[12k/13k] - 1} × 1000; 12k and 13k = rates of 12C and 13C fixation) range from 18 to 29‰, explaining the variation in biomass ?13C values of autotrophs utilizing these enzymes. Isotope discrimination by form IC RubisCO has not been measured, despite the presence of this enzyme in many proteobacteria of ecological interest, including marine manganese-oxidizing bacteria, some nitrifying and nitrogen-fixing bacteria, and extremely metabolically versatile organisms such as Rhodobacter sphaeroides and Ralstonia eutropha. The purpose of this work was to determine the ? values for form IC RubisCO enzymes from R. sphaeroides and R. eutropha. Recombinant form IC RubisCOs were purified by conventional column chromatography procedures. Assay conditions (pH, dissolved inorganic carbon concentration) were tested to determine which parameters were conducive to the high rates of carbon fixation necessary for ? determination. Under standard conditions (pH 8.5 and 5 mM DIC), form IC RubisCO activities were sufficient for ? determination. Experiments are currently being conducted to measure the ? values of these enzymes. Sampling the full phylogenetic breadth of RubisCO enzymes for isotopic discrimination makes it possible to constrain the range of ?13C values of organisms fixing carbon via the Calvin-Benson cycle. These results are critical for determining the degree to which Calvin cycle carbon fixation contributes to primary and secondary productivity in microbially-dominated food webs.

  17. Identification of Multiple Metabolic Enzymes from Mice Cochleae Tissue Using a Novel Functional Proteomics Technology

    PubMed Central

    Wang, David L.; Li, Hui; Liang, Ruqiang; Bao, Jianxin

    2015-01-01

    A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment. PMID:25811366

  18. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases

    PubMed Central

    Caspi, Ron; Altman, Tomer; Billington, Richard; Dreher, Kate; Foerster, Hartmut; Fulcher, Carol A.; Holland, Timothy A.; Keseler, Ingrid M.; Kothari, Anamika; Kubo, Aya; Krummenacker, Markus; Latendresse, Mario; Mueller, Lukas A.; Ong, Quang; Paley, Suzanne; Subhraveti, Pallavi; Weaver, Daniel S.; Weerasinghe, Deepika; Zhang, Peifen; Karp, Peter D.

    2014-01-01

    The MetaCyc database (MetaCyc.org) is a comprehensive and freely accessible database describing metabolic pathways and enzymes from all domains of life. MetaCyc pathways are experimentally determined, mostly small-molecule metabolic pathways and are curated from the primary scientific literature. MetaCyc contains >2100 pathways derived from >37 000 publications, and is the largest curated collection of metabolic pathways currently available. BioCyc (BioCyc.org) is a collection of >3000 organism-specific Pathway/Genome Databases (PGDBs), each containing the full genome and predicted metabolic network of one organism, including metabolites, enzymes, reactions, metabolic pathways, predicted operons, transport systems and pathway-hole fillers. Additions to BioCyc over the past 2 years include YeastCyc, a PGDB for Saccharomyces cerevisiae, and 891 new genomes from the Human Microbiome Project. The BioCyc Web site offers a variety of tools for querying and analysis of PGDBs, including Omics Viewers and tools for comparative analysis. New developments include atom mappings in reactions, a new representation of glycan degradation pathways, improved compound structure display, better coverage of enzyme kinetic data, enhancements of the Web Groups functionality, improvements to the Omics viewers, a new representation of the Enzyme Commission system and, for the desktop version of the software, the ability to save display states. PMID:24225315

  19. Enzymes of phenylpropanoid metabolism in the important medicinal plant Melissa officinalis L.

    PubMed

    Weitzel, Corinna; Petersen, Maike

    2010-08-01

    Lemon balm (Melissa officinalis, Lamiaceae) is a well-known medicinal plant. Amongst the biologically active ingredients are a number of phenolic compounds, the most prominent of which is rosmarinic acid. To obtain better knowledge of the biosynthesis of these phenolic compounds, two enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL) and 4-coumarate:coenzyme A-ligase (4CL), were investigated in suspension cultures of lemon balm. MoPAL1 and Mo4CL1 cDNAs were cloned and heterologously expressed in Escherichia coli and the enzymes characterised. Expression analysis of both genes showed a correlation with the enzyme activities and rosmarinic acid content during a cultivation period of the suspension culture. Southern-blot analysis suggested the presence of most probably two gene copies in the M. officinalis genome of both PAL and 4CL. The genomic DNA sequences of MoPAL1 and Mo4CL1 were amplified and sequenced. MoPAL1 contains one phase 2 intron of 836 bp at a conserved site, whilst Mo4CL1 was devoid of introns. PMID:20563822

  20. [The role of angiotensin-converting enzyme gene I/D polymorphism in development of metabolic disorders in patients with cardiovascular pathology].

    PubMed

    Vynohradova, S V

    2005-01-01

    The role of angiotensin-converting enzyme (ACE) gene I/D polymorphism in development of cardiovascular pathology (CVP), metabolic syndrom and insulin-independent diabet associated with such metabolic disorders as glucose intolerance and hyperglicemia, intolerance to insulin and hyperinsulinemia, dyslipiproteinemia (DLP) and obesity is discussed. Most of authors consider D-allel and DD genotype to be assosiated with development of DLP and such CVP as ishemic heart disease and myocardial infarction. PMID:16018179

  1. Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16.

    PubMed

    Karstens, Katja; Zschiedrich, Christopher P; Bowien, Botho; Stülke, Jörg; Görke, Boris

    2014-04-01

    EIIA(Ntr) is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIA(Ntr) regulates K(+) homeostasis through interaction with the K(+) transporter TrkA and sensor kinase KdpD. In the ?-Proteobacterium Ralstonia eutropha H16, EIIA(Ntr) influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA(Ntr) increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA(Ntr), has the opposite effect. To clarify the role of EIIA(Ntr) in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA(Ntr) from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA(Ntr) interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIA(Ntr) with KdpD appears to be absent in R. eutropha, although R. eutropha EIIA(Ntr) can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary 'ancient' task of EIIA(Ntr) that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIA(Ntr) might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha. PMID:24515609

  2. Polyphenols as enzyme inhibitors in different degraded peat soils: Implication for microbial metabolism in rewetted peatlands

    NASA Astrophysics Data System (ADS)

    Zak, Dominik; Roth, Cyril; Gelbrecht, Jörg; Fenner, Nathalie; Reuter, Hendrik

    2015-04-01

    Recently, more than 30,000 ha of drained minerotrophic peatlands (= fens) in NE Germany were rewetted to restore their ecological functions. Due to an extended drainage history, a re-establishment of their original state is not expected in the short-term. Elevated concentrations of dissolved organic carbon, ammonium and phosphate have been measured in the soil porewater of the upper degraded peat layers of rewetted fens at levels of one to three orders higher than the values in pristine systems; an indicator of increased microbial activity in the upper degraded soil layers. On the other hand there is evidence that the substrate availability within the degraded peat layer is lowered since the organic matter has formerly been subject to intense decomposition over the decades of drainage and intense agricultural use of the areas. Previously however, it was suggested that inhibition of hydrolytic enzymes by polyphenolic substances is suspended during aeration of peat soils mainly due to the decomposition of the inhibiting polyphenols by oxidising enzymes such as phenol oxidase. Accordingly we hypothesised a lack of enzyme inhibiting polyphenols in degraded peat soils of rewetted fens compared to less decomposed peat of more natural fens. We collected both peat samples at the soil surface (0-20 cm) and fresh roots of dominating vascular plants and mosses (as peat parent material) from five formerly drained rewetted sites and five more natural sites of NE Germany and NW Poland. Less decomposed peat and living roots were used to obtain an internal standard for polyphenol analysis and to run enzyme inhibition tests. For all samples we determined the total phenolic contents and in addition we distinguished between the contents of hydrolysable and condensed tannic substances. From a methodical perspective the advantage of internal standards compared to the commercially available standards cyanidin chloride and tannic acid became apparent. Quantification with cyanidin or tannic acid led to a considerable underestimation (up to 90%) of polyphenolic concentrations in peat soils. As hypothesised we found that highly degraded peat contains far lower levels of total polyphenolics (factor 8) and condensed tannins (factor 50) than less decomposed peat. In addition we detected large differences between different plant species with highest polyphenolic contents for the roots of Carex appropinquata that were more than 10-fold higher than Sphagnum spp. (450 mg/g dry mass vs. 39 mg/g dry mass). Despite these differences, we did not find a significant correlation between enzyme activities and peat degradation state, indicating that there is no simple linear relationship between polyphenolic contents and microbial activity.

  3. Study of 'Redhaven' peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism

    PubMed Central

    2011-01-01

    Background Carotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. To investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach (Prunus persica L. Batsch.), and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. Two contrasted cultivars, the yellow-fleshed 'Redhaven' (RH) and its white-fleshed mutant 'Redhaven Bianca' (RHB) were examined. Results The two genotypes displayed marked differences in the accumulation of carotenoid pigments in mesocarp tissues. Lower carotenoid levels and higher levels of norisoprenoid volatiles were observed in RHB, which might be explained by differential activity of carotenoid cleavage dioxygenase (CCD) enzymes. In fact, the ccd4 transcript levels were dramatically higher at late ripening stages in RHB with respect to RH. The two genotypes also showed differences in the expression patterns of several carotenoid and isoprenoid transcripts, compatible with a feed-back regulation of these transcripts. Abamine SG - an inhibitor of CCD enzymes - decreased the levels of both isoprenoid and non-isoprenoid volatiles in RHB fruits, indicating a complex regulation of volatile production. Conclusions Differential expression of ccd4 is likely to be the major determinant in the accumulation of carotenoids and carotenoid-derived volatiles in peach fruit flesh. More in general, dioxygenases appear to be key factors controlling volatile composition in peach fruit, since abamine SG-treated 'Redhaven Bianca' fruits had strongly reduced levels of norisoprenoids and other volatile classes. Comparative functional studies of peach carotenoid cleavage enzymes are required to fully elucidate their role in peach fruit pigmentation and aroma. PMID:21269483

  4. Fetal genotype for the xenobiotic metabolizing enzyme NQO1 influences intrauterine growth among infants whose mothers smoked during pregnancy.

    PubMed

    Price, Thomas S; Grosser, Tilo; Plomin, Robert; Jaffee, Sara R

    2010-01-01

    Maternal smoking during pregnancy retards fetal growth and depresses infant birth weight. The magnitude of these effects may be moderated by fetal genotype. The current study investigated maternal smoking, fetal genotype, and fetal growth in a large population sample of dizygotic twins. Maternal smoking retarded fetal growth in a dose-dependent fashion. In a subsample of 497 twin pairs whose mothers smoked during pregnancy, a functional polymorphism in the NAD(P)H:quinone oxidoreductase gene (NQO1 Pro187Ser; rs1800566) was significantly associated with fetal growth within families. The effect was strongest among moderate smokers. This is the first demonstration that fetal genotype for an enzyme involved in tobacco smoke metabolism influences intrauterine growth independent of maternal genotype. Future studies should conduct formal tests of Fetal Genotype x Maternal Smoking interactions. PMID:20331656

  5. Pulmonary metabolism of foreign compounds: Its role in metabolic activation

    SciTech Connect

    Cohen, G.M. )

    1990-04-01

    The lung has the potential of metabolizing many foreign chemicals to a vast array of metabolites with different pharmacological and toxicological properties. Because many chemicals require metabolic activation in order to exert their toxicity, the cellular distribution of the drug-metabolizing enzymes in a heterogeneous tissue, such as the lung, and the balance of metabolic activation and deactivation pathways in any particular cell are key factors in determining the cellular specificity of many pulmonary toxins. Environmental factors such as air pollution, cigarette smoking, and diet markedly affect the pulmonary metabolism of some chemicals and, thereby, possibly affect their toxicity.

  6. Potential risks resulting from fruit/vegetable-drug interactions: effects on drug-metabolizing enzymes and drug transporters.

    PubMed

    Rodríguez-Fragoso, Lourdes; Martínez-Arismendi, José Luis; Orozco-Bustos, Danae; Reyes-Esparza, Jorge; Torres, Eliseo; Burchiel, Scott W

    2011-05-01

    It has been well established that complex mixtures of phytochemicals in fruits and vegetables can be beneficial for human health. Moreover, it is becoming increasingly apparent that phytochemicals can influence the pharmacological activity of drugs by modifying their absorption characteristics through interactions with drug transporters as well as drug-metabolizing enzyme systems. Such effects are more likely to occur in the intestine and liver, where high concentrations of phytochemicals may occur. Alterations in cytochrome P450 and other enzyme activities may influence the fate of drugs subject to extensive first-pass metabolism. Although numerous studies of nutrient-drug interactions have been published and systematic reviews and meta-analyses of these studies are available, no generalizations on the effect of nutrient-drug interactions on drug bioavailability are currently available. Several publications have highlighted the unintended consequences of the combined use of nutrients and drugs. Many phytochemicals have been shown to have pharmacokinetic interactions with drugs. The present review is limited to commonly consumed fruits and vegetables with significant beneficial effects as nutrients and components in folk medicine. Here, we discuss the phytochemistry and pharmacokinetic interactions of the following fruit and vegetables: grapefruit, orange, tangerine, grapes, cranberry, pomegranate, mango, guava, black raspberry, black mulberry, apple, broccoli, cauliflower, watercress, spinach, tomato, carrot, and avocado. We conclude that our knowledge of the potential risk of nutrient-drug interactions is still limited. Therefore, efforts to elucidate potential risks resulting from food-drug interactions should be intensified in order to prevent undesired and harmful clinical consequences. PMID:22417366

  7. Interactions between Urinary 4-tert-Octylphenol Levels and Metabolism Enzyme Gene Variants on Idiopathic Male Infertility

    PubMed Central

    Xu, Bin; Tang, Rong; Chen, Xiaojiao; Du, Guizhen; Lu, Chuncheng; Meeker, John D.; Zhou, Zuomin; Xia, Yankai; Wang, Xinru

    2013-01-01

    Octylphenol (OP) and Trichlorophenol (TCP) act as endocrine disruptors and have effects on male reproductive function. We studied the interactions between 4-tert-Octylphenol (4-t-OP), 4-n- Octylphenol (4-n-OP), 2,3,4-Trichlorophenol (2,3,4-TCP), 2,4,5-Trichlorophenol (2,4,5-TCP) urinary exposure levels and polymorphisms in selected xenobiotic metabolism enzyme genes among 589 idiopathic male infertile patients and 396 controls in a Han-Chinese population. Ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to measure alkylphenols and chlorophenols in urine. Polymorphisms were genotyped using the SNPstream platform and the Taqman method. Among four phenols that were detected, we found that only exposure to 4-t-OP increased the risk of male infertility (Ptrend?=?1.70×10?7). The strongest interaction was between 4-t-OP and rs4918758 in CYP2C9 (Pinter?=?6.05×10?7). It presented a significant monotonic increase in risk estimates for male infertility with increasing 4-t-OP exposure levels among men with TC/CC genotype (low level compared with non-exposed, odds ratio (OR)?=?2.26, 95% confidence intervals (CI)?=?1.06, 4.83; high level compared with non-exposed, OR?=?9.22, 95% CI?=?2.78, 30.59), but no associations observed among men with TT genotype. We also found interactions between 4-t-OP and rs4986894 in CYP2C19, and between rs1048943 in CYP1A1, on male infertile risk (Pinter?=?8.09×10?7, Pinter?=?3.73×10?4, respectively).We observed notable interactions between 4-t-OP exposure and metabolism enzyme gene polymorphisms on idiopathic infertility in Han-Chinese men. PMID:23555028

  8. Total solids content: a key parameter of metabolic pathways in dry anaerobic digestion

    PubMed Central

    2013-01-01

    Background In solid-state anaerobic digestion (AD) bioprocesses, hydrolytic and acidogenic microbial metabolisms have not yet been clarified. Since these stages are particularly important for the establishment of the biological reaction, better knowledge could optimize the process performances by process parameters adjustment. Results This study demonstrated the effect of total solids (TS) content on microbial fermentation of wheat straw with six different TS contents ranging from wet to dry conditions (10 to 33% TS). Three groups of metabolic behaviors were distinguished based on wheat straw conversion rates with 2,200, 1,600, and 1,400 mmol.kgVS-1 of fermentative products under wet (10 and 14% TS), dry (19 to 28% TS), and highly dry (28 to 33% TS) conditions, respectively. Furthermore, both wet and dry fermentations showed acetic and butyric acid metabolisms, whereas a mainly butyric acid metabolism occurred in highly dry fermentation. Conclusion Substrate conversion was reduced with no changes of the metabolic pathways until a clear limit at 28% TS content, which corresponded to the threshold value of free water content of wheat straw. This study suggested that metabolic pathways present a limit of TS content for high-solid AD. PMID:24261971

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

    SciTech Connect

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

    2010-02-26

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

  10. Origin, evolution, and metabolic role of a novel glycolytic GAPDH enzyme recruited by land plant plastids.

    PubMed

    Petersen, Jörn; Brinkmann, Henner; Cerff, Rüdiger

    2003-07-01

    NAD-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a cytosolic marker enzyme of eukaryotes (GapC; EC 1.2.1.12). Land plants possess an additional NADP+-dependent enzyme (EC 1.2.1.13) within their chloroplasts which is composed of two subunits, GapA and GapB. Another plastid GAPDH enzyme (GapCp) was recently discovered in gymnosperms and ferns. This novel GapCp is closely related to cytosolic GapC and displays glycolytic NAD+ cosubstrate specificity. Here we show that this new gene GapCp is also present and actively expressed in angiosperms, mosses, and liverworts. Phylogenetic analyses of the available GapC and GapCp sequences suggest that the gene duplication giving rise to GapCp occurred in ancestral charophyte algae. The data are also consistent with a monophyletic origin of charophytes and land plants and further support the view that land plants arose from a Coleochaete-like green alga. Northern hybridizations were employed to study the expression of the genes GapCp, GapC, GapA, and GapB in green and nongreen tissues from pepper (Capsicum annuum). The results demonstrate that GapCp mRNAs are mainly expressed in red pepper fruit and roots, in which the transcript levels of photosynthetic GapA and GapB are downregulated. This suggests that in flowering plants GapCp plays a specific role in glycolytic energy production of nongreen plastids such as chromoplasts and leukoplasts and that angiosperms may be the only land plants where glycolysis is absent in green chloroplasts. PMID:12962302

  11. Inhibition of enzymes of drug metabolism in rat liver by ultrasound and correction by heparin.

    PubMed

    Legonkova, L F; Bushma, M I; Zverinsky, I V; Eismont, K A

    1999-07-01

    After 6 days following the local effect (during operation) of ultrasound (2 Wt/cm2, 1 min) the microsomal fraction showed decreased total content of cytochromes P-450 (P-450), rate of NADPH oxidation, activity of NADPH-cytochrome P450 reductase and P450 IIE1 (aniline as substrate) by 40, 28, 16 and 42 %, respectively. In addition, after 12 days the activities of P450 IIIA1 (ethylmorphine as substrate) and cytosolic sulphobromophthalein glutathione transferase (SBPh-GT) were decreased by 59 and 26 %. The administration of heparin (intramuscularly, 250 ED/kg, in a day, 3 and 6 times) exerted a normalizing effect. The P450 concentration, NADPH oxidation rate and P450 IIB1 activity (amidopyrine as substrate), IIE1 and IIIA1, SBPh-GT and 1-chloro-2,4-dinitrobenzene-GT in microsomes and cytosol exceeded the corresponding values in untreated animals by 31, 40, 68, 224, 68, 42, 24 and 36 %. The administration of heparin to control animals (intramuscularly, 250 and 500 mg/kg, in a day, 5 times) essentially unaffected both the monooxygenase, glucuro- and glutathione-conjugating systems and the elimination of antipyrine (substrate of preferably P-450 IA2) and SBPh (substrate of GT) from rat blood plasma. The experimental results provide evidence for a possible role of endogenous heparin in maintaing the optimal level of the activities of the enzyme systems of xenobiotics microsomal oxidation and conjugation in liver injury. One of the most important functions of the liver is its ability to execute biotransformation of a wide range of xenobiotics and some endogenous substances [1]. The activities of the enzyme systems catalyzing these reactions are under a sophisticated regulatory control. Among the natural factors capable of changing the function of enzymes involved in the xenobiotic biotransformation are vitamins [2], phospholipids [3], hormones [4] and many others. We studied the effect of heparin on the activities of the monooxygenase, glucuro- and glutathione transferase systems of the intact and ultrasound-treated rat liver. The significance of this study consists in the elucidation of a putative participation of heparin in the control of the activities of the enzyme system of xenobiotic biotransformation in the intact liver and under membranous pathology of the organ. PMID:10445398

  12. Multilocus Genotypes of Relevance for Drug Metabolizing Enzymes and Therapy with Thiopurines in Patients with Acute Lymphoblastic Leukemia

    PubMed Central

    Stocco, Gabriele; Franca, Raffaella; Verzegnassi, Federico; Londero, Margherita; Rabusin, Marco; Decorti, Giuliana

    2013-01-01

    Multilocus genotypes have been shown to be of relevance for using pharmacogenomic principles to individualize drug therapy. As it relates to thiopurine therapy, genetic polymorphisms of TPMT are strongly associated with the pharmacokinetics and clinical effects of thiopurines (mercaptopurine and azathioprine), influencing their toxicity and efficacy. We have recently demonstrated that TPMT and ITPA genotypes constitute a multilocus genotype of pharmacogenetic relevance for children with acute lymphoblastic leukemia (ALL) receiving thiopurine therapy. The use of high-throughput genomic analysis allows identification of additional candidate genetic factors associated with pharmacogenetic phenotypes, such as TPMT enzymatic activity: PACSIN2 polymorphisms have been identified by a genome-wide analysis, combining evaluation of polymorphisms and gene expression, as a significant determinant of TPMT activity in the HapMap CEU cell lines and the effects of PACSIN2 on TPMT activity and mercaptopurine induced adverse effects were confirmed in children with ALL. Combination of genetic factors of relevance for thiopurine metabolizing enzyme activity, based on the growing understanding of their association with drug metabolism and efficacy, is particularly promising for patients with pediatric ALL. The knowledge basis and clinical applications for multilocus genotypes of importance for therapy with mercaptopurine in pediatric ALL is discussed in the present review. PMID:23335936

  13. The Effects of Space Flight on Some Liver Enzymes Concerned with Carbohydrate and Lipid Metabolism in Rats

    NASA Technical Reports Server (NTRS)

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

    1978-01-01

    The activities of about 30 enzymes concerned with carbohydrate and lipid metabolism and the levels of glycogen and of individual fatty acids were measured in livers of rats ex- posed to prolonged space flight (18.5 days) aboard COSMOS 986 Biosatellite. When flight stationary, (FS) and flight centrifuged (FC) rats were compared at recovery (R(sub 0)), decrceases in the activities of glycogen phosphorylase, alpha glycerphosphate, acyl transferase, diglyceride acyl transferase, acconitase and Epsilon-phosphogluconate dehydrogenase were noted in the weightless group (FS). The significance of these findings was strengthened since all activities, showing alterations at R(sub 0), returned to normal 25 days post-flight. Differences were also seen in levels of two liver constituents. When glycogen and total fatty acids of the two groups of flight animals were determined, differences that could be attributed to reduced gravity were observed, the FS group at R(sub 0) contained, on the average, more than twice the amount of glycogen than did controls ad a remarkable shift in the ratio of palmitate to palmitoleate were noted. These metabolic alterations appear to be unique to the weightless condition. Our data justify the conclusion that centrifugation during space flight is equivalent to terrestrial gravity.

  14. Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus.

    PubMed

    English, T E; Storey, K B

    2000-04-01

    AMP deaminase (AMPD) and adenylate kinase (AK) were purified from skeletal muscle of the white-tailed prairie dog, Cynomus leucurus, and enzyme properties were assayed at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C) to analyze their role in adenylate metabolism during hibernation. Total adenylates decreased in muscle of torpid individuals from 6.97 +/- 0. 31 to 4.66 +/- 0.58 micromol/g of wet weight due to a significant drop in ATP but ADP, AMP, IMP, and energy charge were unchanged. The affinity of prairie dog AMPD for AMP was not affected by temperature and did not differ from that of rabbit muscle AMPD, used for comparison. However, both prairie dog and rabbit AMPD showed much stronger inhibition by ions and GTP at 5 degrees C, versus 37 degrees C, and inhibition by inorganic phosphate, NH(4)Cl, and (NH(4))(2)SO(4) was much stronger at 5 degrees C for the prairie dog enzyme. Furthermore, ATP and ADP, which activated AMPD at 37 degrees C, were strong inhibitors of prairie dog AMPD at 5 degrees C, with I(50) values of 1 and 14 microM, respectively. ATP also inhibited rabbit AMPD at 5 degrees C (I(50) = 103 microM). Strong inhibition of AMPD at 5 degrees C by several effectors suggests that enzyme function is specifically suppressed in muscle of hibernating animals. By contrast, AK showed properties that would maintain or even enhance its function at low temperature. K(m) values for substrates (ATP, ADP, AMP) decreased with decreasing temperature, the change in K(m) ATP paralleling the decrease in muscle ATP concentration. AK inhibition by ions was also reduced at 5 degrees C. The data suggest that adenylate degradation via AMPD is blocked during hibernation but that AK maintains its function in stabilizing energy charge. PMID:10729194

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

    PubMed Central

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

    2015-01-01

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

  16. The multifaceted roles of metabolic enzymes in the Paracoccidioides species complex

    PubMed Central

    Marcos, Caroline M.; de Oliveira, Haroldo C.; da Silva, Julhiany de F.; Assato, Patrícia A.; Fusco-Almeida, Ana M.; Mendes-Giannini, Maria J. S.

    2014-01-01

    Paracoccidioides species are dimorphic fungi and are the etiologic agents of paracoccidioidomycosis, which is a serious disease that involves multiple organs. The many tissues colonized by this fungus suggest a variety of surface molecules involved in adhesion. A surprising finding is that most enzymes in the glycolytic pathway, tricarboxylic acid (TCA) cycle and glyoxylate cycle in Paracoccidioides spp. have adhesive properties that aid in interacting with the host extracellular matrix and thus act as ‘moonlighting’ proteins. Moonlighting proteins have multiple functions, which adds a dimension to cellular complexity and benefit cells in several ways. This phenomenon occurs in both eukaryotes and prokaryotes. For example, moonlighting proteins from the glycolytic pathway or TCA cycle can play a role in bacterial pathogenesis by either acting as proteins secreted in a conventional pathway and/or as cell surface components that facilitate adhesion or adherence. This review outlines the multifunctionality exhibited by many Paracoccidioides spp. enzymes, including aconitase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate lyase, malate synthase, triose phosphate isomerase, fumarase, and enolase. We discuss the roles that moonlighting activities play in the virulence characteristics of this fungus and several other human pathogens during their interactions with the host. PMID:25566229

  17. Effects of supplemental zinc source and level on antioxidant ability and fat metabolism-related enzymes of broilers.

    PubMed

    Liu, Z H; Lu, L; Wang, R L; Lei, H L; Li, S F; Zhang, L Y; Luo, X G

    2015-11-01

    The objective of the present study was to investigate the effects of dietary supplemental Zinc (Zn) source and level on antioxidant ability and fat metabolism-related enzymes of broilers. Dietary treatments included the Zn-unsupplemented corn-soybean meal basal diet (control) and basal diets supplemented with 60, 120, or 180 mg Zn/kg as Zn sulfate, Zn amino acid chelate with a weak chelation strength of 6.5 quotient of formation (Qf) (11.93% Zn) (Zn-AA W), Zn proteinate with a moderate chelation strength of 30.7 Qf (13.27% Zn) (Zn-Pro M), or Zn proteinate with an extremely strong chelation strength of 944.0 Qf (18.61% Zn) (Zn-Pro S). The results showed that dietary supplemental Zn increased (P < 0.01) Zn contents in the liver, breast, and thigh muscles of broilers, and up-regulated mRNA expressions of copper and Zn containing superoxide dismutase (CuZnSOD) and metallothioneins (MT) in the liver (P < 0.01) and thigh muscle (P < 0.05), and also enhanced (P < 0.05) CuZnSOD activities in the breast and thigh muscles, which exerted antioxidant ability and a decreased malondialdehyde (MDA) level in the liver (P < 0.01) and breast and thigh muscles (P < 0.05) of broilers. Furthermore, supplemental Zn increased activities of malate dehydrogenase (MDH) and lipoprotein lipase (LPL) in the abdominal fat (P < 0.05), and fatty acid synthetase (FAS) and LPL in the liver (P < 0.01), which were accompanied with up-regulation (P < 0.01) of the mRNA expressions levels of these enzymes in the abdominal fat and liver of broilers. Dietary Zn source, and an interaction between Zn source and level, had no effects on any measurements. It is concluded that dietary Zn supplementation improved Zn status and resulted in promoting antioxidant ability and activities and gene expressions of fat metabolism-related enzymes of broilers regardless of Zn source and level, and the addition of 60 mg Zn/kg to the corn-soybean meal basal diet (a total dietary Zn of approximately 90 mg/kg) was appropriate for improving the above aspects of broilers. PMID:26500268

  18. The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions

    PubMed Central

    Adler, Philipp; Frey, Lasse Jannis; Berger, Antje; Bolten, Christoph Josef; Hansen, Carl Erik

    2014-01-01

    Acetic acid bacteria (AAB) play an important role during cocoa fermentation, as their main product, acetate, is a major driver for the development of the desired cocoa flavors. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. A carefully designed combination of parallel 13C isotope labeling experiments allowed the elucidation of intracellular fluxes in the complex environment of cocoa pulp, when lactate and ethanol were included as primary substrates among undefined ingredients. We demonstrate that AAB exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for the formation of acetoin and biomass building blocks. Although this is suboptimal for cellular energetics, this allows maximized growth and conversion rates. The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. In fact, gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. As lactate and ethanol are individually supplied by lactic acid bacteria and yeasts during the initial phase of cocoa fermentation, respectively, this underlines the importance of a well-balanced microbial consortium for a successful fermentation process. Indeed, AAB performed the best and produced the largest amounts of acetate in mixed culture experiments when lactic acid bacteria and yeasts were both present. PMID:24837393

  19. The use of enzyme therapy to regulate the metabolic and phenotypic consequences of adenosine deaminase deficiency in mice. Differential impact on pulmonary and immunologic abnormalities.

    PubMed

    Blackburn, M R; Aldrich, M; Volmer, J B; Chen, W; Zhong, H; Kelly, S; Hershfield, M S; Datta, S K; Kellems, R E

    2000-10-13

    Adenosine deaminase (ADA) deficiency results in a combined immunodeficiency brought about by the immunotoxic properties of elevated ADA substrates. Additional non-lymphoid abnormalities are associated with ADA deficiency, however, little is known about how these relate to the metabolic consequences of ADA deficiency. ADA-deficient mice develop a combined immunodeficiency as well as severe pulmonary insufficiency. ADA enzyme therapy was used to examine the relative impact of ADA substrate elevations on these phenotypes. A "low-dose" enzyme therapy protocol prevented the pulmonary phenotype seen in ADA-deficient mice, but did little to improve their immune status. This treatment protocol reduced metabolic disturbances in the circulation and lung, but not in the thymus and spleen. A "high-dose" enzyme therapy protocol resulted in decreased metabolic disturbances in the thymus and spleen and was associated with improvement in immune status. These findings suggest that the pulmonary and immune phenotypes are separable and are related to the severity of metabolic disturbances in these tissues. This model will be useful in examining the efficacy of ADA enzyme therapy and studying the mechanisms underlying the immunodeficiency and pulmonary phenotypes associated with ADA deficiency. PMID:10908569

  20. Enzyme oscillation can enhance the thermodynamic efficiency of cellular metabolism: Consequence of anti-phase coupling between reaction flux and affinity

    E-print Network

    Yusuke Himeoka; Kunihiko Kaneko

    2015-11-12

    Cells generally convert nutrient resources to useful products via energy transduction. Accordingly, the thermodynamic efficiency of this conversion process is one of the most essential characteristics of living organisms. However, although these processes occur under conditions of dynamic metabolism, most studies of cellular thermodynamic efficiency have been restricted to examining steady states; thus, the relevance of dynamics to this efficiency has not yet been elucidated. Here, we develop a simple model of metabolic reactions with anabolism-catabolism coupling catalysed by enzymes. Through application of external oscillation in the enzyme abundances, the thermodynamic efficiency of metabolism was found to be improved. This result is in strong contrast with that observed in the oscillatory input, in which the efficiency always decreased with oscillation. This improvement was effectively achieved by separating the anabolic and catabolic reactions, which tend to disequilibrate each other, and taking advantage of the temporal oscillations so that each of the antagonistic reactions could progress near equilibrium. In this case, anti-phase oscillation between the reaction flux and chemical affinity through oscillation of enzyme abundances is essential. This improvement was also confirmed in a model capable of generating autonomous oscillations in enzyme abundances. Finally, the possible relevance of the improvement in thermodynamic efficiency is discussed with respect to the potential for manipulation of metabolic oscillations in microorganisms.

  1. Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Opportunitistic Enzymes, Catalytic Promiscuity and the Evolution of chemodiversity in Nature (2010 JGI User Meeting)

    ScienceCinema

    Noel, Joseph

    2011-04-25

    Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature" on March 26, 2010 at the 5th Annual DOE JGI User Meeting

  2. THE IMPORTANCE OF OBTAINING INFORMATION ON THE SPECIFIC CONTENT OF TISSUE ENZYMES METABOLIZING ORGANOPHOSPHORUS PESTICIDES, PRIOR TO DETERMINE VMAX, KM VALUES FOR USE IN PBPK MODELS

    EPA Science Inventory

    Physiological pharmacokinetic/pharmacodynamic models require Vmax, Km values for the metabolism of OPs by tissue enzymes. Current literature values cannot be easily used in OP PBPK models (i.e., parathion and chlorpyrifos) because standard methodologies were not used in their ...

  3. THE IMPORTANCE OF OBTAINING INFORMATION ON THE SPECIFIC CONTENT OF TISSUE ENZYMES METABOLIZING ORGANOPHOSPHORUS PESTICIDES, PRIOR TO DETERMINING VMAX, KM VALUES FOR USE IN PBPK MODELS

    EPA Science Inventory

    Physiological pharmacokinetic\\pharmacodynamic models require Vmax, Km values for the metabolism of OPs by tissue enzymes. Current literature values cannot be easily used in OP PBPK models (i.e., parathion and chlorpyrifos) because standard methodologies were not used in their ...

  4. Genetic basis and implications of cross-sensitivity in Zea mays L. (sweet corn) to multiple herbicides metabolized by cytochrome P-450 enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Certain Zea mays L. (sweet corn) hybrids and inbreds can be injured or killed following postemergence applications of several herbicides metabolized by cytochrome P-450 enzymes. Identification of sensitive hybrids in annual screening trials, the primary means of guiding weed management decisions, i...

  5. COMPARISON OF IN VIVO AND IN VITRO METHODS FOR ASSESSING THE EFFECTS OF REPEATED DOSING WITH CARBON TETRACHLORIDE ON THE HEPATIC DRUG-METABOLIZING ENZYME SYSTEM (JOURNAL VERSION)

    EPA Science Inventory

    The effect of a single i.p. injection of 0, 20, 200, and 1000 microliters/kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay, employing lindane (gamma-hexachlorocyclohexane), and by a battery o...

  6. Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Opportunitistic Enzymes, Catalytic Promiscuity and the Evolution of chemodiversity in Nature (2010 JGI User Meeting)

    SciTech Connect

    Noel, Joseph

    2010-03-26

    Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature" on March 26, 2010 at the 5th Annual DOE JGI User Meeting

  7. Effects of oregano essential oil with or without feed enzymes on growth performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal diets.

    PubMed

    Basmacio?lu Malayo?lu, H; Baysal, S; Misirlio?lu, Z; Polat, M; Yilmaz, H; Turan, N

    2010-02-01

    1. The study was conducted to determine the effects of dietary supplementation of enzyme and oregano essential oil at two levels, alone or together, on performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal based diets. 2. The following dietary treatments were used from d 0 to 21. Diet 1 (control, CONT): a commercial diet containing no enzyme or oregano essential oil, diet 2 (ENZY): supplemented with enzyme, diet 3 (EO250): supplemented with essential oil at 250 mg/kg feed, diet 4 (EO500): supplemented with essential oil at 500 mg/kg feed, diet 5 (ENZY + EO250): supplemented with enzyme and essential oil at 250 mg/kg, and diet 6 (ENZY + EO500): supplemented with enzyme and essential oil at 500 mg/kg. 3. Birds fed on diets containing ENZY, EO250 and ENZY + EO250 had significantly higher weight gain than those given CONT diet from d 0 to 7. No significant effects on feed intake, feed conversion ratio, mortality, organ weights except for jejunum weight and intestinal lengths was found with either enzyme or essential oil, alone or in combination, over the 21-d growth period. The supplementation of essential oil together with enzyme decreased jejunum weight compared with essential oil alone. 4. Supplementation with enzyme significantly decreased viscosity and increased dry matter of digesta, but did not alter pH of digesta. There was no effect of essential oil alone at either concentration on viscosity, dry matter or pH of digesta. A significant decrease in viscosity of digesta appeared when essential oil was used with together enzyme. 5. The supplementation of essential oil at both levels with or without enzyme significantly increased chymotrypsin activity in the digestive system, and improved crude protein digestibility. 6. The higher concentration of essential oil with and without enzyme significantly increased serum total cholesterol concentrations. No significant effect on immune response was found with either enzyme or essential oil, alone or together. 7. Enzymes and essential oil had different modes of actions. The supplementation of enzyme with essential oil in diets is likely more effective in view of performance, nutrient digestibility, enzyme activities and immune system. PMID:20390571

  8. Alterations in antioxidant metabolism and associated enzymes in pea (Pisum sativum) exposed to sulfur dioxide

    SciTech Connect

    Madamanchi, N.R.; Alscher, R.G. )

    1990-05-01

    The response of glutathione and ascorbate and the enzymes glutathione reductase (GR) and superoxide dismutase (SOD) was studied in two cultivars of pea known to be differentially sensitive to SO{sub 2} (0.8 ppm). Total glutathione accumulated more rapidly on exposure to SO{sub 2} in insensitive cultivar Progress compared to the sensitive cultivar Nugget, confirming our previous results. However, corresponding changes in oxidized glutathione were not observed and ascorbate levels did not change over the course of the exposure. Changes in the activity of GR corresponded to the changes in total glutatione levels. Preliminary results indicate that SOD activity increased to a significantly higher extent in Progress than in Nugget. These data suggest a significant role for GR and possibly SOD in resistance to oxidative stress.

  9. Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

    PubMed Central

    Yamamoto, Y T; Zamski, E; Williamson, J D; Conkling, M A; Pharr, D M

    1997-01-01

    Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown. PMID:9414553

  10. Effect of the triazolobenzodiazepine estazolam on hepatic drug-metabolizing enzyme activity in rats.

    PubMed

    Down, W H; Hawkins, D R; Chasseaud, L F

    1982-07-01

    Oral doses of the sedative/hypnotic estazolam (500 mg kg-1 day-1) to rats for 21 days caused statistically significant increases in liver weight, ascorbate excretion, cytochrome P-450 concentrations, and in aniline hydroxylase, ethylmorphine N-demethylase and glutathione S-transferase activities, as did approximately equivalent doses of flurazepam hydrochloride. Histologically, the centrilobular hepatocytes were enlarged. Some of these parameters were also increased after doses of estazolam of 100 mg kg-1 day-1, but not after 5 mg kg-1 day-1, which is about 50-fold greater than a clinical dose. Estazolam was a much less potent enzyme inducer than phenobarbitone under the conditions of these studies. PMID:6126539

  11. Enzyme inhibition of dopamine metabolism alters 6-[18F]FDOPA uptake in orthotopic pancreatic adenocarcinoma

    PubMed Central

    2013-01-01

    Background An unknown location hampers removal of pancreatic tumours. We studied the effects of enzyme inhibitors on the uptake of 6-[18F]fluoro-l-3,4-dihydroxyphenylalanine ([18F]FDOPA) in the pancreas, aiming at improved imaging of pancreatic adenocarcinoma. Methods Mice bearing orthotopic BxPC3 pancreatic adenocarcinoma were injected with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) and scanned with positron emission tomography/computed tomography (PET/CT). For [18F]FDOPA studies, tumour-bearing mice and sham-operated controls were pretreated with enzyme inhibitors of aromatic amino acid decarboxylase (AADC), catechol-O-methyl transferase (COMT), monoamine oxidase A (MAO-A) or a combination of COMT and MAO-A. Mice were injected with [18F]FDOPA and scanned with PET/CT. The absolute [18F]FDOPA uptake was determined from selected tissues using a gamma counter. The intratumoural biodistribution of [18F]FDOPA was recorded by autoradiography. The main [18F]FDOPA metabolites present in the pancreata were determined with radio-high-performance liquid chromatography. Results [18F]FDG uptake was high in pancreatic tumours, while [18F]FDOPA uptake was highest in the healthy pancreas and significantly lower in tumours. [18F]FDOPA uptake in the pancreas was lowest with vehicle pretreatment and highest with pretreatment with the inhibitor of AADC. When mice received COMT?+?MAO-A inhibitors, the uptake was high in the healthy pancreas but low in the tumour-bearing pancreas. Conclusions Combined use of [18F]FDG and [18F]FDOPA is suitable for imaging pancreatic tumours. Unequal pancreatic uptake after the employed enzyme inhibitors is due to the blockade of metabolism and therefore increased availability of [18F]FDOPA metabolites, in which uptake differs from that of [18F]FDOPA. Pretreatment with COMT?+?MAO-A inhibitors improved the differentiation of pancreas from the surrounding tissue and healthy pancreas from tumour. Similar advantage was not achieved using AADC enzyme inhibitor, carbidopa. PMID:23497589

  12. Enzyme oscillation can enhance the thermodynamic efficiency of cellular metabolism: Consequence of anti-phase coupling between reaction flux and affinity

    E-print Network

    Himeoka, Yusuke

    2015-01-01

    Cells generally convert nutrient resources to useful products via energy transduction. Accordingly, the thermodynamic efficiency of this conversion process is one of the most essential characteristics of living organisms. However, although these processes occur under conditions of dynamic metabolism, most studies of cellular thermodynamic efficiency have been restricted to examining steady states; thus, the relevance of dynamics to this efficiency has not yet been elucidated. Here, we develop a simple model of metabolic reactions with anabolism-catabolism coupling catalysed by enzymes. Through application of external oscillation in the enzyme abundances, the thermodynamic efficiency of metabolism was found to be improved. This result is in strong contrast with that observed in the oscillatory input, in which the efficiency always decreased with oscillation. This improvement was effectively achieved by separating the anabolic and catabolic reactions, which tend to disequilibrate each other, and taking advantag...

  13. Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, Leads to Altered Carbohydrate Metabolism in Cancer Cells.

    PubMed

    Tan, Bo; Dong, Sucai; Shepard, Robert L; Kays, Lisa; Roth, Kenneth D; Geeganage, Sandaruwan; Kuo, Ming-Shang; Zhao, Genshi

    2015-06-19

    Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD(+) biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD(+) biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500-3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed. PMID:25944913

  14. The protein kinase Sch9 is a key regulator of sphingolipid metabolism in Saccharomyces cerevisiae

    PubMed Central

    Swinnen, Erwin; Wilms, Tobias; Idkowiak-Baldys, Jolanta; Smets, Bart; De Snijder, Pepijn; Accardo, Sabina; Ghillebert, Ruben; Thevissen, Karin; Cammue, Bruno; De Vos, Dirk; Bielawski, Jacek; Hannun, Yusuf A.; Winderickx, Joris

    2014-01-01

    The Saccharomyces cerevisiae protein kinase Sch9 is an in vitro and in vivo effector of sphingolipid signaling. This study examines the link between Sch9 and sphingolipid metabolism in S. cerevisiae in vivo based on the observation that the sch9? mutant displays altered sensitivity to different inhibitors of sphingolipid metabolism, namely myriocin and aureobasidin A. Sphingolipid profiling indicates that sch9? cells have increased levels of long-chain bases and long-chain base-1 phosphates, decreased levels of several species of (phyto)ceramides, and altered ratios of complex sphingolipids. We show that the target of rapamycin complex 1–Sch9 signaling pathway functions to repress the expression of the ceramidase genes YDC1 and YPC1, thereby revealing, for the first time in yeast, a nutrient-dependent transcriptional mechanism involved in the regulation of sphingolipid metabolism. In addition, we establish that Sch9 affects the activity of the inositol phosphosphingolipid phospholipase C, Isc1, which is required for ceramide production by hydrolysis of complex sphingolipids. Given that sphingolipid metabolites play a crucial role in the regulation of stress tolerance and longevity of yeast cells, our data provide a model in which Sch9 regulates the latter phenotypes by acting not only as an effector but also as a regulator of sphingolipid metabolism. PMID:24196832

  15. Intestinal bile acid sensing is linked to key endocrine and metabolic signalng pathways

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acids have historically been considered to mainly function in cholesterol homeostasis and facilitate fat digestion in the gastrointestinal tract. Recent discoveries show that bile acids also function as signaling molecules that exert diverse endocrine and metabolic actions by activating G prote...

  16. Regulation of the expression of key genes involved in HDL metabolism by unsaturated fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine the effects, and possible mechanisms of action, of unsaturated fatty acids on the expression of genes involved in HDL metabolism in HepG2 cells. The mRNA concentration of target genes was assessed by real time PCR. Protein concentrations were determined by wes...

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

    USGS Publications Warehouse

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

    2002-01-01

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

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

    USGS Publications Warehouse

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

    2002-01-01

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

  19. Sodium nitroprusside affects the level of photosynthetic enzymes and glucose metabolism in Phaseolus aureus (mung bean).

    PubMed

    Lum, Hon-Kei; Lee, Chi-Ho; Butt, Yoki Kwok-Chu; Lo, Samuel Chun-Lap

    2005-06-01

    Nitric oxide (NO) is an important signaling molecule in plants. The present study aims to investigate the downstream signaling pathways of NO in plants using a proteomic approach. Phaseolus aureus (mung bean) leaf was treated with sodium nitroprusside (SNP), which releases nitric oxide in the form of nitrosonium cation (NO+) upon light irradiation. Changes in protein expression profiles of the SNP treated mung bean leaf were analyzed by two-dimensional gel electrophoresis (2-DE). Comparison of 2-DE electropherograms revealed seven down-regulated and two up-regulated proteins after treatment with 0.5 mM SNP for 6 h. The identities of these proteins were analyzed by a combination of peptide mass fingerprinting and post-source decay using a matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometer. Six out of these nine proteins found are involved in either photosynthesis or cellular metabolism. We have taken our investigation further by studying the effect of NO+ on glucose contents in mung bean leaves. Our results clearly demonstrated that NO+ rapidly and drastically decrease the amount of glucose in mung bean leaves. Moreover, four out of nine of these proteins are chloroplastic isoforms. These results suggested that chloroplasts might be one of the main sub-cellular targets of NO in plants. PMID:15917215

  20. Identification of the putative tumor suppressor Nit2 as omega-amidase, an enzyme metabolically linked to glutamine and asparagine transamination.

    PubMed

    Krasnikov, Boris F; Chien, Chin-Hsiang; Nostramo, Regina; Pinto, John T; Nieves, Edward; Callaway, Myrasol; Sun, Jin; Huebner, Kay; Cooper, Arthur J L

    2009-09-01

    The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is omega-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated omega-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are alpha-ketoglutaramate and alpha-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of alpha-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating alpha-keto-gamma-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess alpha-ketoglutaramate may be neurotoxic. Moreover, alpha-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of omega-amidase is to remove potentially toxic intermediates by converting alpha-ketoglutaramate and alpha-ketosuccinamate to biologically useful alpha-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of omega-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology. PMID:19595734

  1. Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering1[W][OA

    PubMed Central

    Pang, Yongzhen; Abeysinghe, I. Sarath B.; He, Ji; He, Xianzhi; Huhman, David; Mewan, K. Mudith; Sumner, Lloyd W.; Yun, Jianfei; Dixon, Richard A.

    2013-01-01

    Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (?)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (?)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols. PMID:23288883

  2. Toxicity of xanthene food dyes by inhibition of human drug-metabolizing enzymes in a noncompetitive manner.

    PubMed

    Mizutani, Takaharu

    2009-01-01

    The synthetic food dyes studied were rose bengal (RB), phroxine (PL), amaranth, erythrosine B (ET), allura red, new coccine, acid red (AR), tartrazine, sunset yellow FCF, brilliant blue FCF, and indigo carmine. First, data confirmed that these dyes were not substrates for CYP2A6, UGT1A6, and UGT2B7. ET inhibited UGT1A6 (glucuronidation of p-nitrophenol) and UGT2B7 (glucuronidation of androsterone). We showed the inhibitory effect of xanthene dye on human UGT1A6 activity. Basic ET, PL, and RB in those food dyes strongly inhibited UGT1A6 activity, with IC(50) values = 0.05, 0.04, and 0.015 mM, respectively. Meanwhile, AR of an acidic xanthene food dye showed no inhibition. Next, we studied the inhibition of CYP3A4 of a major phase I drug-metabolizing enzyme and P-glycoprotein of a major transporter by synthetic food dyes. Human CYP3A4 and P-glycoprotein were also inhibited by basic xanthene food dyes. The IC(50) values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the inhibition level of UGT1A6 by three halogenated xanthene food dyes (ET, PL, and RB) described above, except AR, like the results with UGT1A6 and UGT2B7. We also confirmed the noninhibition of CYP3A4 and P-gp by other synthetic food dyes. Part of this inhibition depended upon the reaction of (1)O(2) originating on xanthene dyes by light irradiation, because inhibition was prevented by (1)O(2) quenchers. We studied the influence of superoxide dismutase and catalase on this inhibition by dyes and we found prevention of inhibition by superoxide dismutase but not catalase. This result suggests that superoxide anions, originating on dyes by light irradiation, must attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins on skin under lighting and may lead to rough skin. PMID:20041016

  3. Toxicity of Xanthene Food Dyes by Inhibition of Human Drug-Metabolizing Enzymes in a Noncompetitive Manner

    PubMed Central

    Mizutani, Takaharu

    2009-01-01

    The synthetic food dyes studied were rose bengal (RB), phroxine (PL), amaranth, erythrosine B (ET), allura red, new coccine, acid red (AR), tartrazine, sunset yellow FCF, brilliant blue FCF, and indigo carmine. First, data confirmed that these dyes were not substrates for CYP2A6, UGT1A6, and UGT2B7. ET inhibited UGT1A6 (glucuronidation of p-nitrophenol) and UGT2B7 (glucuronidation of androsterone). We showed the inhibitory effect of xanthene dye on human UGT1A6 activity. Basic ET, PL, and RB in those food dyes strongly inhibited UGT1A6 activity, with IC50 values = 0.05, 0.04, and 0.015 mM, respectively. Meanwhile, AR of an acidic xanthene food dye showed no inhibition. Next, we studied the inhibition of CYP3A4 of a major phase I drug-metabolizing enzyme and P-glycoprotein of a major transporter by synthetic food dyes. Human CYP3A4 and P-glycoprotein were also inhibited by basic xanthene food dyes. The IC50 values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the inhibition level of UGT1A6 by three halogenated xanthene food dyes (ET, PL, and RB) described above, except AR, like the results with UGT1A6 and UGT2B7. We also confirmed the noninhibition of CYP3A4 and P-gp by other synthetic food dyes. Part of this inhibition depended upon the reaction of 1O2 originating on xanthene dyes by light irradiation, because inhibition was prevented by 1O2 quenchers. We studied the influence of superoxide dismutase and catalase on this inhibition by dyes and we found prevention of inhibition by superoxide dismutase but not catalase. This result suggests that superoxide anions, originating on dyes by light irradiation, must attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins on skin under lighting and may lead to rough skin. PMID:20041016

  4. CYP1D1, pseudogenized in human, is expressed and encodes a functional drug-metabolizing enzyme in cynomolgus monkey.

    PubMed

    Uno, Yasuhiro; Uehara, Shotaro; Murayama, Norie; Yamazaki, Hiroshi

    2011-02-01

    Cytochrome P450 (P450 or CYP) 1 family consists of the CYP1A, CYP1B, CYP1C, and CYP1D subfamilies. In the human genome, CYP1A1, CYP1A2, and CYP1B1 are expressed and encode functional enzymes, whereas CYP1D1P (formerly known as CYP1A8P) is present as a pseudogene due to five nonsense mutations in the putative coding region. In this study, we identified CYP1D1 cDNA, highly identical (nearly 95%) to human CYP1D1P sequence, in cynomolgus monkey, a species frequently used in drug metabolism studies due to its evolutionary closeness to human. The amino acid sequence deduced from cynomolgus monkey CYP1D1 cDNA shared the high sequence identity (91%) with human CYP1D1P (postulated from the gene sequence), and the highest sequence identity (44-45%) with CYP1A1 and CYP1A2 among cynomolgus monkey P450s. CYP1D1 mRNA was most abundantly expressed in liver, followed by kidney, and jejunum. The hepatic expression level of CYP1D1 mRNA was comparable to that of CYP1A1 mRNA and much higher than that of CYP1A2 mRNA. CYP1D1 was barely detectable in immunoblots of cynomolgus monkey liver. Cynomolgus monkey CYP1D1 mRNA was induced in primary hepatocytes with omeprazole. Cynomolgus monkey CYP1D1 protein heterologously expressed in Escherichia coli catalyzed ethoxyresorufin O-deethylation and caffeine 8-hydroxylation, which CYP1As also catalyze. Finally, no nonsense mutations, corresponding to those found in human CYP1D1P, were found in the 20 cynomolgus monkeys and 10 rhesus monkeys used in this study. These results suggest that CYP1D1 plays a role as a functional, drug-metabolizing enzyme in cynomolgus monkey liver. PMID:21070747

  5. Unlocking the Key to Personalized Cancer Medicine Using Tumor Metabolism | Physical Sciences in Oncology

    Cancer.gov

    Identifying genetic alterations in cancer patients to predict clinical outcome has been the cornerstone of cancer research for nearly three decades, but now researchers at the Kimmel Cancer Center at the Thomas Jefferson University have come up with a new approach that instead links cancer cell metabolism with poor clinical outcome. This approach can now be applied to virtually any type of human cancer cell.

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

    PubMed

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

    2007-08-01

    Tuberous roots of yacon (Smallanthus sonchifolius) accumulate about 10%, on a fresh weight basis, of inulin-type fructooligosacharides (FOSs), known as a food ingredient with various healthy benefits. However, we have a great difficulty to ensure these benefits because FOSs with a lower degree of polymerization (DP) decreased remarkably, and fructose increased when the tuberous roots were stored after harvesting even under previously recommended storage conditions of low temperature with high humidity. In the present study, to elucidate the involvement of FOS-metabolizing enzymes in FOS reduction during storage at 90% relative humidity and 8 degrees C, we extracted a crude protein from yacon tuberous roots and measured the activities of invertase (beta-fructofuranosidase, EC 3.2.1.26), sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan 1-exohydrolase (1-FEH, EC 3.2.1.80). The enzyme activities acting on sucrose, both invertase and 1-SST, were weakened after storage for a month. In addition, the activity of 1-FEH acting on short FOSs such as 1-kestose (GF(2)) and 1-nystose (GF(3)) was higher than that of 1-FFT. These results suggest that the continuous decline in FOSs of low DP during storage was dependent mainly on the 1-FEH activity. On the other hand, FOSs with a DP of >or= 9 only slightly decreased in stored yacon tuberous roots during storage, though distinct 1-FEH activity was observed in vitro toward a high-DP inulin-type substrate, indicating that highly polymerized FOSs content was unlikely to be closely connected with the 1-FEH activity. PMID:17995694

  7. Energy expenditure in Crocidurinae shrews (Insectivora): is metabolism a key component of the insular syndrome?

    PubMed

    Magnanou, Elodie; Fons, Roger; Blondel, Jacques; Morand, Serge

    2005-11-01

    A cascade of morphological, ecological, demographical and behavioural changes operates within island communities compared to mainland. We tested whether metabolic rates change on islands. Using a closed circuit respirometer, we investigated resting metabolic rate (RMR) of three species of Crocidurinae shrews: Suncus etruscus, Crocidura russula, and C. suaveolens. For the latter, we compared energy expenditure of mainland and island populations. Our measurements agree with those previously reported for others Crocidurinae: the interspecific comparison (ANCOVA) demonstrated an allometric relation between energy requirements and body mass. Energy expenditure also scaled with temperature. Island populations (Corsica and Porquerolles) of C. suaveolens differed in size from mainland (gigantism). A GLM showed a significant relationship between energy expenditure, temperature, body mass and locality. Mass specific RMR allometrically scales body mass, but total RMR does not significantly differ between mainland and island, although island shrews are giant. Our results are consistent with other studies: that demonstrated that the evolution of mammalian metabolism on islands is partially independent of body mass. In relation to the insular syndrome, we discuss how island selective forces (changes in resource availability, decrease in competition and predation pressures) can operate in size and physiological adjustments. PMID:16154371

  8. Coactivator PGC-1{alpha} regulates the fasting inducible xenobiotic-metabolizing enzyme CYP2A5 in mouse primary hepatocytes

    SciTech Connect

    Arpiainen, Satu; Jaervenpaeae, Sanna-Mari; Manninen, Aki; Viitala, Pirkko; Lang, Matti A.; Pelkonen, Olavi; Hakkola, Jukka

    2008-10-01

    The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1{alpha} and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1{alpha} expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1{alpha} expression vector demonstrated that PGC-1{alpha} is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4{alpha} response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1{alpha} binds, together with HNF-4{alpha}, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1{alpha} mediates the expression of CYP2A5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4{alpha}. This strongly suggests that PGC-1{alpha} is the major factor mediating the fasting response of CYP2A5.

  9. Expression of membrane transporters and metabolic enzymes involved in estrone-3-sulphate disposition in human breast tumour tissues.

    PubMed

    Banerjee, Nilasha; Miller, Naomi; Allen, Christine; Bendayan, Reina

    2014-06-01

    Two-thirds of newly diagnosed hormone-dependent (HR?) breast cancers are detected in post-menopausal patients where estrone-3-sulphate (E3S) is the predominant source for tumour estradiol. Understanding intra-tumoral fate of E3S would facilitate in the identification of novel molecular targets for HR? post-menopausal breast cancer patients. Hence this study investigates the clinical expression of (i) organic anion-transporting polypeptides (OATPs), (ii) multidrug resistance protein (MRP-1), breast cancer resistance proteins (BCRP), and (iii) sulphatase (STS), 17?-hydroxysteroid dehydrogenase (17?-HSD-1), involved in E3S uptake, efflux and metabolism, respectively. Fluorescent and brightfield images of stained tumour sections (n = 40) were acquired at 4× and 20× magnification, respectively. Marker densities were measured as the total area of positive signal divided by the surface area of the tumour section analysed and was reported as % area (ImageJ software). Tumour, stroma and non-tumour tissue areas were also quantified (Inform software), and the ratio of optical intensity per histologic area was reported as % area/tumour, % area/stroma and % area/non-tumour. Functional role of OATPs and STS was further investigated in HR? (MCF-7, T47-D, ZR-75) and HR-(MDA-MB-231) cells by transport studies conducted in the presence or absence of specific inhibitors. Amongst all the transporters and enzymes, OATPs and STS have significantly (p < 0.0001) higher expression in HR? tumour sections with highest target signals obtained from the tumour regions of the tissues. Specific OATP-mediated E3S uptake and STS-mediated metabolism were also observed in all HR? breast cancer cells. These observations suggest the potential of OATPs as novel molecular targets for HR? breast cancers. PMID:24831777

  10. UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for puerarin metabolism in human liver microsomes.

    PubMed

    Luo, Cheng-Feng; Cai, Bin; Hou, Ning; Yuan, Mu; Liu, Shi-Ming; Ji, Hong; Xiong, Long-Gen; Xiong, Wei; Luo, Jian-Dong; Chen, Min-Sheng

    2012-11-01

    Puerarin has multiple pharmacological effects and is widely prescribed for patients with cardiovascular diseases, including hypertension, cerebral ischemia, myocardial ischemia, diabetes mellitus, and arteriosclerosis. While puerarin is a useful therapeutic agent, its mechanisms of action have not been well defined. Understanding puerarin metabolism, in particular its interactions with metabolizing enzymes, will contribute to our understanding of its toxic and therapeutic effects and may help to elucidate potential negative drug-drug interactions. In this study, the major metabolite of puerarin was obtained from the urine of rats administered puerarin, by a semi-preparative high-performance liquid chromatography method. The major metabolite was identified as puerarin-7-O-glucuronide. In vitro, we used a UDP-glucuronosyltransferase (UGT) reaction screening method with 12 recombinant human UGTs to demonstrate that formation of puerarin-7-O-glucuronide was catalyzed by UGT1A1, 1A9, 1A10, 1A3, 1A6, 1A7, and 1A8. UGT1A1, 1A9, and 1A10 significantly catalyzed puerarin-7-O-glucuronide formation, and the activity of UGT1A1 was significantly higher than those of 1A9 and 1A10. The V (max) of UGT1A1 was two- to threefold higher than the levels of UGT1A9 or 1A10, with a lower K ( m ) value and a higher V (max)/K ( m ) value. The kinetics of puerarin-7-O-glucuronide formation catalyzed by UGT1A1 were similar to those of the pooled human liver microsomes (HLMs), with V (max) values of 186.3 and 149.2 pmol/min/mg protein, and K ( m ) values of 811.3 and 838.9 ?M, respectively. Furthermore, bilirubin and ?-estradiol, probe substrates for UGT1A1, significantly inhibited the formation of puerarin-7-O-glucuronide in HLMs. PMID:22648071

  11. Thermostable lipoxygenase, a key enzyme in bioconversion of linoleic acid to trihycroxy-octadecenoic acid by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipoxygenases, enzymes that contain non-heme iron, catalyze the oxidation of unsaturated fatty acids with a (1Z,4Z)-pentadiene moiety leading to conjugated (Z,E)-hydroperoxydienoic acids. These enzymes are widely distributed in plants and animals, and a few microorganisms are reported as well. It ...

  12. Correlation between metabolic enzyme GSTP1 polymorphisms and susceptibility to lung cancer

    PubMed Central

    WANG, YUFEI; REN, BU; ZHANG, LEI; GUO, ZHANLIN

    2015-01-01

    The aim of the present study was to determine the frequency distribution and characteristics of polymorphic alleles and genotypes in glutathione S-transferase ? 1 (GSTP1) exon 5, and to explore the correlation between GSTP1 exon 5 polymorphisms and susceptibility to lung cancer using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Patients were diagnosed with lung cancer from May 2006 to October 2008 by postoperative pathological examination. A total of 150 patients, including 115 males and 35 females, aged 31–76 years (mean, 57.1 years) were enrolled. The control group consisted of 152 healthy volunteers who received physical examination at outpatient clinics. Genomic DNA was extracted from the peripheral venous blood of the 302 subjects, and the GSTP1 genotype was determined by PCR-RFLP and restricted enzyme digestion of PCR products. GSTP1 polymorphisms were analyzed in the 302 subjects. The C and G allele frequencies of GSTP1 in the control and lung cancer groups showed no significant difference (P=0.135); the frequencies of three different genotypes, A/A, A/G and G/G, of GSTP1 in the control and lung cancer groups exhibited no significant differences between the two groups (P=0.223). GSTP1 genotype frequencies in the study population fitted the Hardy-Weinberg equilibrium, demonstrating that the genotype results of this study conform to this genetic law. Overall, 50.7% of the subjects in the lung cancer group carried the non-A/A genotype of GSTP1, which was higher than the 43.4% of the control group. The risk of lung cancer in subjects with the non-A/A genotype was 1.43-fold higher than that in those with the A/A genotype, but no statistical significance was found (P=0.138). GSTP1 exon 5 polymorphisms were demonstrated to be associated with lung cancer susceptibility on the whole. However, stratified analysis suggested the correlation of GSTP1 exon 5 polymorphisms with lung squamous cell carcinoma risk, and that exon 5 polymorphisms might increase the risk of lung squamous cell carcinoma. Exon 5 GSTP1 polymorphisms were not found to be a strong influencing factor in lung cancer risk, but may play a certain role.

  13. Identification of key components in the energy metabolism of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus by transcriptome analyses

    PubMed Central

    Hocking, William P.; Stokke, Runar; Roalkvam, Irene; Steen, Ida H.

    2014-01-01

    Energy conservation via the pathway of dissimilatory sulfate reduction is present in a diverse group of prokaryotes, but is most comprehensively studied in Deltaproteobacteria. In this study, whole-genome microarray analyses were used to provide a model of the energy metabolism of the sulfate-reducing archaeon Archaeoglobus fulgidus, based on comparative analysis of litoautotrophic growth with H2/CO2 and thiosulfate, and heterotrophic growth on lactate with sulfate or thiosulfate. Only 72 genes were expressed differentially between the cultures utilizing sulfate or thiosulfate, whereas 269 genes were affected by a shift in energy source. We identified co-located gene cluster encoding putative lactate dehydrogenases (LDHs; lldD, dld, lldEFG), also present in sulfate-reducing bacteria. These enzymes may take part in energy conservation in A. fulgidus by specifically linking lactate oxidation with APS reduction via the Qmo complex. High transcriptional levels of Fqo confirm an important role of F420H2, as well as a menaquinone-mediated electron transport chain, during heterotrophic growth. A putative periplasmic thiosulfate reductase was identified by specific up-regulation. Also, putative genes for transport of sulfate and sulfite are discussed. We present a model for hydrogen metabolism, based on the probable bifurcation reaction of the Mvh:Hdl hydrogenase, which may inhibit the utilization of Fdred for energy conservation. Energy conservation is probably facilitated via menaquinone to multiple membrane-bound heterodisulfide reductase (Hdr) complexes and the DsrC protein—linking periplasmic hydrogenase (Vht) to the cytoplasmic reduction of sulfite. The ambiguous roles of genes corresponding to fatty acid metabolism induced during growth with H2 are discussed. Putative co-assimilation of organic acids is favored over a homologous secondary carbon fixation pathway, although both mechanisms may contribute to conserve the amount of Fdred needed during autotrophic growth with H2. PMID:24672515

  14. Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[W

    PubMed Central

    Beauvoit, Bertrand P.; Colombié, Sophie; Monier, Antoine; Andrieu, Marie-Hélène; Biais, Benoit; Bénard, Camille; Chéniclet, Catherine; Dieuaide-Noubhani, Martine; Nazaret, Christine; Mazat, Jean-Pierre; Gibon, Yves

    2014-01-01

    A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division. PMID:25139005

  15. Protective Effect of Free and Bound Polyphenol Extracts from Ginger (Zingiber officinale Roscoe) on the Hepatic Antioxidant and Some Carbohydrate Metabolizing Enzymes of Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Kazeem, Mutiu Idowu; Akanji, Musbau Adewunmi; Yakubu, Musa Toyin; Ashafa, Anofi Omotayo Tom

    2013-01-01

    This study investigated the hepatoprotective effects of polyphenols from Zingiber officinale on streptozotocin-induced diabetic rats by assessing liver antioxidant enzymes, carbohydrate-metabolizing enzymes and liver function indices. Initial oral glucose tolerance test was conducted using 125?mg/kg, 250?mg/kg, and 500?mg/kg body weight of both free and bound polyphenols from Z. officinale. 28 day daily oral administration of 500?mg/kg body weight of free and bound polyphenols from Z. officinale to streptozotocin-induced (50?mg/kg) diabetic rats significantly reduced (P < 0.05) the fasting blood glucose compared to control groups. There was significant increase (P < 0.05) in the antioxidant enzymes activities in the animals treated with both polyphenols. Similarly, the polyphenols normalised the activities of some carbohydrate metabolic enzymes (hexokinase and phosphofructokinase) in the liver of the rats treated with it and significantly reduced (P < 0.05) the activities of liver function enzymes. The results from the present study have shown that both free and bound polyphenols from Z. officinale especially the free polyphenol could ameliorate liver disorders caused by diabetes mellitus in rats. This further validates the use of this species as medicinal herb and spice by the larger population of Nigerians. PMID:24367390

  16. Biological definition of multiple chemical sensitivity from redox state and cytokine profiling and not from polymorphisms of xenobiotic-metabolizing enzymes

    SciTech Connect

    De Luca, Chiara; Scordo, Maria G.; Cesareo, Eleonora; Pastore, Saveria; Mariani, Serena; Maiani, Gianluca; Stancato, Andrea; Loreti, Beatrice; Valacchi, Giuseppe; Lubrano, Carla; Raskovic, Desanka; De Padova, Luigia; Genovesi, Giuseppe; Korkina, Liudmila G.

    2010-11-01

    Background: Multiple chemical sensitivity (MCS) is a poorly clinically and biologically defined environment-associated syndrome. Although dysfunctions of phase I/phase II metabolizing enzymes and redox imbalance have been hypothesized, corresponding genetic and metabolic parameters in MCS have not been systematically examined. Objectives: We sought for genetic, immunological, and metabolic markers in MCS. Methods: We genotyped patients with diagnosis of MCS, suspected MCS and Italian healthy controls for allelic variants of cytochrome P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A5), UDP-glucuronosyl transferase (UGT1A1), and glutathione S-transferases (GSTP1, GSTM1, and GSTT1). Erythrocyte membrane fatty acids, antioxidant (catalase, superoxide dismutase (SOD)) and glutathione metabolizing (GST, glutathione peroxidase (Gpx)) enzymes, whole blood chemiluminescence, total antioxidant capacity, levels of nitrites/nitrates, glutathione, HNE-protein adducts, and a wide spectrum of cytokines in the plasma were determined. Results: Allele and genotype frequencies of CYPs, UGT, GSTM, GSTT, and GSTP were similar in the Italian MCS patients and in the control populations. The activities of erythrocyte catalase and GST were lower, whereas Gpx was higher than normal. Both reduced and oxidised glutathione were decreased, whereas nitrites/nitrates were increased in the MCS groups. The MCS fatty acid profile was shifted to saturated compartment and IFNgamma, IL-8, IL-10, MCP-1, PDGFbb, and VEGF were increased. Conclusions: Altered redox and cytokine patterns suggest inhibition of expression/activity of metabolizing and antioxidant enzymes in MCS. Metabolic parameters indicating accelerated lipid oxidation, increased nitric oxide production and glutathione depletion in combination with increased plasma inflammatory cytokines should be considered in biological definition and diagnosis of MCS.

  17. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases.

    PubMed

    Caspi, Ron; Billington, Richard; Ferrer, Luciana; Foerster, Hartmut; Fulcher, Carol A; Keseler, Ingrid M; Kothari, Anamika; Krummenacker, Markus; Latendresse, Mario; Mueller, Lukas A; Ong, Quang; Paley, Suzanne; Subhraveti, Pallavi; Weaver, Daniel S; Karp, Peter D

    2016-01-01

    The MetaCyc database (MetaCyc.org) is a freely accessible comprehensive database describing metabolic pathways and enzymes from all domains of life. The majority of MetaCyc pathways are small-molecule metabolic pathways that have been experimentally determined. MetaCyc contains more than 2400 pathways derived from >46 000 publications, and is the largest curated collection of metabolic pathways. BioCyc (BioCyc.org) is a collection of 5700 organism-specific Pathway/Genome Databases (PGDBs), each containing the full genome and predicted metabolic network of one organism, including metabolites, enzymes, reactions, metabolic pathways, predicted operons, transport systems, and pathway-hole fillers. The BioCyc website offers a variety of tools for querying and analyzing PGDBs, including Omics Viewers and tools for comparative analysis. This article provides an update of new developments in MetaCyc and BioCyc during the last two years, including addition of Gibbs free energy values for compounds and reactions; redesign of the primary gene/protein page; addition of a tool for creating diagrams containing multiple linked pathways; several new search capabilities, including searching for genes based on sequence patterns, searching for databases based on an organism's phenotypes, and a cross-organism search; and a metabolite identifier translation service. PMID:26527732

  18. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases

    PubMed Central

    Caspi, Ron; Billington, Richard; Ferrer, Luciana; Foerster, Hartmut; Fulcher, Carol A.; Keseler, Ingrid M.; Kothari, Anamika; Krummenacker, Markus; Latendresse, Mario; Mueller, Lukas A.; Ong, Quang; Paley, Suzanne; Subhraveti, Pallavi; Weaver, Daniel S.; Karp, Peter D.

    2016-01-01

    The MetaCyc database (MetaCyc.org) is a freely accessible comprehensive database describing metabolic pathways and enzymes from all domains of life. The majority of MetaCyc pathways are small-molecule metabolic pathways that have been experimentally determined. MetaCyc contains more than 2400 pathways derived from >46 000 publications, and is the largest curated collection of metabolic pathways. BioCyc (BioCyc.org) is a collection of 5700 organism-specific Pathway/Genome Databases (PGDBs), each containing the full genome and predicted metabolic network of one organism, including metabolites, enzymes, reactions, metabolic pathways, predicted operons, transport systems, and pathway-hole fillers. The BioCyc website offers a variety of tools for querying and analyzing PGDBs, including Omics Viewers and tools for comparative analysis. This article provides an update of new developments in MetaCyc and BioCyc during the last two years, including addition of Gibbs free energy values for compounds and reactions; redesign of the primary gene/protein page; addition of a tool for creating diagrams containing multiple linked pathways; several new search capabilities, including searching for genes based on sequence patterns, searching for databases based on an organism's phenotypes, and a cross-organism search; and a metabolite identifier translation service. PMID:26527732

  19. Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling.

    PubMed

    Zhdanov, Alexander V; Waters, Alicia H C; Golubeva, Anna V; Papkovsky, Dmitri B

    2015-01-01

    Changes in availability and utilisation of O2 and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O2. Upon 2-4h moderate hypoxia, HIF-? protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1? dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2? levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2h anoxia, HIF-2? levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O2 and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2? decreased after 24h glucose deprivation. This effect, associated with increased AMPK? phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2? accumulation, which became mainly glucose-dependent. Overall, the availability of O2 and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-?. PMID:25447307

  20. Dysregulated metabolism contributes to oncogenesis.

    PubMed

    Hirschey, Matthew D; DeBerardinis, Ralph J; Diehl, Anna Mae E; Drew, Janice E; Frezza, Christian; Green, Michelle F; Jones, Lee W; Ko, Young H; Le, Anne; Lea, Michael A; Locasale, Jason W; Longo, Valter D; Lyssiotis, Costas A; McDonnell, Eoin; Mehrmohamadi, Mahya; Michelotti, Gregory; Muralidhar, Vinayak; Murphy, Michael P; Pedersen, Peter L; Poore, Brad; Raffaghello, Lizzia; Rathmell, Jeffrey C; Sivanand, Sharanya; Vander Heiden, Matthew G; Wellen, Kathryn E

    2015-12-01

    Cancer is a disease characterized by unrestrained cellular proliferation. In order to sustain growth, cancer cells undergo a complex metabolic rearrangement characterized by changes in metabolic pathways involved in energy production and biosynthetic processes. The relevance of the metabolic transformation of cancer cells has been recently included in the updated version of the review "Hallmarks of Cancer", where dysregulation of cellular metabolism was included as an emerging hallmark. While several lines of evidence suggest that metabolic rewiring is orchestrated by the concerted action of oncogenes and tumor suppressor genes, in some circumstances altered metabolism can play a primary role in oncogenesis. Recently, mutations of cytosolic and mitochondrial enzymes involved in key metabolic pathways have been associated with hereditary and sporadic forms of cancer. Together, these results demonstrate that aberrant metabolism, once seen just as an epiphenomenon of oncogenic reprogramming, plays a key role in oncogenesis with the power to control both genetic and epigenetic events in cells. In this review, we discuss the relationship between metabolism and cancer, as part of a larger effort to identify a broad-spectrum of therapeutic approaches. We focus on major alterations in nutrient metabolism and the emerging link between metabolism and epigenetics. Finally, we discuss potential strategies to manipulate metabolism in cancer and tradeoffs that should be considered. More research on the suite of metabolic alterations in cancer holds the potential to discover novel approaches to treat it. PMID:26454069

  1. [Regulation of terpene metabolism]. Annual progress report, March 15, 1988--March 14, 1989

    SciTech Connect

    Croteau, R.

    1989-12-31

    Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

  2. Metabolism

    MedlinePLUS

    Metabolism refers to all the physical and chemical processes in the body that convert or use energy, ... Tortora GJ, Derrickson BH. Metabolism. In: Tortora GJ, Derrickson BH. Principles of Anatomy and Physiology . 14th ed. Hoboken, NJ: John H Wiley and Sons; 2013: ...

  3. Effect of Food Load on Activities of Enzymes of the Main Metabolic Pathways in Blood Lymphocytes in Girls with Different Anthropometric Parameters.

    PubMed

    Fefelova, V V; Fefelova, Yu A; Kazakova, T V; Koloskova, T P; Sergeeva, E Yu

    2015-07-01

    Changes in enzyme activities reflecting functioning of the basic metabolic pathways in cells (Krebs cycle, glycolysis, pentose phosphate pathway) were evaluated in blood lymphocytes of girls of different somatotypes with different body composition under conditions of food load. A common regularity was found: a decrease in succinate dehydrogenase activity after meal in girls of all somatotypes. Specific features of individual somatotypes were also revealed. Only girls of athletic somatotype showed increased lactate dehydrogenase level after food load. Activity of glucose-6-phosphate dehydrogenase increased (more than twice) after food load only in girls of euryplastic somatotype. This somatotype is characterized by maximum values of fat and other components of the body. Glucose-6-phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway; activation of this pathway accompanies enhancement of synthetic processes, including lipid synthesis. This can contribute to accumulation of the fat component (and other components) due to redistribution of substrate flows between metabolic pathways. PMID:26205721

  4. Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

    SciTech Connect

    Zhdanov, Alexander V.; Waters, Alicia H.C.; Golubeva, Anna V.; Papkovsky, Dmitri B.

    2015-01-01

    Changes in availability and utilisation of O{sub 2} and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O{sub 2}. Upon 2–4 h moderate hypoxia, HIF-? protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1? dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2? levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2? levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O{sub 2} and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2? decreased after 24 h glucose deprivation. This effect, associated with increased AMPK? phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2? accumulation, which became mainly glucose-dependent. Overall, the availability of O{sub 2} and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-?. - Highlights: • Gln and Glc regulate HIF levels in hypoxic cells by maintaining low O{sub 2} and high ATP. • HIF-? levels under anoxia correlate with cellular ATP and critically depend on Glc. • Gln and Glc modulate activity of Akt, Erk and AMPK, regulating HIF production. • HIF signalling is differentially inhibited by prolonged Glc and Gln deprivation. • Unlike Glc, Gln plays no major role in HIF signalling in chronically hypoxic cells.

  5. Long-term monitoring reveals carbon–nitrogen metabolism key to microcystin production in eutrophic lakes

    PubMed Central

    Beversdorf, Lucas J.; Miller, Todd R.; McMahon, Katherine D.

    2015-01-01

    The environmental drivers contributing to cyanobacterial dominance in aquatic systems have been extensively studied. However, understanding of toxic vs. non-toxic cyanobacterial population dynamics and the mechanisms regulating cyanotoxin production remain elusive, both physiologically and ecologically. One reason is the disconnect between laboratory and field-based studies. Here, we combined 3 years of temporal data, including microcystin (MC) concentrations, 16 years of long-term ecological research, and 10 years of molecular data to investigate the potential factors leading to the selection of toxic Microcystis and MC production. Our analysis revealed that nitrogen (N) speciation and inorganic carbon (C) availability might be important drivers of Microcystis population dynamics and that an imbalance in cellular C: N ratios may trigger MC production. More specifically, precipitous declines in ammonium concentrations lead to a transitional period of N stress, even in the presence of high nitrate concentrations, that we call the “toxic phase.” Following the toxic phase, temperature and cyanobacterial abundance remained elevated but MC concentrations drastically declined. Increases in ammonium due to lake turnover may have led to down regulation of MC synthesis or a shift in the community from toxic to non-toxic species. While total phosphorus (P) to total N ratios were relatively low over the time-series, MC concentrations were highest when total N to total P ratios were also highest. Similarly, high C: N ratios were also strongly correlated to the toxic phase. We propose a metabolic model that corroborates molecular studies and reflects our ecological observations that C and N metabolism may regulate MC production physiologically and ecologically. In particular, we hypothesize that an imbalance between 2-oxoglutarate and ammonium in the cell regulates MC synthesis in the environment. PMID:26029192

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

    PubMed

    Dzieko?ska, A; Fraser, L; Koziorowska-Gilun, M; Strzezek, J; Koziorowski, M; Kordan, W

    2014-01-01

    This study investigated seasonal changes in the metabolic performance of spermatozoa and activity of the antioxidant enzymes in the seminal plasma of three wild boar/domestic pigs (aged 1.5 to 2.5 years) and the activity of the antioxidant enzymes in fluids of the cauda epididymidis and vesicular glands from 16 wild boar/domestic pig hybrids (aged 1 to 3 years). Parameters of the sperm metabolic activity, such as total motility, mitochondrial functions, and measurements of oxygen uptake, ATP content and L-lactate production, were analyzed during the spring-summer and autumn-winter periods. Besides these sperm metabolic parameters, the sperm membrane integrity was also assessed. Total protein content and activity of the antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were measured in the reproductive tract fluids. There were no marked significant differences (P > 0.05) between the seasonal periods in terms of sperm motility, mitochondrial function and oxygen uptake; however, spermatozoa collected during the autumn-winter period exhibited higher (P < 0.05) ATP content and L-lactate production than those harvested during the spring-summer period. It was found that the vesicular gland fluid exhibited a higher level of SOD activity during the spring-summer period compared with the autumn-winter period. Furthermore, CAT activity in the seminal plasma and vesicular gland fluid was greater during the autumn-winter. Total protein content was significantly higher in the vesicular gland fluid, whereas the cauda epididymidal fluid exhibited greater SOD and GPx activities, irrespective of the seasonal period. The findings of this study confirmed seasonal-related differences in the metabolic performance of spermatozoa and activity of antioxidant enzymes of the reproductive tract of the boar/domestic pig hybrids. PMID:24988857

  7. Levels of microsomal drug-metabolizing enzymes in animals which are highly susceptible to aflatoxin carcinogenicity: the case of the duck.

    PubMed

    Thabrew, M I; Bababunmi, E A

    1980-06-01

    A comparative study of the microsomal fractions of the duck and rat has shown significant differences in the total protein contents in these fractions and also in the activities of 2 phase I drug-metabolizing enzymes, ethylmorphine N-demethylase and aniline hydroxylase. These results are discussed in relation to the high susceptibility of birds (especially ducks) to toxic or carcinogenic substances. PMID:7397687

  8. Oncometabolites: linking altered metabolism with cancer

    PubMed Central

    Yang, Ming; Soga, Tomoyoshi; Pollard, Patrick J.

    2013-01-01

    The discovery of cancer-associated mutations in genes encoding key metabolic enzymes has provided a direct link between altered metabolism and cancer. Advances in mass spectrometry and nuclear magnetic resonance technologies have facilitated high-resolution metabolite profiling of cells and tumors and identified the accumulation of metabolites associated with specific gene defects. Here we review the potential roles of such “oncometabolites” in tumor evolution and as clinical biomarkers for the detection of cancers characterized by metabolic dysregulation. PMID:23999438

  9. LacSubPred: predicting subtypes of Laccases, an important lignin metabolism-related enzyme class, using in silico approaches

    PubMed Central

    2014-01-01

    Background Laccases (E.C. 1.10.3.2) are multi-copper oxidases that have gained importance in many industries such as biofuels, pulp production, textile dye bleaching, bioremediation, and food production. Their usefulness stems from the ability to act on a diverse range of phenolic compounds such as o-/p-quinols, aminophenols, polyphenols, polyamines, aryl diamines, and aromatic thiols. Despite acting on a wide range of compounds as a family, individual Laccases often exhibit distinctive and varied substrate ranges. This is likely due to Laccases involvement in many metabolic roles across diverse taxa. Classification systems for multi-copper oxidases have been developed using multiple sequence alignments, however, these systems seem to largely follow species taxonomy rather than substrate ranges, enzyme properties, or specific function. It has been suggested that the roles and substrates of various Laccases are related to their optimal pH. This is consistent with the observation that fungal Laccases usually prefer acidic conditions, whereas plant and bacterial Laccases prefer basic conditions. Based on these observations, we hypothesize that a descriptor-based unsupervised learning system could generate homology independent classification system for better describing the functional properties of Laccases. Results In this study, we first utilized unsupervised learning approach to develop a novel homology independent Laccase classification system. From the descriptors considered, physicochemical properties showed the best performance. Physicochemical properties divided the Laccases into twelve subtypes. Analysis of the clusters using a t-test revealed that the majority of the physicochemical descriptors had statistically significant differences between the classes. Feature selection identified the most important features as negatively charges residues, the peptide isoelectric point, and acidic or amidic residues. Secondly, to allow for classification of new Laccases, a supervised learning system was developed from the clusters. The models showed high performance with an overall accuracy of 99.03%, error of 0.49%, MCC of 0.9367, precision of 94.20%, sensitivity of 94.20%, and specificity of 99.47% in a 5-fold cross-validation test. In an independent test, our models still provide a high accuracy of 97.98%, error rate of 1.02%, MCC of 0.8678, precision of 87.88%, sensitivity of 87.88% and specificity of 98.90%. Conclusion This study provides a useful classification system for better understanding of Laccases from their physicochemical properties perspective. We also developed a publically available web tool for the characterization of Laccase protein sequences (http://lacsubpred.bioinfo.ucr.edu/). Finally, the programs used in the study are made available for researchers interested in applying the system to other enzyme classes (https://github.com/tweirick/SubClPred). PMID:25350584

  10. Peroxisome Proliferator-Activated Receptor ? and C/EBP? Synergistically Activate Key Metabolic Adipocyte Genes by Assisted Loading

    PubMed Central

    Madsen, Maria Stahl; Siersbæk, Rasmus; Boergesen, Michael; Nielsen, Ronni

    2014-01-01

    Peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer binding protein ? (C/EBP?) are key activators of adipogenesis. They mutually induce the expression of each other and have been reported to cooperate in activation of a few adipocyte genes. Recently, genome-wide profiling revealed a high degree of overlap between PPAR? and C/EBP? binding in adipocytes, suggesting that cooperativeness could be mediated through common binding sites. To directly investigate the interplay between PPAR? and C/EBP? at shared binding sites, we established a fibroblastic model system in which PPAR? and C/EBP? can be independently expressed. Using RNA sequencing, we demonstrate that coexpression of PPAR? and C/EBP? leads to synergistic activation of many key metabolic adipocyte genes. This is associated with extensive C/EBP?-mediated reprogramming of PPAR? binding and vice versa in the vicinity of these genes, as determined by chromatin immunoprecipitation combined with deep sequencing. Our results indicate that this is at least partly mediated by assisted loading involving chromatin remodeling directed by the leading factor. In conclusion, we report a novel mechanism by which the key adipogenic transcription factors, PPAR? and C/EBP?, cooperate in activation of the adipocyte gene program. PMID:24379442

  11. Comparison of in vivo and in vitro methods for assessing the effects of carbon tetrachloride on the hepatic drug-metabolizing enzyme system.

    PubMed

    Chadwick, R W; Copeland, M F; Carlson, G P; Trela, B A; Most, B M

    1988-09-01

    The effect of a single i.p. injection of 0, 20, 200, and 1000 microliter/kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-hexachlorocyclohexane) and by a battery of in vitro enzyme assays. The data in this study indicated that carbon tetrachloride had a biphasic influence on the phase I reactions with the lowest dose inducing a significant increase in enzyme activity while the highest dose produced significant inhibition. Significant CCl4-induced reductions in glucuronyltransferase and sulfotransferase activities were also observed while the effect on glutathione-S-transferase was ambiguous. The in vivo and in vitro assays showed good agreement. PMID:2459810

  12. Determination of Phosphate-activated Glutaminase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry)

    PubMed Central

    Botman, Dennis; Tigchelaar, Wikky

    2014-01-01

    Phosphate-activated glutaminase (PAG) converts glutamine to glutamate as part of the glutaminolysis pathway in mitochondria. Two genes, GLS1 and GLS2, which encode for kidney-type PAG and liver-type PAG, respectively, differ in their tissue-specific activities and kinetics. Tissue-specific PAG activity and its kinetics were determined by metabolic mapping using a tetrazolium salt and glutamate dehydrogenase as an auxiliary enzyme in the presence of various glutamine concentrations. In kidney and brain, PAG showed Michaelis-Menten kinetics with a Km of 0.6 mM glutamine and a Vmax of 1.1 µmol/mL/min when using 5 mM glutamine. PAG activity was high in the kidney cortex and inner medulla but low in the outer medulla, papillary tip, glomeruli and the lis of Henle. In brain tissue sections, PAG was active in the grey matter and inactive in myelin-rich regions. Liver PAG showed allosteric regulation with a Km of 11.6 mM glutamine and a Vmax of 0.5 µmol/mL/min when using 20 mM glutamine. The specificity of the method was shown after incubation of brain tissue sections with the PAG inhibitor 6-diazo-5-oxo-L-norleucine. PAG activity was decreased to 22% in the presence of 2 mM 6-diazo-5-oxo-L-norleucine. At low glutamine concentrations, PAG activity was higher in periportal regions, indicating a lower Km for periportal PAG. When compared with liver, kidney and brain, other tissues showed 3-fold to 6-fold less PAG activity. In conclusion, PAG is mainly active in mouse kidney, brain and liver, and shows different kinetics depending on which type of PAG is expressed. PMID:25163927

  13. Gene expression profiling in Entamoeba histolytica identifies key components in iron uptake and metabolism.

    PubMed

    Hernández-Cuevas, Nora Adriana; Weber, Christian; Hon, Chung-Chau; Guillen, Nancy

    2014-01-01

    Entamoeba histolytica is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in E. histolytica. We compared the respective transcriptomes of E. histolytica trophozoites grown in normal medium (containing around 169 µM iron), low-iron medium (around 123 µM iron), iron-deficient medium (around 91 µM iron), and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters) and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of E. histolytica grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite. PMID:25210888

  14. Uric Acid – Key Ingredient in the Recipe for Cardiorenal Metabolic Syndrome

    PubMed Central

    Chaudhary, Kunal; Malhotra, Kunal; Sowers, James; Aroor, Annayya

    2013-01-01

    Elevated serum uric acid levels are a frequent finding in persons with obesity, hypertension, cardiovascular and kidney disease as well as in those with the cardiorenal metabolic syndrome (CRS). The increased consumption of a fructose-rich Western diet has contributed to the increasing incidence of the CRS, obesity and diabetes especially in industrialized populations. There is also increasing evidence that supports a causal role of high dietary fructose driving elevations in uric acid in association with the CRS. Animal and epidemiological studies support the notion that elevated serum uric acid levels play an important role in promoting insulin resistance and hypertension and suggest potential pathophysiological mechanisms that contribute to the development of the CRS and associated cardiovascular disease and chronic kidney disease. To this point, elevated serum levels of uric acid appear to contribute to impaired nitric oxide production/endothelial dysfunction, increased vascular stiffness, inappropriate activation of the renin-angiotensin-aldosterone system, enhanced oxidative stress, and maladaptive immune and inflammatory responses. These abnormalities, in turn, promote vascular, cardiac and renal fibrosis as well as associated functional abnormalities. Small clinical trials have suggested that uric acid-lowering therapies may be beneficial in such patients; however, a consensus on the treatment of asymptomatic hyperuricemia is lacking. Larger randomized controlled trials need to be performed in order to critically evaluate the beneficial effect of lowering serum uric acid in patients with the CRS and those with diabetes and/or hypertension. PMID:24454316

  15. Gene Expression Profiling in Entamoeba histolytica Identifies Key Components in Iron Uptake and Metabolism

    PubMed Central

    Hernández-Cuevas, Nora Adriana; Weber, Christian; Hon, Chung-Chau; Guillen, Nancy

    2014-01-01

    Entamoeba histolytica is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in E. histolytica. We compared the respective transcriptomes of E. histolytica trophozoites grown in normal medium (containing around 169 µM iron), low-iron medium (around 123 µM iron), iron-deficient medium (around 91 µM iron), and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters) and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of E. histolytica grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite. PMID:25210888

  16. Biochemical study on the protective potential of Nardostachys jatamansi extract on lipid profile and lipid metabolizing enzymes in doxorubicin intoxicated rats.

    PubMed

    Subashini, R; Ragavendran, B; Gnanapragasam, A; Yogeeta, S Kumar; Devaki, T

    2007-05-01

    Nardostachys jatamansi is a medicinally important herb of Indian origin used for centuries in Ayurvedic and Unani systems of medicine for the treatment of various ailments. The aim of the present work is to evaluate the effect of ethanolic extract of Nardostachys jatamansi rhizomes on doxorubicin induced myocardial injury with respect to lipid metabolism in serum and heart of Wistar albino rats. Altered lipid metabolism alters the cardiac function which is mainly due to changes in the property of the cardiac cell membrane. Doxorubicin exhibits cardiotoxicity by inhibition of fatty acid oxidation in the heart. The rats treated with a single dose of doxorubicin (15 mg/kg) intraperitoneally showed an increase in serum and cardiac lipids (cholesterol, triglycerides, free fatty acids and phospholipids), along with a significant rise in serum low density lipoproteins (LDL), very low density lipoproteins (VLDL) and drop in high density lipoproteins (HDL) levels, resulting in alteration of serum and cardiac lipid metabolizing enzymes. Pretreatment with a extract of Nardostachys jatamansi (500 mg/kg) orally for seven days to doxorubicin induced rats showed a significant prevention in the lipid status with the activities of the lipid metabolizing enzymes. Histopathological observations were also in correlation with the biochemical parameters. These findings suggest that the protective and hypolipidemic effect of Nardostachys jatamansi against doxorubicin induced myocardial injury in rats could possibly be mediated through its anti lipid peroxidative properties. PMID:17557749

  17. Post-anthesis alternate wetting and moderate soil drying enhances activities of key enzymes in sucrose-to-starch conversion in inferior spikelets of rice.

    PubMed

    Zhang, Hao; Li, Hongwei; Yuan, Liming; Wang, Zhiqin; Yang, Jianchang; Zhang, Jianhua

    2012-01-01

    This study tested the hypothesis that a post-anthesis moderate soil drying can improve grain filling through regulating the key enzymes in the sucrose-to-starch pathway in the grains of rice (Oryza sativa L.). Two rice cultivars were field grown and two irrigation regimes, alternate wetting and moderate soil drying (WMD) and conventional irrigation (CI, continuously flooded), were imposed during the grain-filling period. The grain-filling rate and activities of four key enzymes in sucrose-to-starch conversion, sucrose synthase (SuSase), adenosine diphosphate-glucose pyrophosphorylase (AGPase), starch synthase (StSase), and starch branching enzyme (SBE), showed no significant difference between WMD and CI regimes for the earlier flowering superior spikelets. However, they were significantly enhanced by the WMD for the later flowering inferior spikelets. The activities of both soluble and insoluble acid invertase in the grains were little affected by the WMD. The two cultivars showed the same tendencies. The activities of SuSase, AGPase, StSase, and SBE in grains were very significantly correlated with the grain-filling rate. The abscisic acid (ABA) concentration in inferior spikelets was remarkably increased in the WMD and very significantly correlated with activities of SuSase, AGPase, StSase, and SBE. Application of ABA on plants under CI produced similar results to those seen in plants receiving WMD. Applying fluridone, an indirect inhibitor of ABA synthesis, produced the opposite effect. The results suggest that post-anthesis WMD could enhance sink strength by regulating the key enzymes involved, and consequently, increase the grain-filling rate and grain weight of inferior spikelets. ABA plays an important role in this process. PMID:21926094

  18. Inhibitory potential of omega-3 fatty and fenugreek essential oil on key enzymes of carbohydrate-digestion and hypertension in diabetes rats

    PubMed Central

    2011-01-01

    Background 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 digestives enzymes-related to starch digestion secreted by pancreas. Methods The formulation omega-3 with fenugreek terpenenes was administrated to surviving diabetic rats. The inhibitory effects of this oil on rat pancreas ?-amylase and maltase and plasma angiotensin-converting enzyme (ACE) were determined. Results the findings revealed that administration of formulation omega-3 with fenugreek terpenenes (Om3/terp) considerably inhibited key enzymes-related to diabetes such as ?-amylase activity by 46 and 52% and maltase activity by 37 and 35% respectively in pancreas and plasma. Moreover, the findings revealed that this supplement helped protect the ?-Cells of the rats from death and damage. Interestingly, the formulation Om3/terp modulated key enzyme related to hypertension such as ACE by 37% in plasma and kidney. Moreover administration of fenugreek essential oil to surviving diabetic rats improved starch and glucose oral tolerance additively. Furthermore, the Om3/terp also decreased significantly the glucose, triglyceride (TG) and total-cholesterol (TC) and LDL-cholesterol (LDL-C) rates in the plasma and liver of diabetic rats and increased the HDL-Cholesterol (HDL-Ch) level, which helped maintain the homeostasis of blood lipid. Conclusion overall, the findings of the current study indicate that this formulation Om3/terp exhibit attractive properties and can, therefore, be considered for future application in the development of anti-diabetic, anti-hypertensive and hypolipidemic foods. PMID:22142357

  19. A Single Host-Derived Glycan Impacts Key Regulatory Nodes of Symbiont Metabolism in a Coevolved Mutualism

    PubMed Central

    Pan, Min; Schwartzman, Julia A.; Dunn, Anne K.; Lu, Zuhong

    2015-01-01

    ABSTRACT Most animal-microbe mutualistic associations are characterized by nutrient exchange between the partners. When the host provides the nutrients, it can gain the capacity to shape its microbial community, control the stability of the interaction, and promote its health and fitness. Using the bioluminescent squid-vibrio model, we demonstrate how a single host-derived glycan, chitin, regulates the metabolism of Vibrio fischeri at key points in the development and maintenance of the symbiosis. We first characterized the pathways for catabolism of chitin sugars by V. fischeri, demonstrating that the Ccr-dependent phosphoenolpyruvate-pyruvate phosphotransferase system (PTS) prioritizes transport of these sugars in V. fischeri by blocking the uptake of non-PTS carbohydrates, such as glycerol. Next, we found that PTS transport of chitin sugars into the bacterium shifted acetate homeostasis toward a net excretion of acetate and was sufficient to override an activation of the acetate switch by AinS-dependent quorum sensing. Finally, we showed that catabolism of chitin sugars decreases the rate of cell-specific oxygen consumption. Collectively, these three metabolic functions define a physiological shift that favors fermentative growth on chitin sugars and may support optimal symbiont luminescence, the functional basis of the squid-vibrio mutualism. PMID:26173698

  20. Metabolomic Analysis of Key Central Carbon Metabolism Carboxylic Acids as Their 3-Nitrophenylhydrazones by UPLC/ESI-MS

    PubMed Central

    Han, Jun; Gagnon, Susannah; Eckle, Tobias; Borchers, Christoph H.

    2014-01-01

    Multiple hydroxy-, keto-, di-, and tri-carboxylic acids are among the cellular metabolites of central carbon metabolism (CCM). Sensitive and reliable analysis of these carboxylates is important for many biological and cell engineering studies. In this work, we examined 3-nitrophenylhydrazine as a derivatizing reagent and optimized the reaction conditions for the measurement of ten CCM related carboxylic compounds, including glycolate, lactate, malate, fumarate, succinate, citrate, isocitrate, pyruvate, oxaloacetate, and ?-ketoglutarate as their 3-nitrophenylhydrazones using LC/MS with electrospray ionization. With the derivatization protocol which we have developed, and using negative-ion multiple reaction monitoring on a triple-quadrupole instrument, all of the carboxylates showed good linearity within a dynamic range of ca. 200 to more than 2000. The on-column limits of detection and quantitation were from high femtomoles to low picomoles. The analytical accuracies for eight of the ten analytes were determined to be between 89.5 to 114.8% (CV?7.4%, n=6). Using a quadrupole time-of-flight instrument, the isotopic distribution patterns of these carboxylates, extracted from a 13C-labeled mouse heart, were successfully determined by UPLC/MS with full-mass detection, indicating the possible utility of this analytical method for metabolic flux analysis. In summary, this work demonstrates an efficient chemical derivatization LC/MS method for metabolomic analysis of these key CCM intermediates in a biological matrix. PMID:23580203

  1. Metabolism

    MedlinePLUS

    ... Some metabolic diseases and conditions include: Hyperthyroidism (pronounced: hi-per-THIGH-roy-dih-zum). Hyperthyroidism is caused ... or through surgery or radiation treatments. Hypothyroidism (pronounced: hi-po-THIGH-roy-dih-zum) . Hypothyroidism is caused ...

  2. Lipoxygenase, a key enzyme in bioconversion of linoleic acid into trihydroxy-octadecenoic acid by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipoxygenases catalyze the oxidation of unsaturated fatty acids with a (1Z,4Z)-pentadiene structure leading to the formation of conjugated (Z,E)-hydroperoxydienoic acids, which in turn result in production of hydroxy lipid. These enzymes are widely distributed in plants, animals, and microorganisms...

  3. Rooibos flavonoids inhibit the activity of key adrenal steroidogenic enzymes, modulating steroid hormone levels in H295R cells.

    PubMed

    Schloms, Lindie; Swart, Amanda C

    2014-01-01

    Major rooibos flavonoids--dihydrochalcones, aspalathin and nothofagin, flavones--orientin and vitexin, and a flavonol, rutin, were investigated to determine their influence on the activity of adrenal steroidogenic enzymes, 3?-hydroxysteroid dehydrogenase (3?HSD2) and cytochrome P450 (P450) enzymes, P450 17?-hydroxylase/17,20-lyase (CYP17A1), P450 21-hydroxylase (CYP21A2) and P450 11?-hydroxylase (CYP11B1). All the flavonoids inhibited 3?HSD2 and CYP17A1 significantly, while the inhibition of downstream enzymes, CYP21A2 and CYP11B1, was both substrate and flavonoid specific. The dihydrochalcones inhibited the activity of CYP21A2, but not that of CYP11B1. Although rutin, orientin and vitexin inhibited deoxycortisol conversion by CYP11B1 significantly, inhibition of deoxycorticosterone was <20%. These three flavonoids were unable to inhibit CYP21A2, with negligible inhibition of deoxycortisol biosynthesis only. Rooibos inhibited substrate conversion by CYP17A1 and CYP21A2, while the inhibition of other enzyme activities was <20%. In H295R cells, rutin had the greatest inhibitory effect on steroid production upon forskolin stimulation, reducing total steroid output 2.3-fold, while no effect was detected under basal conditions. Nothofagin and vitexin had a greater inhibitory effect on overall steroid production compared to aspalathin and orientin, respectively. The latter compounds contain two hydroxyl groups on the B ring, while nothofagin and vitexin contain a single hydroxyl group. In addition, all of the flavonoids are glycosylated, albeit at different positions--dihydrochalcones at C3' and flavones at C8 on ring A, while rutin, a larger molecule, has a rutinosyl moiety at C3 on ring C. Structural differences regarding the number and position of hydroxyl and glucose moieties as well as structural flexibility could indicate different mechanisms by which these flavonoids influence the activity of adrenal steroidogenic enzymes. PMID:24662082

  4. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  5. Codeine Ultra-rapid Metabolizers: Age Appears to be a Key Factor in Adverse Effects of Codeine.

    PubMed

    Heintze, K; Fuchs, W

    2015-12-01

    Codeine is widely used as an analgesic drug. Taking into account the high consumption of codeine, only few fatal adverse events have been published. A number of reports, where neonates and children showed serious or fatal adverse reactions, led to a restriction of the use of codeine in this patient group. Therefore, we reviewed the safety of codeine in adults. PubMed was systematically searched for clinical studies and case reports, with a special focus on CYP2D6, the enzyme that converts codeine to morphine and exhibits genetic polymorphism.181 cases were identified in adults in conjunction with serious or lethal effects of codeine. In the vast majority of cases, codeine was used in combination with other drugs by drug-dependent individuals or with a suicidal intent. Only 2 cases were found where ultra-rapid metabolizers experienced severe non-lethal adverse events. This is far less than would be predicted from the number of cases reported in children. The discrepancy may be explained by developmental changes in the disposition of codeine.The strategy of regulatory authorities to restrict access to codeine for infants and young children, the apparent highest risk group, has a factual and pharmacological rationale. By the same standards, there is no need for restrictions for adult use of codeine. PMID:25585351

  6. Important roles of the AKR1C2 and SRD5A1 enzymes in progesterone metabolism in endometrial cancer model cell lines.

    PubMed

    Sinreih, Maša; Anko, Maja; Zukunft, Sven; Adamski, Jerzy; Rižner, Tea Lanišnik

    2015-06-01

    Endometrial cancer is the most frequently diagnosed gynecological malignancy. It is associated with prolonged exposure to estrogens that is unopposed by progesterone, whereby enhanced metabolism of progesterone may decrease its protective effects, as it can deprive progesterone receptors of their active ligand. Furthermore, the 5?-pregnane metabolites formed can stimulate proliferation and may thus contribute to carcinogenesis. The aims of our study were to: (1) identify and quantify progesterone metabolites formed in the HEC-1A and Ishikawa model cell lines of endometrial cancer; and (2) pinpoint the enzymes involved in progesterone metabolism, and delineate their roles. Progesterone metabolism studies combined with liquid chromatography-tandem mass spectrometry enabled identification and quantification of the metabolites formed in these cells. Further quantitative PCR analysis and small-interfering-RNA-mediated gene silencing identified individual progesterone metabolizing enzymes and their relevant roles. In Ishikawa and HEC-1A cells, progesterone was metabolized mainly to 20?-hydroxy-pregn-4-ene-3-one, 20?-hydroxy-5?-pregnane-3-one, and 5?-pregnane-3?/?,20?-diol. The major difference between these cell lines was rate of progesterone metabolism, which was faster in HEC-1A cells. In the Ishikawa and HEC-1A cells, expression of AKR1C2 was 110-fold and 6800-fold greater, respectively, than expression of AKR1C1, which suggests that 20-ketosteroid reduction of 5?-pregnanes and 4-pregnenes is catalyzed mainly by AKR1C2. AKR1C1/AKR1C2 gene silencing showed decreased progesterone metabolism in both cell lines, thus further supporting the significant role of AKR1C2. SRD5A1 was also expressed in these cells, and its silencing confirmed that 5?-reduction is catalyzed by 5?-reductase type 1. Silencing of SRD5A1 also had the most pronounced effects, with decreased rate of progesterone metabolism, and consequently higher concentrations of unmetabolized progesterone. Our data confirm that in model cell lines of endometrial cancer, AKR1C2 and SRD5A1 have crucial roles in progesterone metabolism, and may represent novel targets for treatment. PMID:25463305

  7. Products of enzymatic reduction of benzoyl-CoA, a key reaction in anaerobic aromatic metabolism.

    PubMed

    Koch, J; Eisenreich, W; Bacher, A; Fuchs, G

    1993-02-01

    Benzoyl-coenzyme A is the most common central intermediate of anaerobic aromatic metabolism. Studies with whole cells of different bacteria and in vitro had shown that benzoyl-CoA is reduced to alicyclic compounds, possibly via cyclohexadiene intermediates. This reaction is considered a 'biological Birch reduction'. We have elucidated by NMR techniques the structures of six products of [ring-13C6]benzoate reduction. The reaction is catalyzed by extracts from cells of a denitrifying Pseudomonas strain K172 anaerobically grown with benzoate and nitrate as sole carbon and energy sources. The assay mixture contained [ring-13C6]benzoate plus traces of [U-14C]benzoate, Mg2+, ATP, coenzyme A (CoA), and Ti(III) as reductant. The use of the multiply 13C-labelled precursor increases the sensitivity of NMR detection and allows the analysis of crude product mixtures by two-dimensional coherence transfer procedures such as total correlation 13C-NMR spectroscopy and 13C-filtered 1H-NMR spectroscopy. The time course of product formation is consistent with the following order of events. Benzoyl-CoA is formed from benzoate via benzoate-CoA ligase. The first ring reduction product observed is cyclohex-1,5-diene-1-carboxyl-CoA. The next intermediate is 6-hydroxycyclohex-1-ene-1-carboxyl-CoA which is derived from the diene by addition of water. Part of the diene seems to be reduced to cyclohex-1-ene-1-carboxyl-CoA which becomes hydrated to trans-2-hydroxycyclohexane-1-carboxyl-CoA; these two intermediates may be side products in vitro. The first non-cyclic intermediate formed by beta-oxidation is 3-hydroxypimelyl-CoA. This aliphatic C7 dicarboxylic acid is proposed to be oxidized via glutaryl-CoA and crotonyl-CoA to three molecules of acetyl-CoA and one molecule of CO2. A similar product pattern was observed in the benzoate-degrading phototrophic bacterium Rhodopseudomonas palustris. This indicates that the enzymatic reduction of benzoyl-CoA may be mechanistically similar in different anaerobes. PMID:8436125

  8. Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

    PubMed Central

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

    2014-01-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

  9. Faster Rubisco Is the Key to Superior Nitrogen-Use Efficiency in NADP-Malic Enzyme Relative to NAD-Malic Enzyme C4 Grasses1

    PubMed Central

    Ghannoum, Oula; Evans, John R.; Chow, Wah Soon; Andrews, T. John; Conroy, Jann P.; von Caemmerer, Susanne

    2005-01-01

    In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (kcat; 3.8 versus 5.7 s?1 at 25°C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)?1 in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster kcat. PMID:15665246

  10. Silymarin protects PBMC against B(a)P induced toxicity by replenishing redox status and modulating glutathione metabolizing enzymes-An in vitro study

    SciTech Connect

    Kiruthiga, P.V.; Pandian, S. Karutha; Devi, K. Pandima

    2010-09-01

    PAHs are a ubiquitous class of environmental contaminants that have a large number of hazardous consequences on human health. An important prototype of PAHs, B(a)P, is notable for being the first chemical carcinogen to be discovered and the one classified by EPA as a probable human carcinogen. It undergoes metabolic activation to QD, which generate ROS by redox cycling system in the body and oxidatively damage the macromolecules. Hence, a variety of antioxidants have been tested as possible protectors against B(a)P toxicity. Silymarin is one such compound, which has high human acceptance, used clinically and consumed as dietary supplement around the world for its strong anti-oxidant efficacy. Silymarin was employed as an alternative approach for treating B(a)P induced damage and oxidative stress in PBMC, with an emphasis to provide the molecular basis for the effect of silymarin against B(a)P induced toxicity. PBMC cells exposed to either benzopyrene (1 {mu}M) or silymarin (2.4 mg/ml) or both was monitored for toxicity by assessing LPO, PO, redox status (GSH/GSSG ratio), glutathione metabolizing enzymes GR and GPx and antioxidant enzymes CAT and SOD. This study also investigated the protective effect of silymarin against B(a)P induced biochemical alteration at the molecular level by FT-IR spectroscopy. Our findings were quite striking that silymarin possesses substantial protective effect against B(a)P induced oxidative stress and biochemical changes by restoring redox status, modulating glutathione metabolizing enzymes, hindering the formation of protein oxidation products, inhibiting LPO and further reducing ROS mediated damages by changing the level of antioxidant enzymes. The results suggest that silymarin exhibits multiple protections and it should be considered as a potential protective agent for environmental contaminant induced immunotoxicity.

  11. Two similar gene clusters coding for enzymes of a new type of aerobic 2-aminobenzoate (anthranilate) metabolism in the bacterium Azoarcus evansii.

    PubMed

    Schühle, K; Jahn, M; Ghisla, S; Fuchs, G

    2001-09-01

    In the beta-proteobacterium Azoarcus evansii, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways. The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form. The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced. The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme). Besides the genes for the initial two enzymes, genes for three enzymes of a beta-oxidation pathway were found. A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster. The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by beta-oxidation. This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically. Surprisingly, the cluster of eight genes, which form an operon, is duplicated. The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions. Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate. PMID:11514509

  12. Two Similar Gene Clusters Coding for Enzymes of a New Type of Aerobic 2-Aminobenzoate (Anthranilate) Metabolism in the Bacterium Azoarcus evansii

    PubMed Central

    Schühle, Karola; Jahn, Martina; Ghisla, Sandro; Fuchs, Georg

    2001-01-01

    In the ?-proteobacterium Azoarcus evansii, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways. The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form. The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced. The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme). Besides the genes for the initial two enzymes, genes for three enzymes of a ?-oxidation pathway were found. A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster. The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by ?-oxidation. This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically. Surprisingly, the cluster of eight genes, which form an operon, is duplicated. The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions. Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate. PMID:11514509

  13. Differential metabolism of organic nitrates by aldehyde dehydrogenase 1a1 and 2: substrate selectivity, enzyme inactivation, and active cysteine sites.

    PubMed

    Tsou, Pei-Suen; Page, Nathaniel A; Lee, Sean G; Fung, Sun Mi; Keung, Wing Ming; Fung, Ho-Leung

    2011-12-01

    Organic nitrate vasodilators (ORN) exert their pharmacologic effects through the metabolic release of nitric oxide (NO). Mitochondrial aldehyde dehydrogenase (ALDH2) is the principal enzyme responsible for NO liberation from nitroglycerin (NTG), but lacks activity towards other ORN. Cytosolic aldehyde dehydrogenase (ALDH1a1) can produce NO from NTG, but its activity towards other ORN is unknown. Using purified enzymes, we showed that both isoforms could liberate NO from NTG, isosorbide dinitrate (ISDN), and nicrorandil, while only ALDH1a1 metabolized isosorbide-2-mononitrate and isosorbide-5-mononitrate (IS-5-MN). Following a 10-min incubation with purified enzyme, 0.1 mM NTG and 1 mM ISDN potently inactivated ALDH1a1 (to 21.9%?±?11.1% and 0.44%?±?1.04% of control activity, respectively) and ALDH2 (no activity remaining and 4.57%?±?7.92% of control activity, respectively), while 1 mM IS-5-MN exerted only modest inactivation of ALDH1a1 (reduced to 89%?±?4.3% of control). Cytosolic ALDH in hepatic homogenates incubated at the vascular EC(50) concentrations of ORN was inactivated by NTG (to 45.1%?±?8.1% of control activity) while mitochondrial ALDH was inactivated by NTG and nicorandil (to 68.2%?±?10.0% and 78.7%?±?19.8% of control, respectively). Via site-directed mutagenesis, the active sites of ORN metabolism of ALDH2 (Cys-319) and ALDH1a1 (Cys-303) were found to be identical to those responsible for their dehydrogenase activity. Cysteine-302 of ALDH1a1 and glutamate-504 of ALDH2 were found to modulate the rate of ORN metabolism. These studies provide further characterization of the substrate selectivity, inactivation, and active sites of ALDH2 and ALDH1a1 toward ORN. PMID:21818694

  14. Insulin resistance as a key link for the increased risk of cognitive impairment in the metabolic syndrome

    PubMed Central

    Kim, Bhumsoo; Feldman, Eva L

    2015-01-01

    Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors that includes obesity, diabetes, and dyslipidemia. Accumulating evidence implies that MetS contributes to the development and progression of Alzheimer's disease (AD); however, the factors connecting this association have not been determined. Insulin resistance (IR) is at the core of MetS and likely represent the key link between MetS and AD. In the central nervous system, insulin plays key roles in learning and memory, and AD patients exhibit impaired insulin signaling that is similar to that observed in MetS. As we face an alarming increase in obesity and T2D in all age groups, understanding the relationship between MetS and AD is vital for the identification of potential therapeutic targets. Recently, several diabetes therapies that enhance insulin signaling are being tested for a potential therapeutic benefit in AD and dementia. In this review, we will discuss MetS as a risk factor for AD, focusing on IR and the recent progress and future directions of insulin-based therapies. PMID:25766618

  15. Hemerythrins in the microaerophilic bacterium Campylobacter jejuni help protect key iron–sulphur cluster enzymes from oxidative damage

    PubMed Central

    Kendall, John J; Barrero-Tobon, Angelica M; Hendrixson, David R; Kelly, David J

    2014-01-01

    Microaerophilic bacteria are adapted to low oxygen environments, but the mechanisms by which their growth in air is inhibited are not well understood. The citric acid cycle in the microaerophilic pathogen Campylobacter jejuni is potentially vulnerable, as it employs pyruvate and 2-oxoglutarate:acceptor oxidoreductases (Por and Oor), which contain labile (4Fe-4S) centres. Here, we show that both enzymes are rapidly inactivated after exposure of cells to a fully aerobic environment. We investigated the mechanisms that might protect enzyme activity and identify a role for the hemerythrin HerA (Cj0241). A herA mutant exhibits an aerobic growth defect and reduced Por and Oor activities after exposure to 21% (v/v) oxygen. Slow anaerobic recovery of these activities after oxygen damage was observed, but at similar rates in both wild-type and herA strains, suggesting the role of HerA is to prevent Fe-S cluster damage, rather than promote repair. Another hemerythrin (HerB; Cj1224) also plays a protective role. Purified HerA and HerB exhibited optical absorption, ligand binding and resonance Raman spectra typical of ?-oxo-bridged di-iron containing hemerythrins. We conclude that oxygen lability and poor repair of Por and Oor are major contributors to microaerophily in C. jejuni; hemerythrins help prevent enzyme damage microaerobically or during oxygen transients. PMID:24245612

  16. Community metabolism in shallow coral reef and seagrass ecosystems, Lower Florida Keys

    NASA Astrophysics Data System (ADS)

    Turk, Daniela; Yates, Kimberly; Vega-Rodriguez, Maria; Toro-Farmer, Gerardo; L'Esperance, Chris; Melo, Nelson; Ramsevak, Deanesh; Cerdeira-Estrada, Sergio; Muller-Karger, Frank; Herwitz, Stanley; McGillis, Wade

    2015-04-01

    Diurnal variation of net community production (NEP) and net community calcification (NEC) were measured in coral reef and seagrass biomes during October 2012 in the lower Florida Keys using a mesocosm enclosure and the oxygen gradient flux technique. Seagrass and coral reef sites showed diurnal variations of NEP and NEC with positive values at near-bottom light levels above 100-300 ?Einstein m-2 s-1. During daylight hours, seagrasses showed an average NEP of 12.3 mmol O2 m-2 h-1 compared to daylight coral reef NEP of 8.6 mmol O2 m-2 h-1. At night, NEP at the seagrass was relatively constant, while in the coral reef, net respiration was highest immediately after dusk and decreasing during the rest of the night. NEC values were ranging from 0.20 g CaCO3 m-2 h-1 during daylight to -0.15 g CaCO3 m-2 h-1 at night at the seagrass site, and from 0.17 to -0.10 g CaCO3 m-2 h-1 at the coral reef site. Similar NEC:NEP ratios were observed at the seagrass site and the coral site at the time of maximum daily irradiance. Average photosynthetic quotient (PQ) at the seagrass site was slightly lower in the morning and early afternoon than at the coral reef site, and higher at the seagrass site in the late afternoon. There were no significant differences in pH and aragonite saturation states (?ar) between the seagrass and coral reef sites. Decrease in light levels during thunderstorms significantly decreased NEP, transforming the system from net autotrophic to net heterotrophic.

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

    NASA Astrophysics Data System (ADS)

    Thuesen, Erik V.; Childress, James J.

    1993-05-01

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

  18. A Comparative Study for the Evaluation of Two Doses of Ellagic Acid on Hepatic Drug Metabolizing and Antioxidant Enzymes in the Rat

    PubMed Central

    Celik, Gurbet; Semiz, Asl?; Karakurt, Serdar; Arslan, Sevki; Adali, Orhan; Sen, Alaattin

    2013-01-01

    The present study was designed to evaluate different doses of ellagic acid (EA) in vivo in rats for its potential to modulate hepatic phases I, II, and antioxidant enzymes. EA (10 or 30?mg/kg/day, intragastrically) was administered for 14 consecutive days, and activity, protein, and mRNA levels were determined. Although the cytochrome P450 (CYP) 2B and CYP2E enzyme activities were decreased significantly, the activities of all other enzymes were unchanged with the 10?mg/kg/day EA. In addition, western-blot and qRT-PCR results clearly corroborated the above enzyme expressions. On the other hand, while the NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were increased significantly, CYP1A, 2B, 2C, 2E, and 19 enzyme activities were reduced significantly with 30?mg/kg/day EA. In addition, CYP2B, 2C6, 2E1, and 19 protein and mRNA levels were substantially decreased by the 30?mg/kg/day dose of EA, but the CYP1A protein, and mRNA levels were not changed. CYP3A enzyme activity, protein and mRNA levels were not altered by neither 10 nor 30?mg/kg/day ellagic acid. These results indicate that EA exerts a dose-dependent impact on the metabolism of chemical carcinogens and drugs by affecting the enzymes involved in xenobiotics activation/detoxification and antioxidant pathways. PMID:23971029

  19. Differences in the drug-metabolizing enzyme activities among fish and bivalves living in waters near industrial and non-industrial areas

    SciTech Connect

    Oshima, Y.; Kobayashi, K.; Hidaka, C.; Izu, S.; Imada, N. )

    1994-07-01

    Fish and shellfishes, living in coastal areas receiving agricultural, industrial and domestic wastewaters, have been exposed to various chemicals. Identifing the various harmful chemicals in the environments and accumulated in aquatic organisms is difficult. Even if concentrations of pollutants are low so that no mortality of fish and shellfishes occurs, the pollutants may affect the biochemistry and physiology of aquatic organisms. Activities of some drug-metabolizing enzymes, especially the cytochrome P-450 dependent monooxygenase (MO) in fish livers, increase when fish are exposed to environmental pollutants such as polycyclic aromatic hydrocarbons, halogenated organic chemicals. However, most studies have been done on the field evaluation only by MO induction in fish as a monitor for marine pollution with crude-oil and halogenated organic compounds, without regard for other chemicals. In a previous paper, the activity of benzo(a)pyrene hydroxylase (AHH) was induced by 22 times at 2-wk, although the cytochrome P-450 content increased only twice. Activity of phenol-sulfate transferase in the mid-gut gland of short-necked clam was induced by exposure to some phenolic compounds, especially pentachlorophenol (PCP), resulting in the increase of the enzyme activity by approximately 7 times the control after 5 wk exposure. Induced activity was maintained at least for 3 wk, even after the clam had been transferred to running clean sea water, although PCP accumulated in its body is rapidly excreted. Although the activity of this enzyme in the clam is easily induced by exposure to phenols, the induction of this enzyme activity in fish is very low as compared with that of clam. This paper examines the activities of drug-metabolizing enzymes of fish and bivalves living in waters near industrial and non-industrial areas to elucidate the applicability of the sulfate transferase activity in bivalves as a monitor for marine pollution, as well as the MO activity in fish.

  20. Identification and characterization of Sulfolobus solfataricus D-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

    PubMed Central

    Kim, Seonghun; Lee, Sun Bok

    2004-01-01

    The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway. PMID:15509194

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

    PubMed Central

    He, Jin-Lian; Zhou, Zhi-Wei; Yin, Juan-Juan; He, Chang-Qiang; Zhou, Shu-Feng; Yu, Yang

    2015-01-01

    Drug metabolizing enzymes (DMEs) and drug transporters are regulated via epigenetic, transcriptional, posttranscriptional, and translational and posttranslational modifications. Phase I and II DMEs and drug transporters play an important role in the disposition and detoxification of a large number of endogenous and exogenous compounds. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a critical regulator of a variety of important cytoprotective genes that are involved in disposition and detoxification of xenobiotics. Schisandra chinensis (SC) is a commonly used traditional Chinese herbal medicine that has been primarily used to protect the liver because of its potent antioxidative and anti-inflammatory activities. SC can modulate some DMEs and drug transporters, but the underlying mechanisms are unclear. In this study, we aimed to explore the role of Nrf2 in the regulatory effect of SC extract (SCE) on selected DMEs and drug transporters in human hepatocellular liver carcinoma cell line (HepG2) cells. The results showed that SCE, schisandrin A, and schisandrin B significantly increased the expression of NAD(P)H: Nicotinamide Adenine Dinucleotide Phosphate-oxidase or:quinone oxidoreductase 1, heme oxygenase-1, glutamate–cysteine ligase, and glutathione S-transferase A4 at both transcriptional and posttranscriptional levels. Incubation of HepG2 cells with SCE resulted in a significant increase in the intracellular level of glutathione and total glutathione S-transferase content. SCE significantly elevated the messenger ribonucleic acid and protein levels of P-glycoprotein and multidrug resistance-associated protein 2 and 4, whereas the expression of organic anion transporting peptide 1A2 and 1B1 was significantly downregulated by SCE. Knockdown of Nrf2 by small interfering ribonucleic acid attenuated the regulatory effect of SCE on these DMEs and drug transporters. SCE significantly upregulated Nrf2 and promoted the translocation of Nrf2 from cytoplasm to the nuclei. Additionally, SCE significantly suppressed the expression of cytosolic Kelch-like ECH-associated protein 1 (the repressor of Nrf2) and remarkably increased Nrf2 stability in HepG2 cells. Taken together, our findings suggest that the hepatoprotective effects of SCE may be partially ascribed to the modulation of DMEs and drug transporters via Nrf2-mediated signaling pathway. SCE may alter the pharmacokinetics of other coadministered drugs that are substrates of these DMEs and transporters and thus cause unfavorable herb–drug interactions. PMID:25552902

  2. Hepatocyte Nuclear Factors 4? and 1? Regulate Kidney Developmental Expression of Drug-Metabolizing Enzymes and Drug Transporters

    PubMed Central

    Martovetsky, Gleb; Tee, James B.

    2013-01-01

    The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule (PT) is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors, which are thought to “sense” deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including hepatocyte nuclear factor 4? (Hnf4a) and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), peroxisome proliferator–activated receptor ? (Ppar?), and Tp53. Motif analysis of cis-regulatory enhancers further suggested that Hnf4a and Hnf1a are the main transcriptional regulators of DMEs in the PT. Available expression data from tissue-specific Hnf4a knockout tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. Chromatin immunoprecipitation combined with massively parallel DNA sequencing was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1 (Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8 (Oat3), and Slc47a1 (Mate1). Conversely, overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts, sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-enriched brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT-like cellular identity. We discuss our data in the context of the “remote sensing and signaling hypothesis” (Ahn and Nigam, 2009; Wu et al., 2011). PMID:24038112

  3. Inhibition of Key Enzymes Linked to Type 2 Diabetes and Sodium Nitroprusside Induced Lipid Peroxidation in Rats’ Pancreas by Phenolic Extracts of Avocado Pear Leaves and Fruit

    PubMed Central

    Oboh, Ganiyu; Isaac, Adelusi Temitope; Akinyemi, Ayodele Jacobson; Ajani, Richard Akinlolu

    2014-01-01

    Persea americana fruit and leaves had been known in folk medicine for their anti-diabetic prowess. Therefore, this study sought to investigate the inhibitory effect of phenolic extract from avocado pear (Persea americana) leaves and fruits on some key enzymes linked to type 2 diabetes (?-amylase and ?-glucosidase); and sodium nitroprusside (SNP) induced lipid peroxidation in rats’ pancreas in vitro. The phenolic extracts of Persea americana fruit and leaves were extracted using methanol and 1M HCl (1:1 v/v). Thereafter, their inhibitory effects on sodium nitroprusside induced lipid peroxidation and key enzymes linked to type 2 diabetes (?-amylase and ?-glucosidase) were determined in vitro. The result revealed that the leaves had fruit of avocado pear inhibit both ?-amylase and ?-glucosidase activities in a dose dependent manner. However, the Peel had the highest ?-amylase inhibitory activity while the leaf had the highest ?-glucosidase inhibitory activity as revealed by their IC50 value. Furthermore, incubation of the rat pancreas in the presence of 5 mM SNP caused an increase in the malondialdehyde (MDA) content in the tissue, however, introduction of the phenolic extracts inhibited MDA produced in a dose dependent manner. The additive and/or synergistic action of major phenolic compounds such as syringic acid, eugenol, vnillic acid, isoeugenol, guaiacol, kaemferol, catechin, ?-hydroxybenzoic acid, ferulic acid, apigenin, naringenin, epigallocatechin, epicatechin, lupeol and epigallocatechin-3-O-gallate in avocado pear using gas chromatography (GC) could have contributed to the observed medicinal properties of the plant. Therefore, inhibition of some key enzymes linked to type 2 diabetes and prevention of oxidative stress in the pancreas could be some of the possible mechanism by which they exert their anti-diabetic properties PMID:25324703

  4. Strategies for strain improvement in Fusarium fujikuroi: overexpression and localization of key enzymes of the isoprenoid pathway and their impact on gibberellin biosynthesis.

    PubMed

    Albermann, Sabine; Linnemannstöns, Pia; Tudzynski, Bettina

    2013-04-01

    The rice pathogen Fusarium fujikuroi is known to produce a wide range of secondary metabolites, such as the pigments bikaverin and fusarubins, the mycotoxins fusarins and fusaric acid, and the phytohormones gibberellic acids (GAs), which are applied as plant growth regulators in agri- and horticulture. The development of high-producing strains is a prerequisite for the efficient biotechnological production of GAs. In this work, we used different molecular approaches for strain improvement to directly affect expression of early isoprenoid genes as well as GA biosynthetic genes. Overexpression of the first GA pathway gene ggs2, encoding geranylgeranyl diphosphate synthase 2, or additional integration of ggs2 and cps/ks, the latter encoding the bifunctional ent-copalyldiphosphate synthase/ent-kaurene synthase, revealed an enhanced production level of 150%. However, overexpression of hmgR and fppS, encoding the key enzymes of the mevalonate pathway, hydroxymethylglutaryl coenzyme A reductase, and farnesyldiphosphate synthase, resulted in a reduced production level probably due to a negative feedback regulation of HmgR. Subsequent deletion of the transmembrane domains of HmgR and overexpression of the remaining catalytic domain led to an increased GA content (250%). Using green fluorescent protein and mCherry fusion constructs, we localized Cps/Ks in the cytosol, Ggs2 in small point-like structures, which are not the peroxisomes, and HmgR at the endoplasmatic reticulum. In summary, it was shown for the first time that amplification or truncation of key enzymes of the isoprenoid and GA pathway results in elevated production levels (2.5-fold). Fluorescence microscopy revealed localization of the key enzymes in different compartments. PMID:22983595

  5. Inhibition of key enzymes linked to type 2 diabetes and sodium nitroprusside induced lipid peroxidation in rats' pancreas by phenolic extracts of avocado pear leaves and fruit.

    PubMed

    Oboh, Ganiyu; Isaac, Adelusi Temitope; Akinyemi, Ayodele Jacobson; Ajani, Richard Akinlolu

    2014-09-01

    Persea americana fruit and leaves had been known in folk medicine for their anti-diabetic prowess. Therefore, this study sought to investigate the inhibitory effect of phenolic extract from avocado pear (Persea americana) leaves and fruits on some key enzymes linked to type 2 diabetes (?-amylase and ?-glucosidase); and sodium nitroprusside (SNP) induced lipid peroxidation in rats' pancreas in vitro. The phenolic extracts of Persea americana fruit and leaves were extracted using methanol and 1M HCl (1:1 v/v). Thereafter, their inhibitory effects on sodium nitroprusside induced lipid peroxidation and key enzymes linked to type 2 diabetes (?-amylase and ?-glucosidase) were determined in vitro. The result revealed that the leaves had fruit of avocado pear inhibit both ?-amylase and ?-glucosidase activities in a dose dependent manner. However, the Peel had the highest ?-amylase inhibitory activity while the leaf had the highest ?-glucosidase inhibitory activity as revealed by their IC50 value. Furthermore, incubation of the rat pancreas in the presence of 5 mM SNP caused an increase in the malondialdehyde (MDA) content in the tissue, however, introduction of the phenolic extracts inhibited MDA produced in a dose dependent manner. The additive and/or synergistic action of major phenolic compounds such as syringic acid, eugenol, vnillic acid, isoeugenol, guaiacol, kaemferol, catechin, ?-hydroxybenzoic acid, ferulic acid, apigenin, naringenin, epigallocatechin, epicatechin, lupeol and epigallocatechin-3-O-gallate in avocado pear using gas chromatography (GC) could have contributed to the observed medicinal properties of the plant. Therefore, inhibition of some key enzymes linked to type 2 diabetes and prevention of oxidative stress in the pancreas could be some of the possible mechanism by which they exert their anti-diabetic properties. PMID:25324703

  6. Growth-dependent release of carbohydrate metabolism-related and antioxidant enzymes from Staphylococcus aureus strain 6 as determined by proteomic analysis

    PubMed Central

    DONENKO, F.V.; GRUBER, I.M.; SEMENOVA, I.B.; PRIYATKIN, R.G.; ZIGANSHIN, R.H.; ZARYADYEVA, E.A.; IGNATOVA, O.M.; KURBATOVA, E.A.; KISELEVSKY, M.V.; EFFERTH, T.

    2011-01-01

    Proteins released into the culture medium by Staphylococcus aureus (S. aureus) strain 6 were determined at the end of the exponential growth phase (4.5 h). Eleven proteins were identified by liquid chromatography coupled with mass spectrometry. Three proteins were predicted to have signal peptides indicating their extracellular localization. The other proteins were presumably located in the cytoplasm of the bacteria. Five out of the 11 proteins were involved in carbohydrate metabolism. Other intracellular proteins of S. aureus were not detected in the culture medium. This indicates that the release of these 11 proteins was specific and that unspecific protein release due to damaged or dying bacteria did not play a role. It is suggested that enzymes associated with carbohydrate metabolism may provide the energy necessary for the transition of bacteria from a resting to a proliferative state. Another enzyme released by S. aureus, superoxide dismutase, may catalyze redox reactions in this context. The production of other proteolytic enzymes and toxins may take place at later stages of bacterial growth. A cocktail of these 11 proteins was used for the immunization of mice. Indeed, vaccination with these proteins prolonged the survival times of mice upon infection with S. aureus strain 6. Therefore, these proteins may have implications for the development of novel strategies for the prevention and therapy of S. aureus infections. PMID:22977644

  7. Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism

    PubMed Central

    Egeblad, Louise; Welin, Martin; Flodin, Susanne; Gräslund, Susanne; Wang, Liya; Balzarini, Jan; Eriksson, Staffan; Nordlund, Pär

    2012-01-01

    To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ?Tagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design. PMID:22662200

  8. Type 2 Diabetic Rats on Diet Supplemented With Chromium Malate Show Improved Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism

    PubMed Central

    Feng, Weiwei; Zhao, Ting; Mao, Guanghua; Wang, Wei; Feng, Yun; Li, Fang; Zheng, Daheng; Wu, Huiyu; Jin, Dun; Yang, Liuqing; Wu, Xiangyang

    2015-01-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism in type 2 diabetic rats. Our results showed that fasting blood glucose, serum insulin level, insulin resistance index and C-peptide level in the high dose group had a significant downward trend when compared with the model group, chromium picolinate group and chromium trichloride group. The hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, Glut4, phosphor-AMPK?1 and Akt levels in the high dose group were significantly higher than those of the model, chromium picolinate and chromium trichloride groups. Chromium malate in a high dose group can significantly increase high density lipoprotein cholesterol level while decreasing the total cholesterol, low density lipoprotein cholesterol and triglyceride levels when compared with chromium picolinate and chromium trichloride. The serum chromium content in chromium malate and chromium picolinate group is significantly higher than that of the chromium trichloride group. The results indicated that the curative effects of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism changes are better than those of chromium picolinate and chromium trichloride. Chromium malate contributes to glucose uptake and transport in order to improved glycometabolism and glycometabolism-related enzymes. PMID:25942313

  9. METABOLISM OF CAPSAICINOIDS BY P450 ENZYMES: A REVIEW OF RECENT FINDINGS ON REACTION MECHANISMS, BIO-ACTIVATION, AND DETOXIFICATION PROCESSES

    PubMed Central

    Reilly, Christopher A.; Yost, Garold S.

    2008-01-01

    Capsaicinoids are botanical irritants present in chili peppers. Chili pepper extracts and capsaicinoids are common dietary constituents and important pharmaceutical agents. Use of these substances in modern consumer products and medicinal preparations occurs worldwide. Capsaicinoids are the principals of pepper spray self-defense weapons and several over-the-counter pain treatments as well as the active component of many dietary supplements. Capsaicinoids interact with the capsaicin receptor (a.k.a., VR1 or TRPV1) to produce acute pain and cough as well as long-term analgesia. Capsaicinoids are also toxic to many cells via TRPV1-dependent and independent mechanisms. Chemical modifications to capsaicinoids by P450 enzymes decreases their potency at TRPV1 and reduces the pharmacological and toxicological phenomena associated with TRPV1 stimulation. Metabolism of capsaicinoids by P450 enzymes also produces reactive electrophiles capable of modifying biological macromolecules. This review highlights data describing specific mechanisms by which P450 enzymes convert the capsaicinoids to novel products and explores the relationship between capsaicinoid metabolism and its effects on capsaicinoid pharmacology and toxicology. PMID:17145696

  10. Expression of peroxisome proliferator-activated receptor-gamma in key neuronal subsets regulating glucose metabolism and energy homeostasis.

    PubMed

    Sarruf, David A; Yu, Fang; Nguyen, Hong T; Williams, Diana L; Printz, Richard L; Niswender, Kevin D; Schwartz, Michael W

    2009-02-01

    In addition to increasing insulin sensitivity and adipogenesis, peroxisome proliferator-activated receptor (PPAR)-gamma agonists cause weight gain and hyperphagia. Given the central role of the brain in the control of energy homeostasis, we sought to determine whether PPARgamma is expressed in key brain areas involved in metabolic regulation. Using immunohistochemistry, PPARgamma distribution and its colocalization with neuron-specific protein markers were investigated in rat and mouse brain sections spanning the hypothalamus, the ventral tegmental area, and the nucleus tractus solitarius. In several brain areas, nuclear PPARgamma immunoreactivity was detected in cells that costained for neuronal nuclei, a neuronal marker. In the hypothalamus, PPARgamma immunoreactivity was observed in a majority of neurons in the arcuate (including both agouti related protein and alpha-MSH containing cells) and ventromedial hypothalamic nuclei and was also present in the hypothalamic paraventricular nucleus, the lateral hypothalamic area, and tyrosine hydroxylase-containing neurons in the ventral tegmental area but was not expressed in the nucleus tractus solitarius. To validate and extend these histochemical findings, we generated mice with neuron-specific PPARgamma deletion using nestin cre-LoxP technology. Compared with littermate controls, neuron-specific PPARgamma knockout mice exhibited dramatic reductions of both hypothalamic PPARgamma mRNA levels and PPARgamma immunoreactivity but showed no differences in food intake or body weight over a 4-wk study period. We conclude that: 1) PPARgamma mRNA and protein are expressed in the hypothalamus, 2) neurons are the predominant source of PPARgamma in the central nervous system, although it is likely expressed by nonneuronal cell types as well, and 3) arcuate nucleus neurons that control energy homeostasis and glucose metabolism are among those in which PPARgamma is expressed. PMID:18845632

  11. Enzymatic reduction of benzoyl-CoA to alicyclic compounds, a key reaction in anaerobic aromatic metabolism.

    PubMed

    Koch, J; Fuchs, G

    1992-04-01

    Different anaerobic bacteria can oxidize a variety of aromatic compounds completely to CO2 via one common aromatic intermediate, benzoyl-CoA. It has been postulated that anaerobically the aromatic nucleus of benzoyl-CoA becomes reduced. An oxygen-sensitive enzyme system is described catalyzing the reduction of benzoyl-CoA to trans-2-hydroxycyclohexanecarboxyl-CoA in a denitrifying Pseudomonas species grown anaerobically on benzoate plus nitrate. The assay mixture consists of cell extract, [U-14C]benzoyl-CoA, a [U-14C]benzoyl-CoA-generating system (consisting of [U-14C]benzoate, purified benzoate-CoA ligase, Mg(2+)-ATP, coenzyme A), an ATP-regenerating system (consisting of phosphoenolpyruvate, pyruvate kinase, myokinase), and a low-potential reductant [titanium(III) citrate]. The optimal pH is about 7, the specific activity 10 nmol benzoyl-CoA reduced min-1 x mg-1 protein. The apparent Km for benzoyl-CoA is below 50 microM. Five major products were found. One product is cyclohex-1-enecarboxyl-CoA which must have been formed by a benzoyl-CoA reductase. The other product is probably trans-2-hydroxycyclohexanecarboxyl-CoA rather than the cis-stereoisomer; this product must have been formed by a cyclohex-1-enecarboxyl-CoA hydratase. Two other products are likely to be intermediates of benzoyl-CoA reduction to cyclohex-1-enecarboxyl-CoA, suggesting that the reduction reaction is more complex. An early formed fifth product is more polar than cyclohexanecarboxyl- or cyclohex-1-enecarboxyl-CoA. The enzyme system is under oxygen control since it was not found in cells grown aerobically on benzoate. It is induced by aromatic compounds since its activity is low in cells grown anaerobically on acetate. The actual inducer is probably benzoyl-CoA rather than benzoate. This conclusion is drawn from the fact that the system is also present in cells grown anaerobically on phenol, phenylacetate, 4-hydroxybenzoate, or 2-aminobenzoate; the anaerobic metabolism of these compounds has been shown in this organism to proceed directly via benzoyl-CoA rather than via free benzoate. PMID:1555579

  12. GENE EXPRESSION PROFILING OF XENOBIOTIC METABOLIZING ENZYMES (XMES) THROUGH THE LIFE STAGES OF THE MALE C57BL/6 MOUSE

    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. Oxidised low density lipoprotein causes human macrophage cell death through oxidant generation and inhibition of key catabolic enzymes.

    PubMed

    Katouah, Hanadi; Chen, Alpha; Othman, Izani; Gieseg, Steven P

    2015-10-01

    Oxidised low density lipoprotein (oxLDL) is thought to be a significant contributor to the death of macrophage cells observed in advanced atherosclerotic plaques. Using human-derived U937 cells we have examined the effect of cytotoxic oxLDL on oxidative stress and cellular catabolism. Within 3h of the addition of oxLDL, there was a rapid, concentration dependent rise in cellular reactive oxygen species followed by the loss of cellular GSH, and the enzyme activity of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aconitase. The loss of these catabolic enzymes was accompanied by the loss of cellular ATP and lower lactate generation. Addition of the macrophage antioxidant 7,8-dihydroneopterin inhibited the ROS generation, glutathione loss and catabolic inactivation. NOX was shown to be activated by oxLDL addition while apocynin inhibited the loss of GSH and cell viability. The data suggests that oxLDL triggers an excess of ROS production through NOX activation, and catabolic failure through thiol oxidation resulting in cell death. PMID:26255116

  14. Alteration of vitamin D metabolic enzyme expression and calcium transporter abundance in kidney involved in type 1 diabetes-induced bone loss

    PubMed Central

    Papasian, C. J.; Deng, H.-W.

    2015-01-01

    Summary This study aimed to delineate the mechanism involved in type 1 diabetes-induced bone loss. The results revealed the alteration of vitamin D metabolic enzyme expression and the downregulation of renal calcium transporter abundance in type 1 diabetic mice. Introduction The purpose of this study was to investigate the changes of the expression of vitamin D metabolic enzymes and transcellular calcium-transporting proteins in kidneys from mice with experimentally induced diabetes. Methods Male DBA/2J mice were injected with either vehicle (control) or streptozotocin (STZ) daily for five consecutive days. Bone mineral density was measured by peripheral quantitative computerized tomography, and bone histomorphology was analyzed by Safranin O staining. Real-time PCR and Western blotting were applied to determine the expression of target genes and proteins. Results Type 1 diabetes produced high urinary calcium excretion and loss of trabecular bone measured at the proximal metaphysis of the tibia and the distal femur. Bone loss was associated with deterioration of trabecular bone microstructure. Quantified PCR results showed that mRNA expression level in the kidney of diabetic mice for 25-hydroxyvitamin D-24-hydroxylase was downregulated at week 10, while those for 25-hydroxyvitamin D-1?-hydroxylase were upregulated at week 20. In addition, mRNA expression levels for renal transient receptor potential V6, plasma membrane Ca-ATPase (PMCA)1b, and vitamin D receptor (VDR) genes were decreased in STZ-treated mice. Western blot analysis showed that protein expression of PMCA1b and VDR was significantly decreased in kidneys from STZ-treated mice compared to that of controls. Conclusions The limitation in this study is the lack of vitamin D, parathyroid hormone, and phosphorus levels in serum. However, the present study supports the conclusion that the underlying mechanism contributing to type 1 diabetes-associated bone loss may be alterations of vitamin D metabolic enzyme expression and associated decreases in expression of renal calcium transporters. PMID:20878391

  15. Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes.

    PubMed

    Shi, Rong; Ma, Bingliang; Wu, Jiasheng; Wang, Tianming; Ma, Yueming

    2015-10-01

    The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high-throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin-O deethylation, coumarin-7 hydroxylation, bupropion hydroxylation, taxol-6 hydroxylation, omeprazole-5 hydroxylation, dextromethorphan-O demethylation, tolbutamide-4 hydroxylation, chlorzoxazone-6 hydroxylation, testosterone-6? hydroxylation, and midazolam-1 hydroxylation in rat liver microsomes. PMID:26256777

  16. Structure-based drug design studies of UDP-N-acetylglucosamine pyrophosphosrylase, a key enzyme for the control of witches’ broom disease

    PubMed Central

    2013-01-01

    Background The witches’ broom disease is a plague caused by Moniliophthora perniciosa in the Theobroma cacao, which has been reducing the cocoa production since 1989. This issue motivated a genome project that has showing several new molecular targets, which can be developed inhibitors in order to control the plague. Among the molecular targets obtained, the UDP-N-acetylglucosamine pyrophosphorylase (UNAcP) is a key enzyme to construct the fungal cell wall. The inhibition of this enzyme results in the fungal cell death. Results The results show that the molecular recognition of the enzyme with the substrates occurs mainly by hydrogen bonds between ligands and Arg116, Arg383, Gly381, and Lys408 amino acids; and few hydrophobic interactions with Tyr382 and Lys123 residues. Conclusions Among the compounds analyzed, the NAG5 showed the best binding energy (?95.2 kcal/mol). The next steps for the control of witches’ broom plague involve the synthesis and biological evaluation of these compounds, which are in progress. PMID:23497581

  17. Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea

    PubMed Central

    Cao, Shiyun; Hepowit, Nathaniel; Maupin-Furlow, Julie A.

    2015-01-01

    Ubiquitin/ubiquitin-like (Ub/Ubl) proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1) and a JAMM/MPN+ metalloprotease (HvJAMM1). Molybdopterin (MPT) synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE) of MPT synthase. HvJAMM1 was shown to cleave the covalently linked inactive form of SAMP1-MoaE to the free functional individual SAMP1 and MoaE subunits of MPT synthase, suggesting reactivation of MPT synthase by this metalloprotease. Overall, this study provides new insight into the broad idea that Ub/Ubl modification is a post-translational process that can directly and reversibly regulate the activity of metabolic enzymes. In particular, we show that Ub/Ubl linkages on the active site residues of an enzyme (MPT synthase) can inhibit its catalytic activity and that the enzyme can be reactivated through cleavage by a JAMM/MPN+ metalloprotease. PMID:26010867

  18. An Enhanced In Vivo Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Model for Quantification of Drug Metabolism Enzymes*

    PubMed Central

    MacLeod, A. Kenneth; Fallon, Padraic G.; Sharp, Sheila; Henderson, Colin J.; Wolf, C. Roland; Huang, Jeffrey T.-J.

    2015-01-01

    Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug–drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models. PMID:25561501

  19. Novel Roles for the Polyphenol Oxidase Enzyme in Secondary Metabolism and the Regulation of Cell Death in Walnut1[W][OPEN

    PubMed Central

    Araji, Soha; Grammer, Theresa A.; Gertzen, Ross; Anderson, Stephen D.; Mikulic-Petkovsek, Maja; Veberic, Robert; Phu, My L.; Solar, Anita; Leslie, Charles A.; Dandekar, Abhaya M.; Escobar, Matthew A.

    2014-01-01

    The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut. PMID:24449710

  20. Synthesis and characterization of mononuclear oxovanadium(IV) complexes and their enzyme inhibition studies with a carbohydrate metabolic enzyme phosphodiesterase I.

    PubMed

    Mahroof-Tahir, Mohammad; Brezina, Dan; Fatima, Naheed; Choudhary, Muhammad Iqbal; Atta-ur-Tahman

    2005-02-01

    The increasing interest in vanadium coordination chemistry is based on its well-established chemical and biological functions. A beta-diketonato complex of oxovanadium(IV) is known to be having numerous catalytic applications and also exhibits promising insulin mimetic properties. In continuation of our structure activity relationship studies of metal complexes, we report herein the synthesis and characterization of the vanadium complexes of beta-diketonato ligand system with systematic variations of electronic and steric factors. Two complexes, VO(tmh)(2) (tmh = 2,2,6,6,-tetramethyl-3,5-heptanedione), and VO(hd)(2) (hd = 3,5-heptanedione) were synthesized and characterized by using different spectroscopic techniques. Elemental and mass spectral analysis supports the presence of two beta-diketonato ligands per VO(2+) unit. UV-Vis spectra in different solvents indicate coordination of coordinating solvent molecules at sixth position resulting in red shift of the band I transition. NMR and IR spectra reveal binding of coordinating solvent molecule at vacant sixth position trans to oxo group without releasing beta-diketonato ligands. Enzyme inhibition studies of these and other related oxovanadium(IV) complexes with beta-diketonato ligand system are conducted with snake venom phosphodiesterase I (SPVDE). All of these complexes showed significant inhibitory potential and were found to be non-competitive inhibitors against this enzyme. PMID:15702518

  1. Purification and Identification of Naringenin 7-O-Methyltransferase, a Key Enzyme in Biosynthesis of Flavonoid Phytoalexin Sakuranetin in Rice*

    PubMed Central

    Shimizu, Takafumi; Lin, Fengqiu; Hasegawa, Morifumi; Okada, Kazunori; Nojiri, Hideaki; Yamane, Hisakazu

    2012-01-01

    Sakuranetin, the major flavonoid phytoalexin in rice, is induced by ultraviolet (UV) irradiation, CuCl2 treatment, jasmonic acid treatment, and infection by phytopathogens. It was recently demonstrated that sakuranetin has anti-inflammatory activity, anti-mutagenic activity, anti-pathogenic activities against Helicobacter pylori, Leishmania, and Trypanosoma and contributes to the maintenance of glucose homeostasis in animals. Thus, sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). In previous research, rice NOMT (OsNOMT) was purified to apparent homogeneity from UV-treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT) activity and not NOMT activity. In this study, we found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and we purified OsNOMT using the oscomt1 mutant. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps, resulting in a 400-fold purification from the crude enzyme preparation. Using SDS-PAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa. Two O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties to OsNOMT. On the basis of these results, we conclude that Os12g0240900 encodes an OsNOMT. PMID:22493492

  2. Regulation and Localization of Key Enzymes during the Induction of Kranz-Less, C4-Type Photosynthesis in Hydrilla verticillata.

    PubMed Central

    Magnin, N. C.; Cooley, B. A.; Reiskind, J. B.; Bowes, G.

    1997-01-01

    Kranz-less, C4-type photosynthesis was induced in the submersed monocot Hydrilla verticillata (L.f.) Royle. During a 12-d induction period the CO2 compensation point and O2 inhibition of photosynthesis declined linearly. Phosphoenolpyruvate carboxylase (PEPC) activity increased 16-fold, with the major increase occurring within 3 d. Asparagine and alanine aminotransferases were also induced rapidly. Pyruvate orthophosphate dikinase (PPDK) and NADP-malic enzyme (ME) activities increased 10-fold but slowly over 15 d. Total ribulose-1,5-bisphosphate carboxylase/oxygenase activity did not increase, and its activation declined from 82 to 50%. Western blots for PEPC, PPDK, and NADP-ME indicated that increased protein levels were involved in their induction. The H. verticillata NADP-ME polypeptide was larger (90 kD) than the maize C4 enzyme (62 kD). PEPC and PPDK exhibited up-regulation in the light. Subcellular fractionation of C4-type leaves showed that PEPC was cytosolic, whereas PPDK and NADP-ME were located in the chloroplasts. The O2 inhibition of photosynthesis was doubled when C4-type but not C3-type leaves were exposed to diethyl oxalacetate, a PEPC inhibitor. The data are consistent with a C4-cycle concentrating CO2 in H. verticillata chloroplasts and indicate that Kranz anatomy is not obligatory for C4-type photosynthesis. H. verticillata predates modern terrestrial C4 monocots; therefore, this inducible CO2-concentrating mechanism may represent an ancient form of C4 photosynthesis. PMID:12223888

  3. Overexpression of metabolic enzymes at the junction of glycolysis and the TCA cycle in Escherichia coli: physiological effects and application 

    E-print Network

    Spitzer, Richard G.

    1999-01-01

    the enzyme phosphoenolpyruvate synthase (Pps) has increased the yield of the aromatic precursor 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) from glucose by two- fold compared to previous methods. In this study, Pps overexpression is again applied...

  4. Phenotypic flexibility in passerine birds: Seasonal variation of aerobic enzyme activities in skeletal muscle

    E-print Network

    Swanson, David L.

    Phenotypic flexibility in passerine birds: Seasonal variation of aerobic enzyme activities activities of the key aerobic enzymes citrate synthase (CS) and b-hydroxyacyl CoA-dehydrogenase (HOAD and cellular aerobic capacity in muscle contribute to seasonal metabolic flexibility in some species

  5. Variation in Sulfur and Selenium Accumulation Is Controlled by Naturally Occurring Isoforms of the Key Sulfur Assimilation Enzyme ADENOSINE 5?-PHOSPHOSULFATE REDUCTASE2 across the Arabidopsis Species Range1[W][OPEN

    PubMed Central

    Chao, Dai-Yin; Baraniecka, Patrycja; Danku, John; Koprivova, Anna; Lahner, Brett; Luo, Hongbing; Yakubova, Elena; Dilkes, Brian; Kopriva, Stanislav; Salt, David E.

    2014-01-01

    Natural variation allows the investigation of both the fundamental functions of genes and their role in local adaptation. As one of the essential macronutrients, sulfur is vital for plant growth and development and also for crop yield and quality. Selenium and sulfur are assimilated by the same process, and although plants do not require selenium, plant-based selenium is an important source of this essential element for animals. Here, we report the use of linkage mapping in synthetic F2 populations and complementation to investigate the genetic architecture of variation in total leaf sulfur and selenium concentrations in a diverse set of Arabidopsis (Arabidopsis thaliana) accessions. We identify in accessions collected from Sweden and the Czech Republic two variants of the enzyme ADENOSINE 5?-PHOSPHOSULFATE REDUCTASE2 (APR2) with strongly diminished catalytic capacity. APR2 is a key enzyme in both sulfate and selenate reduction, and its reduced activity in the loss-of-function allele apr2-1 and the two Arabidopsis accessions Hodonín and Shahdara leads to a lowering of sulfur flux from sulfate into the reduced sulfur compounds, cysteine and glutathione, and into proteins, concomitant with an increase in the accumulation of sulfate in leaves. We conclude from our observation, and the previously identified weak allele of APR2 from the Shahdara accession collected in Tadjikistan, that the catalytic capacity of APR2 varies by 4 orders of magnitude across the Arabidopsis species range, driving significant differences in sulfur and selenium metabolism. The selective benefit, if any, of this large variation remains to be explored. PMID:25245030

  6. Phenolic Extract from Moringa oleifera Leaves Inhibits Key Enzymes Linked to Erectile Dysfunction and Oxidative Stress in Rats' Penile Tissues.

    PubMed

    Oboh, Ganiyu; Ademiluyi, Adedayo O; Ademosun, Ayokunle O; Olasehinde, Tosin A; Oyeleye, Sunday I; Boligon, Aline A; Athayde, Margareth L

    2015-01-01

    This study was designed to determine the antioxidant properties and inhibitory effects of extract from Moringa oleifera leaves on angiotensin-I-converting enzyme (ACE) and arginase activities in vitro. The extract was prepared and phenolic (total phenols and flavonoid) contents, radical (nitric oxide (NO), hydroxyl (OH)) scavenging abilities, and Fe(2+)-chelating ability were assessed. Characterization of the phenolic constituents was done via high performance liquid chromatography-diode array detection (HPLC-DAD) analysis. Furthermore, the effects of the extract on Fe(2+)-induced MDA production in rats' penile tissue homogenate as well as its action on ACE and arginase activities were also determined. The extract scavenged NO (?) , OH (?) , chelated Fe(2+), and inhibited MDA production in a dose-dependent pattern with IC50 values of 1.36, 0.52, and 0.38?mg/mL and 194.23?µg/mL, respectively. Gallic acid, chlorogenic acid, quercetin, and kaempferol were the most abundant phenolic compounds identified in the leaf extract. The extract also inhibited ACE and arginase activities in a dose-dependent pattern and their IC50 values were 303.03 and 159.59?µg/mL, respectively. The phenolic contents, inhibition of ACE, arginase, and Fe(2+)-induced MDA production, and radical (OH (?) , NO (?) ) scavenging and Fe(2+)-chelating abilities could be some of the possible mechanisms by which M. oleifera leaves could be used in the treatment and/or management of erectile dysfunction. PMID:26557995

  7. Phenolic Extract from Moringa oleifera Leaves Inhibits Key Enzymes Linked to Erectile Dysfunction and Oxidative Stress in Rats' Penile Tissues

    PubMed Central

    Oboh, Ganiyu; Ademiluyi, Adedayo O.; Ademosun, Ayokunle O.; Olasehinde, Tosin A.; Oyeleye, Sunday I.; Boligon, Aline A.; Athayde, Margareth L.

    2015-01-01

    This study was designed to determine the antioxidant properties and inhibitory effects of extract from Moringa oleifera leaves on angiotensin-I-converting enzyme (ACE) and arginase activities in vitro. The extract was prepared and phenolic (total phenols and flavonoid) contents, radical (nitric oxide (NO), hydroxyl (OH)) scavenging abilities, and Fe2+-chelating ability were assessed. Characterization of the phenolic constituents was done via high performance liquid chromatography-diode array detection (HPLC-DAD) analysis. Furthermore, the effects of the extract on Fe2+-induced MDA production in rats' penile tissue homogenate as well as its action on ACE and arginase activities were also determined. The extract scavenged NO?, OH?, chelated Fe2+, and inhibited MDA production in a dose-dependent pattern with IC50 values of 1.36, 0.52, and 0.38?mg/mL and 194.23?µg/mL, respectively. Gallic acid, chlorogenic acid, quercetin, and kaempferol were the most abundant phenolic compounds identified in the leaf extract. The extract also inhibited ACE and arginase activities in a dose-dependent pattern and their IC50 values were 303.03 and 159.59?µg/mL, respectively. The phenolic contents, inhibition of ACE, arginase, and Fe2+-induced MDA production, and radical (OH?, NO?) scavenging and Fe2+-chelating abilities could be some of the possible mechanisms by which M. oleifera leaves could be used in the treatment and/or management of erectile dysfunction. PMID:26557995

  8. Structural insights into a key carotenogenesis related enzyme phytoene synthase of P. falciparum: a novel drug target for malaria.

    PubMed

    Agarwal, Shalini; Sharma, Vijeta; Phulera, Swastik; Abdin, M Z; Ayana, R; Singh, Shailja

    2015-12-01

    Carotenoids represent a diverse group of pigments derived from the common isoprenoid precursors and fulfill a variety of critical functions in plants and animals. Phytoene synthase (PSY), a transferase enzyme that catalyzes the first specific step in carotenoid biosynthesis plays a central role in the regulation of a number of essential functions mediated via carotenoids. PSYs have been deeply investigated in plants, bacteria and algae however in apicomplexans it is poorly studied. In an effort to characterize PSY in apicomplexans especially the malaria parasite Plasmodium falciparum (P. falciparum), a detailed bioinformatics analysis is undertaken. We have analysed the Phylogenetic relationship of PSY also referred to as octaprenyl pyrophosphate synthase (OPPS) in P. falciparum with other taxonomic groups. Further, we in silico characterized the secondary and tertiary structures of P. falciparum PSY/OPPS and compared the tertiary structures with crystal structure of Thermotoga maritima (T. maritima) OPPS. Our results evidenced the resemblance of P. falciparum PSY with the active site of T. maritima OPPS. Interestingly, the comparative structural analysis revealed an unconserved unique loop in P. falciparum OPPS/PSY. Such structural insights might contribute novel accessory functions to the protein thus, offering potential drug targets. PMID:26702306

  9. Proteomics of Fusarium oxysporum Race 1 and Race 4 Reveals Enzymes Involved in Carbohydrate Metabolism and Ion Transport That Might Play Important Roles in Banana Fusarium Wilt

    PubMed Central

    Peng, Ming; Zeng, Huicai; Wang, Dan; Li, Bo; Tong, Zheng; Chang, Lili; Jin, Xiang; Wang, Xuchu

    2014-01-01

    Banana Fusarium wilt is a soil–spread fungal disease caused by Fusarium oxysporum. In China, the main virulence fungi in banana are F. oxysporum race 1 (F1, weak virulence) and race 4 (F4, strong virulence). To date, no proteomic analyses have compared the two races, but the difference in virulence between F1 and F4 might result from their differentially expressed proteins. Here we report the first comparative proteomics of F1 and F4 cultured under various conditions, and finally identify 99 protein species, which represent 59 unique proteins. These proteins are mainly involved in carbohydrate metabolism, post-translational modification, energy production, and inorganic ion transport. Bioinformatics analysis indicated that among the 46 proteins identified from F4 were several enzymes that might be important for virulence. Reverse transcription PCR analysis of the genes for 15 of the 56 proteins revealed that their transcriptional patterns were similar to their protein expression patterns. Taken together, these data suggest that proteins involved in carbohydrate metabolism and ion transport may be important in the pathogenesis of banana Fusarium wilt. Some enzymes such as catalase-peroxidase, galactosidase and chitinase might contribute to the strong virulence of F4. Overexpression or knockout of the genes for the F4-specific proteins will help us to further understand the molecular mechanism of Fusarium-induced banana wilt. PMID:25460190

  10. Proteomics of Fusarium oxysporum race 1 and race 4 reveals enzymes involved in carbohydrate metabolism and ion transport that might play important roles in banana Fusarium wilt.

    PubMed

    Sun, Yong; Yi, Xiaoping; Peng, Ming; Zeng, Huicai; Wang, Dan; Li, Bo; Tong, Zheng; Chang, Lili; Jin, Xiang; Wang, Xuchu

    2014-01-01

    Banana Fusarium wilt is a soil-spread fungal disease caused by Fusarium oxysporum. In China, the main virulence fungi in banana are F. oxysporum race 1 (F1, weak virulence) and race 4 (F4, strong virulence). To date, no proteomic analyses have compared the two races, but the difference in virulence between F1 and F4 might result from their differentially expressed proteins. Here we report the first comparative proteomics of F1 and F4 cultured under various conditions, and finally identify 99 protein species, which represent 59 unique proteins. These proteins are mainly involved in carbohydrate metabolism, post-translational modification, energy production, and inorganic ion transport. Bioinformatics analysis indicated that among the 46 proteins identified from F4 were several enzymes that might be important for virulence. Reverse transcription PCR analysis of the genes for 15 of the 56 proteins revealed that their transcriptional patterns were similar to their protein expression patterns. Taken together, these data suggest that proteins involved in carbohydrate metabolism and ion transport may be important in the pathogenesis of banana Fusarium wilt. Some enzymes such as catalase-peroxidase, galactosidase and chitinase might contribute to the strong virulence of F4. Overexpression or knockout of the genes for the F4-specific proteins will help us to further understand the molecular mechanism of Fusarium-induced banana wilt. PMID:25460190

  11. CYP82Y1 Is N-Methylcanadine 1-Hydroxylase, a Key Noscapine Biosynthetic Enzyme in Opium Poppy*

    PubMed Central

    Dang, Thu-Thuy T.; Facchini, Peter J.

    2014-01-01

    Noscapine is a phthalideisoquinoline alkaloid investigated for its potent pharmacological properties. Although structurally elucidated more than a century ago, the biosynthesis of noscapine has not been established. Radiotracer studies have shown that noscapine is derived from the protoberberine alkaloid (S)-scoulerine and has been proposed to proceed through (S)-N-methylcanadine. However, pathway intermediates involved in the conversion of N-methylcanadine to noscapine have not been identified. We report the isolation and characterization of the cytochrome P-450 CYP82Y1, which catalyzes the 1-hydroxylation of N-methylcanadine to 1-hydroxy-N-methylcanadine. Comparison of transcript and metabolite profiles of eight opium poppy chemotypes revealed four cytochrome P-450s, three from the CYP82 and one from the CYP719 families, that were tightly correlated with noscapine accumulation. Recombinant CYP82Y1 was the only enzyme that accepted (R,S)-N-methylcanadine as a substrate with strict specificity and high affinity. As expected, CYP82Y1 was abundantly expressed in opium poppy stems where noscapine accumulation is highest among plant organs. Suppression of CYP82Y1 using virus-induced gene silencing caused a significant reduction in the levels of noscapine, narcotoline, and a putative downstream secoberbine intermediate and also resulted in increased accumulation of the upstream pathway intermediates scoulerine, tetrahydrocolum-bamine, canadine, and N-methylcanadine. The combined biochemical and physiological data support the 1-hydroxylation of (S)-N-methylcanadine catalyzed by CYP82Y1 as the first committed step in the formation of noscapine in opium poppy. PMID:24324259

  12. CYP82Y1 is N-methylcanadine 1-hydroxylase, a key noscapine biosynthetic enzyme in opium poppy.

    PubMed

    Dang, Thu-Thuy T; Facchini, Peter J

    2014-01-24

    Noscapine is a phthalideisoquinoline alkaloid investigated for its potent pharmacological properties. Although structurally elucidated more than a century ago, the biosynthesis of noscapine has not been established. Radiotracer studies have shown that noscapine is derived from the protoberberine alkaloid (S)-scoulerine and has been proposed to proceed through (S)-N-methylcanadine. However, pathway intermediates involved in the conversion of N-methylcanadine to noscapine have not been identified. We report the isolation and characterization of the cytochrome P-450 CYP82Y1, which catalyzes the 1-hydroxylation of N-methylcanadine to 1-hydroxy-N-methylcanadine. Comparison of transcript and metabolite profiles of eight opium poppy chemotypes revealed four cytochrome P-450s, three from the CYP82 and one from the CYP719 families, that were tightly correlated with noscapine accumulation. Recombinant CYP82Y1 was the only enzyme that accepted (R,S)-N-methylcanadine as a substrate with strict specificity and high affinity. As expected, CYP82Y1 was abundantly expressed in opium poppy stems where noscapine accumulation is highest among plant organs. Suppression of CYP82Y1 using virus-induced gene silencing caused a significant reduction in the levels of noscapine, narcotoline, and a putative downstream secoberbine intermediate and also resulted in increased accumulation of the upstream pathway intermediates scoulerine, tetrahydrocolum-bamine, canadine, and N-methylcanadine. The combined biochemical and physiological data support the 1-hydroxylation of (S)-N-methylcanadine catalyzed by CYP82Y1 as the first committed step in the formation of noscapine in opium poppy. PMID:24324259

  13. Molecular Insight into Substrate Recognition and Catalysis of Baeyer–Villiger Monooxygenase MtmOIV, the Key Frame Modifying Enzyme in the Biosynthesis of Anticancer Agent Mithramycin

    PubMed Central

    Buchanan, Susan K.; Rohr, Jürgen

    2013-01-01

    Baeyer-Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C-C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed us to probe the substrate binding pocket of MtmOIV, and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues. PMID:23992662

  14. Interplay between epigenetics & cancer metabolism.

    PubMed

    Gupta, Vibhor; Gopinath, P; Iqbal, Mohd Askandar; Mazurek, Sybille; Wellen, Kathryn E; Bamezai, Rameshwar N K

    2014-01-01

    Nutrient utilization is dramatically altered when cells receive signals to proliferate. Characteristic metabolic changes enable cells to meet the large biosynthetic demands associated with cell growth and division. Changes in rate-limiting glycolytic enzymes redirect metabolism to support growth and proliferation. Metabolic reprogramming in cancer is controlled largely by oncogenic activation of signal transduction pathways and transcription factors. Although less well understood, epigenetic mechanisms may seem to contribute to the regulation of metabolic gene expression in cancer. Reciprocally, accumulating evidence suggests that metabolic alterations may affect the epigenome. Understanding the relation between metabolism and epigenetics in cancer cells may open new avenues for anti-cancer strategies. In multi-cellular systems, molecular signals promoting cell growth and proliferation mediate the switch between catabolism and anabolism. Both normal proliferating and cancer cells must achieve high levels of macromolecular biosynthesis to provide the raw materials needed to produce new daughter cells. From a therapeutic view point, it is of great interest to determine metabolic differences that exist between normal proliferating cells and cancer cells. Cancer cells also exhibit significant alterations in the epigenome. Recent data indicate that cellular metabolism and epigenetic phenomenon are engaged in crosstalk. Considering current efforts to target both cancer metabolism and epigenetics, an understanding of the relationship between these two key features is of paramount importance. Here we discuss the role of cellular metabolism in regulation of the epigenome. Moreover, we discuss how epigenetic changes may contribute to establish cancer-specific metabolic features. PMID:23888952

  15. Effect of PCB153 on BaP-induced genotoxicity in HepG2 cells via modulation of metabolic enzymes.

    PubMed

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

    2009-04-30

    Benzo(a)pyrene (BaP) is a representative environmental carcinogen and is metabolically activated by several cytochrome P450 (CYP) enzymes to become the ultimate carcinogen. Numerous studies have indicated that 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) could effectively alter the activity of xenobiotic metabolizing enzymes (XMEs). Therefore, we propose that PCB153 may affect BaP-induced genotoxicity mediated by XMEs. In the present study, we treated HepG2 cells with BaP (50 microM) or PCB153 (0.1, 1, 10 and 100 microM), or pretreated the cells with PCB153 for 48 h followed by treatment with a combination of both BaP and PCB153. CYP1A1 activity was dramatically increased in cells treated with either BaP or PCB153. Glutathione-S-transferase (GST) activity was increased in BaP-treated cells, but decreased in PCB153-treated cells. In parallel to studies on enzyme activity, the micronuclei (MN) assay was used to assess the genotoxic damage caused by BaP and PCB153. BaP and PCB153 at 100 microM enhanced MN formation. In contrast to BaP treatment alone, treatment with both BaP and PCB153 significantly enhanced the activity of CYP1A1 and the formation of MN, but reduced the activity of GST. alpha-Naphthoflavone (ANF), an inhibitor of CYP1A1, inhibited MN formation in the presence of both BaP and PCB153. In addition, there was a positive correlation between CYP1A activity and MN formation (r(2)=0.794, P<0.001). Our observations suggest that co-exposure to BaP and PCB153 may increase BaP-induced genotoxicity, possibly through the induction of CYP1A1 and inhibition of GST. PMID:19386251

  16. Effects of pre- and postnatal exposure to 3,3[prime],4,4-[prime],5-pentachlorobiphenyl on physical development, neurobehavior and xenobiotic metabolizing enzymes in rats

    SciTech Connect

    Bernhoft, A.; Nafstad, I.; Engen, P. ); Skaare, J.U. )

    1994-10-01

    An experiment was conducted to study the effects of the coplanar non-ortho-chlorinated congener 3,3[prime],4,4[prime],5-pentachlorobiphenyl (PCB-126) in rats exposed during fetal development and postnatal suckling period. Two groups of eight dams were administered by gavage six doses of 10 and 20 [mu]g/kg body weight of PCB-126 dissolved in corn oil every second day from days 9 to 19 of gestation. The corresponding control rats were treated with corn oil only. The physical development of the offspring was observed. The effects of PCB-126 on hepatic xenobiotic metabolizing enzyme activities and the concentrations of PCB in the liver and brain were investigated in samples from pups of different age and from their mothers. The litter size, the body weights, and the survival of the exposed sucklings were reduced, and the onset of spontaneous movement and neuromuscular maturation were delayed, whereas the development of reflexes was not affected. The body weight was still reduced in a dose-related manner up to 18 weeks postpartum. Also, the postpartum body weight of the PCB-exposed mothers was reduced as compared to controls, but the difference disappeared at weaning. The hepatic enzyme activities of cytochrome P450 1A1 examined by ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) toward 1-chloro-2,4-dinitrobenzene (CDNB) were increased in both the exposed pups and their mothers, and the relative liver weight was increased in the exposed pups. Hepatic PCB-126 residues were detected in samples collected throughout the experiment, whereas no detectable concentration was found in the brain. The authors conclude that exposure of this PCB congener in utero and through lactation showed fetotoxic effects, delayed physical maturation, and induced liver xenobiotic metabolizing enzymes without causing neurobehavioral effects.

  17. 2008 GRC Iron Sulfur Enzymes-Conference to be held June 8-13, 2008

    SciTech Connect

    Stephen Cramer, Nancy Ryan Gray

    2009-01-01

    Iron-sulfur proteins are among the most common and ancient enzymes and electron-transfer agents in nature. They play key roles in photosynthesis, respiration, and the metabolism of small molecules such as H2, CO, and N2. The Iron Sulfur Enzyme Gordon Research Conference evolved from an earlier GRC on Nitrogen Fixation that began in 1994. The scope of the current meeting has broadened to include all enzymes or metalloproteins in which Fe-S bonds play a key role. This year's meeting will focus on the biosynthesis of Fe-S clusters, as well as the structure and mechanism of key Fe-S enzymes such as hydrogenase, nitrogenase and its homologues, radical SAM enzymes, and aconitase-related enzymes. Recent progress on the role of Fe-S enzymes in health, disease, DNA/RNA-processing, and alternative bio-energy systems will also be highlighted. This conference will assemble a broad, diverse, and international group of biologists and chemists who are investigating fundamental issues related to Fe-S enzymes, on atomic, molecular, organism, and environmental scales. The topics to be addressed will include: Biosynthesis & Genomics of Fe-S Enzymes; Fundamental Fe-S Chemistry; Hydrogen and Fe-S Enzymes; Nitrogenase & Homologous Fe-S Enzymes; Fe-S Enzymes in Health & Disease; Radical SAM and Aconitase-Related Fe-S Enzymes; Fe-S Enzymes and Synthetic Analogues in BioEnergy; and Fe-S Enzymes in Geochemistry and the Origin of Life.

  18. Inhibition of lipopolysaccharide-induced liver injury in rats treated with a hepatic drug-metabolizing enzyme inducer p,p'-DDT.

    PubMed

    Shimada, Yuko; Tomita, Mariko; Yoshida, Toshinori; Fukuyama, Tomoki; Katoh, Yoshitaka; Ohnuma-Koyama, Aya; Takahashi, Naofumi; Soma, Katsumi; Kojima, Sayuri; Ohtsuka, Ryoichi; Takeda, Makio; Kuwahara, Maki; Harada, Takanori

    2015-03-01

    Hepatocellular hypertrophy in association with drug-metabolizing enzyme induction is considered to be an adaptive change associated with drug metabolism. To improve our understanding of liver hypertrophy, we determined the effect of a single ip injection of either lipopolysaccharide (LPS) or vehicle in male F344 rats with hepatocellular hypertrophy induced by oral delivery of p,p'-DDT for 2 weeks. The rats were sacrificed 3h or 24h after LPS or vehicle injection. LPS induced a focal hepatocellular necrosis in rats fed the control diet. When rats pre-treated with p,p'-DDT were injected with LPS, necrotic foci surrounded by ballooned hepatocytes were observed in the liver. The change was consistent with reduced LPS-mediated increases in plasma hepatic biomarkers, neutrophil influx, and apoptosis, and also associated with hepatic mRNA levels of TNF-?, CYPs, and NOS2. By contrast, when combined with p,p'-DDT and LPS, faint hepatocellular fatty change was extended, together with a synergistic increase in total blood cholesterol. These results suggest that hepatocytes exposed to p,p'-DDT are protected from the cell-lethal toxic effects of an exogenous stimulus, resulting in cell ballooning rather than necrosis in association with reduced inflammation and apoptosis, but compromised by an adverse effect on lipid metabolism. PMID:25577727

  19. Sterol Side Chain Reductase 2 Is a Key Enzyme in the Biosynthesis of Cholesterol, the Common Precursor of Toxic Steroidal Glycoalkaloids in Potato[W][OPEN

    PubMed Central

    Sawai, Satoru; Ohyama, Kiyoshi; Yasumoto, Shuhei; Seki, Hikaru; Sakuma, Tetsushi; Yamamoto, Takashi; Takebayashi, Yumiko; Kojima, Mikiko; Sakakibara, Hitoshi; Aoki, Toshio; Muranaka, Toshiya; Saito, Kazuki; Umemoto, Naoyuki

    2014-01-01

    Potatoes (Solanum tuberosum) contain ?-solanine and ?-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels. PMID:25217510

  20. Metabolism of cydiastatin 4 and analogues by enzymes associated with the midgut and haemolymph of Manduca sexta larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The degradation of synthetic cydiastatin 4 (ARPYSFGL-amide) and cydiastatin 4 analogues cydiastatin 4a (PPPPPARPYSFGL-amide) and cydiastatin 4b (PPPPPARPYSF[Acpc]L-amide) by enzymes associated with the midgut and/or haemolymph of the tobacco hawkmoth moth, Manduca sexta were investigated using rever...