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Sample records for hepatic enzyme activity

  1. Hepatic biotransformation and antioxidant enzyme activities in Mediterranean fish from different habitat depths.

    PubMed

    Ribalta, C; Sanchez-Hernandez, J C; Sole, M

    2015-11-01

    Marine fish are threatened by anthropogenic chemical discharges. However, knowledge on adverse effects on deep-sea fish or their detoxification capabilities is limited. Herein, we compared the basal activities of selected hepatic detoxification enzymes in several species (Solea solea, Dicentrarchus labrax, Trachyrhynchus scabrus, Mora moro, Cataetix laticeps and Alepocehalus rostratus) collected from the coast, middle and lower slopes of the Blanes Canyon region (Catalan continental margin, NW Mediterranean Sea). The xenobiotic-detoxifying enzymes analysed were the phase-I carboxylesterases (CbEs), and the phase-II conjugation activities uridine diphosphate glucuronyltransferase (UDPGT) and glutathione S-transferase (GST). Moreover, some antioxidant enzyme activities, i.e., catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR), were also included in this comparative study. Because CbE activity is represented by multiple isoforms, the substrates α-naphthyl acetate (αNA) and ρ-nitrophenyl acetate (ρNPA) were used in the enzyme assays, and in vitro inhibition kinetics with dichlorvos were performed to compare interspecific CbE sensitivity. Activity of xenobiotic detoxification enzymes varied among the species, following a trend with habitat depth and body size. Thus, UDPGT and some antioxidant enzyme activities decreased in fish inhabiting lower slopes of deep-sea, whereas UDPGT and αNA-CbE activities were negatively related to fish size. A trend between CbE activities and the IC50 values for dichlorvos suggested S. solea and M. moro as potentially more sensitive to anticholinesterasic pesticides, and T. scabrus as the most resistant one. A principal component analysis considering all enzyme activities clearly identified the species but this grouping was not related to habitat depth or phylogeny. Although these results can be taken as baseline levels of the main xenobiotic detoxification enzymes in Mediterranean fish, further research is

  2. Acute effects of oral and intravenous ethanol on rat hepatic enzyme activities.

    PubMed

    Stifel, F B; Greene, H L; Lufkin, E G; Wrensch, M R; Hagler, L; Herman, R H

    1976-05-28

    1. Oral administration of ethanol (3 ml) of 95% in 12 ml total volume over a two day period) significantly decrease plasma glucose and insulin levels and the activities of two key gluconeogenic enzymes, pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1) and fructose diphosphatase, (D-Fru-1,6-P2 1-phosphohydrolase, EC 3.1.3.11), and one glycolytic enzyme, fructose-1,6-P2 aldolase (Fru-1,6-P2 D-glyceraldehyde-3-P lyase, EC 4.1.2.13). In each instance, the administration of 2400 mug daily of oral folate in conjuction with the ethanol prevented these alterations in carbohydrate metabolism. 2. Intravenous injection of ethanol produced a rapid decrease (within 10--15 min) in the activities of hepatic phosphofructokinase, (ATP:D-fructose-6-phosphate 6-phosphotransferase, EC 2.7.1.11), pyruvate kinase, (ATP:pyruvate phosphotransferase, EC 2.7.1.40), fructose diphosphatase and fructose-1,6-P2 aldolase. 3. Intravenous ethanol significantly increased hepatic cyclic AMP concentration approximately 60% within 10 min, while oral ethanol did not alter hepatic cyclic AMP concentrations. 4. These data confirm the known antagonism ethanol and folate and suggest that oral folate might offer a protective effect against hypoglycemia in rats receiving ethanol. PMID:179581

  3. Age-Related Changes in Hepatic Activity and Expression of Detoxification Enzymes in Male Rats

    PubMed Central

    Vyskočilová, Erika; Szotáková, Barbora; Skálová, Lenka; Bártíková, Hana; Hlaváčová, Jitka

    2013-01-01

    Process of aging is accompanied by changes in the biotransformation of xenobiotics and impairment of normal cellular functions by free radicals. Therefore, this study was designed to determine age-related differences in the activities and/or expressions of selected drug-metabolizing and antioxidant enzymes in young and old rats. Specific activities of 8 drug-metabolizing enzymes and 4 antioxidant enzymes were assessed in hepatic subcellular fractions of 6-week-old and 21-month-old male Wistar rats. Protein expressions of carbonyl reductase 1 (CBR1) and glutathione S-transferase (GST) were determined using immunoblotting. Remarkable age-related decrease in specific activities of CYP2B, CYP3A, and UDP-glucuronosyl transferase was observed, whereas no changes in activities of CYP1A2, flavine monooxygenase, aldo-keto reductase 1C, and antioxidant enzymes with advancing age were found. On the other hand, specific activity of CBR1 and GST was 2.4 folds and 5.6 folds higher in the senescent rats compared with the young ones, respectively. Interindividual variability in CBR1 activity increased significantly with rising age. We suppose that elevated activities of GST and CBR1 may protect senescent rats against xenobiotic as well as eobiotic electrophiles and reactive carbonyls, but they may alter metabolism of drugs, which are CBR1 and especially GSTs substrates. PMID:23971034

  4. Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

    PubMed Central

    Deliconstantinos, G; Ramantanis, G

    1983-01-01

    A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration. PMID:6309144

  5. Activity-based protein profiling identifies a host enzyme, carboxylesterase 1, which is differentially active during hepatitis C virus replication.

    PubMed

    Blais, David R; Lyn, Rodney K; Joyce, Michael A; Rouleau, Yanouchka; Steenbergen, Rineke; Barsby, Nicola; Zhu, Lin-Fu; Pegoraro, Adrian F; Stolow, Albert; Tyrrell, David L; Pezacki, John Paul

    2010-08-13

    Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation. PMID:20530478

  6. Oxidative Modification of Rat Sulfotransferase 1A1 Activity in Hepatic Tissue Slices Correlates with Effects on the Purified Enzyme

    PubMed Central

    Dammanahalli, Jagadeesha K.

    2012-01-01

    Mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of xenobiotics as well as numerous endogenous molecules. The major aryl (phenol) SULT in rat liver, rSULT1A1, has been used extensively as a model enzyme for understanding the catalytic function of SULTs. Previous studies showed that purified rSULT1A1 displays significant catalytic changes in the presence of GSSG and other oxidants. In the present study, the effects of diamide [1,1′-azobis(N,N-dimethylformamide)] and tert-butyl hydroperoxide (TBHP) on the activity of rSULT1A1 in rat hepatic slices were compared with the effects of these oxidants on a homogeneous preparation of the enzyme. Precision-cut hepatic slices were incubated with 10 μM 7-hydroxycoumarin (7-HC) in the presence of varied concentrations of either diamide or TBHP. Analysis of the 7-hydroxycoumarin sulfate released into the incubation medium indicated that both oxidants significantly increased the sulfation of 7-HC, and this occurred at optimal concentrations of 5 and 10 μM, respectively. Cellular GSH and GSSG levels in the hepatic slices were not significantly altered from control values at these concentrations of diamide and TBHP. Exposure of homogeneous rSULT1A1 to diamide or TBHP also increased the rate of sulfation of 7-HC, although the optimal concentrations of diamide and TBHP were lower (50- and 100-fold, respectively) than those required for effects with the hepatic slices. These results indicate that both diamide and TBHP may modify the rSULT1A1 in intact cells in a manner similar to that observed with the homogeneous purified enzyme. PMID:22041107

  7. Activity of hepatic but not skeletal muscle carnitine palmitoyltransferase enzyme is depressed by intravenous glucose infusions in lactating dairy cows.

    PubMed

    Al-Trad, B; Wittek, T; Gäbel, G; Fürll, M; Reisberg, K; Aschenbach, J R

    2010-12-01

    A positive energy balance in dairy cows pre-partum may decrease hepatic carnitine palmitoyltransferase (CPT) enzyme activity, which might contribute to disturbances of lipid metabolism post-partum. The purpose of this study was to investigate whether skeletal muscle CPT activity can also be downregulated during positive energy balance. Mid-lactating dairy cows were maintained on intravenous infusion of either saline (control) or glucose solutions that increased linearly over 24 days, remained at the 24-day level until day 28 and were suspended thereafter. Liver and skeletal muscle biopsies, as well as four diurnal blood samples, were taken on days 0, 8, 16, 24, and 32, representing infusion levels equivalent to 0%, 10%, 20%, 30% and 0% of the net energy for lactation (NE(L)) requirement respectively. Glucose infusion increased serum insulin concentrations on day 16 and 24 while plasma glucose levels were increased at only a single time point on day 24. Serum beta-hydroxybutyric acid concentrations decreased between day 8 and 24; whereas changes in non-esterified fatty acids were mostly insignificant. Total lipid contents of liver and skeletal muscle were not affected by treatment. Hepatic CPT activity decreased with glucose infusion (by 35% on day 24) and remained decreased on day 32. Hepatic expression levels of CPT-1A and CPT-2 mRNA were not significantly altered but tended to reflect the changes in enzyme activity. In contrast to the liver, no effect of glucose infusion was observed on skeletal muscle CPT activity. We conclude that suppression of CPT activity by positive energy balance appears to be specific for the liver in mid-lactating dairy cows. PMID:20546068

  8. Angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis

    PubMed Central

    Cao, Xi; Yang, Fangyuan; Shi, Tingting; Yuan, Mingxia; Xin, Zhong; Xie, Rongrong; Li, Sen; Li, Hongbing; Yang, Jin-Kui

    2016-01-01

    The classical axis of renin-angiotensin system (RAS), angiotensin (Ang)-converting enzyme (ACE)/Ang II/AT1, contributes to the development of non-alcoholic fatty liver disease (NAFLD). However, the role of bypass axis of RAS (Angiotensin-converting enzyme 2 (ACE2)/Ang-(1–7)/Mas) in hepatic steatosis is still unclear. Here we showed that deletion of ACE2 aggravates liver steatosis, which is correlated with the increased expression of hepatic lipogenic genes and the decreased expression of fatty acid oxidation-related genes in the liver of ACE2 knockout (ACE2−/y) mice. Meanwhile, oxidative stress and inflammation were also aggravated in ACE2−/y mice. On the contrary, overexpression of ACE2 improved fatty liver in db/db mice, and the mRNA levels of fatty acid oxidation-related genes were up-regulated. In vitro, Ang-(1–7)/ACE2 ameliorated hepatic steatosis, oxidative stress and inflammation in free fatty acid (FFA)-induced HepG2 cells, and what’s more, Akt inhibitors reduced ACE2-mediated lipid metabolism. Furthermore, ACE2-mediated Akt activation could be attenuated by blockade of ATP/P2 receptor/Calmodulin (CaM) pathway. These results indicated that Ang-(1–7)/ACE2/Mas axis may reduce liver lipid accumulation partly by regulating lipid-metabolizing genes through ATP/P2 receptor/CaM signaling pathway. Our findings support the potential role of ACE2/Ang-(1–7)/Mas axis in prevention and treatment of hepatic lipid metabolism. PMID:26883384

  9. Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis.

    PubMed

    Cao, Xi; Yang, Fangyuan; Shi, Tingting; Yuan, Mingxia; Xin, Zhong; Xie, Rongrong; Li, Sen; Li, Hongbing; Yang, Jin-Kui

    2016-01-01

    The classical axis of renin-angiotensin system (RAS), angiotensin (Ang)-converting enzyme (ACE)/Ang II/AT1, contributes to the development of non-alcoholic fatty liver disease (NAFLD). However, the role of bypass axis of RAS (Angiotensin-converting enzyme 2 (ACE2)/Ang-(1-7)/Mas) in hepatic steatosis is still unclear. Here we showed that deletion of ACE2 aggravates liver steatosis, which is correlated with the increased expression of hepatic lipogenic genes and the decreased expression of fatty acid oxidation-related genes in the liver of ACE2 knockout (ACE2(-/y)) mice. Meanwhile, oxidative stress and inflammation were also aggravated in ACE2(-/y) mice. On the contrary, overexpression of ACE2 improved fatty liver in db/db mice, and the mRNA levels of fatty acid oxidation-related genes were up-regulated. In vitro, Ang-(1-7)/ACE2 ameliorated hepatic steatosis, oxidative stress and inflammation in free fatty acid (FFA)-induced HepG2 cells, and what's more, Akt inhibitors reduced ACE2-mediated lipid metabolism. Furthermore, ACE2-mediated Akt activation could be attenuated by blockade of ATP/P2 receptor/Calmodulin (CaM) pathway. These results indicated that Ang-(1-7)/ACE2/Mas axis may reduce liver lipid accumulation partly by regulating lipid-metabolizing genes through ATP/P2 receptor/CaM signaling pathway. Our findings support the potential role of ACE2/Ang-(1-7)/Mas axis in prevention and treatment of hepatic lipid metabolism. PMID:26883384

  10. Effect of Antiviral Therapy on Serum Activity of Angiotensin Converting Enzyme in Patients with Chronic Hepatitis C

    PubMed Central

    Husic-Selimovic, Azra; Sofic, Amela; Huskic, Jasminko; Bulja, Deniz

    2016-01-01

    Introduction: Renin-angiotenzin system (RAS) is frequently activated in patients with chronic liver disease. Angiotenzin - II (AT-II), produced by angiotenzin converting enzyme (ACE), has many physiological effects, including an important role in liver fibrogenesis. Combined antiviral therapy with PEG-IFN and ribavirin besides its antiviral effect also leads to a reduction in liver parenchyma fibrosis. Aim of the study: Determining the value of ACE in serum of patients with chronic hepatitis C before and after combined antiviral therapy, as well as the value of ACE activities in sera of the control group. Materials and methods: We studied 50 patients treated at Gastroenterohepatology Department, in the time-period of four years. Value of ACE in serum was determined by Olympus AU 400 device, with application of kit “Infinity TN ACE Liquid Stable Reagent”. HCV RNA levels in sera were measured by real time PCR. HCV RNA test was performed with modular analysis of AMPLICOR and COBAS AMPLICOR HCV MONITOR test v2.0, which has proved infection and was used for quantification of the viruses and monitoring of the patients’ response to therapy. Liver histology was evaluated in accordance with the level of necroinflammation activity and stage of fibrosis. Results: Serum activities of ACE in chronic hepatitis C patients is statistically higher than the values in the control group (p=0.02). Antiviral therapy in chronic hepatitis C patients statistically decreases serum activities of ACE (p= 0.02) and indirectly affects fibrogenesis of the liver parenchyma. Correlation between ACE and ALT activity after the therapy was proved (0.3934). Conclusion: Our findings suggest that the activity of ACE in serum is a good indirect parameter of the liver damage, and could be used as an indirect prognostic factor of the level of liver parenchyma damage. Serum activity of ACE can be used as a parameter for non-invasive assessment of intensity of liver damage. PMID:27147779

  11. Characterization of hepatic enzyme activity in older adults with dementia: potential impact on personalizing pharmacotherapy

    PubMed Central

    Campbell, Noll L; Skaar, Todd C; Perkins, Anthony J; Gao, Sujuan; Li, Lang; Khan, Babar A; Boustani, Malaz A

    2015-01-01

    Objective To determine the frequency of pharmacogenomic variants and concurrent medications that may alter the efficacy and tolerability of acetylcholinesterase inhibitors (AChEIs). Materials and methods A multisite cross-sectional study was carried out across four memory care practices in the greater Indianapolis area. Participants were adults aged 65 years and older with a diagnosis of probable or possible Alzheimer’s disease (AD) (n=105). Blood samples and self-reported medication data were collected. Since two of the three AChEIs are metabolized by cytochrome P450 (CYP)-2D6, we determined the frequency of functional genetic variants in the CYP2D6 gene and calculated their predicted CYP2D6-activity scores. Concurrent medication data were collected from self-reported medication surveys, and their predicted effect on the pharmacokinetics of AChEIs was determined based on their known effects on CYP2D6 and CYP3A4/5 enzyme activities. Results Among the 105 subjects enrolled, 72% were female and 36% were African American. Subjects had a mean age of 79.6 years. The population used a mean of eight medications per day (prescription and nonprescription). The CYP2D6 activity score frequencies were 0 (3.8%), 0.5 (4.8%), 1.0 (36.2%), 1.5–2.0 (51.4%), and >2.0 (3.8%). Nineteen subjects (18.1%) used a medication considered a strong or moderate inhibitor of CYP2D6, and eight subjects (7.6%) used a medication considered a strong or moderate inhibitor of CYP3A4/5. In total, 28.6% of the study population was predicted to have reduced activity of the CYP2D6 or CYP3A4/5 enzymes due to either genetic variants or concomitant medications. Conclusion Both pharmacogenetic variants and concurrent drug therapies that are predicted to alter the pharmacokinetics of AChEIs should be evaluated in older adults with AD. Pharmacogenetic and drug-interaction data may help personalize AD therapy and increase adherence by improving tolerability. PMID:25609939

  12. POLYCHLORINATED BIPHENYLS AS INDUCERS OF HEPATIC MICROSOMAL ENZYMES: STRUCTURE-ACTIVITY RULES

    EPA Science Inventory

    A number of highly purified polychlorinated biphenyl (PCB) isomers and congeners were synthesized and administered to male Wistar rats at dosage levels of 30 and 150 micromol/kg. The effects of this in vivo treatment on the drug-metabolizing enzymes were determined by measuring t...

  13. Short-term fasting induces intra-hepatic lipid accumulation and decreases intestinal mass without reduced brush-border enzyme activity in mink (Mustela vison) small intestine.

    PubMed

    Bjornvad, C R; Elnif, J; Sangild, P T

    2004-11-01

    For many mammalian species short-term fasting is associated with intestinal atrophy and decreased digestive capacity. Under natural conditions, strictly carnivorous animals often experience prey scarcity during winter, and they may therefore be particularly well adapted to short-term food deprivation. To examine how the carnivorous gastrointestinal tract is affected by fasting, small-intestinal structure, brush-border enzyme activities and hepatic structure and function were examined in fed mink (controls) and mink that had been fasted for 1-10 days. During the first 1-2 days of fasting, intestinal mass decreased more rapidly than total body mass and villus heights were reduced 25-40%. In contrast, tissue-specific activity of the brush-border enzymes sucrase, maltase, lactase, aminopeptidase A and dipeptidylpeptidase IV increased 0.5- to 1.5-fold at this time, but returned to prefasting levels after 6 days of fasting. After 6-10 days of fasting there was a marked increase in the activity of hepatic enzymes and accumulation of intra-hepatic lipid vacuoles. Thus, mink may be a useful model for studying fasting-induced intestinal atrophy and adaptation as well as mechanisms involved in accumulation of intra-hepatic lipids following food deprivation in strictly carnivorous domestic mammals, such as cats and ferrets. PMID:15503054

  14. Comparative toxicology of tetrachlorobiphenyls in mink and rats. I. Changes in hepatic enzyme activity and smooth endoplasmic reticulum volume

    SciTech Connect

    Gillette, D.M.; Corey, R.D.; Helferich, W.G.; McFarland, J.M.; Lowenstine, L.J.; Moody, D.E.; Hammock, B.D.; Shull, L.R.

    1987-01-01

    Mink have been shown previously to be extraordinarily sensitive to polychlorinated biphenyls (PCBs) and related classes of halogenated hydrocarbons. This study explored several aspects of the acute response of mink to two purified tetrachlorobiphenyl (TCB) congeners and compared their response with that of the rat, a less sensitive and more thoroughly studied species. Young female pastel mink and young female Sprague-Dawley rats received three daily intraperitoneal injections with equimolar doses of either 2,4,2',4'-TCB or 3,4,3',4'-TCB, and were sacrificed after 7 days. Two control groups were used for each species; one was allowed free access to food and the other was pair-fed to the 3,4,3',4'-TCB treatment group. Rats remained clinically normal, while mink treated with 3,4,3',4'-TCB developed severe anorexia, diarrhea, and melena. Both species had significant increases in hepatic cytochrome P-450 content and the characteristic shift in the spectral maxima from 450 to 448 nm in the 3,4,3',4'-TCB- but not in the 2,4,2',4'-TCB-treated animals. Rats but not mink had increased activities of several hepatic monooxygenases in response to both congeners while microsomal epoxide hydrolase was increased in rats after 2,4,2',4'-TCB and in mink after 3,4,3',4'-TCB. Significant increases in the relative volume of smooth endoplasmic reticulum within hepatocytes of 2,4,2',4'-TCB-treated rats but not mink were confirmed by ultrastructural morphometry. Accumulation of both congeners was greater in adipose tissue than in the liver of either species. In both species, concentrations in adipose tissue were much greater for 2,4,2',4'-TCB than for 3,4,3',4'-TCB. PCB toxicosis in mink, as in other species, appeared to be dependent on isomeric arrangement of chlorine substituents. However, unlike other species, the toxicosis was not associated with biochemical or morphological evidence of hepatic enzyme induction.

  15. Hepatic enzyme activity after combined administration of methylmercury, lead and cadmium in the pekin duck

    SciTech Connect

    Jordan, S.A.; Bhatnagar, M.K. )

    1990-04-01

    In order to assess adequately the environmental impact of heavy metals it is important to consider that they may occur simultaneously in the environment, where they may interact to alter their individual toxicities on living systems. Metals such as mercury (Hg), lead (Pb) and cadmium (Cd) can be found in all levels of the polluted ecosystem, and in animals inhabiting such areas. In the polluted aquatic environment waterfowl have been noted to accumulate high levels of these metals in their tissues. A major toxic manifestation of heavy metal exposure is the perturbation of a wide range of enzyme systems in virtually all subcellular compartments. With the exception of lead, little data is available on the effects of metals on avian enzyme systems. The present report describes the effects observed in vivo on acid phosphatase (AP), glutathione S-transferase (GST) and cytochrome c oxidase (cyt c ox) in the liver of pekin ducks exposed to combinations of methylmercury (MeHg), lead and cadmium.

  16. Multiple plasma enzyme activities in liver disease

    PubMed Central

    Hargreaves, T.; Janota, I.; Smith, M. J. H.

    1961-01-01

    The measurement of the plasma activities of glutamic-oxaloacetic and glutamic-pyruvic transaminases, aldolase, cholinesterase, and isocitric, lactic, and phosphogluconic dehydrogenases in random samples of blood was found to be of no value in the differential diagnosis of hepatitis, obstructive jaundice, hepatic cirrhosis, and neoplastic conditions involving the liver. Serial determinations of the enzyme activities provided useful information about the course of certain hepatic disorders, particularly acute viral hepatitis. PMID:13711559

  17. Comparison among Different Gilthead Sea Bream (Sparus aurata) Farming Systems: Activity of Intestinal and Hepatic Enzymes and 13C-NMR Analysis of Lipids

    PubMed Central

    Coco, Laura Del; Papadia, Paride; Pascali, Sandra A. De; Bressani, Giorgia; Storelli, Carlo; Zonno, Vincenzo; Fanizzi, Francesco Paolo

    2009-01-01

    In order to evaluate differences in general health and nutritional values of gilthead sea bream (Sparus aurata), the effects of semi-intensive, land-based tanks and sea-cages intensive rearing systems were investigated, and results compared with captured wild fish. The physiological state was determined by measuring the activity of three different intestinal digestive enzymes: alkaline phosphatase (ALP), leucine aminopeptidase (LAP) and maltase; and the activity of the hepatic ALP. Also, the hepatic content in protein, cholesterol, and lipid were assessed. 13C-NMR analysis for qualitative and quantitative characterization of the lipid fraction extracted from fish muscles for semi-intensive and land based tanks intensive systems was performed. The lipid fraction composition showed small but significant differences in the monounsaturated/saturated fatty acid ratio, with the semi-intensive characterized by higher monounsaturated and lower saturated fatty acid content with respect to land based tanks intensive rearing system. PMID:22253985

  18. E2 potentializes benzo(a)pyrene-induced hepatic cytochrome P450 enzyme activities in Nile tilapia at high concentrations.

    PubMed

    Rodrigues, Aline Cristina Ferreira; Moneró, Tatiana de Oliveira; Frighetto, Rosa Toyoko Shiraishi; de Almeida, Eduardo Alves

    2015-11-01

    In the aquatic environment, biotransformation enzymes are established biomarkers for assessing PAH exposure in fish, but little is known about the effect of 17β-estradiol (E2) on these enzymes during exposure to benzo(a)pyrene (BaP). In this study, Nile tilapia (Oreochromis niloticus) were exposed for 3, 5, and 10 days to BaP (300 μg L(-1)) and E2 (5 μg L(-1)). These substances were applied isolated or mixed. In the mixture experiment, fish were analyzed pre- and postexposure in order to better understand whether preexposure to the hormone masks the responses activated by PAH or vice versa. Phase I enzymes ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD) activities as well as the phase II enzyme glutathione S-transferase (GST) were analyzed. Isolated E2 treatment decreased EROD activity after 3 days, but this enzyme activity returned to control values after 5 and 10 days of exposure. Isolated BaP treatment significantly induced EROD activity after 3 and 5 days, and the activity returned to control levels after ten exposure days. Combined treatment (E2 + Bap) significantly increased EROD activity, both in the pre- and postexposure. This increase was even higher than in the isolated BaP treatment, suggesting a synergism between these two compounds. When E2 and BaP were used singly, they did not change BROD and PROD activities. However, combined treatment (E2 + Bap) significantly increased PROD activity. Isolated BaP treatment increased GST activity after 10 days. However, this response was not observed in the mixture treatment, suggesting that E2 suppressed the GST induction modulated by BaP. The results put together indicated that E2 altered the biotransformation pathway regarding enzymes activated by BaP in Nile tilapia. PMID:25280508

  19. Mechanism-based inhibitory and peroxisome proliferator-activated receptor α-dependent modulating effects of silybin on principal hepatic drug-metabolizing enzymes.

    PubMed

    Wang, Hong; Yan, Tingting; Xie, Yuan; Zhao, Min; Che, Yuan; Zhang, Jun; Liu, Huiying; Cao, Lijuan; Cheng, Xuefang; Xie, Yang; Li, Feiyan; Qi, Qu; Wang, Guangji; Hao, Haiping

    2015-04-01

    Silybin, a major pharmacologically active compound in silymarin, has been widely used in combination with other prescriptions in the clinic to treat hepatitis and a host of other diseases. Previous studies suggested that silybin is a potential inhibitor of multiple drug-metabolizing enzymes (DMEs); however, the in vitro to in vivo translation and the mechanisms involved remain established. The aim of this study was to provide a mechanistic understanding of the regulatory effects of silybin on principal DMEs. Silybin (50 or 150 mg/kg/d) was administered to mice for a consecutive 14 days. The plasma and hepatic exposure of silybin were detected; the mRNA, protein levels, and enzyme activities of principal DMEs were determined. The results demonstrated that the enzyme activities of CYP1A2, CYP2C, CYP3A11, and UGT1A1 were significantly repressed, whereas little alteration of the mRNA and protein levels was observed. Silybin inhibits these DMEs in a mechanism-based and/or substrate-competitive manner. More importantly, silybin was found to be a weak agonist of peroxisome proliferator-activated receptor (PPAR)α, as evidenced from the molecular docking, reporter gene assay, and the targeting gene expression analysis. However, silybin could significantly compromise the activation of PPARα by fenofibrate, characterized with significantly repressed expression of PPARα targeting genes, including L-FABP, ACOX1, and UGT1A6. This study suggests that silybin, despite its low bioavailability, may inhibit enzyme activities of multiple DMEs in a mechanism-based mode, and more importantly, may confer significant drug-drug interaction with PPARα agonists via the repression of PPARα activation in a competitive mode. PMID:25587127

  20. Hepatitis and activity

    PubMed Central

    Krikler, Dennis M.

    1971-01-01

    The effects of physical activity during an attack of infectious hepatitis are discussed. There is no evidence that activity during convalescence produces any ill-effects. On the other hand, strenuous physical activity in the acute stage may be dangerous, possibly because hepatic blood-flow is reduced. PMID:5560143

  1. Effects of dietary tannic acid on the growth, hepatic gene expression, and antioxidant enzyme activity in Brandt's voles (Microtus brandti).

    PubMed

    Ye, Man-Hong; Nan, Yan-Lei; Ding, Meng-Meng; Hu, Jun-Bang; Liu, Qian; Wei, Wan-Hong; Yang, Sheng-Mei

    2016-01-01

    This study was designed to investigate the physiological and biochemical responses of Brandt's voles to the persistent presence of dietary tannic acid. The diet for animals in the experimental group was supplemented with 3% dietary tannic acid for 5weeks. The control group received a commercial lab chow. No significant differences were detected in body weight, organ (heart, kidney, and liver) weights, and organ parameters between animals from two groups. However, voles in the experimental group had significantly higher daily food intake, increased contents of proline and histidine in saliva and feces after protein hydrolysis, and elevated hepatic expression of transferrin than the control. Our results suggested the existence of adaptive strategies developed in Brandt's voles to overcome the adverse effects of dietary tannic acid. (1) Food consumption was increased to satisfy their nutritional demands. (2) The secretion of tannic-acid-binding salivary proteins was promoted. (3) The absorption of iron was enhanced. These alterations contributed to neutralize the negative effects of tannic acid and maintain body mass in animals supplemented with tannic acid. As the result of the consumption of tannic acid, hepatic expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase was significantly decreased, while the overall potential of the antioxidant system, characterized by increased hepatic enzymatic activities of catalase and glutathione peroxidase, was enhanced. Our results also implied the involvement of tannic acid in the regulation of lipid metabolism and oxidative stress in voles. PMID:26850644

  2. Dietary ɛ-Polylysine Decreased Serum and Liver Lipid Contents by Enhancing Fecal Lipid Excretion Irrespective of Increased Hepatic Fatty Acid Biosynthesis-Related Enzymes Activities in Rats

    PubMed Central

    Hosomi, Ryota; Yamamoto, Daiki; Otsuka, Ren; Nishiyama, Toshimasa; Yoshida, Munehiro; Fukunaga, Kenji

    2015-01-01

    ɛ-Polylysine (EPL) is used as a natural preservative in food. However, few studies have been conducted to assess the beneficial functions of dietary EPL. The purpose of this study was to elucidate the mechanism underlying the inhibition of neutral and acidic sterol absorption and hepatic enzyme activity-related fatty acid biosynthesis following EPL intake. EPL digest prepared using an in vitro digestion model had lower lipase activity and micellar lipid solubility and higher bile acid binding capacity than casein digest. Male Wistar rats were fed an AIN-93G diet containing 1% (wt/wt) EPL or l-lysine. After 4 weeks of feeding these diets, the marked decrease in serum and liver triacylglycerol contents by the EPL diet was partly attributed to increased fecal fatty acid excretion. The activities of hepatic acetyl-coenzyme A carboxylase and glucose-6-phosphate dehydrogenase, which are key enzymes of fatty acid biosynthesis, were enhanced in rats fed EPL diet. The increased fatty acid biosynthesis activity due to dietary EPL may be prevented by the enhancement of fecal fatty acid excretion. The hypocholesterolemic effect of EPL was mediated by increased fecal neutral and acidic sterol excretions due to the EPL digest suppressing micellar lipid solubility and high bile acid binding capacity. These results show that dietary EPL has beneficial effects that could help prevent lifestyle-related diseases such as hyperlipidemia and atherosclerosis. PMID:25866749

  3. Dietary ɛ-Polylysine Decreased Serum and Liver Lipid Contents by Enhancing Fecal Lipid Excretion Irrespective of Increased Hepatic Fatty Acid Biosynthesis-Related Enzymes Activities in Rats.

    PubMed

    Hosomi, Ryota; Yamamoto, Daiki; Otsuka, Ren; Nishiyama, Toshimasa; Yoshida, Munehiro; Fukunaga, Kenji

    2015-03-01

    ɛ-Polylysine (EPL) is used as a natural preservative in food. However, few studies have been conducted to assess the beneficial functions of dietary EPL. The purpose of this study was to elucidate the mechanism underlying the inhibition of neutral and acidic sterol absorption and hepatic enzyme activity-related fatty acid biosynthesis following EPL intake. EPL digest prepared using an in vitro digestion model had lower lipase activity and micellar lipid solubility and higher bile acid binding capacity than casein digest. Male Wistar rats were fed an AIN-93G diet containing 1% (wt/wt) EPL or l-lysine. After 4 weeks of feeding these diets, the marked decrease in serum and liver triacylglycerol contents by the EPL diet was partly attributed to increased fecal fatty acid excretion. The activities of hepatic acetyl-coenzyme A carboxylase and glucose-6-phosphate dehydrogenase, which are key enzymes of fatty acid biosynthesis, were enhanced in rats fed EPL diet. The increased fatty acid biosynthesis activity due to dietary EPL may be prevented by the enhancement of fecal fatty acid excretion. The hypocholesterolemic effect of EPL was mediated by increased fecal neutral and acidic sterol excretions due to the EPL digest suppressing micellar lipid solubility and high bile acid binding capacity. These results show that dietary EPL has beneficial effects that could help prevent lifestyle-related diseases such as hyperlipidemia and atherosclerosis. PMID:25866749

  4. SENESCENCE-ASSOCIATED DECLINE IN HEPATIC PEROXISOMAL ENZYME ACTIVITIES CORRESPONDS WITH DIMINISHED LEVELS OF RETINOID X RECEPTOR ALPHA, BUT NOT PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA1

    EPA Science Inventory

    Abstract

    Aging is associated with alterations in hepatic peroxisomal metabolism and susceptibility to hepatocarcinogenecity produced by agonists of peroxisome proliferator-activated receptor alpha (PPARa). Mechanisms involved in these effects are not well understood. Howev...

  5. [Activity of the sphingomyelin cycle enzymes and concentration of products of sphingomyelin degradation in the rat liver in the course of acute toxic hepatitis].

    PubMed

    Serebrov, V Iu; Kuz'menko, D I; Burov, P G; Sapugol'tseva, O B

    2010-01-01

    Activity of key enzymes of a sphingomyelin cycle and the maintenance of its components (sphingomyelin, ceramide and sphingosine-1-phosphate) have been studied in livers of rats in dynamics of the acute toxic hepatitis caused by hypodermic introduction of an oil solution of CCl4. Sphingomyelinase activity significally increased already on early terms and remained increased over the whole period of observation. Activity of ceramidase insignificantly differed from the control level. The levels of sphingomyelin and sphingosine-1-phosphate did not undergo marked changes while ceramide content significally increased. Thus, balance between liver content of ceramide (proapoptotic) and the sphingosine-1-phosphate, being the antiapoptotic factor, was shifted towards ceramide. In sphingomyelin molecules there was a significant decrease in the content of fatty acids C18: and C22:2, while in ceramide molecules and sphingosine-1-phosphate only fatty acid C22:2 changed. In spite of significant decrease in content of some unsaturated fatty acids, calculated unsaturation coefficients of the fatty acid component of the sphingomyelin cycle metabolites. Thus, our results together with literature data suggests involvement of ceramide-mediated apoptosis in the pathogenesis of acute toxic hepatitis. Elimination of damaged hepatocytes facilitates realization of repair processes and optimization of cellular community of a liver. PMID:21341516

  6. A method for the determination of the hepatic enzyme activity catalyzing bile acid acyl glucuronide formation by high-performance liquid chromatography with pulsed amperometric detection.

    PubMed

    Ikegawa, S; Oohashi, J; Murao, N; Goto, J

    2000-05-01

    A method for the determination of the activity of hepatic glucuronyltransferase catalyzing formation of bile acid 24-glucuronides using high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD) has been developed. Bile acid 24-glucuronides were simultaneously separated on a semimicrobore column, Capcell Pak C18UG120, using 20 mM ammonium phosphate (pH 6.0)-acetonitrile (27:10 and 16:10) as the mobile phase in the stepwise gradient elution mode. A 1 M potassium hydroxide solution for the hydrolysis of the 24-glucuronides, which liberates the corresponding bile acids and glucuronic acid, was mixed with the mobile phase in a post-column mode, and the resulting eluant was heated at 90 degrees C, the 24-glucuronides being monitored using a pulsed amperometric detector; the limit of detection was 10 ng. The proposed method was applied to the determination of the hepatic enzyme activity catalyzing bile acid 24-glucuronide formation and the result exhibited the efficient 24-glucuronide formation of the monohydroxylated bile acid, lithocholic acid. PMID:10850616

  7. Chronic alcohol intake up-regulates hepatic expressions of carotenoid cleavage enzymes and peroxisomal proliferator-activated receptors in rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Excessive and chronic alcohol intake leads to a lower hepatic vitamin A status by interfering with vitamin A metabolism.Dietary provitamin A carotenoids can be converted into vitamin A mainly by carotenoid 15,15’-monooxygenase 1 (CMO1) and, to a lesser degree, carotenoid 9910’-monooxygenase 2 (CMO2)...

  8. Dietary chia seed induced changes in hepatic transcription factors and their target lipogenic and oxidative enzyme activities in dyslipidaemic insulin-resistant rats.

    PubMed

    Rossi, Andrea S; Oliva, Maria E; Ferreira, Maria R; Chicco, Adriana; Lombardo, Yolanda B

    2013-05-01

    The present study analyses the effect of dietary chia seed rich in n-3 α-linolenic acid on the mechanisms underlying dyslipidaemia and liver steatosis developed in rats fed a sucrose-rich diet (SRD) for either 3 weeks or 5 months. The key hepatic enzyme activities such as fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), glucose-6-phosphate dehydrogenase (G-6-PDH), carnitine palmitoyltransferase-1 (CPT-1) and fatty acid oxidase (FAO) involved in lipid metabolism and the protein mass levels of sterol regulatory element-binding protein-1 (SREBP-1) and PPARα were studied. (1) For 3 weeks, Wistar rats were fed either a SRD with 11 % of maize oil (MO) as dietary fat or a SRD in which chia seed replaced MO (SRD+Chia). (2) A second group of rats were fed a SRD for 3 months. Afterwards, half the rats continued with the SRD while for the other half, MO was replaced by chia for 2 months (SRD+Chia). In a control group, maize starch replaced sucrose. Liver TAG and the aforementioned parameters were analysed in all groups. The replacement of MO by chia in the SRD prevented (3 weeks) or improved/normalised (5 months) increases in dyslipidaemia, liver TAG, FAS, ACC and G-6-PDH activities, and increased FAO and CPT-1 activities. Protein levels of PPARα increased, and the increased mature form of SREBP-1 protein levels in the SRD was normalised by chia in both protocols (1 and 2). The present study provides new data regarding some key mechanisms related to the fate of hepatic fatty acid metabolism that seem to be involved in the effect of dietary chia seed in preventing and normalising/improving dyslipidaemia and liver steatosis in an insulin-resistant rat model. PMID:22947172

  9. Interaction of cadmium with hepatic and testicular microsomal enzymes

    SciTech Connect

    Wetzel, L.T.

    1982-01-01

    Cadmium, a ubiquitous environmental pollutant, inhibits or activates a number of microsomal enzymes. Among the enzymes affected by cadmium are cytochrome P-450 containing mixed-function oxidases (MFO) which are present in both the liver and testis. Cadmium affects MFO activity, and as a result, cadmium-induced alterations in BP metabolism might alter BP toxicity in the liver or testis. In addition, MFO essential for testosterone production are located in the testis and cadmium-MFO interactions in the testis might alter androgen production. Therefore studies were carried out to evaluate the interaction of cadmium with heptic and testicular MFO. The results indicated that cadmium affected the activities of hepatic and testicular MFO and in so doing may influence the toxicity of BP and other chemicals in liver and testes. In addition, exposure to metals may also compromise testicular androgen biosynthesis.

  10. Effect of High Dietary Carbohydrate on the Growth Performance, Blood Chemistry, Hepatic Enzyme Activities and Growth Hormone Gene Expression of Wuchang Bream (Megalobrama amblycephala) at Two Temperatures

    PubMed Central

    Zhou, Chuanpeng; Ge, Xianping; Liu, Bo; Xie, Jun; Chen, Ruli; Ren, Mingchun

    2015-01-01

    The effects of high carbohydrate diet on growth, serum physiological response, and hepatic heat shock protein 70 expression in Wuchang bream were determined at 25°C and 30°C. At each temperature, the fish fed the control diet (31% CHO) had significantly higher weight gain, specific growth rate, protein efficiency ratio and hepatic glucose-6-phosphatase activities, lower feed conversion ratio and hepatosomatic index (HSI), whole crude lipid, serum glucose, hepatic glucokinase (GK) activity than those fed the high-carbohydrate diet (47% CHO) (p<0.05). The fish reared at 25°C had significantly higher whole body crude protein and ash, serum cholesterol and triglyceride, hepatic G-6-Pase activity, lower glycogen content and relative levels of hepatic growth hormone (GH) gene expression than those reared at 30°C (p<0.05). Significant interaction between temperature and diet was found for HSI, condition factor, hepatic GK activity and the relative levels of hepatic GH gene expression (p<0.05). PMID:25557816

  11. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state.

    PubMed

    Alarcon, Valentina; Hernández, Sergio; Rubio, Lorena; Alvarez, Francisca; Flores, Yvo; Varas-Godoy, Manuel; De Ferrari, Giancarlo V; Kann, Michael; Villanueva, Rodrigo A; Loyola, Alejandra

    2016-01-01

    With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients. PMID:27174370

  12. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state

    PubMed Central

    Alarcon, Valentina; Hernández, Sergio; Rubio, Lorena; Alvarez, Francisca; Flores, Yvo; Varas-Godoy, Manuel; De Ferrari, Giancarlo V.; Kann, Michael; Villanueva, Rodrigo A.; Loyola, Alejandra

    2016-01-01

    With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients. PMID:27174370

  13. Insulin regulates enzyme activity, malonyl-CoA sensitivity and mRNA abundance of hepatic carnitine palmitoyltransferase-I.

    PubMed Central

    Park, E A; Mynatt, R L; Cook, G A; Kashfi, K

    1995-01-01

    The regulation of hepatic mitochondrial carnitine palmitoyltransferase-I (CPT-I) was studied in rats during starvation and insulin-dependent diabetes and in rat H4IIE cells. The Vmax. for CPT-I in hepatic mitochondrial outer membranes isolated from starved and diabetic rats increased 2- and 3-fold respectively over fed control values with no change in Km values for substrates. Regulation of malonyl-CoA sensitivity of CPT-I in isolated mitochondrial outer membranes was indicated by an 8-fold increase in Ki during starvation and by a 50-fold increase in Ki in the diabetic state. Peroxisomal and microsomal CPT also had decreased sensitivity to inhibition by malonyl-CoA during starvation. CPT-I mRNA abundance was 7.5 times greater in livers of 48-h-starved rats and 14.6 times greater in livers of insulin-dependent diabetic rats compared with livers of fed rats. In H4IIE cells, insulin increased CPT-I sensitivity to inhibition by malonyl-CoA in 4 h, and sensitivity continued to increase up to 24 h after insulin addition. CPT-I mRNA levels in H4IIE cells were decreased by insulin after 4 h and continued to decrease so that at 24 h there was a 10-fold difference. The half-life of CPT-I mRNA was 4 h in the presence of actinomycin D or with actinomycin D plus insulin. These results suggest that insulin regulates CPT-I by inhibiting transcription of the CPT-I gene. Images Figure 2 Figure 4 PMID:7575418

  14. Nelumbo nucifera leaves protect hydrogen peroxide-induced hepatic damage via antioxidant enzymes and HO-1/Nrf2 activation.

    PubMed

    Je, Jae-Young; Lee, Da-Bin

    2015-06-01

    Naturally occurring phenolic compounds are widely found in plants. Here, the phenolic composition and hepatoprotective effect of the butanolic extract (BE) from Nelumbo nucifera leaves against H2O2-induced hepatic damage in cultured hepatocytes were investigated. BE showed high total phenol and flavonoid contents, and major phenolic compounds are quercetin, catechin, ferulic acid, rutin, and protocatechuic acid by HPLC analysis. BE effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) cation radicals (IC50 values of 5.21 μg mL(-1) for DPPH and 6.22 μg mL(-1) for ABTS(+)) and showed strong reducing power. Pretreatment of BE prior to 650 μM H2O2 exposure markedly increased cell viability and suppressed H2O2-induced intracellular reactive oxygen species generation and AAPH-induced cell membrane lipid peroxidation. In addition, BE up-regulated intracellular glutathione levels under normal and oxidative stress conditions. Notably, the hepatoprotective effect of BE was directly correlated with the increased expression of superoxide dismutase-1 (SOD-1) by 0.62-fold, catalase (CAT) by 0.42-fold, and heme oxygenase-1 (HO-1) by 2.4-fold. Pretreatment of BE also increased the nuclear accumulation of Nrf2 by 8.1-fold indicating that increased SOD-1, CAT, and HO-1 expressions are Nrf2-mediated. PMID:25962859

  15. Photoperiodism and Enzyme Activity

    PubMed Central

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  16. Pericholecystic hepatic activity in cholescintigraphy

    SciTech Connect

    Smith, R.; Rosen, J.M.; Gallo, L.N.; Alderson, P.O.

    1985-09-01

    Gallbladder nonvisualization in cholescintigraphy has been shown to be a reliable finding in acute cholecystitis. In some cholescintigrams, the authors have observed faintly increased pericholecystic hepatic activity in conjunction with gallbladder nonvisualization. To determine the frequency and significance of the pericholecystic hepatic activity finding, they evaluated 334 consecutive adult patients who had cholescintigrams with technetium-99m diisopropylphenylcarboamoyl iminodiacetic acid. Pericholecystic hepatic activity was seen in 21% of the abnormal scans demonstrating gallbladder nonvisualization but in none of the other scans. Thirteen of these patients underwent surgery; 11 (85%) were found to have acute cholecystitis, and two (15%) had chronic cholecystitis. The pericholecystic hepatic activity sign is not specific for gangrenous cholecystitis or gallbladder perforation but does reliably indicate inflammatory gallbladder disease and is associated with a relatively high incidence of cholecystitis complicated by perforation.

  17. [Plasma cholinesterase activity in hepatic diseases].

    PubMed

    Araoud, Manel; Mhenni, Hamida; Hellara, Ilhem; Hellara, Olfa; Neffati, Fadoua; Douki, Wahiba; Mili, Marwa; Saffar, Hammouda; Najjar, Mohamed Fadhel

    2013-01-01

    Plasma cholinesterase activity (ChE) may vary in some pathological circumstances. We studied the changes in activity of this enzyme according to the type of liver injury, to assess the interest of this parameter in the diagnosis of liver diseases. Our study was performed on 102 patients with different liver diseases and 53 healthy controls. The ChE activity was lower in patients compared to control group (p < 0.0001), and more pronounced in cirrhotic patients compared to those suffering from hepatitis. Elevated activities of AST, ALT, GGT and ALP and bilirubinemia, and decreased albuminemia were noted in patients compared to controls (p < 0.001). Hypoalbuminemia was significantly important in cirrhotic patients compared to those suffering from cholestasis or hepatitis. A correlation between ChE and bilirubin, albumin and serum protein was found in patients with cirrhosis or those with chronic hepatitis. A significantly lower activity of ChE was found in patients with hepatic insufficiency (HI). In case of suspicion of HI, the prescription of ChE activity could guide or confirm the diagnosis of the impairment. PMID:23747666

  18. Platycodi radix saponin inhibits α-glucosidase in vitro and modulates hepatic glucose-regulating enzyme activities in C57BL/KsJ-db/db mice.

    PubMed

    Lee, Jeom-Sook; Choi, Myung-Sook; Seo, Kown-Il; Lee, Jin; Lee, Hae-In; Lee, Ju-Hye; Kim, Myung-Joo; Lee, Mi-Kyung

    2014-06-01

    This study investigated anti-diabetic activity of a concentrated saponin fraction from Platycodi radix (SK1) in C57BL/KsJ-db/db mice and its underlying mechanism. Mice were fed diet with 0.5 % SK1 (w/w) for 6 weeks. SK1 significantly lowered the blood glucose and glycosylated hemoglobin levels and improved glucose and insulin tolerance. The plasma and pancreatic insulin and C-peptide levels and fecal cholesterol content were increased, whereas plasma urea nitrogen, free fatty acid and triglyceride levels were decreased by SK1 supplementation. Glucokinase (GK) activity in the liver was significantly higher in the SK1 group than the control group, whereas the glucose-6-phosphatase (G6Pase) activity was lower. SK1 significantly down-regulated GK mRNA expression compared to the control group but did not affect G6Pase and glucose transporter 2 mRNA. Phosphoenolpyruvate carboxykinase activity and mRNA levels did not differ between groups. SK1 also markedly inhibited the small intestinal disaccharidases activities compared to those of control db/db mice. Furthermore, SK1 was a more effective α-glucosidase inhibitor than acarbose in vitro. Overall, these findings suggest that SK1 is a potential glucose-lowering agent that functions via inhibition of carbohydrate digestive enzyme activities and modulation of glucose-regulating enzyme activities in db/db mice. PMID:24105419

  19. Effects of ursodeoxycholic acid on serum liver enzymes and bile acid metabolism in chronic active hepatitis: a dose-response study.

    PubMed

    Crosignani, A; Battezzati, P M; Setchell, K D; Camisasca, M; Bertolini, E; Roda, A; Zuin, M; Podda, M

    1991-02-01

    The effect of ursodeoxycholic acid administration on liver function tests and on bile acid metabolism was investigated in 18 patients with chronic active hepatitis. Three different doses of ursodeoxycholic acid--250 mg, 500 mg and 750 mg--were administered daily to each patient for consecutive 2-mo periods. The order of doses was randomly assigned according to a replicated Latin-square design. A significant decrease in serum transaminases and gamma-glutamyl transpeptidase occurred with the lowest dose of ursodeoxycholic acid, which corresponded to 4 mg/kg body wt/day, and no further significant decrease with the higher doses was seen. Biliary bile acid composition was determined by high-performance liquid chromatography and gas chromatography-mass spectrometry. At entry the relative proportions of major bile acids were similar to those observed in normal individuals. During treatment the mean percentage of ursodeoxycholic acid in bile (22% with the 250 mg dose, 32% with the 500 mg dose and 34% with the 750 mg dose) was lower than values previously reported for patients with gallstones and normal liver function. The major bile acids were cholic, chenodeoxycholic and deoxycholic acids. A number of unusual bile acids were identified by gas chromatography-mass spectrometry, but these accounted for only 3% to 5% of the total and did not change during ursodeoxycholic acid therapy. No correlation between the improvement in liver function tests and the percentage of ursodeoxycholic acid in bile existed. These data suggest that even a slight enrichment of bile with ursodeoxycholic acid, as is attained with 250 mg/day, is effective in improving biochemical markers of liver function in patients with chronic active hepatitis. PMID:1671665

  20. Effects of methapyrilene on rat hepatic xenobiotic metabolizing enzymes and liver morphology.

    PubMed

    Graichen, M E; Neptun, D A; Dent, J G; Popp, J A; Leonard, T B

    1985-02-01

    Short-term treatment of rats with hepatocarcinogens elicits a consistent pattern of phenotypic changes in hepatic drug metabolizing enzymes, the most striking of which is a marked increase in microsomal epoxide hydrolase (EH) activity. The antihistaminic drug methapyrilene induces a high incidence of hepatocellular carcinoma in F-344 rats. The studies reported here were designed to assess the effects of methapyrilene on hepatic EH activity, cytochrome P-450-dependent mixed-function oxidase activities, liver morphology, and liver-derived serum enzymes. Male F-344 rats were treated with three daily oral doses of methapyrilene-HCl, up to 300 mg/kg/day, and were sacrificed 48 hr after the last dose. Hepatic microsomal EH and cytosolic DT-diaphorase activities were increased in a dose-related fashion, to 420 and 230% of control, respectively. Cytochrome P-450 content and benzphetamine-N-demethylase and ethoxycoumarin-O-deethylase activities were concomitantly decreased to 35-50% of control. Serum gamma-glutamyl transpeptidase and alanine aminotransferase activities were elevated 22- to 27-fold, and serum bile acids to 36-fold by treatment with methapyrilene. Periportal lesions, characterized by inflammation, nuclear and nucleolar enlargement, bile duct hyperplasia, and hepatocellular necrosis, were observed following methapyrilene administration. The severity of the periportal lesion correlated with elevations in the serum chemistry parameters. The increases noted in microsomal EH activity supports the suggestion that this enzyme may be a useful biochemical marker for exposure to hepatocarcinogens. PMID:2859228

  1. Leflunomide Induces Pulmonary and Hepatic CYP1A Enzymes via Aryl Hydrocarbon Receptor.

    PubMed

    Patel, Ananddeep; Zhang, Shaojie; Paramahamsa, Maturu; Jiang, Weiwu; Wang, Lihua; Moorthy, Bhagavatula; Shivanna, Binoy

    2015-12-01

    Emerging evidence indicates that the aryl hydrocarbon receptor (AhR) plays a crucial role in normal physiologic homeostasis. Additionally, aberrant AhR signaling leads to several pathologic states in the lung and liver. Activation of AhR transcriptionally induces phase I (CYP1A) detoxifying enzymes. Although the effects of the classic AhR ligands such as 3-methylcholanthrene and dioxins on phase 1 enzymes are well studied in rodent lung, liver, and other organs, the toxicity profiles limit their use as therapeutic agents in humans. Hence, there is a need to identify and investigate nontoxic AhR ligands not only to understand the AhR biology but also to develop the AhR as a clinically relevant therapeutic target. Leflunomide is a Food and Drug Administration-approved drug in humans that is known to have AhR agonist activity in vitro. Whether it activates AhR and induces phase 1 enzymes in vivo is unknown. Therefore, we tested the hypothesis that leflunomide will induce pulmonary and hepatic CYP1A enzymes in C57BL/6J wild-type mice, but not in AhR-null mice. We performed real-time reverse-transcription polymerase chain reaction analyses for CYP1A1/2 mRNA expression, western blot assays for CYP1A1/2 protein expression, and ethoxyresorufinO-deethylase assay for CYP1A1 catalytic activity. Leflunomide increased CYP1A1/A2 mRNA, protein, and enzymatic activities in wild-type mice. In contrast, leflunomide failed to increase pulmonary and hepatic CYP1A enzymes in AhR-null mice. In conclusion, we provide evidence that leflunomide induces pulmonary and hepatic CYP1A enzymes via the AhR. PMID:26417045

  2. Enzyme activity determination using ultrasound

    NASA Astrophysics Data System (ADS)

    Holmes, M. J.; Southworth, T.; Watson, N. G.; Povey, M. J. W.

    2014-04-01

    Here are presented the results of a novel approach to the measurement of enzyme reaction rates in which ultrasound velocity measurement is used. Our results show enzyme activity is observable, in the acoustic context, and that furthermore this offers the potential to estimate the rate of reaction over different substrate concentrations and temperatures. Findings are corroborated with optical microscopy and rheological measurements. Ultrasound velocity measurement can be performed without the need for aliquot extraction and offers an efficient, non-invasive and dynamic method to monitor enzyme activity.

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

    PubMed

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

    2001-03-01

    Possible chemoprotective effects of the naturally occurring alkaloid boldine, a major alkaloid of boldo (Peumus boldus Mol.) leaves and bark, including in vitro modulations of drug-metabolizing enzymes in mouse hepatoma Hepa-1 cell line and mouse hepatic microsomes, were investigated. Boldine manifested inhibition activity on hepatic microsomal CYP1A-dependent 7-ethoxyresorufin O-deethylase and CYP3A-dependent testosterone 6 beta-hydroxylase activities and stimulated glutathione S-transferase activity in Hepa-1 cells. In addition to the known antioxidant activity, boldine could decrease the metabolic activation of other xenobiotics including chemical mutagens. PMID:11265593

  4. Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis.

    PubMed

    Kang, Soon Ah; Hong, Kyunghee; Jang, Ki-Hyo; Kim, Yun-Young; Choue, Ryowon; Lim, Yoongho

    2006-06-01

    Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression. PMID:16214330

  5. 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

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

    PubMed Central

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

    2014-01-01

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

  7. Pharmacological Intervention in Hepatic Stellate Cell Activation and Hepatic Fibrosis

    PubMed Central

    Schon, Hans-Theo; Bartneck, Matthias; Borkham-Kamphorst, Erawan; Nattermann, Jacob; Lammers, Twan; Tacke, Frank; Weiskirchen, Ralf

    2016-01-01

    The activation and transdifferentiation of hepatic stellate cells (HSCs) into contractile, matrix-producing myofibroblasts (MFBs) are central events in hepatic fibrogenesis. These processes are driven by autocrine- and paracrine-acting soluble factors (i.e., cytokines and chemokines). Proof-of-concept studies of the last decades have shown that both the deactivation and removal of hepatic MFBs as well as antagonizing profibrogenic factors are in principle suitable to attenuate ongoing hepatic fibrosis. Although several drugs show potent antifibrotic activities in experimental models of hepatic fibrosis, there is presently no effective pharmaceutical intervention specifically approved for the treatment of liver fibrosis. Pharmaceutical interventions are generally hampered by insufficient supply of drugs to the diseased liver tissue and/or by adverse effects as a result of affecting non-target cells. Therefore, targeted delivery systems that bind specifically to receptors solely expressed on activated HSCs or transdifferentiated MFBs and delivery systems that can improve drug distribution to the liver in general are urgently needed. In this review, we summarize current strategies for targeted delivery of drugs to the liver and in particular to pro-fibrogenic liver cells. The applicability and efficacy of sequestering molecules, selective protein carriers, lipid-based drug vehicles, viral vectors, transcriptional targeting approaches, therapeutic liver- and HSC-specific nanoparticles, and miRNA-based strategies are discussed. Some of these delivery systems that had already been successfully tested in experimental animal models of ongoing hepatic fibrogenesis are expected to translate into clinically useful therapeutics specifically targeting HSCs. PMID:26941644

  8. Changes in plasma and hepatic lipids, small intestinal histology and pancreatic enzyme activity due to aging and dietary fiber in rats.

    PubMed

    Schneeman, B O; Richter, D

    1993-07-01

    Rats were fed either a control diet or a control diet supplemented with wheat bran, psyllium husk or oat bran to increase intake of fiber. Groups of rats were killed after 3.5, 10, 15, or 18.5 mo of consuming the diets. Plasma cholesterol and triglyceride concentrations were significantly higher in 18.5-mo-old than younger animals. Fiber supplementation did not prevent the age-related increase in lipids. Cecal weight, including contents, was higher in the psyllium husk and oat bran groups than control, and smooth muscle thickness in the ileum of psyllium husk and oat bran animals was greater than control. The score for torn villi in the small intestine was lower than expected in the wheat bran group. Amylase activity in the pancreas declined significantly with age in all groups. In aging animals fiber supplementation may enhance ileal compensation for decreases in proximal intestinal function but does not prevent age-related changes in the gut or in lipid concentrations. PMID:7686573

  9. Chromosome abnormalities in chronic active hepatitis

    PubMed Central

    Stefanescu, D. T.; Moanga, M.; Teodorescu, M.; Brucher, J.

    1972-01-01

    An investigation on human peripheral blood lymphocyte chromosomes in chronic active hepatitis was carried out. A higher percentage of chromatid and chromosome lesions was recorded in all patients studied as compared with control groups—normal individuals, healthy subjects who had suffered from acute viral hepatitis, patients with alcoholic liver disease, and patients with mechanical jaundice due to cancer. The possible origin of these abnormalities is discussed. PMID:5076805

  10. Differential regulation of hepatic enzymes by polycyclic aromatic hydrocarbons and glucocorticoids

    SciTech Connect

    Smith, J.A.; Linder, M.W.; Fernandez, D.; Prough, R.A. )

    1991-03-15

    A putative glucocorticoid (GC) response element has been identified within the first intron of the P450IA1 gene and is apparently necessary for GC-dependent potentiation of polycyclic aromatic hydrocarbon (PAH) induction of P450IA1. In cultured rat hepatocytes, the synthetic GC, dexamethasone (DEX), potentiated PAH induction of both P450IA1 and glutathione S-transferase protein and mRNA. However, DEX caused a small decrease in PAH-dependent induction of NAD(P)H:quinone oxidoreductase (QOR) subunit protein and mRNA in culture. The potentiation of 3-methylcholanthrene (MC) dependent induction of hepatic P450IA1, GST and QOR by low doses of DEX was evaluated in neonatal and adult rats. In neonates, MC induction was potentiated 2-, 1.5-, and 1.4-fold for P450IA1, GST, and QOR activities, respectively, by DEX. However, in adult rats, DEX potentiated MC induction of P450IA1 activity, but repressed MC induction of GST and QOR. Western immunoanalysis and Northern analysis indicated that the changes in these activities were associated with parallel changes in the levels of immunoreactive proteins and mRNA. Glucocorticoids may have an age-dependent influence on the induction of hepatic enzymes by PAH possibly involving other regulatory factors, in addition to Ah and GC receptors.

  11. Effects of ciprofibrate and 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) on the distribution of carnitine and CoA and their acyl-esters and on enzyme activities in rats. Relation between hepatic carnitine concentration and carnitine acetyltransferase activity.

    PubMed Central

    Bhuiyan, A K; Bartlett, K; Sherratt, H S; Agius, L

    1988-01-01

    The effects of feeding the peroxisome proliferators ciprofibrate (a hypolipidaemic analogue of clofibrate) or POCA (2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate) (an inhibitor of CPT I) to rats for 5 days on the distribution of carnitine and acylcarnitine esters between liver, plasma and muscle and on hepatic CoA concentrations (free and acylated) and activities of carnitine acetyltransferase and acyl-CoA hydrolases were determined. Ciprofibrate and POCA increased hepatic [total CoA] by 2 and 2.5 times respectively, and [total carnitine] by 4.4 and 1.9 times respectively, but decreased plasma [carnitine] by 36-46%. POCA had no effect on either urinary excretion of acylcarnitine esters or [acylcarnitine] in skeletal muscle. By contrast, ciprofibrate decreased [acylcarnitine] and [total carnitine] in muscle. In liver, ciprofibrate increased the [carnitine]/[CoA] ratio and caused a larger increase in [acylcarnitine] (7-fold) than in [carnitine] (4-fold), thereby increasing the [short-chain acylcarnitine]/[carnitine] ratio. POCA did not affect the [carnitine]/[CoA] and the [short-chain acylcarnitine]/[carnitine] ratios, but it decreased the [long-chain acylcarnitine]/[carnitine] ratio. Ciprofibrate and POCA increased the activities of acyl-CoA hydrolases, and carnitine acetyltransferase activity was increased 28-fold and 6-fold by ciprofibrate and POCA respectively. In cultures of hepatocytes, ciprofibrate caused similar changes in enzyme activity to those observed in vivo, although [carnitine] decreased with time. The results suggest that: (1) the reactions catalysed by the short-chain carnitine acyltransferases, but not by the carnitine palmitoyltransferases, are near equilibrium in liver both before and after modification of metabolism by administration of ciprofibrate or POCA; (2) the increase in hepatic [carnitine] after ciprofibrate or POCA feeding can be explained by redistribution of carnitine between tissues; (3) the activity of carnitine

  12. Complement activation in discordant hepatic xenotransplantation.

    PubMed

    Tector, A J; Chen, X; Soderland, C; Tchervenkov, J I

    1998-11-01

    Little is known about hyperacute rejection in hepatic xenotransplantation. Information from clinical xenoperfusions suggests that the liver may be rejected by a mechanism less vigorous than either kidney or heart xenografts. We used the in vitro model of porcine hepatic sinusoidal endothelial cells (PHEC) incubated with either complement replete or deficient human serum to determine the relative roles of the classical and alternate pathways of complement in the immediate response to hepatic xenotransplantation. Our results suggest that either the classical or alternate pathways are capable of independently activating the complement cascade upon exposure to the porcine hepatic sinusoidal endothelium. Our results also imply that either pathway alone is capable of initiating similar degrees of injury as the entire cascade. PMID:9915253

  13. Determining Enzyme Activity by Radial Diffusion

    ERIC Educational Resources Information Center

    Davis, Bill D.

    1977-01-01

    Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

  14. (+)-Catechin attenuates activation of hepatic stellate cells.

    PubMed

    Bragança de Moraes, Cristina Machado; Bitencourt, Shanna; de Mesquita, Fernanda Cristina; Mello, Denizar; de Oliveira, Leticia Paranhos; da Silva, Gabriela Viegas; Lorini, Vinicius; Caberlon, Eduardo; de Souza Basso, Bruno; Schmid, Julia; Ferreira, Gabriela Acevedo; de Oliveira, Jarbas Rodrigues

    2014-04-01

    (+)-Catechin is a type of catechin present in large amounts in açaí fruits and cocoa seeds. Besides its antioxidant and anti-inflammatory activities, little is known about its effects in the liver, especially during hepatic fibrosis. We report here the effects of (+)-catechin on hepatic stellate cells. (+)-Catechin induced quiescent phenotype in GRX cells, along with an increase in lipid droplets. Proliferator-activated receptor γ mRNA expression was upregulated, whereas type I collagen mRNA expression was downregulated. Pro-inflammatory cytokines were not influenced by (+)-catechin, whereas the levels of interleukin 10 were significantly increased. The data provide evidence that (+)-catechin can reduce hepatic stellate cell activation. PMID:24353036

  15. Enzyme immunoassay for the detection of antibody to hepatitis E virus based on synthetic peptides.

    PubMed

    Favorov, M O; Khudyakov, Y E; Fields, H A; Khudyakova, N S; Padhye, N; Alter, M J; Mast, E; Polish, L; Yashina, T L; Yarasheva, D M

    1994-02-01

    Five synthetic peptides were prepared based on the nucleotide sequence of open reading frames 2 and 3 encoded in the hepatitis E virus (HEV) genome and were used to develop an enzyme immunoassay (EIA) for the detection of anti-HEV activity in sera. Three different approaches were employed to ascertain the optimal preparation of these peptides as an immunodiagnostic reagent, including (1) a mixture of unconjugated peptides, (2) conjugating individual peptides to bovine serum albumin (BSA) followed by mixing each conjugate at various concentrations, and (3) mixing the peptides before conjugation with BSA to create an artificial antigen complex. The third method was superior in discriminating anti-HEV activity in sera previously tested by Western blot (WB). A frequency distribution of optical density values demonstrated that the peptide-based EIA was able to readily discriminate anti-HEV positive sera from sera devoid of anti-HEV activity. To confirm anti-HEV activity a neutralization test was developed using a mixture of 5 unconjugated peptides. With the exception of sera containing high levels of anti-HEV activity, all sera were neutralized greater than 50%. Strong sera required a higher dilution before a 50% neutralization was achieved. The sensitivity of the WB compared to EIA was 89.5% with and overall concordance of 94.8%. The peptide-EIA was used to determine anti-HEV activity in sera collected from various populations worldwide. In six outbreaks of ET-NANB hepatitis in various geographic regions, anti-HEV activity was demonstrated in 78-100% of cases. The peptide-EIA also detected anti-HEV activity in 14 out of 14 follow-up sera obtained 4-6 months after onset of disease and in 2 of 2 of these patients 5 yr after the acute episode. Anti-HEV activity was found in 8.5% of sera obtain from a healthy population residing in an HEV endemic region and 0.5% in two non-endemic regions (P < 0.001). These data demonstrate that a synthetic peptide-based EIA is sensitive

  16. Transporter-Enzyme Interplay: Deconvoluting Effects of Hepatic Transporters and Enzymes on Drug Disposition Using Static and Dynamic Mechanistic Models.

    PubMed

    Varma, Manthena V; El-Kattan, Ayman F

    2016-07-01

    A large body of evidence suggests hepatic uptake transporters, organic anion-transporting polypeptides (OATPs), are of high clinical relevance in determining the pharmacokinetics of substrate drugs, based on which recent regulatory guidances to industry recommend appropriate assessment of investigational drugs for the potential drug interactions. We recently proposed an extended clearance classification system (ECCS) framework in which the systemic clearance of class 1B and 3B drugs is likely determined by hepatic uptake. The ECCS framework therefore predicts the possibility of drug-drug interactions (DDIs) involving OATPs and the effects of genetic variants of SLCO1B1 early in the discovery and facilitates decision making in the candidate selection and progression. Although OATP-mediated uptake is often the rate-determining process in the hepatic clearance of substrate drugs, metabolic and/or biliary components also contribute to the overall hepatic disposition and, more importantly, to liver exposure. Clinical evidence suggests that alteration in biliary efflux transport or metabolic enzymes associated with genetic polymorphism leads to change in the pharmacodynamic response of statins, for which the pharmacological target resides in the liver. Perpetrator drugs may show inhibitory and/or induction effects on transporters and enzymes simultaneously. It is therefore important to adopt models that frame these multiple processes in a mechanistic sense for quantitative DDI predictions and to deconvolute the effects of individual processes on the plasma and hepatic exposure. In vitro data-informed mechanistic static and physiologically based pharmacokinetic models are proven useful in rationalizing and predicting transporter-mediated DDIs and the complex DDIs involving transporter-enzyme interplay. PMID:27385183

  17. Regulation of hepatic lipase activity by sphingomyelin in plasma lipoproteins.

    PubMed

    Yang, Peng; Subbaiah, Papasani V

    2015-10-01

    Hepatic lipase (HL) is an important enzyme in the clearance of triacylglycerol (TAG) from the circulation, and has been proposed to have pro-atherogenic as well as anti-atherogenic properties. It hydrolyzes both phospholipids and TAG of lipoproteins, and its activity is negatively correlated with HDL levels. Although it is known that HL acts preferentially on HDL lipids, the basis for this specificity is not known, since it does not require any specific apoprotein for activity. In this study, we tested the hypothesis that sphingomyelin (SM), whose concentration is much higher in VLDL and LDL compared to HDL, is an inhibitor of HL, and that this could explain the lipoprotein specificity of the enzyme. The results presented show that the depletion of SM from normal lipoproteins activated the HL roughly in proportion to their SM content. SM depletion stimulated the hydrolysis of both phosphatidylcholine (PC) and TAG, although the PC hydrolysis was stimulated more. In the native lipoproteins, HL showed specificity for PC species containing polyunsaturated fatty acids at sn-2 position, and produced more unsaturated lyso PC species. The enzyme also showed preferential hydrolysis of certain TAG species over others. SM depletion affected the specificity of the enzyme towards PC and TAG species modestly. These results show that SM is a physiological inhibitor of HL activity in lipoproteins and that the specificity of the enzyme towards HDL is at least partly due to its low SM content. PMID:26193433

  18. Evaluation of enzyme immunoassay for anti-HBc IgM in the diagnosis of acute hepatitis B virus infection.

    PubMed

    Govindarajan, S; Ashcavai, M; Chau, K H; Nevalainen, D E; Peters, R L

    1984-09-01

    Corzyme-MTM (Abbott Laboratories, North Chicago, IL), a newly introduced kit for the measurement of serum IgM antihepatitis B core antigen by enzyme immunoassay, was evaluated for the diagnosis of acute B-viral hepatitis (AVH-B). The study included 175 acute viral hepatitis patients with transient hepatitis B surface antigen (HBsAg). Sera from 160 were tested on multiple occasions until their HBsAg cleared. IgM anti-HBc was found in 171 of 175 patients (98.4%) during the acute phase. The serum samples from 42 patients with liver biopsy-proven chronic active hepatitis, type B (CAH-B), and 18 patients with persistent hepatitis, type B (PH-B), were analyzed for the presence of IgM anti-HBc, using the same technic. None of the sera from 42 patients with CAH-B and only 2 of the 18 patients with PHB had IgM anti-HBc. Thus, the measuring IgM anti-HBc using Corzyme-M kit is helpful in the diagnosis of AVH-B and in the discrimination of acute from chronic HBV infections. PMID:6380271

  19. Enzyme Activity Experiments Using a Simple Spectrophotometer

    ERIC Educational Resources Information Center

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

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

  20. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.

    PubMed

    Hall, Angela M; Soufi, Nisreen; Chambers, Kari T; Chen, Zhouji; Schweitzer, George G; McCommis, Kyle S; Erion, Derek M; Graham, Mark J; Su, Xiong; Finck, Brian N

    2014-07-01

    Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol (DAG), a lipid that has been linked to the development of hepatic insulin resistance through activation of protein kinase C (PKC). The expression of genes that encode MGAT enzymes is induced in the livers of insulin-resistant human subjects with nonalcoholic fatty liver disease, but whether MGAT activation is causal of hepatic steatosis or insulin resistance is unknown. We show that the expression of Mogat1, which encodes MGAT1, and MGAT activity are also increased in diet-induced obese (DIO) and ob/obmice. To probe the metabolic effects of MGAT1 in the livers of obese mice, we administered antisense oligonucleotides (ASOs) against Mogat1 to DIO and ob/ob mice for 3 weeks. Knockdown of Mogat1 in liver, which reduced hepatic MGAT activity, did not affect hepatic triacylglycerol content and unexpectedly increased total DAG content. Mogat1 inhibition also increased both membrane and cytosolic compartment DAG levels. However, Mogat1 ASO treatment significantly improved glucose tolerance and hepatic insulin signaling in obese mice. In summary, inactivation of hepatic MGAT activity, which is markedly increased in obese mice, improved glucose tolerance and hepatic insulin signaling independent of changes in body weight, intrahepatic DAG and TAG content, and PKC signaling. PMID:24595352

  1. N-vinylpyrrolidone dimer, a novel formulation excipient, causes hepatic and thyroid hypertrophy through the induction of hepatic microsomal enzymes in rats.

    PubMed

    Yang, Yi; Ciurlionis, Rita; Kowalkowski, Kenneth; Marsh, Kennan C; Bracken, William M; Blomme, Eric A G

    2012-01-01

    N-vinylpyrrolidone dimer (VPD) is a novel experimental formulation excipient intended for preclinical toxicology studies. In a previous 4-week toxicity study, VPD induced dose-dependent hepatocellular and thyroid gland hypertrophy in Sprague-Dawley (SD) rats. The objectives of the current investigation were to define the underlying molecular mechanisms of these changes. Two separate studies were conducted using male SD rats, daily doses of 300, 1000 or 3,000 mg/kg of VPD, and a positive control (phenobarbital at 75 mg/kg/day): (1) a 28-day study to monitor thyroid hormone levels after 7 and 28 days of dosing; (2) a 5-day study to evaluate hepatic and thyroid gland transcriptomic changes, as well as hepatic UGT activity levels. At VPD dosages of 300 mg/kg/day and higher, 2-fold increases of serum thyroid stimulating hormone (TSH) levels were observed in male SD rats after 28 days of dosing, while serum thyroxine (T4) and triiodothyronine (T3) levels were unchanged. Liver UGT enzyme activity levels were increased in VPD-treated rats after 5 days. In addition, in the 5-day study, VPD caused increased hepatic mRNA levels of a panel of drug metabolizing enzymes (DMEs) and transporters, including Cyp3a1, Cyp2b1, Ugt 2b1, and Abcc3. Similar patterns of induction were observed in primary rat hepatocytes exposed to VPD. Transcriptomic changes in the thyroid gland were identified for genes involved in thyroid hormone biosynthesis and in the FAK, PTEN, and Wnt/β-catenin signaling pathways. Collectively, these data indicate that VPD acts as an inducer of hepatic DMEs in SD rats and that this likely leads to enhanced peripheral metabolism of T3/T4, resulting in a feedback response characterized by increased serum TSH levels, and thyroid gland hypertrophy and hyperplasia. PMID:22037397

  2. POLYCHLORINATED BIPHENYLS AS INDUCERS OF HEPATIC MICROSOMAL ENZYMES: EFFECTS OF DI-ORTHO SUBSTITUTION

    EPA Science Inventory

    All of the 13 possible polychlorinated biphenyl (PCB) isomers and congeners substituted at both para positions, at least two meta positions (but not necessarily on the same ring) and at two ortho positions have been synthesized and tested as rat hepatic microsomal enzyme inducers...

  3. Effects of PCBs on plasma enzymes, testosterone level, and hepatic xenobiotic metabolism in the grey partridge, perdix perdlx

    SciTech Connect

    Abiola, F. ); Lorgue, G.; Riviere, J.L. ); Benoit, E. ); Soyez, D. )

    1989-09-01

    The hepatic cytochrome P-450-dependent monooxygenase (MO) system functions in oxidative biotransformation of a wide variety of both endogenous and exogenous (xenobiotic) compounds in many animal species. However, most of the previous studies were carried out with a narrow range of species and investigations on wild species are lacking. In this report, the authors describe the effects of a commercial mixture of PCBs (DP5) on the hepatic MO activities of the grey partridge (Perdix perdix). To more thoroughly investigate the inducing effects of DP5, they used two series of homologous substrates, alkylresorufins and alkoxycoumarins, and an endogenous compound, testosterone, which were shown in mammals to differentiate between different forms of cytochrome P-450. Furthermore, to more carefully assess the effects of DP5, they also measured the activity of two plasma marker enzymes, alanine transpeptidase (ALAT) and gamma-glutamyl transferase (gamma-GT), and the plasmatic concentration of testosterone.

  4. Characterization of Soil Samples of Enzyme Activity

    ERIC Educational Resources Information Center

    Freeland, P. W.

    1977-01-01

    Described are nine enzyme essays for distinguishing soil samples. Colorimetric methods are used to compare enzyme levels in soils from different sites. Each soil tested had its own spectrum of activity. Attention is drawn to applications of this technique in forensic science and in studies of soil fertility. (Author/AJ)

  5. Enzyme Activities in Polarized Cell Membranes

    PubMed Central

    Bass, L.; McIlroy, D. K.

    1968-01-01

    The theoretical pH dependence of enzyme activities in membranes of low dielectric constant is estimated. It is shown that in biological membranes some types of enzymes may attain a limiting pH sensitivity such that an increment of only 0.2 pH unit (sufficient to induce action potentials in squid axons) causes a relative activity change of over 25%. The transients of enzyme activity generated by membrane depolarization and by pH increments in the bathing solution are discussed in relation to the transients of nervous excitation. PMID:5641405

  6. [Muscle enzyme activity and exercise].

    PubMed

    Gojanovic, B; Feihl, F; Gremion, G; Waeber, B

    2009-02-01

    Exercise is classically associated with muscular soreness, presenting one to two days later, delayed onset muscular soreness. Blood muscle enzymes and protein elevations are characteristic, and may cause renal failure. Creatin phosphokinase peak appears on the fourth day and depends on exercise type and individual parameters. This effect is attenuated with repeated bouts, by habituation. Metabolic complications are rare. The knowledge of this reaction, even with common exercises, allows to postpone investigations for a complex metabolic disorder, or to avoid stopping a medication for fear of a side effect, as with statins. Indeed, it is necessary to wait for seven days without any exercise before interpreting an elevated CK result. PMID:19180440

  7. Visualization of enzyme activities inside earthworm pores

    NASA Astrophysics Data System (ADS)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

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

  8. The anticancer drug ellipticine activated with cytochrome P450 mediates DNA damage determining its pharmacological efficiencies: studies with rats, Hepatic Cytochrome P450 Reductase Null (HRN™) mice and pure enzymes.

    PubMed

    Stiborová, Marie; Černá, Věra; Moserová, Michaela; Mrízová, Iveta; Arlt, Volker M; Frei, Eva

    2015-01-01

    Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models. PMID:25547492

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

    PubMed

    Srinivasan, Subramani; Muruganathan, Udaiyar

    2016-04-25

    Diabetes mellitus is a clinically complex disease characterized by chronic hyperglycemia with metabolic disturbances. During diabetes, endogenous hepatic glucose production is increased as a result of impaired activities of the key enzymes of carbohydrate metabolism. The purpose of the present study was to evaluate the antidiabetic efficacy of citronellol, a citrus monoterpene in streptozotocin (STZ)-induced diabetic rats. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (40 mg/kg b.w). STZ induced diabetic rats received citronellol orally at the doses of 25, 50, and 100 mg/kg b.w for 30 days. In this study the levels of plasma glucose, insulin, hemoglobin (Hb), glycated hemoglobin (HbA1C), glycogen, and the activities of carbohydrate metabolic enzymes, liver and kidney markers were evaluated. Oral administration of citronellol (50 mg/kg) for 30 days dose dependently improved the levels of insulin, Hb and hepatic glycogen with significant decrease in glucose and HbA1C levels. The altered activities of carbohydrate metabolic enzymes, hepatic and kidney markers were restored to near normal. Citronellol supplement was found to be effective in preserving the normal histological appearance of hepatic cells and insulin-positive β-cells in STZ-rats. Our results suggest that administration of citronellol attenuates the hyperglycemia in the STZ-induced diabetic rats by ameliorating the key carbohydrate metabolic enzymes and could be developed as a functional and nutraceutical ingredient in combating diabetes mellitus. PMID:26944432

  10. Effect of selenium deficiency on hepatic type I 5-iodothyronine deiodinase activity and hepatic thyroid hormone levels in the rat.

    PubMed Central

    Beckett, G J; Russell, A; Nicol, F; Sahu, P; Wolf, C R; Arthur, J R

    1992-01-01

    Selenium deficiency in rats for a period of up to 6 weeks inhibited both the production of 3,3',5-tri-iodothyronine (T3) from thyroxine (T4) (5'-deiodination) and also the catabolism of T3 to 3,3'-di-iodothyronine (5-deiodination) in liver homogenates. The hepatic stores of T3 were decreased by only 8% in selenium deficiency, despite the T3 production rate from T4 being only 7% of the rate found in selenium-supplemented rats. Hepatic glutathione S-transferase (GST) activity was increased in both hypothyroidism and selenium deficiency, but apparently by different mechanisms, since mRNA expression for this family of enzymes was lowered by hypothyroidism and increased in selenium deficiency. It is concluded that, since both T3 production and catabolism are inhibited by selenium deficiency, there is little change in hepatic T3 stores, and therefore the changes in the activity of certain hepatic enzymes, such as GST, that are found in selenium deficiency are not the result of tissue hypothyroidism. Images Fig. 1. PMID:1546962

  11. Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

    PubMed

    Kimura, Kumi; Tanida, Mamoru; Nagata, Naoto; Inaba, Yuka; Watanabe, Hitoshi; Nagashimada, Mayumi; Ota, Tsuguhito; Asahara, Shun-ichiro; Kido, Yoshiaki; Matsumoto, Michihiro; Toshinai, Koji; Nakazato, Masamitsu; Shibamoto, Toshishige; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2016-03-15

    Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity. PMID:26947072

  12. Proteomic characterization of hepatitis C eradication: enzyme switch in the healing liver.

    PubMed

    Babudieri, S; Soddu, A; Nieddu, P; Tanca, A; Madeddu, G; Addis, M F; Pagnozzi, D; Cossu-Rocca, P; Massarelli, G; Dore, M P; Uzzau, S; Mura, M S

    2013-07-01

    Lipid pathway impairment, decrease in the antioxidant pool and downregulation in amino-acid metabolism are just some of the metabolic variations attributed to chronic HCV infection. All of them have been studied separately, mainly in animal models. Thanks to proteomic analysis we managed to describe (for the fist time to the best of our knowledge), in vivo and in humans, the metabolic alterations caused by HCV, and the recovery of the same alterations during HCV treatment. We performed proteomic analysis on liver specimens of a 28-year-old woman affected by hepatitis C genotype 1a, alcoholism and diabetes mellitus type 1, before and after antiviral treatment with pegylated interferon alpha 2b and ribavirin. The subject, thanks to a patient-tailored therapy, reached Sustained Virological Response. Throughout the treatment period the patient was monitored with subsequent biochemical, clinical and psychological examinations. The data obtained by the patient's close monitoring suggest a direct interaction between insulin resistance and an active HCV genotype 1 infection, with a leading role played by the infection, and not by insulin resistance, as demonstrated by the sharp fall of the insulin units needed per day during treatment. The proteomic analysis showed that after therapy, a downregulation of enzymes involved in amino acid metabolism, glycolysis/gluconeogenesis and alcohol catabolism takes place, the latter probably due to cessation of alcohol abuse. On the contrary, the metabolic pathways linked to metabolism of the reactive oxygen species were upregulated after therapy. Finally, a significant alteration in the pathway regulated by peroxisome proliferator-activated receptor alpha (PPARA), a major regulator of lipid metabolism in the liver, was reported. These "real time" data confirm in vivo, in humans, that during HCV infection, the pathways related to fatty acids, glucose metabolism and free radical scavenging are inhibited. The same enzyme deficit is

  13. Predictors of Hepatitis B Cure Using Gene Therapy to Deliver DNA Cleavage Enzymes: A Mathematical Modeling Approach

    PubMed Central

    Schiffer, Joshua T.; Swan, Dave A.; Stone, Daniel; Jerome, Keith R.

    2013-01-01

    Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA), the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR-associated system 9 (Cas9) proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which will in turn

  14. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  15. Enzyme activity in dialkyl phosphate ionic liquids.

    PubMed

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

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v. PMID:22001053

  16. Enzyme activity in dialkyl phosphate ionic liquids

    SciTech Connect

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

    2011-12-01

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

  17. Effect of the combined probiotics with aflatoxin B₁-degrading enzyme on aflatoxin detoxification, broiler production performance and hepatic enzyme gene expression.

    PubMed

    Zuo, Rui-yu; Chang, Juan; Yin, Qing-qiang; Wang, Ping; Yang, Yu-rong; Wang, Xiao; Wang, Guo-qiang; Zheng, Qiu-hong

    2013-09-01

    In order to degrade aflatoxin B₁ (AFB₁), AFB₁-degrading microbes (probiotics) such as Lactobacillus casei, Bacillus subtilis and Pichia anomala, and the AFB₁-degrading enzyme from Aspergillus oryzae were selected and combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as the basal diet, 400 μg/kg AFB₁ supplement without feed additive, and 200, 400, 800 μg/kg AFB₁ supplement with 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine production performance of broilers. In addition, serum, liver and chest muscle were selected for measuring AFB₁ residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding 0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB₁ on chicken's production performance and nutrient metabolic rates (P<0.05). It could also improve AFB₁ metabolism, hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxidoreductase, apoptosis, cell growth, immune system and metabolic process (P<0.05). It could be concluded that the feed additive was able to degrade AFB₁ and improve animal production. PMID:23831311

  18. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

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

    PubMed

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

    2016-06-01

    In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling. PMID:26944803

  20. Correlation of Tc-99m GSA hepatic studies with biopsies in patients with chronic active hepatitis.

    PubMed

    Tomiguchi, S; Kira, T; Oyama, Y; Nabeshima, M; Nakashima, R; Tsuji, A; Kojima, A; Takahashi, M; Yoshimatsu, S; Sagara, K

    1995-08-01

    To determine whether scintigraphic findings of Tc-99m DTPA-galactosyl-HSA (GSA) correspond to histopathologic findings, Tc-99m GSA hepatic scintigraphy and biopsy were compared in 65 patients with chronic active hepatitis. After injecting 185 MBq of Tc-99m GSA, anterior images were obtained at 5 minutes and 15 minutes. Scintigrams were classified into three grades according to the extent of visualization of the cardiac blood pool on 5 minute and 15 minute images. Biopsies were subjectively graded for findings of necrosis and fibrosis. Scintigraphic grades on 5 minute images were correlated with hepatic necrosis and fibrosis and those on 15-minute images with hepatic fibrosis. Scintigraphic abnormalities of Tc-99m GSA correlated well with histopathologic abnormalities, especially with hepatic fibrosis and necrosis in patients with chronic active hepatitis. PMID:7586877

  1. Human variation and CYP enzyme contribution in benfuracarb metabolism in human in vitro hepatic models.

    PubMed

    Abass, Khaled; Reponen, Petri; Mattila, Sampo; Rautio, Arja; Pelkonen, Olavi

    2014-01-13

    Human responses to the toxicological effects of chemicals are often complicated by a substantial interindividual variability in toxicokinetics, of which metabolism is often the most important factor. Therefore, we investigated human variation and the contributions of human-CYP isoforms to in vitro metabolism of benfuracarb. The primary metabolic pathways were the initial sulfur oxidation to benfuracarb-sulfoxide and the nitrogen-sulfur bond cleavage to carbofuran (activation). The Km, Vmax, and CL(int) values of carbofuran production in ten individual hepatic samples varied 7.3-, 3.4-, and 5.4-fold, respectively. CYP2C9 and CYP2C19 catalyzed benfuracarb sulphur oxidation. Carbofuran formation, representing from 79% to 98% of the total metabolism, was catalyzed predominantly by CYP3A4. The calculated relative contribution of CYP3A4 to carbofuran formation was 93%, while it was 4.4% for CYP2C9. The major contribution of CYP3A4 in benfuracarb metabolism was further substantiated by showing a strong correlation with CYP3A4-selective markers midazolam-1'-hydroxylation and omeprazole-sulfoxidation (r=0.885 and 0.772, respectively). Carbofuran formation was highly inhibited by the CYP3A inhibitor ketoconazole. Moreover, CYP3A4 marker activities were relatively inhibited by benfuracarb. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro activation of benfuracarb and that CYP3A4-catalyzed metabolism is the primary source of interindividual differences. PMID:24016712

  2. Coral calcium hydride prevents hepatic steatosis in high fat diet-induced obese rats: A potent mitochondrial nutrient and phase II enzyme inducer.

    PubMed

    Hou, Chen; Wang, Yongyao; Zhu, Erkang; Yan, Chunhong; Zhao, Lin; Wang, Xiaojie; Qiu, Yingfeng; Shen, Hui; Sun, Xuejun; Feng, Zhihui; Liu, Jiankang; Long, Jiangang

    2016-03-01

    Diet-induced nonalcoholic fatty liver disease (NAFLD) is characterized by profound lipid accumulation and associated with an inflammatory response, oxidative stress and hepatic mitochondrial dysfunction. We previously demonstrated that some mitochondrial nutrients effectively ameliorated high fat diet (HFD)-induced hepatic steatosis and metabolic disorders. Molecular hydrogen in hydrogen-rich liquid or inhaling gas, which has been confirmed in scavenging reactive oxygen species and preventing mitochondrial decay, improved metabolic syndrome in patients and animal models. Coral calcium hydride (CCH) is a new solid molecular hydrogen carrier made of coral calcium. However, whether and how CCH impacts HFD-induced hepatic steatosis remains uninvestigated. In the present study, we applied CCH to a HFD-induced NAFLD rat model for 13 weeks. We found that CCH durably generated hydrogen in vivo and in vitro. CCH treatment significantly reduced body weight gain, improved glucose and lipid metabolism and attenuated hepatic steatosis in HFD-induced obese rats with no influence on food and water intake. Moreover, CCH effectively improved HFD-induced hepatic mitochondrial dysfunction, reduced oxidative stress, and activated phase II enzymes. Our results suggest that CCH is an efficient hydrogen-rich agent, which could prevent HFD-induced NAFLD via activating phase II enzymes and improving mitochondrial function. PMID:26774456

  3. Halophilic enzyme activation induced by salts

    PubMed Central

    Ortega, Gabriel; Laín, Ana; Tadeo, Xavier; López-Méndez, Blanca; Castaño, David; Millet, Oscar

    2011-01-01

    Halophilic archea (halobacteriae) thrive in hypersaline environments, avoiding osmotic shock by increasing the ion concentration of their cytoplasm by up to 3–6 M. To remain folded and active, their constitutive proteins have evolved towards a biased amino acid composition. High salt concentration affects catalytic activity in an enzyme-dependent way and a unified molecular mechanism remains elusive. Here, we have investigated a DNA ligase from Haloferax volcanii (Hv LigN) to show that K+ triggers catalytic activity by preferentially stabilising a specific conformation in the reaction coordinate. Sodium ions, in turn, do not populate such isoform and the enzyme remains inactive in the presence of this co-solute. Our results show that the halophilic amino acid signature enhances the enzyme's thermodynamic stability, with an indirect effect on its catalytic activity. This model has been successfully applied to reengineer Hv LigN into an enzyme that is catalytically active in the presence of NaCl. PMID:22355525

  4. Proteomic analysis for the impact of hypercholesterolemia on expressions of hepatic drug transporters and metabolizing enzymes.

    PubMed

    Liu, Yan; Pu, Qiang-Hong; Wu, Ming-Jun; Yu, Chao

    2016-10-01

    1. Our objective is to investigate the alterations of hepatic drug transporters and metabolizing enzymes in hypercholesterolemia. Male Sprague-Dawley rats were fed high-cholesterol chows for 8 weeks to induce hypercholesterolemia. Protein levels of hepatic drug transporters and metabolizing enzymes were analyzed by iTRAQ labeling coupled with LC TRIPLE-TOF. 2. Total 239 differentially expressed proteins were identified using proteomic analysis. Among those, protein levels of hepatic drug transporters (MRP2, ABCD3, OAT2, SLC25A12, SCL38A3, SLC2A2 and SLC25A5) and metabolizing enzymes (CYP2B3, CYP2C7, CYP2C11, CYP2C13, CYP4A2 and UGT2B) were markedly reduced, but the levels of CYP2C6 and CYP2E1 were increased in hypercholesterolemia group compared to control. Decreased expressions of drug transporters MRP2 and OAT2 were further confirmed by real time quantitative PCR (RT-qPCR) and western blot. 3. Ingenuity pathway analysis revealed that these differentially expressed proteins were regulated by various signaling pathways including nuclear receptors and inflammatory cytokines. One of the nuclear receptor candidates, liver X receptor alpha (LXRα), was further validated by RT-qPCR and western blot. Additionally, LXRα agonist T0901317 rescued the reduced expressions of MRP2 and OAT2 in HepG2 cells in hypercholesterolemic serum treatment. 4. Our present results indicated that hypercholesterolemia affected the expressions of various drug transporters and metabolizing enzymes in liver via nuclear receptors pathway. Especially, decreased function of LXRα contributes to the reduced expressions of MRP2 and OAT2. PMID:26887802

  5. An NMR Study of Enzyme Activity.

    ERIC Educational Resources Information Center

    Peterman, Keith E.; And Others

    1989-01-01

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

  6. Enzyme Specific Activity in Functionalized Nanoporous Supports

    SciTech Connect

    Lei, Chenghong; Soares, Thereza A.; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2008-03-26

    Enzyme specific activity can be increased or decreased to a large extent by changing protein loading density in functionalized nanoporous support, where organophosphorus hydrolase can display a constructive orientation and thus leave a completely open entrance for substrate even at higher protein loading density, but glucose oxidase can not.

  7. 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.

  8. Hepatic Enzyme Alterations in HIV Patients on Antiretroviral Therapy: A Case-Control Study in a Hospital Setting in Ghana

    PubMed Central

    Osakunor, Derick Nii Mensah; Obirikorang, Christian; Fianu, Vincent; Asare, Isaac; Dakorah, Mavis

    2015-01-01

    Background Diagnosing hepatic injury in HIV infection can be a herculean task for clinicians as several factors may be involved. In this study, we sought to determine the effects of antiretroviral therapy (ART) and disease progression on hepatic enzymes in HIV patients. Methods A case-control study conducted from January to May 2014 at the Akwatia Government Hospital, Eastern region, Ghana, The study included 209 HIV patients on ART (designated HIV-ART) and 132 ART-naive HIV patients (designated HIV-Controls). Data gathered included demography, clinical history and results of blood tests for hepatic enzymes. We employed the Fisher’s, Chi-square, unpaired t-test and Pearson’s correlation in analysis, using GraphPad Prism and SPSS. A P value < 0.05 was considered significant. Results Median CD4 lymphocyte count of HIV-ART participants (604.00 cells/mm3) was higher than that of HIV-Controls (491.50 cells/mm3; P = 0.0005). Mean values of ALP, ALT, AST and GGT did not differ between the two groups compared (P > 0.05). There was a significant positive correlation between hepatic enzymes (ALP, ALT, AST and GGT) for both groups (p < 0.01 each). Duration of ART correlated positively with ALT (p < 0.05). The effect size of disease progression on hepatic enzymes for both groups was small. Conclusion Antiretroviral therapy amongst this population has minimal effects on hepatic enzymes and does not suggest modifications in therapy. Hepatic injury may occur in HIV, even in the absence of ART and other traditional factors. Monitoring of hepatic enzymes is still important in HIV patients. PMID:26247879

  9. Chemomodulatory effect of Moringa oleifera, Lam, on hepatic carcinogen metabolising enzymes, antioxidant parameters and skin papillomagenesis in mice.

    PubMed

    Bharali, Rupjyoti; Tabassum, Jawahira; Azad, Mohammed Rekibul Haque

    2003-01-01

    The modulatory effects of a hydro-alcoholic extract of drumsticks of Moringa oliefera Lam at doses of 125 mg/kg bodyweight and 250 mg/ kg body weight for 7 and 14 days, respectively, were investigated with reference to drug metabolising Phase I (Cytochrome b(5) and Cytochrome p(450) ) and Phase II (Glutathione-S- transferase) enzymes, anti-oxidant enzymes, glutathione content and lipid peroxidation in the liver of 6-8 week old female Swiss albino mice. Further, the chemopreventive efficacy of the extract was evaluated in a two stage model of 7,12 - dimethylbenz(a)anthracene induced skin papillomagenesis. Significant increase (p<0.05 to p<0.01) in the activities of hepatic cytochrome b(5), cytochrome p(450), catalase, glutathione peroxidase ( GPx ), glutathione reductase (GR), acid soluble sulfhydryl content (-SH ) and a significant decrease ( p<0.01 ) in the hepatic MDA level were observed at both dose levels of treatment when compared with the control values. Glutathione-S- transferase ( GST )activity was found to be significantly increased (p<0.01 ) only at the higher dose level. Butylated hydroxyanisol (BHA ) fed at a dose of 0.75% in the diet for 7 and 14 days (positive control ) caused a significant increase (p<0.05 to p<0.01) in the levels of hepatic phase I and phase II enzymes, anti- oxidant enzymes, glutathione content and a decrease in lipid peroxidation. The skin papillomagenesis studies demonstrated a significant decrease (p<0.05 ) in the percentage of mice with papillomas, average number of papillomas per mouse and papillomas per papilloma bearing mouse when the animals received a topical application of the extract at a dose of 5mg/ kg body weight in the peri-initiation phase 7 days before and 7 days after DMBA application, Group II ), promotional phase (from the day of croton oil application and continued till the end of the experiment, Group III ) and both peri and post initiation stages (from 7 days prior to DMBA application and continued till the

  10. Hepatitis

    MedlinePlus

    ... Got Homework? Here's Help White House Lunch Recipes Hepatitis KidsHealth > For Kids > Hepatitis Print A A A ... an important digestive liquid called bile . What Is Hepatitis? Hepatitis is an inflammation (say: in-fluh-MAY- ...

  11. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  12. Comparison of Enzyme Immunoassays for Detection of Antibodies to Hepatitis D Virus in Serum.

    PubMed

    Chow, Siu-Kei; Atienza, Ederlyn E; Cook, Linda; Prince, Harry; Slev, Patricia; Lapé-Nixon, Mary; Jerome, Keith R

    2016-08-01

    Serology remains critical for diagnosing hepatitis D virus (HDV) infection, which affects 15 to 20 million people worldwide, but the literature on characterizing commercial enzyme immunoassays (EIAs) dates back to 15 years ago. We evaluated 2 commercial EIAs currently available for detecting anti-HDV antibodies. The DiaSorin assay demonstrated 100% sensitivity and specificity. Using a modified cutoff value, the Cusabio assay demonstrated a sensitivity of 81.3% and specificity of 90.9%. Our data show that recently developed EIAs are reliable for anti-HDV antibody detection. PMID:27280621

  13. The Action of Antidiabetic Plants of the Canadian James Bay Cree Traditional Pharmacopeia on Key Enzymes of Hepatic Glucose Homeostasis

    PubMed Central

    Nachar, Abir; Vallerand, Diane; Musallam, Lina; Lavoie, Louis; Arnason, John; Haddad, Pierre S.

    2013-01-01

    We determined the capacity of putative antidiabetic plants used by the Eastern James Bay Cree (Canada) to modulate key enzymes of gluconeogenesis and glycogen synthesis and key regulating kinases. Glucose-6-phosphatase (G6Pase) and glycogen synthase (GS) activities were assessed in cultured hepatocytes treated with crude extracts of seventeen plant species. Phosphorylation of AMP-dependent protein kinase (AMPK), Akt, and Glycogen synthase kinase-3 (GSK-3) were probed by Western blot. Seven of the seventeen plant extracts significantly decreased G6Pase activity, Abies balsamea and Picea glauca, exerting an effect similar to insulin. This action involved both Akt and AMPK phosphorylation. On the other hand, several plant extracts activated GS, Larix laricina and A. balsamea, far exceeding the action of insulin. We also found a significant correlation between GS stimulation and GSK-3 phosphorylation induced by plant extract treatments. In summary, three Cree plants stand out for marked effects on hepatic glucose homeostasis. P. glauca affects glucose production whereas L. laricina rather acts on glucose storage. However, A. balsamea has the most promising profile, simultaneously and powerfully reducing G6Pase and stimulating GS. Our studies thus confirm that the reduction of hepatic glucose production likely contributes to the therapeutic potential of several antidiabetic Cree traditional medicines. PMID:23864882

  14. Epigenetic Changes during Hepatic Stellate Cell Activation

    PubMed Central

    Götze, Silke; Schumacher, Eva C.; Kordes, Claus; Häussinger, Dieter

    2015-01-01

    Background and Aims Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC. Methods and Results The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism. Conclusions In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism. PMID:26065684

  15. Effect of squalene synthase inhibition on the expression of hepatic cholesterol biosynthetic enzymes, LDL receptor, and cholesterol 7 alpha hydroxylase.

    PubMed

    Ness, G C; Zhao, Z; Keller, R K

    1994-06-01

    Squalene synthase catalyzes the committed step in the biosynthesis of sterols. Treating rats with zaragozic acid A, a potent inhibitor of squalene synthase, caused marked increases in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, squalene synthase, and LDL receptor mRNA levels. The increase in HMG-CoA reductase mRNA fully accounted for the increases seen in enzyme protein and activity. Farnesyl pyrophosphate synthase mRNA and activity were only slightly increased by zaragozic acid A, while cholesterol 7 alpha hydroxylase mRNA levels were decreased substantially. When rats were pretreated with zaragozic acid A, there was no change in mRNA levels for the cholesterol biosynthetic enzymes or cholesterol 7 alpha hydroxylase upon subsequent treatment with mevalonolactone. Under these same conditions, the enzymatic activity of HMG-CoA reductase was also unaffected. Mevalonolactone treatment reduced the zaragozic acid A-mediated increase in hepatic LDL receptor mRNA levels. Feeding cholesterol eliminated the zaragozic acid A-induced increase in HMG-CoA reductase mRNA levels. These results suggest that inhibition of squalene synthase decreases the level of a squalene-derived regulatory product, resulting in altered amounts of several mRNAs and coordinate increases in HMG-CoA reductase mRNA, protein, and activity. The increase in HMG-CoA reductase gene expression was closely related to the degree of inhibition of cholesterol synthesis caused by zaragozic acid A. PMID:7911291

  16. Exercise training down-regulates hepatic lipogenic enzymes in meal-fed rats: fructose versus complex-carbohydrate diets.

    PubMed

    Fiebig, R; Griffiths, M A; Gore, M T; Baker, D H; Oscai, L; Ney, D M; Ji, L L

    1998-05-01

    The maximal activity and mRNA abundance of hepatic fatty acid synthase (FAS) and other lipogenic enzymes were investigated in rats meal-fed either a high fructose (F) or a high cornstarch (C) diet. The diet contained 50% F or C (g/100 g), casein (20%), cornstarch (16.13%), corn oil (5%), minerals (5.37%), vitamins (1%) and Solka-floc (2%). Female Sprague-Dawley rats (n = 44) were randomly divided into C or F groups that were meal-fed for 3 h/d; each group was subdivided into exercise-trained (T) and untrained (U) groups. Treadmill training was performed 4 h after the initiation of the meal at 25 m/min, 10% grade for 2 h/d, 5 d/wk, for 10 wk. Rats were killed 9 h after the meal and 27 h after the last training session. F-fed rats had significantly higher activities of all lipogenic enzymes assayed and mRNA abundance of FAS and acetyl-coenzyme A carboxylase (ACC) than C rats (P < 0.05). Concentrations of plasma insulin and glucose and liver pyruvate were not altered by F feeding. Proportions of the fatty acids 18:2 and 20:4 were lower, whereas those of 16:0 and 16:1 were higher, in livers of F than of C rats (P < 0.05). Training decreased FAS activity by 50% (P < 0.05), without affecting FAS mRNA level in C rats; this down-regulation was absent in the F rats. ACC mRNA abundance tended to be lower in CT than in CU rats (P < 0.075). L-Type pyruvate kinase activity was lower in FT than in FU rats (P < 0.05), whereas other lipogenic enzyme activities did not differ between T and U rats of each diet group. We conclude that hepatic lipogenic enzyme induction by high carbohydrate meal feeding may be inhibited by exercise training and that a fructose-rich diet may attenuate this training-induced down-regulation. PMID:9566986

  17. Hepatic ischemia-reperfusion syndrome after partial liver resection (LR): hepatic venous oxygen saturation, enzyme pattern, reduced and oxidized glutathione, procalcitonin and interleukin-6.

    PubMed

    Kretzschmar, Michael; Krüger, Antie; Schirrmeister, Wulf

    2003-06-01

    The hepatic ischemia-reperfusion syndrome was investigated in 28 patients undergoing elective partial liver resection with intraoperative occlusion of hepatic inflow (Pringle maneuver) using the technique of liver vein catheterization. Hepatic venous oxygen saturation (ShvO2) was monitored continuously up to 24 hours after surgery. Aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyl transpeptidase, pseudocholinesterase, alpha-glutathione S-transferase, reduced and oxidized glutathione, procalcitonine, and interleukin-6 were serially measured both before and after Pringle maneuver during the resection and postoperatively in arterial and/or hepatic venous blood. ShvO2 measurement demonstrated that peri- and postoperative management was suitable to maintain an optimal hepatic oxygen supply. As expected, we were able to demonstrate a typical enzyme pattern of postischemic liver injury. There was a distinct decrease of reduced glutathione levels both in arterial and hepatic venous plasma after LR accompanied by a strong increase in oxidized glutathione concentration during the phase of reperfusion. We observed increases in procalcitonin and interleukin-6 levels both in arterial and hepatic venous blood after declamping. Our data support the view that liver resection in man under conditions of inflow occlusion resulted in ischemic lesion of the liver (loss of glutathione synthesizing capacity with disturbance of protection against oxidative stress) and an additional impairment during reperfusion (liberation of reactive oxygen species, local and systemic inflammation reaction with cytokine production). Additionally, we found some evidence for the assumption that the liver has an export function for reduced glutathione into plasma in man. PMID:12877355

  18. Mitochondrial uncouplers inhibit hepatic stellate cell activation

    PubMed Central

    2012-01-01

    Background Mitochondrial dysfunction participates in the progression of several pathologies. Although there is increasing evidence for a mitochondrial role in liver disease, little is known about its contribution to hepatic stellate cell (HSC) activation. In this study we investigated the role of mitochondrial activity through mild uncoupling during in vitro activation of HSCs. Methods Cultured primary human and mouse HSCs were treated with the chemical uncouplers FCCP and Valinomycin. ATP levels were measured by luciferase assay and production of reactive oxygen species was determined using the fluorescent probe DCFH-DA. Possible cytotoxicity by uncoupler treatment was evaluated by caspase 3/7 activity and cytoplasmic protease leakage. Activation of HSCs and their response to the pro-fibrogenic cytokine TGF-β was evaluated by gene expression of activation markers and signal mediators using RT-qPCR. Proliferation was measured by incorporation of EdU and protein expression of α-smooth muscle actin was analyzed by immunocytochemistry and western blot. Results FCCP and Valinomycin treatment mildly decreased ATP and reactive oxygen species levels. Both uncouplers increased the expression of mitochondrial genes such as Tfam and COXIV while inducing morphological features of quiescent mouse HSCs and abrogating TGF-β signal transduction. Mild uncoupling reduced HSC proliferation and expression of pro-fibrogenic markers of mouse and human HSCs. Conclusions Mild mitochondrial uncoupling inhibits culture-induced HSC activation and their response to pro-fibrogenic cytokines like TGF-β. These results therefore suggest mitochondrial uncoupling of HSCs as a strategy to reduce progression of liver fibrosis. PMID:22686625

  19. Hepatic Expression of Detoxification Enzymes Is Decreased in Human Obstructive Cholestasis Due to Gallstone Biliary Obstruction

    PubMed Central

    Chai, Jin; Feng, Xinchan; Zhang, Liangjun; Chen, Sheng; Cheng, Ying; He, Xiaochong; Yang, Yingxue; He, Yu; Wang, Huaizhi; Wang, Rongquan; Chen, Wensheng

    2015-01-01

    Background & Aims Levels of bile acid metabolic enzymes and membrane transporters have been reported to change in cholestasis. These alterations (e.g. CYP7A1 repression and MRP4 induction) are thought to be adaptive responses that attenuate cholestatic liver injury. However, the molecular mechanisms of these adaptive responses in human obstructive cholestasis due to gallstone biliary obstruction remain unclear. Methods We collected liver samples from cholestatic patients with biliary obstruction due to gallstones and from control patients without liver disease (n = 22 per group). The expression levels of bile acid synthetic and detoxification enzymes, membrane transporters, and the related nuclear receptors and transcriptional factors were measured. Results The levels of bile acid synthetic enzymes, CYP7B1 and CYP8B1, and the detoxification enzyme CYP2B6 were increased in cholestatic livers by 2.4-fold, 2.8-fold, and 1.9-fold, respectively (p<0.05). Conversely, the expression levels of liver detoxification enzymes, UGT2B4/7, SULT2A1, GSTA1-4, and GSTM1-4, were reduced by approximately 50% (p<0.05) in human obstructive cholestasis. The levels of membrane transporters, OSTβ and OCT1, were increased 10.4-fold and 1.8-fold, respectively, (p<0.05), whereas those of OSTα, ABCG2 and ABCG8 were all decreased by approximately 40%, (p<0.05) in human cholestatic livers. Hepatic nuclear receptors, VDR, HNF4α, RXRα and RARα, were induced (approximately 2.0-fold, (p<0.05) whereas FXR levels were markedly reduced to 44% of control, (p<0.05) in human obstructive cholestasis. There was a significantly positive correlation between the reduction in FXR mRNA and UGT2B4/7, SULT2A1, GSTA1, ABCG2/8 mRNA levels in livers of obstructive cholestatic patients (p<0.05). Conclusions The levels of hepatic detoxification enzymes were significantly decreased in human obstructive cholestasis, and these decreases were positively associated with a marked reduction of FXR levels. These findings

  20. Induction of hepatic drug metabolizing enzymes by coal fly ash in rats

    SciTech Connect

    Srivastava, P.K.; Singh, Y.; Tyagi, S.R.; Misra, U.K.

    1987-12-01

    The effect of intratracheal administration of fly ash, its benzene-extracted residue and the benzene extract has been studied on the activities of hepatic mixed-function oxidases in the rat. Fly ash and its fractions significantly increased the levels of cytochrome P-450, cytochrome b/sub 5/, cytochrome b/sub 5/ reductase, NADPH-cytochrome c reductase, aminopyrine N-demethylase, aniline hydroxylase, and glutathione S-transferase in a dose-dependent manner. Phenobarbital or 3-methylcholanthrene treatment along with the administration of fly ash or its fractions showed an additive effect on the activities of the mixed-function oxidases. The observed effects were due to chemical component, i.e., organic and inorganic fractions of fly ash, and not due to its particulate nature. This was shown by the administration of glass beads, which did not cause any alteration in the activities of hepatic mixed-function oxidases.

  1. Chemoprotective influence of Zanthoxylum sps. on hepatic carcinogen metabolizing and antioxidant enzymes and skin papillomagenesis in murine model.

    PubMed

    Rajamani, Paulraj; Banerjeet, Sanjeev; Rao, A Ramesha

    2011-11-01

    In the present study, the putative potential of pericarp of dried fruit of Zanthoxylum (Rutaceae Family), a common spice additive in India's west coast cuisines, in protecting against carcinogenesis has been reported. Extract from dried fruit of Zanthoxylum was orally administered to mice at two dose levels: 100 and 200 mg/kg body wt. for 14 days. Results reveal bifunctional nature of Zanthoxylum species as deduced from its potential to induce phase-I and phase-II enzyme activities associated with carcinogen activation and detoxification in the liver of mice. Hepatic glutathione S-transferase and DT-diaphorase were found significantly elevated by the treatment. Zanthoxylum was also effective in augmenting the antioxidant enzyme activities of glutathione peroxidase, superoxide dismutase and catalase albeit significantly by high dose of the extract (P < 0.05; P < 0.01). Reduced glutathione was also significantly elevated in the liver of treated animals (P < 0.05). The present study also investigated peri-initiation application of acetone extract of Zanthoxylum on initiated mouse skin. Results showed a significant reduction in tumor incidence from 68% to 36% (P < 0.05); as well as, a reduction in tumor burden per effective mouse from 3.87 to 0.72 (P < 0.01). Cumulatively, the findings strongly suggest cancer chemopreventive potential of Zanthoxylum sps. PMID:22126017

  2. RNA-Sequencing Quantification of Hepatic Ontogeny and Tissue Distribution of mRNAs of Phase II Enzymes in Mice

    PubMed Central

    Gunewardena, Sumedha; Cui, Julia Y.; Yoo, Byunggil; Zhong, Xiao-bo; Klaassen, Curtis D.

    2013-01-01

    Phase II conjugating enzymes play key roles in the metabolism of xenobiotics. In the present study, RNA sequencing was used to elucidate hepatic ontogeny and tissue distribution of mRNA expression of all major known Phase II enzymes, including enzymes involved in glucuronidation, sulfation, glutathione conjugation, acetylation, methylation, and amino acid conjugation, as well as enzymes for the synthesis of Phase II cosubstrates, in male C57BL/6J mice. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. Many of these Phase II enzymes were expressed at much higher levels in adult livers than in perinatal livers, such as Ugt1a6b, -2a3, -2b1, -2b5, -2b36, -3a1, and -3a2; Gsta1, -m1, -p1, -p2, and -z1; mGst1; Nat8; Comt; Nnmt; Baat; Ugdh; and Gclc. In contrast, hepatic mRNA expression of a few Phase II enzymes decreased during postnatal liver development, such as mGst2, mGst3, Gclm, and Mat2a. Hepatic expression of certain Phase II enzymes peaked during the adolescent stage, such as Ugt1a1, Sult1a1, Sult1c2, Sult1d1, Sult2as, Sult5a1, Tpmt, Glyat, Ugp2, and Mat1a. In adult mice, the total transcripts for Phase II enzymes were comparable in liver, kidney, and small intestine; however, individual Phase II enzymes displayed marked tissue specificity among the three organs. In conclusion, this study unveils for the first time developmental changes in mRNA abundance of all major known Phase II enzymes in mouse liver, as well as their tissue-specific expression in key drug-metabolizing organs. The age- and tissue-specific expression of Phase II enzymes indicate that the detoxification of xenobiotics is highly regulated by age and cell type. PMID:23382457

  3. Liver Enzymes in Children with beta-Thalassemia Major: Correlation with Iron Overload and Viral Hepatitis

    PubMed Central

    Salama, Khaled M.; Ibrahim, Ola M.; Kaddah, Ahmed M.; Boseila, Samia; Ismail, Leila Abu; Hamid, May M. Abdel

    2015-01-01

    BACKGROUND: Beta Thalassemia is the most common chronic hemolytic anemia in Egypt (85.1%) with an estimated carrier rate of 9-10.2%. Injury to the liver, whether acute or chronic, eventually results in an increase in serum concentrations of Alanine transaminase (ALT) and Aspartate transaminase (AST). AIM: Evaluating the potentiating effect of iron overload & viral hepatitis infection on the liver enzymes. PATIENTS AND METHODS: Eighty (80) thalassemia major patients were studied with respect to liver enzymes, ferritin, transferrin saturation, HBsAg, anti-HCV antibody and HCV-PCR for anti-HCV positive patients. RESULTS: Fifty % of the patients were anti-HCV positive and 55% of them were HCV-PCR positive. Patients with elevated ALT and AST levels had significantly higher mean serum ferritin than those with normal levels. Anti-HCV positive patients had higher mean serum ferritin, serum ALT, AST and GGT levels and higher age and duration of blood transfusion than the negative group. HCV-PCR positive patients had higher mean serum ferritin and serum ALT and also higher age and duration of blood transfusion than the negative group. CONCLUSION: Iron overload is a main leading cause of elevated liver enzymes, and presence of HCV infection is significantly related to the increased iron overload. PMID:27275237

  4. Modulation of insulin degrading enzyme activity and liver cell proliferation

    PubMed Central

    Pivovarova, Olga; von Loeffelholz, Christian; Ilkavets, Iryna; Sticht, Carsten; Zhuk, Sergei; Murahovschi, Veronica; Lukowski, Sonja; Döcke, Stephanie; Kriebel, Jennifer; de las Heras Gala, Tonia; Malashicheva, Anna; Kostareva, Anna; Lock, Johan F; Stockmann, Martin; Grallert, Harald; Gretz, Norbert; Dooley, Steven; Pfeiffer, Andreas FH; Rudovich, Natalia

    2015-01-01

    Diabetes mellitus type 2 (T2DM), insulin therapy, and hyperinsulinemia are independent risk factors of liver cancer. Recently, the use of a novel inhibitor of insulin degrading enzyme (IDE) was proposed as a new therapeutic strategy in T2DM. However, IDE inhibition might stimulate liver cell proliferation via increased intracellular insulin concentration. The aim of this study was to characterize effects of inhibition of IDE activity in HepG2 hepatoma cells and to analyze liver specific expression of IDE in subjects with T2DM. HepG2 cells were treated with 10 nM insulin for 24 h with or without inhibition of IDE activity using IDE RNAi, and cell transcriptome and proliferation rate were analyzed. Human liver samples (n = 22) were used for the gene expression profiling by microarrays. In HepG2 cells, IDE knockdown changed expression of genes involved in cell cycle and apoptosis pathways. Proliferation rate was lower in IDE knockdown cells than in controls. Microarray analysis revealed the decrease of hepatic IDE expression in subjects with T2DM accompanied by the downregulation of the p53-dependent genes FAS and CCNG2, but not by the upregulation of proliferation markers MKI67, MCM2 and PCNA. Similar results were found in the liver microarray dataset from GEO Profiles database. In conclusion, IDE expression is decreased in liver of subjects with T2DM which is accompanied by the dysregulation of p53 pathway. Prolonged use of IDE inhibitors for T2DM treatment should be carefully tested in animal studies regarding its potential effect on hepatic tumorigenesis. PMID:25945652

  5. Contaminants in eggs of colonial waterbirds and hepatic cytochrome P450 enzyme levels in pipped tern embryos, Washington State

    USGS Publications Warehouse

    Blus, L.J.; Melancon, M.J.; Hoffman, D.J.; Henny, C.J.

    1998-01-01

    Eggs of Forster's terns (Sterna forsteri) collected in 1991 from nesting colonies on Crescent Island (Columbia River) and the Potholes Reservoir in south central Washington generally contained low residues of organochlorine pesticides and metabolites, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and polychlorinated biphenyls (PCBs). Hepatic cytochrome P450 enzyme activity in pipped embryos of Forster's terns from the two colonies seemed unaffected by contaminants. At Crescent Island, examination of 23 Forster's tern eggs with large embryos (19 viable [10 pipped] and four dead [two pipped]) revealed developmental abnormalities in two viable pipped embryos (missing maxilla and deformed pelvic girdle) and a viable prepipping embryos (shortened beak). Our limited sample sizes and number of compounds analyzed preclude us from determining whether or not the abnormalities are related to contaminants. No abnormalities were noted in 10 pipped eggs (nine viable and one dead at collection) of Forster's terns collected from the Potholes Reservoir colony. Eggs of Caspian terns (Sterna caspia) collected from Crescent Island in 1991 also contained generally low residues of contaminants, only one developmental abnormality was noted, and limited data indicated that cytochrome P450 enzyme activity apparently was unaffected by contaminants. Organochlorine contaminants were generally low in addled eggs of American white pelicans (Pelecanus erythrorhynchos) collected from Crescent Island in 1994

  6. Contaminants in eggs of colonial waterbirds and hepatic cytochrome P450 enzyme levels in pipped tern embryos, Washington State.

    PubMed

    Blus, L J; Melancon, M J; Hoffman, D J; Henny, C J

    1998-10-01

    Eggs of Forster's terns (Sterna forsteri) collected in 1991 from nesting colonies on Crescent Island (Columbia River) and the Potholes Reservoir in south central Washington generally contained low residues of organochlorine pesticides and metabolites, 2,3,7, 8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and polychlorinated biphenyls (PCBs). Hepatic cytochrome P450 enzyme activity in pipped embryos of Forster's terns from the two colonies seemed unaffected by contaminants. At Crescent Island, examination of 23 Forster's tern eggs with large embryos (19 viable [10 pipped] and four dead [two pipped]) revealed developmental abnormalities in two viable pipped embryos (missing maxilla and deformed pelvic girdle) and a viable prepipping embryo (shortened beak). Our limited sample sizes and number of compounds analyzed preclude us from determining whether or not the abnormalities are related to contaminants. No abnormalities were noted in 10 pipped eggs (nine viable and one dead at collection) of Forster's terns collected from the Potholes Reservoir colony. Eggs of Caspian terns (Sterna caspia) collected from Crescent Island in 1991 also contained generally low residues of contaminants, only one developmental abnormality was noted, and limited data indicated that cytochrome P450 enzyme activity apparently was unaffected by contaminants. Organochlorine contaminants were generally low in addled eggs of American white pelicans (Pelecanus erythrorhynchos) collected from Crescent Island in 1994. PMID:9732482

  7. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Guoxin Lu

    2007-12-01

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

  8. Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate.

    PubMed

    Sayyed, Katia; Vee, Marc Le; Abdel-Razzak, Ziad; Jouan, Elodie; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-07-01

    Smoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRG cells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B1, OATP2B1, OAT2, NTCP, OCT1 and BSEP, and enhanced that of MRP4. Such changes in transporter gene expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reference activator of the aryl hydrocarbon receptor (AhR) pathway, and were counteracted, for some of them, by siRNA-mediated AhR silencing. This suggests that CSC alters hepatic drug transporter levels via activation of the AhR cascade. Importantly, drug transporter expression regulations as well as some transporter activity inhibitions occurred for a range of CSC concentrations similar to those required for inducing drug metabolizing enzymes and may therefore be hypothesized to be relevant for smokers. Taken together, these data established human hepatic transporters as targets of cigarette smoke, which could contribute to known alteration of pharmacokinetics and some liver adverse effects caused by smoking. PMID:27450509

  9. Angiotensin Converting Enzyme Activity in Alopecia Areata

    PubMed Central

    Namazi, Mohammad Reza; Handjani, Farhad; Eftekhar, Ebrahim; Kalafi, Amir

    2014-01-01

    Background. Alopecia areata (AA) is a chronic inflammatory disease of the hair follicle. The exact pathogenesis of AA remains unknown, although recent studies support a T-cell mediated autoimmune process. On the other hand, some studies have proposed that the renin-angiotensin-aldosterone system (RAAS) may play a role in autoimmunity. Therefore, we assessed serum activity of angiotensin converting enzyme (ACE), a component of this system, in AA. Methods. ACE activity was measured in the sera of 19 patients with AA and 16 healthy control subjects. In addition, the relationship between severity and duration of the disease and ACE activity was evaluated. Results. Serum ACE activity was higher in the patient group (55.81 U/L) compared to the control group (46.41 U/L), but the difference was not statistically significant (P = 0.085). Also, there was no correlation between ACE activity and severity (P = 0.13) and duration of disease (P = 0.25) in the patient group. Conclusion. The increased serum ACE activity found in this study may demonstrate local involvement of the RAAS in the pathogenesis of AA. Assessment of ACE in a study with a larger sample size as well as in tissue samples is recommended in order to further evaluate the possible role of RAAS in AA. PMID:25349723

  10. 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

  11. Regulatory effects of testosterone and 17 beta-oestradiol on the metabolism of dimethylnitrosamine by renal and hepatic microsomal enzymes from BALB/c mice.

    PubMed

    Ampy, F R; Asseffa, A

    1988-01-01

    Previous investigations with BALB/c mice have demonstrated that no sex-related differences exist in the ability of liver microsomal fractions (S-9) to biotransform dimethylnitrosamine (DMN) to its active mutagenic metabolites as evidenced by bacterial screening assays. In contrast, kidney microsomal enzymes from adult male BALB/c mice and not from females, castrates, and immature animals, were capable of activating DMN. The present study was designed to test the effects of testosterone and oestradiol on DMN bioactivation by hepatic or renal microsomal enzymes. Mutagenic assays were performed using liver and kidney microsomal enzymes with the histidine deficient mutant Salmonella typhimurium TA100. Results indicate that testosterone treatment of female BALB/c mice resulted in an increase in the ability of their renal microsomal enzymes to metabolize DMN to its active mutagenic intermediates. Renal microsomal enzymes from female mice treated with 17 beta-oestradiol had no effect on DMN metabolism. However, the ability of the renal microsomal enzymes treated with 17 beta-oestradiol to bioactivate DMN was significantly decreased in males. PMID:3229148

  12. Modulation of CYP1A1 and CYP1A2 hepatic enzymes after oral administration of Chios mastic gum to male Wistar rats.

    PubMed

    Katsanou, Efrosini S; Kyriakopoulou, Katerina; Emmanouil, Christina; Fokialakis, Nikolas; Skaltsounis, Alexios-Leandros; Machera, Kyriaki

    2014-01-01

    Chios mastic gum (CMG), a resin derived from Pistacia lentiscus var. chia, is known since ancient times for its pharmacological activities. CYP1A1 and CYP1A2 enzymes are among the most involved in the biotransformation of chemicals and the metabolic activation of pro-carcinogens. Previous studies referring to the modulation of these enzymes by CMG have revealed findings of unclear biological and toxicological significance. For this purpose, the modulation of CYP1A1 and CYP1A2 enzymes in the liver of male Wistar rats following oral administration of CMG extract (CMGE), at the levels of mRNA and CYP1A1 enzyme activity, was compared to respective enzyme modulation following oral administration of a well-known bioactive natural product, caffeine, as control compound known to involve hepatic enzymes in its metabolism. mRNA levels of Cyp1a1 and Cyp1a2 were measured by reverse transcription real-time polymerase chain reaction and their relative quantification was calculated. CYP1A1 enzyme induction was measured through the activity of ethoxyresorufin-O-deethylase (EROD). The results indicated that administration of CMGE at the recommended pharmaceutical dose does not induce significant transcriptional modulation of Cyp1a1/2 and subsequent enzyme activity induction of CYP1A1 while effects of the same order of magnitude were observed in the same test system following the administration of caffeine at the mean daily consumed levels. The outcome of this study further confirms the lack of any toxicological or biological significance of the specific findings on liver following the administration of CMGE. PMID:24950217

  13. Antioxidant enzymes activities in obese Tunisian children

    PubMed Central

    2013-01-01

    Background The oxidant stress, expected to increase in obese adults, has an important role in the pathogenesis of many diseases. It results when free radical formation is greatly increased or protective antioxidant mechanisms are compromised. The main objective of this study is to evaluate the antioxidant response to obesity-related stress in healthy children. Methods A hundred and six healthy children (54 obese and 52 controls), aged 6–12 years old, participated in this study. The collected data included anthropometric measures, blood pressure, fasting glucose, total cholesterol, triglycerides and enzymatic antioxidants (Superoxide dismutase: SOD, Catalase: CAT and Glutathione peroxidase: GPx). Results The first step antioxidant response, estimated by the SOD activity, was significantly higher in obese children compared with normal-weight controls (p < 0.05). Mean activities of anti-radical GPx and CAT enzymes were not affected by the BMI increase. Although, total cholesterol levels were statistically higher in the obese group, there was no significant association with the SOD activity. Conclusions The obesity-related increase of the oxidant stress can be observed even in the childhood period. In addition to the complications of an increased BMI, obesity itself can be considered as an independent risk factor of free radical production resulting in an increased antioxidant response. PMID:23360568

  14. 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, Aslı; 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

  15. HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPAR{gamma}

    SciTech Connect

    Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin; Park, Min Jung; Kim, Kwang Jin; Cheong, JaeHun . E-mail: molecule85@pusan.ac.kr

    2007-04-20

    Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPAR{gamma} (peroxisome proliferators-activated receptor {gamma}) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPAR{gamma}. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfected with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPAR{gamma} transcriptional activity. However, HCV core protein had no effect on PPAR{gamma} gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection.

  16. Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake.

    PubMed

    Labrie, Marilyne; Lalonde, Simon; Najyb, Ouafa; Thiery, Maxime; Daneault, Caroline; Des Rosiers, Chrisitne; Rassart, Eric; Mounier, Catherine

    2015-01-01

    Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver. PMID:26083030

  17. Hepatic Glycogen Supercompensation Activates AMP-Activated Protein Kinase, Impairs Insulin Signaling, and Reduces Glycogen Deposition in the Liver

    PubMed Central

    Winnick, Jason J.; An, Zhibo; Ramnanan, Christopher J.; Smith, Marta; Irimia, Jose M.; Neal, Doss W.; Moore, Mary Courtney; Roach, Peter J.; Cherrington, Alan D.

    2011-01-01

    OBJECTIVE The objective of this study was to determine how increasing the hepatic glycogen content would affect the liver’s ability to take up and metabolize glucose. RESEARCH DESIGN AND METHODS During the first 4 h of the study, liver glycogen deposition was stimulated by intraportal fructose infusion in the presence of hyperglycemic-normoinsulinemia. This was followed by a 2-h hyperglycemic-normoinsulinemic control period, during which the fructose infusion was stopped, and a 2-h experimental period in which net hepatic glucose uptake (NHGU) and disposition (glycogen, lactate, and CO2) were measured in the absence of fructose but in the presence of a hyperglycemic-hyperinsulinemic challenge including portal vein glucose infusion. RESULTS Fructose infusion increased net hepatic glycogen synthesis (0.7 ± 0.5 vs. 6.4 ± 0.4 mg/kg/min; P < 0.001), causing a large difference in hepatic glycogen content (62 ± 9 vs. 100 ± 3 mg/g; P < 0.001). Hepatic glycogen supercompensation (fructose infusion group) did not alter NHGU, but it reduced the percent of NHGU directed to glycogen (79 ± 4 vs. 55 ± 6; P < 0.01) and increased the percent directed to lactate (12 ± 3 vs. 29 ± 5; P = 0.01) and oxidation (9 ± 3 vs. 16 ± 3; P = NS). This change was associated with increased AMP-activated protein kinase phosphorylation, diminished insulin signaling, and a shift in glycogenic enzyme activity toward a state discouraging glycogen accumulation. CONCLUSIONS These data indicate that increases in hepatic glycogen can generate a state of hepatic insulin resistance, which is characterized by impaired glycogen synthesis despite preserved NHGU. PMID:21270252

  18. Enzyme and root activities in surface-flow constructed wetlands.

    PubMed

    Kong, Ling; Wang, Yu-Bin; Zhao, Li-Na; Chen, Zhang-He

    2009-07-01

    Sixteen small-scale wetlands planted with four plant species were constructed for domestic wastewater purification. The objective of this study was to determine the correlations between contaminant removal and soil enzyme activity, root activity, and growth in the constructed wetlands. The results indicated that correlations between contaminant removal efficiency and enzyme activity varied depending on the contaminants. The removal efficiency of NH4+ was significantly correlated with both urease and protease activity in all wetlands, and the removal of total phosphorus and soluble reactive phosphorus was significantly correlated with phosphatase activity in most wetlands, while the removal of total nitrogen, NO3(-) , and chemical oxygen demand (COD) was significantly correlated with enzyme activity only in a few instances. Correlations between soil enzyme activity and root activity varied among species. Activities of all enzymes were significantly correlated with root activity in Vetiveria zizanioides and Phragmites australis wetlands, but not in Hymenocallis littoralis wetlands. Significant correlations between enzyme activity and root biomass and between enzyme activity and root growth were found mainly in Cyperus flabelliformis wetlands. Root activity was significantly correlated with removal efficiencies of all contaminants except NO3(-) and COD in V. zizanioides wetlands. Enzyme activities and root activity showed single-peak seasonal patterns. Activities of phosphatase, urease, and cellulase were significantly higher in the top layer of the substrate than in the deeper layers, and there were generally no significant differences between the deeper layers (deeper than 15 cm). PMID:19497608

  19. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    SciTech Connect

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  20. Alterations in hepatic mRNA expression of phase II enzymes and xenobiotic transporters after targeted disruption of hepatocyte nuclear factor 4 alpha.

    PubMed

    Lu, Hong; Gonzalez, Frank J; Klaassen, Curtis

    2010-12-01

    Hepatocyte nuclear factor 4 alpha (HNF4a) is a liver-enriched master regulator of liver function. HNF4a is important in regulating hepatic expression of certain cytochrome P450s. The purpose of this study was to use mice lacking HNF4a expression in liver (HNF4a-HNull) to elucidate the role of HNF4a in regulating hepatic expression of phase II enzymes and transporters in mice. Compared with male wild-type mice, HNF4a-HNull male mouse livers had (1) markedly lower messenger RNAs (mRNAs) encoding the uptake transporters sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide (Oatp) 1a1, Oatp2b1, organic anion transporter 2, sodium phosphate cotransporter type 1, sulfate anion transporter 1, sodium-dependent vitamin C transporter 1, the phase II enzymes Uridine 5'-diphospho (UDP)-glucuronosyltransferase (Ugt) 2a3, Ugt2b1, Ugt3a1, Ugt3a2, sulfotransferase (Sult) 1a1, Sult1b1, Sult5a1, the efflux transporters multidrug resistance-associated protein (Mrp) 6, and multidrug and toxin extrusion 1; (2) moderately lower mRNAs encoding Oatp1b2, organic cation transporter (Oct) 1, Ugt1a5, Ugt1a9, glutathione S-transferase (Gst) m4, Gstm6, and breast cancer resistance protein; but (3) higher mRNAs encoding Oatp1a4, Octn2, Ugt1a1, Sult1e1, Sult2a2, Gsta4, Gstm1-m3, multidrug resistance protein (Mdr) 1a, Mrp3, and Mrp4. Hepatic signaling of nuclear factor E2-related factor 2 and pregnane X receptor appear to be activated in HNF4a-HNull mice. In conclusion, HNF4a deficiency markedly alters hepatic mRNA expression of a large number of phase II enzymes and transporters, probably because of the loss of HNF4a, which is a transactivator and a determinant of gender-specific expression and/or adaptive activation of signaling pathways important in hepatic regulation of these phase II enzymes and transporters. PMID:20935164

  1. Virus-specific mRNA capping enzyme encoded by hepatitis E virus.

    PubMed

    Magden, J; Takeda, N; Li, T; Auvinen, P; Ahola, T; Miyamura, T; Merits, A; Kääriäinen, L

    2001-07-01

    Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m(7)GTP or m(7)GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m(7)GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m(7)GMP, in the presence of AdoMet and GTP, because radioactivity from both [alpha-(32)P]GTP and [(3)H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m(7)GTP, m(7)GDP, et(2)m(7)GMP, and m(2)et(7)GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families. PMID:11413290

  2. Hepatitis

    MedlinePlus

    ... has been associated with drinking contaminated water. Hepatitis Viruses Type Transmission Prognosis A Fecal-oral (stool to ... risk for severe disease. Others A variety of viruses can affect the liver Signs and Symptoms Hepatitis ...

  3. Evaluation of a novel chemiluminescent microplate enzyme immunoassay for hepatitis B surface antigen detection.

    PubMed

    Yang, Lin; Song, Liu-Wei; Fang, Lin-Lin; Wu, Yong; Ge, Sheng-Xiang; Li, Hui; Yuan, Quan; Zhang, Jun; Xia, Ning-Shao

    2016-02-01

    Hepatitis B virus surface antigen (HBsAg) is an important biomarker used in the diagnosis of hepatitis B virus (HBV) infection, but false-negative results are still reported in the detection of HBsAg using commercial assays. In this study, we evaluated the qualitative properties of a novel HBsAg chemiluminescence enzyme immunoassay (CLEIA) assay--WTultra. WHO standard sample dilution series and samples from low-level HBsAg carriers (<1 ng/mL) were used to evaluate the sensitivity of the WTultra assay. Boston Biomedica, Inc. (BBI) hepatitis B seroconversion panels were used to assess the ability of the WTultra assay to detect the window period. In addition, dilution series of 22 serum samples with different genotypes, serotypes and HBsAg mutations were used to assess the WTultra assay, and these were compared with other commercial assays. The lower detection limit of the WTultra assay was 0.012 IU/mL, and it showed a high sensitivity (97.52%, 95% CI, 94.95-99.00) in the detection of 282 low-level HBsAg carriers (<1 ng/mL). In samples with various HBV genotypes, serotypes and HBsAg mutations, the WTultra assay yielded 117 positive results in 132 samples, which was significantly higher than the results with the other four commercial assays (89, 83, 65 and 45, respectively, p<0.01). In the assays of mutant strains, the WTultra assay detected 82 positive results in 90 samples, which was significantly better than the results for the Hepanostika HBsAg Ultra (58 positive) and Architect (55 positive) (p<0.01) assays, which in turn were significantly better than the Murex V.3 (41 positive, p=0.026) and AxSYM V2 (29 positive, p<0.01) assays. However, in the detection of 42 samples of wild-type strains with various genotypes and serotypes, no significant differences were observed among the WTultra (35 positive), Architect (28 positive) and Hepanostika HBsAg Ultra (31 positive) assays. However, the WTultra assay detected significantly more samples than the Murex V.3 (24

  4. Hepatic Enzyme Decline after Pediatric Blunt Trauma: A Tool for Timing Child Abuse?

    ERIC Educational Resources Information Center

    Baxter, Amy L.; Lindberg, Daniel M.; Burke, Bonnie L.; Shults, Justine; Holmes, James F.

    2008-01-01

    Objectives: Previous research in adult patients with blunt hepatic injuries has suggested a pattern of serum hepatic transaminase concentration decline. Evaluating this decline after pediatric blunt hepatic trauma could establish parameters for estimating the time of inflicted injuries. Deviation from a consistent transaminase resolution pattern…

  5. Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control?

    PubMed Central

    Kholodenko, B N; Brown, G C

    1996-01-01

    It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes. Paradoxical control behaviour occurs widely when enzyme activity is altered by changing Km (if an enzyme is already close to saturation by its substrate), but may also occur with changes in Vmax. when the elasticity to the linking metabolite increases with its concentration (as in some cases of sigmoidal and exponential kinetics or for reactions catalysed by isoenzymes). These findings suggest that enzymes with sigmoidal kinetics may have low control in the absence of activation, but may gain control with activation, and thus have beneficial regulatory properties. PMID:8615766

  6. Single-antibody in situ enzyme immunoassay for infectivity titration of hepatitis A virus.

    PubMed

    Borovec, S; Uren, E

    1997-10-01

    Hepatitis A virus (HAV) establishes a persistent infection in cultured cells, with minimal effect on host cell metabolism. As a result, the virus produces very little, if any, cytopathic effect (CPE), even with cell culture-adapted strains. This feature precludes the use of a plaque or standard endpoint assay (using CPE as an indicator of infection) for the titration of infectious virus. The radioimmunofocus assay (RIFA) is the standard method for HAV titration, though this method is labour intensive and requires the use of radioisotopes. To this end, a single-antibody in situ enzyme immunoassay (EIA) has been developed, using binding of a perioxidase-labelled monoclonal antibody to fixed cell monolayers as an indicator of infection. This novel assay is highly reproducible, can be read by eye, and is suitable for high throughput situations. Furthermore, the assay has been validated against the RIFA making it suitable for use in studies validating the safety of therapeutic biologicals for human use. PMID:9395142

  7. Spatial distribution of enzyme activities in the rhizosphere

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  8. Self-Assembly of Amyloid Fibrils That Display Active Enzymes

    PubMed Central

    Zhou, Xiao-Ming; Entwistle, Aiman; Zhang, Hong; Jackson, Antony P; Mason, Thomas O; Shimanovich, Ulyana; Knowles, Tuomas P J; Smith, Andrew T; Sawyer, Elizabeth B; Perrett, Sarah

    2014-01-01

    Enzyme immobilization is an important strategy to enhance the stability and recoverability of enzymes and to facilitate the separation of enzymes from reaction products. However, enzyme purification followed by separate chemical steps to allow immobilization on a solid support reduces the efficiency and yield of the active enzyme. Here we describe polypeptide constructs that self-assemble spontaneously into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We measured the steady-state kinetic parameters for the appended enzymes in situ within fibrils and compare these with the identical protein constructs in solution. Finally, we demonstrated that the fibrils can be recycled and reused in functional assays both in conventional batch processes and in a continuous-flow microreactor. PMID:25937845

  9. Liver Enzymes Are Associated With Hepatic Insulin Resistance, Insulin Secretion, and Glucagon Concentration in Healthy Men and Women

    PubMed Central

    Bonnet, Fabrice; Ducluzeau, Pierre-Henri; Gastaldelli, Amalia; Laville, Martine; Anderwald, Christian H.; Konrad, Thomas; Mari, Andrea; Balkau, Beverley

    2011-01-01

    OBJECTIVE The pathophysiological mechanisms to explain the association between risk of type 2 diabetes and elevated concentrations of γ-glutamyltransferase (GGT) and alanineaminotransferase (ALT) remain poorly characterized. We explored the association of liver enzymes with peripheral and hepatic insulin resistance, insulin secretion, insulin clearance, and glucagon concentration. RESEARCH DESIGN AND METHODS We studied 1,309 nondiabetic individuals from the Relationship between Insulin Sensitivity and Cardiovascular disease (RISC) study; all had a euglycemic-hyperinsulinemic clamp and an oral glucose tolerance test (OGTT) with assessment of insulin secretion and hepatic insulin extraction. The hepatic insulin resistance index was calculated in 393 individuals. RESULTS In both men and women, plasma concentrations of GGT and ALT were inversely related with insulin sensitivity (M/I) (all P < 0.01). Likewise, the hepatic insulin resistance index was positively correlated with both GGT (r = 0.37, P < 0.0001, men; r = 0.36, P < 0.0001, women) and ALT (r = 0.25, P = 0.0005, men; r = 0.18, P = 0.01, women). These associations persisted in multivariable models. Increased GGT and ALT were significantly associated with higher insulin secretion rates and with both reduced endogenous clearance of insulin and hepatic insulin extraction during the OGTT (P = 0.0005 in men; P = 0.003 in women). Plasma fasting glucagon levels increased over ALT quartiles (men, quartile 4 vs. quartile 1 11.2 ± 5.1 vs. 9.3 ± 3.8 pmol/L, respectively, P = 0.0002; women, 9.0 ± 4.3 vs. 7.6 ± 3.1, P = 0.001). CONCLUSIONS In healthy individuals, increased GGT and ALT were biomarkers of both systemic and hepatic insulin resistance with concomitant increased insulin secretion and decreased hepatic insulin clearance. The novel finding of a positive correlation between ALT and fasting glucagon level concentrations warrants confirmation in type 2 diabetes. PMID:21521874

  10. Intracellular localization of mevalonate-activating enzymes in plant cells

    PubMed Central

    Rogers, L. J.; Shah, S. P. J.; Goodwin, T. W.

    1966-01-01

    Mevalonate-activating enzymes are shown to be present in the chloroplasts of French-bean leaves. The chloroplast membrane is impermeable to mevalonic acid. Mevalonate-activating enzymes also appear to be found outside the chloroplast. These results support the view that terpenoid biosynthesis in the plant cell is controlled by a combination of enzyme segregation and specific membrane permeability. ImagesFig. 1.Fig. 2. PMID:5947149

  11. Activities of biotransformation enzymes in pheasant (Phasianus colchicus) and their modulation by in vivo administration of mebendazole and flubendazole.

    PubMed

    Savlík, M; Polácková, L; Szotáková, B; Lamka, J; Velík, J; Skálová, L

    2007-08-01

    Basal activities of certain pheasant hepatic and intestinal biotransformation enzymes and modulation of their activities by anthelmintics flubendazole (FLBZ) and mebendazole (MBZ) were investigated in subcellular fractions that were prepared from liver and small intestine of control and FLBZ or MBZ treated birds. Several oxidation, reduction and conjugation enzyme activities were assessed. In the liver, treatment of pheasants by FLBZ or MBZ caused very slight or no changes in monooxygenase activities and conjugation enzymes. More significative changes were detected in small intestine. Metyrapone and daunorubicin reductase activities were increased by both substances in the liver. This is the first evidence that certain benzimidazoles modulate reductases of carbonyl group. With respect to the relatively slight extent of the changes caused by FLBZ or MBZ we can assume that repeated administration of therapeutic doses of both FLBZ and MBZ has probably no serious influence on pheasant biotransformation enzyme system. PMID:17316720

  12. Experiment K304: Studies of specific hepatic enzymes and liver constituents involved in the conversion of carbohydrates to lipids in rats exposed to prolonged space flight

    NASA Technical Reports Server (NTRS)

    Abraham, S.; Klein, H. P.; Lin, C. Y.; Volkmann, C.; Tigranyan, R. A.; Vetrova, E. G.

    1981-01-01

    The effects of space flight on the activities of 26 enzymes concerned with carbohydrate and lipid metabolism in hepatic tissue taken from male Wistar rats are investigated. These activities were measured in the various hepatic cell compartments, i.e., cytosol, mitochondria and microsomes. In addition, the levels of glycogen, total lipids, phospholipids, triglycerides, cholesterol, cholesterol esters, and the fatty acid composition of the rat livers were also examined and quantified. A similar group of ground-based rats treated in an identical manner served as controls. Both flight and synchronous control rats were sacrificed at three time intervals: R+0, 7-11 hours after recovery; R+6, after 6 days; R+6(S), after 6 days (having undergone 2-5 hour periods of fixed stress in a "backupward" position on days 0, 3, 4, 5 and 6) and R+29, after 29 days post-flight. Although most of the enzyme activities and the amounts of liver constituents studied were unaffected by the period of weightlessness, some significant differences were observed.

  13. Hepatic steroid inactivating enzymes, hepatic portal blood flow, and corpus luteum blood perfusion in lactating dairy cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In ruminants, a decrease in pregnancy rates may be due to decreased concentrations of progesterone (P4). It is important to note that both production from the corpus luteum and/or hepatic steroid inactivation impacts peripheral concentrations of P4. Cattle with an elevated dry matter intake have inc...

  14. Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays.

    PubMed

    Funeriu, Daniel P; Eppinger, Jörg; Denizot, Lucile; Miyake, Masato; Miyake, Jun

    2005-05-01

    The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, K(i)(app) values and inhibitor profiles were confirmed and refined. PMID:15821728

  15. Hepatitis B virus e antigen induces activation of rat hepatic stellate cells

    SciTech Connect

    Zan, Yanlu; Zhang, Yuxia; Tien, Po

    2013-06-07

    Highlights: •HBeAg expression in HSCs induced production of ECM protein and liver fibrotic markers. •The activation and proliferation of HSCs were mediated by TGF-β. •HBeAg protein purified from cell medium directly activated HSCs. -- Abstract: Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs.

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

    ERIC Educational Resources Information Center

    Yip, Din-Yan

    1997-01-01

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

  17. Eucommia ulmoides Oliver Extract, Aucubin, and Geniposide Enhance Lysosomal Activity to Regulate ER Stress and Hepatic Lipid Accumulation

    PubMed Central

    Lee, Hwa-Young; Lee, Geum-Hwa; Lee, Mi-Rin; Kim, Hye-Kyung; Kim, Nan-young; Kim, Seung-Hyun; Lee, Yong-Chul; Kim, Hyung-Ryong; Chae, Han-Jung

    2013-01-01

    Eucommia ulmoides Oliver is a natural product widely used as a dietary supplement and medicinal plant. Here, we examined the potential regulatory effects of Eucommia ulmoides Oliver extracts (EUE) on hepatic dyslipidemia and its related mechanisms by in vitro and in vivo studies. EUE and its two active constituents, aucubin and geniposide, inhibited palmitate-induced endoplasmic reticulum (ER) stress, reducing hepatic lipid accumulation through secretion of apolipoprotein B and associated triglycerides and cholesterol in human HepG2 hepatocytes. To determine how EUE diminishes the ER stress response, lysosomal and proteasomal protein degradation activities were analyzed. Although proteasomal activity was not affected, lysosomal enzyme activities including V-ATPase were significantly increased by EUE as well as aucubin and geniposide in HepG2 cells. Treatment with the V-ATPase inhibitor, bafilomycin, reversed the inhibition of ER stress, secretion of apolipoprotein B, and hepatic lipid accumulation induced by EUE or its component, aucubin or geniposide. In addition, EUE was determined to regulate hepatic dyslipidemia by enhancing lysosomal activity and to regulate ER stress in rats fed a high-fat diet. Together, these results suggest that EUE and its active components enhance lysosomal activity, resulting in decreased ER stress and hepatic dyslipidemia. PMID:24349058

  18. Polar bear hepatic cytochrome P450: Immunochemical quantitation, EROD/PROD activity and organochlorines

    SciTech Connect

    Letcher, R.J.; Norstrom, R.J. |

    1994-12-31

    Polar bears (Ursus maritimus) are an ubiquitous mammal atop the arctic marine food chain and bioaccumulate lipophilic environmental contaminants. Antibodies prepared against purified rat liver cytochrome P450-1 Al, -1 A2, -2Bl and -3Al enzymes have been found to cross-react with structurally-related orthologues present in the hepatic microsomes of wild polar bears, immunochemically determined levels of P450-1 A and -2B proteins in polar bear liver relative to liver of untreated rats suggested enzyme induction, probably as a result of exposure to xenobiotic contaminants. Optical density quantitation of the most immunochemically responsive isozymes P450-I Al, -IA2 and -2Bi to polygonal rabbit anti-rat P450-IA/IA2 sera and -2BI antibodies in hepatic microsomes of 13 adult male polar bars from the Resolute Bay area of the Canadian Arctic is presented. Correlations with EROD and PROD catalytic activities and levels of organochlorines, such as polychlorinated biphenyls (PCBs), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene (p,p-DDE) and their methyl sulfone (MeSO2-) metabolites are made to determine if compound-specific enzyme induction linkages exist. Inter-species immunochemical quantitation of isozymic P450 cytochromes can serve as an indicator of exposure to biologically active contaminant.

  19. Chronic active hepatitis experience of Groote Schuur Hospital, 1964 - 1977.

    PubMed

    Poreh, S; Kirsch, R E; Terblanche, J; Saunders, S J

    1980-06-14

    From 1964 to 1977, 54 patients with clinical, biochemical and histological criteria of chronic active hepatitis were seen at Groote Schuur Hospital, Cape Town. Our experience with this disease is reviewed, and the diagnosis and mangement are commented on. PMID:7404076

  20. Hepatitis

    MedlinePlus

    ... be serious. Some can lead to scarring, called cirrhosis, or to liver cancer. Sometimes hepatitis goes away by itself. If it does not, it can be treated with drugs. Sometimes hepatitis lasts a lifetime. Vaccines can help prevent some viral forms.

  1. Successful Interferon Therapy Reverses Enhanced Hepatic Progenitor Cell Activation in Patients with Chronic Hepatitis C.

    PubMed

    Noritake, Hidenao; Kobayashi, Yoshimasa; Ooba, Yukimasa; Matsunaga, Erika; Ohta, Kazuyoshi; Shimoyama, Shin; Yamazaki, Satoru; Chida, Takeshi; Kawata, Kazuhito; Sakaguchi, Takanori; Suda, Takafumi

    2015-12-01

    The enhanced accumulation of hepatic progenitor cells (HPCs) is related to the risk of progression to hepatocellular carcinoma (HCC). Interferon (IFN) treatment reduces HCC risk in patients with chronic hepatitis C virus (HCV) infection. However, the underlying mechanisms remain unclear. The aim of this study was to examine the effects of IFN treatment on HPC activation in HCV patients. Immunohistochemical detection and computer-assisted quantitative image analyses of cytokeratin 7 (CK7) were performed to evaluate HPC activation in paired pre- and post-treatment liver biopsies from 18 HCV patients with sustained virological response (SVR) to IFN-based therapy and from 23 patients without SVR, as well as normal liver tissues obtained from surgical resection specimens of 10 patients. Pretreatment HCV livers showed increased CK7 immunoreactivity, compared with normal livers (HCV: median, 1.38%; normal: median, 0.69%, P=0.006). IFN treatment reduced hepatic CK7 immunoreactivity (median, 1.57% pre-IFN vs. 0.69% post-IFN, P=0.006) in SVR patients, but not in non-SVR patients. The development of HCC following IFN treatment was encountered in 3 non-SVR patients who showed high post-IFN treatment CK7 immunoreactivity (>4%). Successful IFN therapy can reverse enhanced HPC activation in HCV patients, which may contribute to the reduced risk of HCC development in these patients. PMID:26308703

  2. Activated farnesoid X receptor attenuates apoptosis and liver injury in autoimmune hepatitis.

    PubMed

    Lian, Fan; Wang, Yu; Xiao, Youjun; Wu, Xiwen; Xu, Hanshi; Liang, Liuqin; Yang, Xiuyan

    2015-10-01

    Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease associated with interface hepatitis, the presence of autoantibodies, regulatory T‑cell dysfunction and raised plasma liver enzyme levels. The present study assessed the hepatoprotective and antiapoptotic role of farnesoid X receptor (FXR) in AIH. a mouse model of AIH was induced by treatment with concanavalin A (ConA). The FXR agonist, chenodeoxycholic acid (CDCA), was administered to mice exhibiting ConA‑induced liver injury and a normal control. Blood samples were obtained to detect the levels of aminotransferases and inflammatory cytokines. Liver specimens were collected, and hematoxylin‑eosin staining was used for histopathological examination and detection. Apoptosis was evaluated using the terminal deoxynucleotidyl-transferase‑mediated dUTP nick end labeling (TUNEL) method. The expression levels of apoptosis‑associated genes and proteins were determined by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. The results demonstrated that FXR was downregulated at the mRNA and protein level in the liver specimens of mice induced with ConA‑induced hepatitis. Increased levels of aminotransferases and inflammatory cytokines, including interferon‑γ, tumor necrosis factor‑α, interleukin (IL)‑4 and IL‑2, were detected in ConA‑treated mice. The mice pretreated with the FXR agonist, CDCA, were more resistant to ConA hepatitis, as indicated by reduced levels of alanine transaminase/aspartate aminotransferase and aminotransferases. The activation of FXR ameliorated hepatocyte apoptosis, as demonstrated by TUNEL analysis and downregulation of the Fas/Fas ligand, tumor necrosis factor‑related apoptosis‑inducing ligand and caspase‑3. Taken together, FXR activation ameliorated liver injury and suppressed inflammatory cytokines in ConA‑induced hepatitis. FXR, therefore, exerts a protective role against ConA-induced apoptosis. PMID

  3. 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 ...

  4. Activation and stabilization of enzymes in ionic liquids.

    PubMed

    Moniruzzaman, Muhammad; Kamiya, Noriho; Goto, Masahiro

    2010-06-28

    As environmentally benign "green" solvents, room temperature ionic liquids (ILs) have been used as solvents or (co)solvents in biocatalytic reactions and processes for a decade. The technological utility of enzymes can be enhanced greatly by their use in ionic liquids (ILs) rather than in conventional organic solvents or in their natural aqueous reaction media. In fact, the combination of green properties and unique tailor-made physicochemical properties make ILs excellent non-aqueous solvents for enzymatic catalysis with numerous advantages over other solvents, including high conversion rates, high selectivity, better enzyme stability, as well as better recoverability and recyclability. However, in many cases, particularly in hydrophilic ILs, enzymes show relative instability and/or lower activity compared with conventional solvents. To improve the enzyme activity as well as stability in ILs, various attempts have been made by modifying the form of the enzymes. Examples are enzyme immobilization onto support materials via adsorption or multipoint attachment, lyophilization in the presence of stabilizing agents, chemical modification with stabilizing agents, formation of cross-linked enzyme aggregates, pretreatment with polar organic solvents or enzymes combined with suitable surfactants to form microemulsions. The use of these enzyme preparations in ILs can dramatically increase the solvent tolerance, enhance activity as well as stability, and improve enantioselectivity. This perspective highlights a number of pronounced strategies being used successfully for activation and stabilization of enzymes in non-aqueous ILs media. This review is not intended to be comprehensive, but rather to present a general overview of the potential approaches to activate enzymes for diverse enzymatic processes and biotransformations in ILs. PMID:20445940

  5. Enzyme activities along a latitudinal transect in Western Siberia

    NASA Astrophysics Data System (ADS)

    Schnecker, Jörg; Wild, Birgit; Eloy Alves, Ricardo J.; Gentsch, Norman; Gittel, Antje; Knoltsch, Anna; Lashchinskiy, Nikolay; Mikutta, Robert; Takriti, Mounir; Richter, Andreas

    2014-05-01

    Decomposition of soil organic matter (SOM) and thus carbon and nutrient cycling in soils is mediated by the activity of extracellular enzymes. The specific activities of these enzymes and their ratios to each other represent the link between the composition of soil organic matter and the nutrient demand of the microbial community. Depending on the difference between microbial nutrient demand and substrate availability, extracellular enzymes can enhance or slow down different nutrient cycles in the soil. We investigated activities of six extracellular enzymes (cellobiohydrolase, leucine-amino-peptidase, N-acetylglucosaminidase, chitotriosidase, phosphatase and phenoloxidase) in the topsoil organic horizon, topsoil mineral horizon and subsoil horizon in seven ecosystems along a 1,500 km-long North-South transect in Western Siberia. The transect included sites in the southern tundra, northern taiga, middle taiga, southern taiga, forest-steppe (in forested patches as well as in adjacent meadows) and Steppe. We found that enzyme patterns varied stronger with soil depth than between ecosystems. Differences between horizons were mainly based on the increasing ratio of oxidative enzymes to hydrolytic enzymes. Differences between sites were more pronounced in topsoil than in subsoil mineral horizons, but did not reflect the north-south transect and the related gradients in temperature and precipitation. The observed differences between sites in topsoil horizons might therefore result from differences in vegetation rather than climatic factors. The decreasing variability in the enzyme pattern with depth might also indicate that the composition of soil organic matter becomes more similar with soil depth, most likely by an increasing proportion of microbial remains compared to plant derived constituents of SOM. This also indicates, that SOM becomes less divers the more it is processed by soil microorganisms. Our findings highlight the importance of soil depth on enzyme

  6. How should enzyme activities be used in fish growth studies?

    PubMed

    Pelletier; Blier; Dutil; Guderley

    1995-01-01

    The activity of glycolytic and oxidative enzymes was monitored in the white muscle of Atlantic cod Gadus morhua experiencing different growth rates. A strong positive relationship between the activity of two glycolytic enzymes and individual growth rate was observed regardless of whether the enzyme activity was expressed as units per gram wet mass, units per gram dry mass or with respect to muscle protein and DNA content. The most sensitive response to growth rate was observed when pyruvate kinase and lactate dehydrogenase activities were expressed as units per microgram DNA, and this may be useful as an indicator of growth rate in wild fish. In contrast, no relationship between the activities of oxidative enzymes and growth rate was observed when cytochrome c oxidase and citrate synthase activities were expressed as units per gram protein. Apparently, the aerobic capacity of white muscle in cod is not specifically increased to match growth rate. PMID:9319392

  7. Effect of cadmium, mercury, and zinc on the hepatic microsomal enzymes of Channa punctatus

    SciTech Connect

    Dalal, R.; Bhattacharya, S. )

    1994-06-01

    The increased use of heavy metals like cadmium and mercury in industry and agriculture, and their subsequent intrusion in indeterminate amounts into the environment has caused ecological and biological changes. In vivid contrast, zinc, one of the essential elements, and used in the cosmetic industry, is known to play a pivotal roles in various cellular processes. The seriousness and longevity of these metals in the environment are compounded by the fact that they are non-degradable with significant oxidizing capacity and substantial affinity for electronegative nucleophilic species in proteins and enzymes. Exposure of aquatic animals, especially fish, to these toxic metals for a prolonged period produces an intrinsic toxicity in relation to susceptible organs and/or tissues, although no serious morphological or anatomical changes in the animal or even their feeding behavior may occur. The p-hydroxylation of aniline by aniline hydroxylase (AH) and the N-demethylation of amines to generate formaldehyde (HCHO) by aminopyrine demethylase (APD) are the two oxygen-dependent reactions of microsomal mixed-function oxidase (MFOs) which control the pharmacological and toxicological activities of xenobiotics in mammalian and other species. While both these classical enzymes in fish are reported to demonstrate relatively low specific activity, they are used as criteria for delineating polluted areas. Unlike mammalian species, however, intoxication and interference of MFO enzymes by metal toxicants, especially during prolonged exposure, has not been investigated. The present report describes the results of studies from the concurrent exposure for 28 d to cadmium (CdCl[sub 2]), mercury (HgCl[sub 2]) or zinc (ZnCl[sub 2]) individually, on the AH and APD activities and microsomal protein content in liver of freshwater teleost Channa punctatus.

  8. The hormonal regulation of hepatic microsomal 11beta-hydroxysteroid dehydrogenase activity in the rat.

    PubMed

    Lax, E R; Ghraf, R; Schriefers, H

    1978-10-01

    Hepatic microsomal 11beta-hydroxysteroid dehydrogenase activity is higher in male than in female rat liver. Gonadectomy on day 25 of life only affects the activity in the adult male animal, causing a decrease towards the normal female level. Administration of testosterone to gonadectomized rats of either sex causes the induction of typical male activity levels. On the basis of these experiments, this enzyme activity may be classified as an drogen-dependent. However, 11beta-hydroxysteroid dehydrogenase differs from other known androgen-dependent activities in that administration of oestradiol to gonadectomized animals of either sex causes a further significant repression of the activity to levels close to the limits of detection. Hypophysectomy on day 50 of life does not affect the activity in 75 day-old male rats, but causes the appearance of typically male activity levels in females. These results indicate that the hypophysis exerts a repressive influence on hepatic 11beta-hydroxysteroid dehydrogenase in female rats. The facts that this activity is not influenced by androgen or oestrogen administration once the pituitary has been removed demonstrates the obligatory role of the hypophysis for sex hormone action. PMID:696183

  9. Influence of age and caloric restriction on liver glycolytic enzyme activities and metabolite concentrations in mice.

    PubMed

    Hagopian, Kevork; Ramsey, Jon J; Weindruch, Richard

    2003-03-01

    The influence of caloric restriction (CR) from 2 months of age on the activities of liver glycolytic enzymes and metabolite levels was studied in young and old mice. Livers were sampled 48 h after the last scheduled feeding time. Old mice on CR showed significant decreases in the activities of all the enzymes studied, except for aldolase, triosephosphate isomerase and phosphoglycerate mutase, which were unchanged. The metabolites glucose, glucose-6-phosphate, fructose-6-phosphate, pyruvate and lactate were lower while fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate and phosphoenolpyruvate were increased in old CR. Young mice on CR also showed reduced enzyme activities, except for aldolase, triosephosphate isomerase and enolase which were unchanged when compared with young controls. The metabolites glucose, glucose-6-phosphate, fructose-6-phosphate and pyruvate were decreased when compared with young controls, while phosphoenolpyruvate was increased. Ketone bodies increased (65%) in old, but not young, CR mice while fructose-2,6-bisphosphate decreased in both young (22%) and old CR (28%) mice. The results indicate that decreased hepatic glucose levels in CR mice are associated with decreased enzyme activities but not a uniform decrease in metabolite levels. Increased ketone body levels indicate increased utilization of non-carbohydrate fuels while decreased fructose-2,6-bisphosphate level suggests its importance in the control of glycolysis in CR. PMID:12581789

  10. TREATABILITY STUDY BULLETIN: ENZYME-ACTIVATED CELLULOSE TECHNOLOGY - THORNECO, INC

    EPA Science Inventory

    The Enzyme-Activated Cellulose Technology developed by Thorneco, Inc. uses cellulose placed into one or more cylindrical towers to remove metals and organic compounds from an aqueous solution. The cellulose is coated with a proprietary enzyme. Operating parameters that can affe...

  11. Photoreactivating enzyme activity in the rat tapeworm, Hymenolepis diminuta

    SciTech Connect

    Woodhead, A.D.; Achey, P.M.

    1981-01-01

    There has been considerable speculation about the occurrence of photoreactivating enzyme in different organisms and about its biologic purpose. We have developed a simple, sensitive assay for estimating pyrimidine dimers in DNA which is useful in making a rapid survey for the presence of the enzyme. Using this method, we have found photoreactivating enzyme activity in the tissues of the rat tapeworm, Hymenolepis diminuta. This parasite spends the majority of its life span in the bodies of its definitive or intermediate hosts, but a period is spent externally. We suggest that photoreactivating enzyme may be important in perserving the integrity of embryonic DNA during this free-living stage.

  12. Photoreactivating enzyme activity in the rat tapeworm, Hymenolepis diminuta

    SciTech Connect

    Woodhead, A.D.; Achey, P.M.

    1981-06-01

    There has been considerable speculation about the occurrence of photoreactivating enzyme in different organisms and about its biological purpose. We have developed a simple, sensitive assay for estimating pyrimidine dimers in DNA which is useful in making a rapid survey for the presence of the enzyme. Using this method, we have found photoreactivating enzyme activity in the tissues of the rat tapeworm Hymenolepis diminuta. This parasite spends the majority of its life span in the bodies of its definitive or intermediate hosts, but a period is spent externally. We suggest that photoreactivating enzyme may be important in preserving the integrity of embryonic DNA during this free-living stage.

  13. Activation of immobilized enzymes by acoustic wave resonance oscillation.

    PubMed

    Nishiyama, Hiroshi; Watanabe, Tomoya; Inoue, Yasunobu

    2014-12-01

    Acoustic wave resonance oscillation has been used successfully in the development of methods to activate immobilized enzyme catalysts. In this study, resonance oscillation effects were demonstrated for enzyme reactions on galactose oxidase (GAD), D-amino acid oxidase (DAAO), and L-amino acid oxidase (LAAO), all of which were immobilized covalently on a ferroelectric lead zirconate titanate (PZT) device that could generate thickness-extensional resonance oscillations (TERO) of acoustic waves. For galactose oxidation on immobilized GAD in a microreactor, TERO generation immediately increased enzyme activity 2- to 3-fold. Eliminating TERO caused a slight decrease in the activity, with ∼90% of the enhanced activity retained while the reaction proceeded. Contact of the enhanced enzyme with a galactose-free solution caused almost complete reversion of the activity to the original low level before TERO generation, indicating that, not only TERO-induced GAD activation, but also preservation of the increased activity, required a galactose substrate. Similar activity changes with TERO were observed for enzyme reactions on DAAO and LAAO. Kinetic analysis demonstrated that TERO helped strengthen the interactions of the immobilized enzyme with the reactant substrate and promoted formation of an activation complex. PMID:25442945

  14. The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme.

    PubMed Central

    Stempniak, M; Hostomska, Z; Nodes, B R; Hostomsky, Z

    1997-01-01

    NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn2+ (and, to a lesser degree, by Cd2+, Pb2+, and Co2+) and inhibited by Cu2+ and Hg2+ ions. Elemental analysis of the purified NS3 proteinase domain revealed the presence of zinc in an equimolar ratio. The zinc content was unchanged in a mutated NS3 proteinase in which active-site residues His-57 and Ser-139 were replaced with Ala, suggesting that the zinc atom is not directly involved in catalysis but rather may have a structural role. Based on data from site-directed mutagenesis combined with zinc content determination, we propose that Cys-97, Cys-99, Cys-145, and His-149 coordinate the structural zinc in the HCV NS3 proteinase. A similar metal binding motif is found in 2A proteinases of enteroviruses and rhinoviruses, suggesting that these 2A proteinases and HCV NS3 proteinase are structurally related. PMID:9060645

  15. Superoxide dismutase activity as a measure of hepatic oxidative stress in cattle following ethionine administration.

    PubMed

    Abd Ellah, Mahmoud R; Okada, Keiji; Goryo, Masanobu; Oishi, Akihiro; Yasuda, Jun

    2009-11-01

    The goal of this study was to assess if oxidative stress, as measured by alterations in the concentrations of antioxidant enzymes in the liver and erythrocytes of cattle, could be induced following dl-ethionine administration. Whole blood, serum and liver biopsy samples were collected 0, 4, 7 and 10 days after intra-peritoneal ethionine administration to five cows. The activities of the antioxidant enzymes copper zinc superoxide dismutase (Cu, Zn SOD) and catalase were assessed in the liver biopsies which were also examined histopathologically. Significant increases in hepatic Cu, Zn SOD concentrations (P<0.01) were noted on days 7 and 10 post-treatment. Hepatic catalase activity decreased significantly (P<0.01) on days 4, 7 and 10 post-treatment and erythrocyte Cu, Zn SOD activity was significantly increased on day 10. Serum biochemical analysis revealed a significant increase (P<0.01) in non-esterified fatty acid concentrations on day 4 and significant decreases in total cholesterol and phospholipid levels on days 4 (P<0.05), 7 (P<0.01) and 10 (P<0.01). In this model system, dl-ethionine administration was effective in inducing oxidative stress particularly reflected in the liver. PMID:18585936

  16. Function and biotechnology of extremophilic enzymes in low water activity

    PubMed Central

    2012-01-01

    Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology. PMID:22480329

  17. Lipid metabolizing enzyme activities modulated by phospholipid substrate lateral distribution.

    PubMed

    Salinas, Dino G; Reyes, Juan G; De la Fuente, Milton

    2011-09-01

    Biological membranes contain many domains enriched in phospholipid lipids and there is not yet clear explanation about how these domains can control the activity of phospholipid metabolizing enzymes. Here we used the surface dilution kinetic theory to derive general equations describing how complex substrate distributions affect the activity of enzymes following either the phospholipid binding kinetic model (which assumes that the enzyme molecules directly bind the phospholipid substrate molecules), or the surface-binding kinetic model (which assumes that the enzyme molecules bind to the membrane before binding the phospholipid substrate). Our results strongly suggest that, if the enzyme follows the phospholipid binding kinetic model, any substrate redistribution would increase the enzyme activity over than observed for a homogeneous distribution of substrate. Besides, enzymes following the surface-binding model would be independent of the substrate distribution. Given that the distribution of substrate in a population of micelles (each of them a lipid domain) should follow a Poisson law, we demonstrate that the general equations give an excellent fit to experimental data of lipases acting on micelles, providing reasonable values for kinetic parameters--without invoking special effects such as cooperative phenomena. Our theory will allow a better understanding of the cellular-metabolism control in membranes, as well as a more simple analysis of the mechanisms of membrane acting enzymes. PMID:21108012

  18. Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development

    PubMed Central

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.

    2016-01-01

    Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography–mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage–dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics. PMID:27084891

  19. Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development.

    PubMed

    Sadler, Natalie C; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo; Ansong, Charles; Anderson, Lindsey N; Smith, Jordan N; Corley, Richard A; Wright, Aaron T

    2016-07-01

    Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography-mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics. PMID:27084891

  20. Hepatic ERK activity plays a role in energy metabolism.

    PubMed

    Jiao, Ping; Feng, Bin; Li, Yujie; He, Qin; Xu, Haiyan

    2013-08-15

    Mitogen activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK) and P38, have been reported to play important roles in energy homeostasis. In this study, we show that the activity of extracellular signal-regulated kinase (ERK) is increased in the livers of diet induced and genetically obese mice. Activation of ERK in the livers of lean mice by over-expressing the constitutively active MAPK kinase 1 (MEK CA) results in decreased energy expenditure, lowered expression of genes involved in fatty acid oxidation, increases fasting hyperglycemia and causes systemic insulin resistance. Interestingly, hepatic glycogen content is markedly increased and expression of G6Pase gene is decreased in mice over-expressing MEK CA compared to control mice expressing green fluorescent protein (GFP), therefore hepatic glucose output is not likely the major contributor of hyperglycemia. One potential mechanism of decreased expression of G6Pase gene by MEK CA is likely due to ERK mediated phosphorylation and cytosolic retention of FOXO1. Adipocytes isolated from MEK CA mice display increased lipolysis. Circulating levels of free fatty acids (FFAs) in these mice are also increased, which possibly contribute to systemic insulin resistance and subsequent hyperglycemia. Consistent with these results, knocking down ERK expression in the liver of diet induced obese (DIO) mice improves systemic insulin and glucose tolerance. These results indicate that increased hepatic ERK activity in DIO mice may contribute to increased liver glycogen content and decreased energy expenditure in obesity. PMID:23732116

  1. Profiling Kinase Activity during Hepatitis C Virus Replication Using a Wortmannin Probe.

    PubMed

    Desrochers, Geneviève F; Sherratt, Allison R; Blais, David R; Nasheri, Neda; Ning, Zhibin; Figeys, Daniel; Goto, Natalie K; Pezacki, John Paul

    2015-09-11

    To complete its life cycle, the hepatitis C virus (HCV) induces changes to numerous aspects of its host cell. As kinases act as regulators of many pathways utilized by HCV, they are likely enzyme targets for virally induced inhibition or activation. Herein, we used activity-based protein profiling (ABPP), which allows for the identification of active enzymes in complex protein samples and the quantification of their activity, to identify kinases that displayed differential activity in HCV-expressing cells. We utilized an ABPP probe, wortmannin-yne, based on the kinase inhibitor wortmannin, which contains a pendant alkyne group for bioconjugation using bioorthogonal chemistry. We observed changes in the activity of kinases involved in the mitogen-activated protein kinase pathway, apoptosis pathways, and cell cycle control. These results establish changes to the active kinome, as reported by wortmannin-yne, in the proteome of human hepatoma cells actively replicating HCV. The observed changes include kinase activity that affect viral entry, replication, assembly, and secretion, implying that HCV is regulating the pathways that it uses for its life cycle through modulation of the active kinome. PMID:27617927

  2. Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates

    NASA Astrophysics Data System (ADS)

    Fuhrmann, Gregor; Grotzky, Andrea; Lukić, Ružica; Matoori, Simon; Luciani, Paola; Yu, Hao; Zhang, Baozhong; Walde, Peter; Schlüter, A. Dieter; Gauthier, Marc A.; Leroux, Jean-Christophe

    2013-07-01

    Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

  3. Hepatitis C virus-specific T-cell response correlates with hepatitis activity and donor IL28B genotype early after liver transplantation.

    PubMed

    Tsuzaki, Ryuichiro; Takaki, Akinobu; Yagi, Takahito; Ikeda, Fusao; Koike, Kazuko; Iwasaki, Yoshiaki; Shiraha, Hidenori; Miyake, Yasuhiro; Sadamori, Hiroshi; Shinoura, Susumu; Umeda, Yuzo; Yoshida, Ryuichi; Nobuoka, Daisuke; Utsumi, Masashi; Nakayama, Eiichi; Fujiwara, Toshiyoshi; Yamamoto, Kazuhide

    2014-01-01

    It is not known how the immune system targets hepatitis C virus (HCV)-infected HLA-mismatched hepatocytes under immune-suppressed conditions after orthotopic liver transplantation (OLT). In addition, the relationship between the HCV-specific immune response and IL28B variants as predictors of HCV clearance has not been well-characterized. We determined the IL28B polymorphisms for 57 post-OLT HCV carriers, and we assessed the HCV-specific immune responses by measuring the peripheral blood mononuclear cell-derived HCV-specific interferon-gamma (IFN-γ) response using an enzyme-linked immunospot assay. At 1-3 years after OLT, patients with no active hepatitis showed higher total spots on the immunospot assay. At>3 years after OLT, patients with resolved HCV showed higher levels of core, NS3, NS5A, and total spots compared to the chronic hepatitis patients. The IL28B major genotype in the donors correlated with higher spot counts for NS5A and NS5B proteins at 1-3 years after OLT. In the post-OLT setting, the HCV-specific immune response could be strongly induced in patients with no active hepatitis with an IL28B major donor or sustained virological response. Strong immune responses in the patients with no active hepatitis could only be maintained for 3 years and diminished later. It may be beneficial to administer IFN treatment starting 3 years after OLT, to induce the maximum immunological effect. PMID:25338486

  4. Using shotgun sequence data to find active restriction enzyme genes.

    PubMed

    Zheng, Yu; Posfai, Janos; Morgan, Richard D; Vincze, Tamas; Roberts, Richard J

    2009-01-01

    Whole genome shotgun sequence analysis has become the standard method for beginning to determine a genome sequence. The preparation of the shotgun sequence clones is, in fact, a biological experiment. It determines which segments of the genome can be cloned into Escherichia coli and which cannot. By analyzing the complete set of sequences from such an experiment, it is possible to identify genes lethal to E. coli. Among this set are genes encoding restriction enzymes which, when active in E. coli, lead to cell death by cleaving the E. coli genome at the restriction enzyme recognition sites. By analyzing shotgun sequence data sets we show that this is a reliable method to detect active restriction enzyme genes in newly sequenced genomes, thereby facilitating functional annotation. Active restriction enzyme genes have been identified, and their activity demonstrated biochemically, in the sequenced genomes of Methanocaldococcus jannaschii, Bacillus cereus ATCC 10987 and Methylococcus capsulatus. PMID:18988632

  5. Silk microgels formed by proteolytic enzyme activity.

    PubMed

    Samal, Sangram K; Dash, Mamoni; Chiellini, Federica; Kaplan, David L; Chiellini, Emo

    2013-09-01

    The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMGs) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer-scaled crystals in native silkworm fibers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zeta potential results demonstrated that α-chymotrypsin utilized only the non-amorphous domains or segments of the heavy chain of SFP to form negatively charged SMGs. The SMGs were characterized in terms of size, charge, structure, morphology, crystallinity, swelling kinetics, water content and thermal properties. The results suggest that the present technique of preparing SMGs by α-chymotrypsin is simple and efficient, and that the prepared SMGs have useful features for studies related to biomaterial and pharmaceutical needs. This process is also an easy way to obtain the amorphous peptide chains for further study. PMID:23756227

  6. Hepatic ATF6 Increases Fatty Acid Oxidation to Attenuate Hepatic Steatosis in Mice Through Peroxisome Proliferator-Activated Receptor α.

    PubMed

    Chen, Xuqing; Zhang, Feifei; Gong, Qi; Cui, Aoyuan; Zhuo, Shu; Hu, Zhimin; Han, Yamei; Gao, Jing; Sun, Yixuan; Liu, Zhengshuai; Yang, Zhongnan; Le, Yingying; Gao, Xianfu; Dong, Lily Q; Gao, Xin; Li, Yu

    2016-07-01

    The endoplasmic reticulum quality control protein activating transcription factor 6 (ATF6) has emerged as a novel metabolic regulator. Here, we show that adenovirus-mediated overexpression of the dominant-negative form of ATF6 (dnATF6) increases susceptibility to develop hepatic steatosis in diet-induced insulin-resistant mice and fasted mice. Overexpression of dnATF6 or small interfering RNA-mediated knockdown of ATF6 decreases the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα)/retinoid X receptor complex, and inhibits oxygen consumption rates in hepatocytes, possibly through inhibition of the binding of PPARα to the promoter of its target gene. Intriguingly, ATF6 physically interacts with PPARα, enhances the transcriptional activity of PPARα, and triggers activation of PPARα downstream targets, such as CPT1α and MCAD, in hepatocytes. Furthermore, hepatic overexpression of the active form of ATF6 promotes hepatic fatty acid oxidation and protects against hepatic steatosis in diet-induced insulin-resistant mice. These data delineate the mechanism by which ATF6 controls the activity of PPARα and hepatic mitochondria fatty acid oxidation. Therefore, strategies to activate ATF6 could be used as an alternative avenue to improve liver function and treat hepatic steatosis in obesity. PMID:27207533

  7. Hepatic and extrahepatic distribution of ornithine urea cycle enzymes in holocephalan elephant fish (Callorhinchus milii).

    PubMed

    Takagi, Wataru; Kajimura, Makiko; Bell, Justin D; Toop, Tes; Donald, John A; Hyodo, Susumu

    2012-04-01

    Cartilaginous fish comprise two subclasses, the Holocephali (chimaeras) and Elasmobranchii (sharks, skates and rays). Little is known about osmoregulatory mechanisms in holocephalan fishes except that they conduct urea-based osmoregulation, as in elasmobranchs. In the present study, we examined the ornithine urea cycle (OUC) enzymes that play a role in urea biosynthesis in the holocephalan elephant fish, Callorhinchus milii (cm). We obtained a single mRNA encoding carbamoyl phosphate synthetase III (cmCPSIII) and ornithine transcarbamylase (cmOTC), and two mRNAs encoding glutamine synthetases (cmGSs) and two arginases (cmARGs), respectively. The two cmGSs were structurally and functionally separated into two types: brain/liver/kidney-type cmGS1 and muscle-type cmGS2. Furthermore, two alternatively spliced transcripts with different sizes were found for cmgs1 gene. The longer transcript has a putative mitochondrial targeting signal (MTS) and was predominantly expressed in the liver and kidney. MTS was not found in the short form of cmGS1 and cmGS2. A high mRNA expression and enzyme activities were found in the liver and muscle. Furthermore, in various tissues examined, mRNA levels of all the enzymes except cmCPSIII were significantly increased after hatching. The data show that the liver is the important organ for urea biosynthesis in elephant fish, but, extrahepatic tissues such as the kidney and muscle may also contribute to the urea production. In addition to the role of the extrahepatic tissues and nitrogen metabolism, the molecular and functional characteristics of multiple isoforms of GSs and ARGs are discussed. PMID:22227372

  8. Characterization of human liver enzymes involved in the biotransformation of boceprevir, a hepatitis C virus protease inhibitor.

    PubMed

    Ghosal, Anima; Yuan, Yuan; Tong, Wei; Su, Ai-Duen; Gu, Chunyan; Chowdhury, Swapan K; Kishnani, Narendra S; Alton, Kevin B

    2011-03-01

    Boceprevir (SCH 503034), a protease inhibitor, is under clinical development for the treatment of human hepatitis C virus infections. In human liver microsomes, formation of oxidative metabolites after incubations with [(14)C]boceprevir was catalyzed by CYP3A4 and CYP3A5. In addition, the highest turnover was observed in recombinant CYP3A4 and CYP3A5. After a single radiolabeled dose to human, boceprevir was subjected to two distinct pathways, namely cytochrome P450-mediated oxidation and ketone reduction. Therefore, attempts were made to identify the enzymes responsible for the formation of carbonyl-reduced metabolites. Human liver S9 and cytosol converted ∼ 28 and ∼ 68% of boceprevir to M28, respectively, in the presence of an NADPH-generating system. Screening of boceprevir with recombinant human aldo-keto reductases (AKRs) revealed that AKR1C2 and AKR1C3 exhibited catalytic activity with respect to the formation of M+2 metabolites (M28 and M31). The formation of M28 was inhibited by 100 μM flufenamic acid (80.3%), 200 μM mefenamic acid (83.7%), and 100 μM phenolphthalein (86.1%), known inhibitors of AKRs, suggesting its formation through carbonyl reduction pathway. Formation of M28 was also inhibited by 100 μM diazepam (75.1%), 1 mM ibuprofen (70%), and 200 μM diflunisal (89.4%). These data demonstrated that CYP3A4 and CYP3A5 are primarily responsible for the formation of oxidative metabolites and the formation of M28 and M31, the keto-reduced metabolites, are most likely mediated by AKR1C2 and AKR1C3. Because the biotransformation and clearance of boceprevir involves two different enzymatic pathways, boceprevir is less likely to be a victim of significant drug-drug interaction with concomitant medication affecting either of these pathways. PMID:21123164

  9. Effect of Boswellia serrata supplementation on blood lipid, hepatic enzymes and fructosamine levels in type2 diabetic patients

    PubMed Central

    2014-01-01

    Background Type 2 diabetes is an endocrine disorder that affects a large percentage of patients. High blood glucose causes fatty deposits in the liver which is likely to increase in SGOT and SGPT activities. Significant increase in SGOT/SGPT and low HDL levels is observed in patients with diabetes. Serum fructosamine concentration reflects the degree of blood glucose control in diabetic patients. This study was aimed to investigate the antidiabetic, hypolipidemic and hepatoprotective effects of supplementation of Boswellia serrata in type2 diabetic patients. Methods 60 type 2 diabetic patients from both sexes (30 males and 30 females) were dedicated to the control and intervention groups (30 subjects per group). Boswellia serrata gum resin in amount of 900 mg daily for 6 weeks were orally administered (as three 300 mg doses) in intervention group and the control group did not receive anything. Blood samples were taken at the beginning of the study and after 6 weeks. Blood levels of fructosamine, lipid profiles as well as hepatic enzyme in type 2 diabetic patients were measured. Results Treatment of diabetic patient with Boswellia serrata was caused to significant increase in blood HDL levels as well as a remarkable decrease in cholesterol, LDL, fructosamine (p < 0.05) SGPT and SGOT levels after 6 weeks (p < 0.01). In spite of reduction of serum triglyceride, VLDL levels in intervention group, we did not detect a significant difference after 6 weeks. Conclusion This study showed that Boswellia serrata supplementation can be beneficial in controlling blood parameters in patients with type 2 diabetes. Therefore, its use can be useful in patients with medicines. PMID:24495344

  10. Effect of Commiphora mukul gum resin on hepatic marker enzymes, lipid peroxidation and antioxidants status in pancreas and heart of streptozotocin induced diabetic rats

    PubMed Central

    Ramesh, B; Karuna, R; Sreenivasa, Reddy S; Haritha, K; Sai, Mangala D; Sasi, Bhusana Rao B; Saralakumari, D

    2012-01-01

    Objective To study the antioxidant efficacy of Commiphora mukul (C. mukul) gum resin ethanolic extract in streptozotocin (STZ) induced diabetic rats. Methods The male Wistar albino rats were randomly divided into four groups of eight animals each: Control group (C), CM-treated control group (C+CMEE), Diabetic control group (D), CM- treated diabetic group (D+CMEE). Diabetes was induced by intraperitoneal injection of STZ (55 mg/kg/ bwt). After being confirmed the diabetic rats were treated with C. mukul gum resin ethanolic extract (CMEE) for 60 days. The biochemical estimations like antioxidant, oxidative stress marker enzymes and hepatic marker enzymes of tissues were performed. Results The diabetic rats showed increased level of enzymatic activities aspartate aminotransaminase (AST), alanine aminotransaminase (ALT) in liver and kidney and oxidative markers like lipid peroxidation (LPO) and protein oxidation (PO) in pancreas and heart. Antioxidant enzyme activities were significantly decreased in the pancreas and heart compared to control group. Administration of CMEE (200 mg/kg bw) to diabetic rats for 60 days significantly reversed the above parameters towards normalcy. Conclusions In conclusion, our data indicate the preventive role of C. mukul against STZ-induced diabetic oxidative stress; hence this plant could be used as an adjuvant therapy for the prevention and/or management of diabetes and aggravated antioxidant status. PMID:23569867

  11. Synergetic Effects of Nanoporous Support and Urea on Enzyme Activity

    SciTech Connect

    Lei, Chenghong; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2007-02-01

    Here we report that synergetic effects of functionalized nanoporous support and urea on enzyme activity enhancement. Even in 8.0 M urea, the specific activity of GI entrapped in FMS was still higher than the highest specific activity of GI free in solution, indicating the strong tolerance of GI in FMS to the high concentration of urea.

  12. Inhibition of existing denitrification enzyme activity by chloramphenicol

    USGS Publications Warehouse

    Brooks, M.H.; Smith, R.L.; Macalady, D.L.

    1992-01-01

    Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

  13. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  14. Evaluation of the assumptions of an ontogeny model of rat hepatic cytochrome P450 activity.

    PubMed

    Alcorn, Jane; Elbarbry, Fawzy A; Allouh, Mohammed Z; McNamara, Patrick J

    2007-12-01

    We previously reported an ontogeny model of hepatic cytochrome P450 (P450) activity that predicts in vivo P450 elimination from in vitro intrinsic clearance. The purpose of this study was to conduct investigations into key assumptions of the P450 ontogeny model using the developing rat model system. We used two developmentally dissimilar enzymes, CYP2E1 and CYP1A2, and male rats (n = 4) at age groups representing critical developmental stages. Total body and liver weights and hepatic microsomal protein contents were measured. Following high-performance liquid chromatography analysis, apparent K(M) and V(max) estimates were calculated using nonlinear regression analysis for CYP2E1- and CYP1A2-mediated chlorzoxazone 6-hydroxylation and methoxyresorufin O-dealkylation, and V(max) estimates for p-nitrophenol and phenacetin hydroxylations, respectively. Hepatic scaling factors and V(max) values provided estimates for infant scaling factors (ISF). The data show microsomal protein contents increased with postnatal age and reached adult values after postnatal day (PD) 7. Apparent K(M) values were similar at all developmental stages except at < or =PD7. Developmental increases in probe substrate V(max) values did not correlate with the biphasic increase in immunoquantifiable P450. The activity of two different probe substrates for each P450 covaried as a function of age. A plot of observed ISF values as a function of age reflected the developmental pattern of rat hepatic P450. In summation, these observations diverge from several of the model's assumptions. Further investigations are required to explain these inconsistencies and to investigate whether the developing rat may provide a predictive paradigm for pediatric risk assessment for P450-mediated elimination processes. PMID:17881659

  15. Activity of selected hydrolytic enzymes in Allium sativum L. anthers.

    PubMed

    Winiarczyk, Krystyna; Gębura, Joanna

    2016-05-01

    The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant. PMID:26901781

  16. Interfacial activation-based molecular bioimprinting of lipolytic enzymes.

    PubMed Central

    Mingarro, I; Abad, C; Braco, L

    1995-01-01

    Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template. PMID:7724558

  17. Nonlinear (amplified) relationship between nuclear occupancy by triiodothyronine and the appearance rate of hepatic alpha-glycerophosphate dehydrogenase and malic enzyme in the rat.

    PubMed Central

    Oppenheimer, J H; Coulombe, P; Schwartz, H L; Gutfeld, N W

    1978-01-01

    Three separate approaches were applied to examine the general relationship between R, the rate of induction of specific enzymes (mitochondrial alpha-glycero-phosphate dehydrogenase and cytosolic malic enzyme) and q, the fractional nuclear occupancy by triiodothyronine in male Sprague-Dawley rats. Daily 200-microgram injections of triiodothyronine per 10u g body wt for 7 days resulted in saturation of the hepatic nuclear sites and the achievement of an apparent new steady state of enzyme levels. The increase achieved over base-line hypothyroid levels was then compared with the increment over hypothyroid base line characteristic of intact euthyroid animals with 47% of nuclear sites occupied. The maximal theoretical reate of steady-state enzyme induction could be protected on the basis of the observed maximal increase in enzyme activity observed 1 day after the injection of graded doses of hormone and lambda, the known fractional rate of enzyme dissipation. The 24-h dose-response studies were used to generate R as a continuous function of q, both in hypothyroid as well as in euthyroid animals. This approach involved the numerical solution of an ordinary differential equation describing the rate of change of enzyme as a function of R, which was assumed to be uniquely related to q. Results of these analyses indicated that the ratio of the maximal rate of induction of enzyme at full occupancy to the rate of induction under euthyroid conditions assumes a value between 9.0 and 19.5, depending on the precise analytic and experimental approach applied. This value is far in excess of the theoretical ratio 2.13 which on would anticipate if R were linearly related to q and 47% of the nuclear sites occupied under physiological conditions. Thus, the signal for enzyme induction appears to undergo progressjive amplification with increasing nuclear occupancy. Moreover, the curve describing the relationship between R and q appears highly nonlinear throughout (concave upwards

  18. Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.

    PubMed

    Nakatani, Kazuki; Seki, Shuichi; Kawada, Norifumi; Kitada, Takuya; Yamada, Takao; Sakaguchi, Hiroki; Kadoya, Hirokazu; Ikeda, Kazuo; Kaneda, Kenji

    2002-11-01

    Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries. PMID:12447677

  19. Cadmium effect on microsomal drug-metabolizing enzyme activity in rat livers with respect to differences in age and sex

    SciTech Connect

    Ando, M.

    1982-04-01

    The effect of cadmium on the hepatic microsomal drug-metabolizing enzyme system was investigated. Cadmium chloride caused the conversion of cytochrome P-450 to P-420 in rat liver microsomes. The destruction of cytochrome P-450 by cadmium caused the reduction of microsomal drug-metabolizing enzyme activity and prolonged the pentobarbital sleeping time. There is a sex-related difference in the ability of cadmium to inhibit the hepatic drug metabolism in rats: male rats are more sensitive to cadmium than females. The effective period when cadmium prolonged their sleep depended upon the age of rats; older rats were more sensitive to cadmium than younger ones. The maximum increase of sleeping time depended upon the dose level of cadium, and the rate constant of the equations seems to depend upon the age of the animals.

  20. Activation of hepatic branched-chain 2-oxoacid dehydrogenase by rat liver cytosolic supernatant.

    PubMed

    Hauschildt, S

    1986-10-29

    Hepatic branched-chain 2-oxoacid dehydrogenase is inactivated by nutritional alterations. Reactivation occurs during preincubation of intact mitochondria in the presence of rat liver cytosolic supernatant. Cytosolic supernatant contains two factors capable of reactivating the enzyme. On gel-filtration (Sephadex G-100), one factor (AF1) elutes in the molecular range of 35,000-40,000 and the other factor (AF2) elutes slightly later than inorganic phosphate. AF2 is stable against heat denaturation and treatment with proteinases. It is destroyed by alkaline phosphatase and in the presence of Ap5A, atractyloside, CaCl2 and NaF its stimulatory effect on branched-chain 2-oxoacid dehydrogenase activity is abolished. Inhibition of activation by NaF suggests that a phosphatase might be involved in the activation process. PMID:3768411

  1. DEHP reduces thyroid hormones via interacting with hormone synthesis-related proteins, deiodinases, transthyretin, receptors, and hepatic enzymes in rats.

    PubMed

    Liu, Changjiang; Zhao, Letian; Wei, Li; Li, Lianbing

    2015-08-01

    Di-(2-ethylhexyl) phthalate (DEHP) is used extensively in many personal care and consumer products, resulting in widespread nonoccupational human exposure through multiple routes and media. Limited studies suggest that exposure to DEHP may be associated with altered thyroid function, but detailed mechanisms are unclear. In order to elucidate potential mechanisms by which DEHP disturbs thyroid hormone homeostasis, Sprague-Dawley (SD) rats were dosed with DEHP by gavage at 0, 250, 500, and 750 mg/kg/day for 30 days and sacrificed within 24 h after the last dose. Gene expressions of thyroid hormone receptors, deiodinases, transthyretin, and hepatic enzymes were measured by RT-PCR; protein levels of transthyretin were also analyzed by Western blot. Results showed that DEHP caused histological changes in the thyroid and follicular epithelial cell hypertrophy and hyperplasia were observed. DEHP significantly reduced thyroid hormones (T3, T4) and thyrotropin releasing hormone (TRH) levels, whereas thyroid stimulating hormone (TSH) was not affected. After exposure to DEHP, biosynthesis of thyroid hormones was suppressed, and sodium iodide symporter (NIS) and thyroid peroxidase (TPO) levels were significantly reduced. Additionally, levels of deiodinases and transthyretin were also affected. TSH receptor (TSHr) level was downregulated, while TRH receptor (TRHr) level was upregulated. Metabolism of thyroid hormones was accelerated due to elevated gene expression of hepatic enzymes (UDPGTs and CYP2B1) by DEHP. Taken together, observed findings indicate that DEHP could reduce thyroid hormones through influencing biosynthesis, biotransformation, biotransport, receptor levels, and metabolism of thyroid hormones. PMID:25913319

  2. PCB153 and p,p'-DDE disorder thyroid hormones via thyroglobulin, deiodinase 2, transthyretin, hepatic enzymes and receptors.

    PubMed

    Liu, Changjiang; Ha, Mei; Li, Lianbing; Yang, Kedi

    2014-10-01

    Polychlorinated biphenyls (PCBs) and DDT are widespread environmental persistent organic pollutants that have various adverse effects on reproduction, development and endocrine function. In order to elucidate effects of PCBs and DDT on thyroid hormone homeostasis, Sprague-Dawley rats were dosed with PCB153 and p,p'-DDE intraperitoneally (ip) for five consecutive days and sacrificed within 24 h after the last dose. Results indicated that after combined exposure to PCB153 and p,p'-DDE, total thyroxine , free thyroxine, total triiodothyronine, and thyroid-stimulating hormone in serum were decreased, whereas free triiodothyronine and thyrotropin-releasing hormone were not affected. Thyroglobulin and transthyretin levels in serum were significantly reduced. mRNA expression of deiodinases 2 (D2) was also suppressed, while D1 and D3 levels were not significantly influenced after combined exposure. PCB153 and p,p'-DDE induced hepatic enzymes, UDPGTs, CYP1A1, CYP2B1, and CYP3A1 mRNA expressions being significantly elevated. Moreover, TRα1, TRβ1, and TRHr expressions in the hypothalamus displayed increasing trends after combined exposure to PCB153 and p,p'-DDE. Taken together, observed results indicate that PCB153 and p,p'-DDE could disorder thyroid hormone homeostasis via thyroglobulin, deiodinase 2, transthyretin, hepatic enzymes, and hormone receptors. PMID:24878560

  3. Effect of Nigella sativa fixed and essential oils on antioxidant status, hepatic enzymes, and immunity in streptozotocin induced diabetes mellitus

    PubMed Central

    2014-01-01

    Background Nigella sativa fixed (NSFO) and essential (NSEO) oils have been used to treat diabetes mellitus and its complications. Present study was undertaken to explore and validate these folkloric uses. Methods Sprague dawley rats having streptozotocin (STZ) induced diabetes mellitus were used to assess the role of NSFO and NSEO in the management of diabetes complications. Parameters investigated were antioxidant potential, oxidative stress, and the immunity by in vivo experiments. Results The results indicated that STZ decreased the glutathione contents (25.72%), while NSFO and NSEO increased the trait significantly (P < 0.05). Experimental diets increased the tocopherol contents (P < 0.01) and enhanced the expression of hepatic enzymes (P < 0.01). Correlation matrix further indicated that antioxidant potential is positively associated (P < 0.05) responsible for the modulation of hepatic enzymes and the decrease of the nitric oxide production thus controlling the diabetes complications. Conclusions Overall, results of present study supported the traditional use of N. sativa and its derived products as a treatment for hyperglycemia and allied abnormalities. Moreover, N. sativa fixed and essential oils significantly ameliorate free radicals and improve antioxidant capacity thus reducing the risk of diabetic complications. PMID:24939518

  4. Chokeberry (Aronia melanocarpa) juice modulates 7,12-dimethylbenz[a]anthracene induced hepatic but not mammary gland phase I and II enzymes in female rats.

    PubMed

    Szaefer, Hanna; Krajka-Kuźniak, Violetta; Ignatowicz, Ewa; Adamska, Teresa; Baer-Dubowska, Wanda

    2011-03-01

    Chokeberry is a rich source of procyanidins known to have several types of biological activity including anticarcinogenic potential in experimental models. In this study we examined the effect of chokeberry juice on the hepatic and mammary gland carcinogen metabolizing enzyme expression altered by the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA). Sprague-Dawley rats were gavaged with chokeberry juice (8 ml/kg b.w.) for 28 consecutive days. DMBA was administered i.p. on the 27th and the 28th days. Pretreatment with chokeberry juice reduced the activity of CYP1A1 and increased that of CYP2B involved in metabolic activation/detoxication of DMBA in rat liver, as well as expression and activity of phase II enzymes. Chokeberry juice had no effect on these parameters in the mammary gland and DMBA induced DNA damage in rat blood cells. These results together with our earlier observations indicate that metabolic alterations induced by chokeberry feeding are tissue specific and depend on the class of carcinogen. PMID:21787703

  5. Screening for enzyme activity in turbid suspensions with scattered light.

    PubMed

    Huber, Robert; Wulfhorst, Helene; Maksym, Lukas; Stehr, Regina; Pöhnlein, Martin; Jäger, Gernot; Spiess, Antje C; Büchs, Jochen

    2011-01-01

    New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition. PMID:21302369

  6. Enzyme-polymer composites with high biocatalytic activity and stability

    SciTech Connect

    Kim, Jungbae; Kosto, Timothy J.; Manimala, Joseph C.; Nauman, E B.; Dordick, Jonathan S.

    2004-08-22

    We have applied vacuum-spraying and electrospinning to incorporate an enzyme into a polymer matrix, creating a novel and highly active biocatalytic composite. As a unique technical approach, enzymes were co-dissolved in toluene with polymers, and the solvent was then rapidly removed by injecting the mixture into a vacuum chamber or by electrospinning. Subsequent crosslinking of the enzyme with glutaraldehyde resulted in stable entrapped enzyme within the polymeric matrices. For example, an amorphous composite of alpha-chymotrypsin and polyethylene showed no significant loss of enzymatic activity in aqueous buffer for one month. Nanofibers of alpha-chymotrypsin and polystyrene also showed no decrease in activity for more than two weeks. The normalized activity of amorphous composite in organic solvents was 3-13 times higher than that of native alpha-chymotrypsin. The activity of nanofibers was 5-7 times higher than that of amorphous composite in aqueous buffer solution. The composites of alpha-chymotrypsin and polymers demonstrate the feasibility of obtaining a wide variety of active and stable biocatalytic materials with many combinations of enzymes and polymers.

  7. Chimeric enzymes with improved cellulase activities

    SciTech Connect

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  8. Relative potency based on hepatic enzyme induction predicts immunosuppressive effects of a mixture of PCDDS/PCDFS and PCBS

    SciTech Connect

    Smialowicz, R.J.; DeVito, M.J. Williams, W.C.; Birnbaum, L.S.

    2008-03-15

    The toxic equivalency factor (TEF) approach was employed to compare immunotoxic potency of mixtures containing polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), using the antibody response to sheep erythrocytes (SRBC). Mixture-1 (MIX-1) contained TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), and 1,2,3,4,6,7,8,9-octachlorodibenzofuran (OCDF). Mixture-2 (MIX-2) contained MIX-1 and the following PCBs, 3,3',4,4'-tetrachlorobiphenyl (IUPAC No. 77), 3,3',4,4',5-pentachlorobiphenyl (126), 3,3',4,4',5,5N-hexachlorobiphenyl (169), 2,3,3',4,4'-pentachlorobiphenyl (105), 2,3',4,4',5-pentachlorobiphenyl (118), and 2,3,3',4,4',5-hexachlorobiphenyl (156). The mixture compositions were based on relative chemical concentrations in food and human tissues. TCDD equivalents (TEQ) of the mixture were estimated using relative potency factors from hepatic enzyme induction in mice [DeVito, M.J., Diliberto, J.J., Ross, D.G., Menache, M.G., Birnbaum, L.S., 1997. Dose-response relationships for polyhalogenated dioxins and dibenzofurans following subchronic treatment in mice. I .CYP1A1 and CYP1A2 enzyme activity in liver, lung and skin. Toxicol. Appl. Pharmacol. 130, 197-208; DeVito, M.J., Menache, G., Diliberto, J.J., Ross, D.G., Birnbaum L.S., 2000. Dose-response relationships for induction of CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin in female mice following subchronic exposure to polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 167, 157-172] Female mice received 0, 1.5, 15, 150 or 450 ng TCDD/kg/day or approximately 0, 1.5, 15, 150 or 450 ng TEQ/kg/day of MIX-1 or MIX-2 by gavage 5 days per week for 13 weeks. Mice were immunized 3 days after the last exposure and 4 days later, body, spleen, thymus, and liver weights were measured

  9. Stereochemical aspects of vinylcyclohexene bioactivation in rodent hepatic microsomes and purified human cytochrome P450 enzyme systems.

    PubMed

    Fontaine, S M; Mash, E A; Hoyer, P B; Sipes, I G

    2001-02-01

    The racemic mixture of 4-vinylcyclohexene (VCH) forms ovotoxic epoxides [VCH-1,2-epoxide, VCH-7,8-epoxide, and vinylcyclohexene diepoxide (VCD)] by cytochrome P450 (CYP) in B6C3F(1) female mice. These epoxides deplete primordial and primary follicles. The current studies compared in vitro epoxidation of (R)-VCH with that of (S)-VCH in hepatic microsomes prepared from adult female B6C3F(1) mice and Fischer 344 rats. Bioactivation of VCH in the rat was significantly less compared with that in the mouse. (R)-VCH formed significantly more VCH-1,2-epoxide as compared with (S)-VCH in both species, and less VCH-7,8-epoxide in the mouse. Neither of the enantiomers formed detectable amounts of VCD in the mouse or rat. Hepatic microsomes prepared from mice and rats pretreated with CYP-inducing agents (phenobarbital and acetone) were also incubated with (R)-VCH or (S)-VCH. Although monoepoxide formation was not increased enantioselectively in the mouse, VCD was formed preferentially from (R)-VCH as compared with (S)-VCH. Pretreatment with VCH resulted in nonstereoselective increases in both monoepoxide and diepoxide formation. In the rat, these pretreatments resulted in nonstereoselective increases in monoepoxide formation, but VCD formation was not detectable. Incubations with human CYP2E1 enzyme revealed that (R)-VCH formed significantly more VCH-1,2-epoxide and less VCH-7,8-epoxide than (S)-VCH. Human CYP2A6 was limited in its ability to form epoxides from either enantiomer of VCH. Human CYP2B6 preferentially formed VCH-7,8-epoxide compared with VCH-1,2-epoxide, and to a greater extent from (R)-VCH than from (S)-VCH. These results demonstrate regioselectivity and enantioselectivity in the bioactivation of VCH in rodent hepatic microsomes as well as in expressed human CYP enzymes. PMID:11159809

  10. Interactions of the hepatitis C virus protease inhibitor faldaprevir with cytochrome P450 enzymes: in vitro and in vivo correlation.

    PubMed

    Sabo, John P; Kort, Jens; Ballow, Charles; Kashuba, Angela D M; Haschke, Manuel; Battegay, Manuel; Girlich, Birgit; Ting, Naitee; Lang, Benjamin; Zhang, Wei; Cooper, Curtis; O'Brien, Drané; Seibert, Eleanore; Chan, Tom S; Tweedie, Donald; Li, Yongmei

    2015-04-01

    The potential inhibition of the major human cytochrome P450 (CYP) enzymes by faldaprevir was evaluated both in vitro and in clinical studies (healthy volunteers and hepatitis C virus [HCV] genotype 1-infected patients). In vitro studies indicated that faldaprevir inhibited CYP2B6, CYP2C9, and CYP3A, and was a weak-to-moderate inactivator of CYP3A4. Faldaprevir 240 mg twice daily in healthy volunteers demonstrated moderate inhibition of hepatic and intestinal CYP3A (oral midazolam: 2.96-fold increase in AUC(0-24 h)), weak inhibition of hepatic CYP3A (intravenous midazolam: 1.56-fold increase in AUC(0-24 h)), weak inhibition of CYP2C9 ([S]-warfarin: 1.29-fold increase in AUC(0-120 h)), and had no relevant effects on CYP1A2, CYP2B6, or CYP2D6. Faldaprevir 120 mg once daily in HCV-infected patients demonstrated weak inhibition of hepatic and intestinal CYP3A (oral midazolam: 1.52-fold increase in AUC(0-∞)), and had no relevant effects on CYP2C9 or CYP1A2. In vitro drug-drug interaction predictions based on inhibitor concentration ([I])/inhibition constant (Ki) ratios tended to overestimate clinical effects and a net-effect model provided a more accurate approach. These studies suggest that faldaprevir shows a dose-dependent inhibition of CYP3A and CYP2C9, and does not induce CYP isoforms. PMID:25449227

  11. Improving Activity of Salt-Lyophilized Enzymes in Organic Media

    NASA Astrophysics Data System (ADS)

    Borole, Abhijeet P.; Davison, Brian H.

    Lyophilization with salts has been identified as an important method of activating enzymes in organic media. Using salt-activated enzymes to transform molecules tethered to solid surfaces in organic phase requires solubilization of enzymes in the solvents. Methods of improving performance of salt-lyophilized enzymes, further, via chemical modification, and use of surfactants and surfactants to create fine emulsions prior to lyophilization are investigated. The reaction system used is transesterification of N-acetyl phenylalanine ethyl ester with methanol or propanol. Initial rate of formation of amino acid esters by subtilisin Carlsberg (SC) was studied and found to increase two to sevenfold by either chemical modification or addition of surfactants in certain solvents, relative to the salt (only)-lyophilized enzyme. The method to prepare highly dispersed enzymes in a salt-surfactant milieu also improved activity by two to threefold. To test the effect of chemical modification on derivatization of drug molecules, acylation of bergenin was investigated using chemically modified SC.

  12. Eletriptan metabolism by human hepatic CYP450 enzymes and transport by human P-glycoprotein.

    PubMed

    Evans, David C; O'Connor, Desmond; Lake, Brian G; Evers, Raymond; Allen, Christopher; Hargreaves, Richard

    2003-07-01

    "Reaction phenotyping" studies were performed with eletriptan (ETT) to determine its propensity to interact with coadministered medications. Its ability to serve as a substrate for human P-glycoprotein (P-gp) was also investigated since a central mechanism of action has been proposed for this "triptan" class of drug. In studies with a characterized bank of human liver microsome preparations, a good correlation (r2 = 0.932) was obtained between formation of N-desmethyl eletriptan (DETT) and CYP3A4-catalyzed testosterone 6 beta-hydroxylation. DETT was selected to be monitored in our studies since it represents a significant ETT metabolite in humans, circulating at concentrations 10 to 20% of those observed for parent drug. ETT was metabolized to DETT by recombinant CYP2D6 (rCYP2D6) and rCYP3A4, and to a lesser extent by rCYP2C9 and rCYP2C19. The metabolism of ETT to DETT in human liver microsomes was markedly inhibited by troleandomycin, erythromycin, miconazole, and an inhibitory antibody to CYP3A4, but not by inhibitors of other major P450 enzymes. ETT had little inhibitory effect on any of the P450 enzymes investigated. ETT was determined to be a good substrate for human P-gp in vitro. In bidirectional transport studies across LLC-MDR1 and LLC-Mdr1a cell monolayers, ETT had a BA/AB transport ratio in the range 9 to 11. This finding had significance in vivo since brain exposure to ETT was reduced 40-fold in Mdr1a+/+ relative to Mdr1a-/- mice. ETT metabolism to DETT is therefore catalyzed primarily by CYP3A4, and plasma concentrations are expected to be increased when coadministered with inhibitors of CYP3A4 and P-gp activity. PMID:12814962

  13. Distribution and activity of hydrogenase enzymes in subsurface sediments

    NASA Astrophysics Data System (ADS)

    Adhikari, R.; Nickel, J.; Glombitza, C.; Spivack, A. J.; D'Hondt, S. L.; Kallmeyer, J.

    2013-12-01

    Metabolically active microbial communities are present in a wide range of subsurface environments. Techniques like enumeration of microbial cells, activity measurements with radiotracer assays and the analysis of porewater constituents are currently being used to explore the subsurface biosphere, alongside with molecular biological analyses. However, many of these techniques reach their detection limits due to low microbial activity and abundance. Direct measurements of microbial turnover not just face issues of insufficient sensitivity, they only provide information about a single specific process rather than an overall microbial activity. Since hydrogenase enzymes are intracellular and ubiquitous in subsurface microbial communities, the enzyme activity represents a measure of total activity of the entire microbial community. A hydrogenase activity assay could quantify total metabolic activity without having to identify specific processes. This would be a major advantage in subsurface biosphere studies, where several metabolic processes can occur simultaneously. We quantified hydrogenase enzyme activity and distribution in sediment samples from different aquatic subsurface environments (Lake Van, Barents Sea, Equatorial Pacific and Gulf of Mexico) using a tritium-based assay. We found enzyme activity at all sites and depths. Volumetric hydrogenase activity did not show much variability between sites and sampling depths, whereas cell-specific activity ranged from 10-5 to 1 nmol H2 cell-1 d-1. Activity was lowest in sediment layers where nitrate was detected. Higher activity was associated with samples in which sulfate was the predominant electron acceptor. We found highest activity in samples from environments with >10 ppm methane in the pore water. The results show that cell-specific hydrogenase enzyme activity increases with decreasing energy yield of the electron acceptor used. It is not possible to convert volumetric or cell-specific hydrogenase activity into a

  14. Patterns of functional enzyme activity in fungus farming ambrosia beetles

    PubMed Central

    2012-01-01

    Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose

  15. Effects of hepatic enzyme inducers on thyroxine (T4) catabolism in primary rat hepatocytes

    EPA Science Inventory

    Nuclear receptor agonists such as phenobarbital (PB), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and 3-methylcholantrene (3-MC) decrease circulating thyroxine (T4) concentrations in rats. It is suspected that this decrease occurs through the induction of hepatic metabolizing en...

  16. Evaluation of the Novel HISCL Chemiluminescence Enzyme Immunoassay for Laboratory Screening of Hepatitis C Virus.

    PubMed

    Feng, Shu; Wei, Bin; Liu, Qianqian; Wang, Tingting; Li, Dongdong; Rao, Chenli; Tao, Chuanmin; Wang, Lanlan

    2016-07-01

    The hepatitis C virus (HCV) antibody assay remains the first-line screening test to identify HCV infection. The newly arrived HISCL Anti-HCV assay had a satisfactory seroconversion sensitivity. Its sensitivity and specificity were 98.97 and 100% for clinical samples. In general, the HISCL Anti-HCV assay may be a novel choice for clinical HCV screening. PMID:27170643

  17. INFLUENCE OF OZONE AND NITROGEN DIOXIDE ON HEPATIC MICROSOMAL ENZYMES IN MICE

    EPA Science Inventory

    Since ambient concentrations of ozone and nitrogen dioxide increase drug-induced sleeping time in female mice, potential mechanisms were sought by investigating the effects of these gases on hepatic microsomal mixed-function oxidases in female CD-1 mice. Exposure to ozone did not...

  18. Effects of selenium dietary enhancement on hatchery-reared coho salmon, Oncorhynchus kisutch (Walbaum), when compared with wild coho: hepatic enzymes and seawater adaptation evaluated.

    USGS Publications Warehouse

    Felton, S.P.; Landolt, M.L.; Grace, R.; Palmisano, A.N.

    1996-01-01

    Hatchery-reared coho salmon, Oncorhynchus kisutch (Walbaum), were fed elevated levels of selenium (as Na2SeO3) to raise eviscerated body burdens to the level measured in wild counterparts. The goal was to find a dietary concentration that would achieve the desired effect without causing damage to growth and normal development. To measure some indices of health, the detoxifying enzymes chosen were hepatic glutathione peroxidase (GSH-Px) and hepatic superoxide dismutase (SOD). Eviscerated body selenium (Se) concentration, GSH-Px and SOD levels were measured during and at the end of the 9 month freshwater feeding trial. Selenium retention and enzyme activity were also measured during 6 months’residence in sea water (SW). Selenium supplements were added to a commercial ration to give final concentrations of 1.1, 8.6, 11.1, 13.6 μg g-1 Se in the four respective diets. The results indicated that a dietary concentration of 8.6 μg g-1selenium was capable of inducing eviscerated body burdens similar to those found in wild fish. The elevated selenium levels persisted throughout the freshwater (FW) rearing phase, but declined when the fish were fed an unsupplemented ration upon SW entry. Superoxide dismutase levels did not increase above control levels. Glutathione peroxidase levels increased in fish fed the supplemented diets. GSH-Px activity declined in the higher supplemented dietary groups when all groups were reduced to the control group level of 1.1 μg g-1. Cumulative mortality in SW was 20% in fish fed either the 1.1 or the 8.6 μg g-1 Se diets. The 8.6 μg g-1 Se supplemented diets did produce healthy coho, comparable to their wild counterparts.

  19. Regulation of Human Hepatic Drug Transporter Activity and Expression by Diesel Exhaust Particle Extract

    PubMed Central

    Le Vee, Marc; Jouan, Elodie; Stieger, Bruno; Lecureur, Valérie; Fardel, Olivier

    2015-01-01

    Diesel exhaust particles (DEPs) are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe) may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC) uptake transporters organic anion-transporting polypeptides (OATP) 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC) efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT) 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP), whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP). Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a reference activator of the aryl hydrocarbon receptor (AhR) pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute to their

  20. Regulation of human hepatic drug transporter activity and expression by diesel exhaust particle extract.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Stieger, Bruno; Lecureur, Valérie; Fardel, Olivier

    2015-01-01

    Diesel exhaust particles (DEPs) are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe) may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC) uptake transporters organic anion-transporting polypeptides (OATP) 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC) efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT) 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP), whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP). Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a reference activator of the aryl hydrocarbon receptor (AhR) pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute to their

  1. Modulation of hepatic drug metabolizing enzymes by dietary doses of thymoquinone in female New Zealand White rabbits.

    PubMed

    Elbarbry, Fawzy; Ragheb, Ahmed; Marfleet, Travis; Shoker, Ahmed

    2012-11-01

    Herbal medicines can affect drug metabolizing enzymes. Therefore the effect of thymoquinone (TQ), the active ingredient of black seeds, was examined on rabbit liver drug metabolizing enzymes. Two groups of New Zealand female rabbits received TQ at 10 and 20 mg/kg/day orally and a control group of six animals each were killed after 8 weeks. Blood and livers were harvested and the activity of cytochrome P450 (CYP) and phase II enzymes in the microsomal and cytosolic preparations were measured by HPLC and ELISA methods. The liver enzymes ALT/AST and albumin were similar in the three groups. CYP1A2, CYP3A4, but not CYP2E1, were significantly diminished by TQ treatment. Of the phase II enzymes, glutathione-S-transferase (GST) and glutathione peroxidase (GPx) were significantly induced by the high TQ dose, while the total glutathione levels were unaffected. Glutathione reductase (GR), on the other hand, was significantly induced in the two experimental groups. Thymoquinone has differential effects on CYP and phase II enzymes. Inhibition of some CYP enzyme activities may lead to a hazardous herb-drug interaction. Induction of GR activity may explain the salutatory effect of the black seeds in inhibiting the generation of bioactive metabolites known to promote carcinogenesis and oxidative cell damage. PMID:22422469

  2. Chemoproteomic profiling of host and pathogen enzymes active in cholera

    PubMed Central

    Hatzios, Stavroula K.; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A.; Qadri, Firdausi; Ryan, Edward T.; Davis, Brigid M.; Weerapana, Eranthie; Waldor, Matthew K.

    2016-01-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human cholera stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, while genetic disruption of all four proteases increased the abundance and binding of an intestinal lectin—intelectin—to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialogue in an animal model of infection. PMID:26900865

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  5. Chronic administration of caderofloxacin, a new fluoroquinolone, increases hepatic CYP2E1 expression and activity in rats

    PubMed Central

    Liu, Li; Miao, Ming-xing; Zhong, Ze-yu; Xu, Ping; Chen, Yang; Liu, Xiao-dong

    2016-01-01

    Aim: Caderofloxacin is a new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of caderofloxacin interacting with co-administered drugs. Methods: Male rats were treated with caderofloxacin (9 mg/kg, ig) once or twice daily for 14 consecutive days. The effects of caderofloxacin on CYP3A, 2D6, 2C19, 1A2, 2E1 and 2C9 were evaluated using a “cocktail” of 6 probes (midazolam, dextromethorphan, omeprazole, theophylline, chlorzoxazone and diclofenac) injected on d 0 (prior to caderofloxacin exposure) and d 15 (after caderofloxacin exposure). Hepatic microsomes from the caderofloxacin-treated rats were used to assess CYP2E1 activity and chlorzoxazone metabolism. The expression of CYP2E1 mRNA and protein in hepatic microsomes was analyzed with RT-PCR and Western blotting, respectively. Results: Fourteen-day administration of caderofloxacin significantly increased the activity of hepatic CYP2E1, leading to enhanced metabolism of chlorzoxazone. In vitro microsomal study confirmed that CYP2E1 was a major metabolic enzyme involved in chlorzoxazone metabolism, and the 14-d administration of caderofloxacin significantly increased the activity of CYP2E1 in hepatic microsomes, resulting in increased formation of 6-hydroxychlorzoxazone. Furthermore, the 14-d administration of caderofloxacin significantly increased the expression of CYP2E1 mRNA and protein in liver microsomes, which was consistent with the pharmacokinetic results. Conclusion: Fourteen-day administration of caderofloxacin can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a potential drug-drug interaction mediated by CYP2E1 induction should be considered. PMID:26838075

  6. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    SciTech Connect

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A.

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  7. A DNA enzyme with N-glycosylase activity

    NASA Technical Reports Server (NTRS)

    Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

  8. Activation Energy of Extracellular Enzymes in Soils from Different Biomes

    PubMed Central

    Steinweg, J. Megan; Jagadamma, Sindhu; Frerichs, Joshua; Mayes, Melanie A.

    2013-01-01

    Enzyme dynamics are being incorporated into soil carbon cycling models and accurate representation of enzyme kinetics is an important step in predicting belowground nutrient dynamics. A scarce number of studies have measured activation energy (Ea) in soils and fewer studies have measured Ea in arctic and tropical soils, or in subsurface soils. We determined the Ea for four typical lignocellulose degrading enzymes in the A and B horizons of seven soils covering six different soil orders. We also elucidated which soil properties predicted any measurable differences in Ea. β-glucosidase, cellobiohydrolase, phenol oxidase and peroxidase activities were measured at five temperatures, 4, 21, 30, 40, and 60°C. Ea was calculated using the Arrhenius equation. β-glucosidase and cellobiohydrolase Ea values for both A and B horizons in this study were similar to previously reported values, however we could not make a direct comparison for B horizon soils because of the lack of data. There was no consistent relationship between hydrolase enzyme Ea and the environmental variables we measured. Phenol oxidase was the only enzyme that had a consistent positive relationship between Ea and pH in both horizons. The Ea in the arctic and subarctic zones for peroxidase was lower than the hydrolases and phenol oxidase values, indicating peroxidase may be a rate limited enzyme in environments under warming conditions. By including these six soil types we have increased the number of soil oxidative enzyme Ea values reported in the literature by 50%. This study is a step towards better quantifying enzyme kinetics in different climate zones. PMID:23536898

  9. Genetic inhibition of hepatic acetyl-CoA carboxylase activity increases liver fat and alters global protein acetylationa

    PubMed Central

    Chow, Jenny D.Y.; Lawrence, Robert T.; Healy, Marin E.; Dominy, John E.; Liao, Jason A.; Breen, David S.; Byrne, Frances L.; Kenwood, Brandon M.; Lackner, Carolin; Okutsu, Saeko; Mas, Valeria R.; Caldwell, Stephen H.; Tomsig, Jose L.; Cooney, Gregory J.; Puigserver, Pere B.; Turner, Nigel; James, David E.; Villén, Judit; Hoehn, Kyle L.

    2014-01-01

    Lipid deposition in the liver is associated with metabolic disorders including fatty liver disease, type II diabetes, and hepatocellular cancer. The enzymes acetyl-CoA carboxylase 1 (ACC1) and ACC2 are powerful regulators of hepatic fat storage; therefore, their inhibition is expected to prevent the development of fatty liver. In this study we generated liver-specific ACC1 and ACC2 double knockout (LDKO) mice to determine how the loss of ACC activity affects liver fat metabolism and whole-body physiology. Characterization of LDKO mice revealed unexpected phenotypes of increased hepatic triglyceride and decreased fat oxidation. We also observed that chronic ACC inhibition led to hyper-acetylation of proteins in the extra-mitochondrial space. In sum, these data reveal the existence of a compensatory pathway that protects hepatic fat stores when ACC enzymes are inhibited. Furthermore, we identified an important role for ACC enzymes in the regulation of protein acetylation in the extra-mitochondrial space. PMID:24944901

  10. Effects of humic acid-metal complexes on hepatic carnitine palmitoyltransferase, carnitine acetyltransferase and catalase activities

    SciTech Connect

    Fungjou Lu; Youngshin Chen . Dept. of Biochemistry); Tienshang Huang . Dept. of Medicine)

    1994-03-01

    A significant increase in activities of hepatic carnitine palmitoyltransferase and carnitine acetyltransferase was observed in male Balb/c mice intraperitoneally injected for 40 d with 0.125 mg/0.1 ml/d humic acid-metal complexes. Among these complexes, the humic acid-As complex was relatively effective, whereas humic acid-25 metal complex was more effective, and humic acid-26 metal complex was most effective. However, humic acid or metal mixtures, or metal such as As alone, was not effective. Humic acid-metal complexes also significantly decreased hepatic catalase activity. A marked decrease of 60-kDa polypeptide in liver cytoplasm was also observed on SDS-polyacrylamide gel electrophoresis after the mice had been injected with the complexes. Morphological analysis of a histopathological biopsy of such treated mice revealed several changes in hepatocytes, including focal necrosis and cell infiltration, mild fatty changes, reactive nuclei, and hypertrophy. Humic acid-metal complexes affect activities of metabolic enzymes of fatty acids, and this results in accumulation of hydrogen peroxide and increase of the lipid peroxidation. The products of lipid peroxidation may be responsible for liver damage and possible carcinogenesis. Previous studies in this laboratory had shown that humic acid-metal complex altered the coagulation system and that humic acid, per se, caused vasculopathy. Therefore, humic acid-metal complexes may be main causal factors of not only so-called blackfoot disease, but also the liver cancer prevailing on the southwestern coast of Taiwan.

  11. Targeting Cellular Squalene Synthase, an Enzyme Essential for Cholesterol Biosynthesis, Is a Potential Antiviral Strategy against Hepatitis C Virus

    PubMed Central

    Saito, Kyoko; Shirasago, Yoshitaka; Suzuki, Tetsuro; Aizaki, Hideki; Hanada, Kentaro; Wakita, Takaji; Nishijima, Masahiro

    2014-01-01

    ABSTRACT Hepatitis C virus (HCV) exploits host membrane cholesterol and its metabolism for progeny virus production. Here, we examined the impact of targeting cellular squalene synthase (SQS), the first committed enzyme for cholesterol biosynthesis, on HCV production. By using the HCV JFH-1 strain and human hepatoma Huh-7.5.1-derived cells, we found that the SQS inhibitors YM-53601 and zaragozic acid A decreased viral RNA, protein, and progeny production in HCV-infected cells without affecting cell viability. Similarly, small interfering RNA (siRNA)-mediated knockdown of SQS led to significantly reduced HCV production, confirming the enzyme as an antiviral target. A metabolic labeling study demonstrated that YM-53601 suppressed the biosynthesis of cholesterol and cholesteryl esters at antiviral concentrations. Unlike YM-53601, the cholesterol esterification inhibitor Sandoz 58-035 did not exhibit an antiviral effect, suggesting that biosynthesis of cholesterol is more important than that of cholesteryl esters for HCV production. YM-53601 inhibited transient replication of a JFH-1 subgenomic replicon and entry of JFH-1 pseudoparticles, suggesting that at least suppression of viral RNA replication and entry contributes to the antiviral effect of the drug. Collectively, our findings highlight the importance of the cholesterol biosynthetic pathway in HCV production and implicate SQS as a potential target for antiviral strategies against HCV. IMPORTANCE Hepatitis C virus (HCV) is known to be closely associated with host cholesterol and its metabolism throughout the viral life cycle. However, the impact of targeting cholesterol biosynthetic enzymes on HCV production is not fully understood. We found that squalene synthase, the first committed enzyme for cholesterol biosynthesis, is important for HCV production, and we propose this enzyme as a potential anti-HCV target. We provide evidence that synthesis of free cholesterol is more important than that of esterified

  12. Modulating enzyme activity using ionic liquids or surfactants.

    PubMed

    Goldfeder, Mor; Fishman, Ayelet

    2014-01-01

    One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme-water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems. PMID:24281758

  13. Diminution of Hepatic Response to 7, 12-dimethylbenz(α)anthracene by Ethyl Acetate Fraction of Acacia catechu Willd. through Modulation of Xenobiotic and Anti-Oxidative Enzymes in Rats

    PubMed Central

    Kumar, Rakesh; Kaur, Rajbir; Singh, Amrit Pal; Arora, Saroj

    2014-01-01

    Background Liver is the primary metabolizing site of body and is prone to damage by exogenous as well as endogenous intoxicants. Polycyclic aromatic hydrocarbons such as 7, 12- dimethylbenz(α)anthracene (DMBA) is an exogenous hepatotoxin, which is well known for modulating phase I, II and anti-oxidative enzymes of liver. Plants contain plethora of polyphenolic compounds which can reverse the damaging effect of various xenobiotics. The present study investigated protective role of the ethyl acetate fraction of Acacia catechu Willd. (EAF) against DMBA induced alteration in hepatic metabolizing and anti-oxidative enzymes in rats. Methodology and Principal Findings The rats were subjected to hepatic damage by treating with DMBA for 7 weeks on alternative days and treatment schedule was terminated at the end of 14 weeks. The rats were euthanized at the end of protocol and livers were homogenized. The liver homogenates were used to analyse phase I (NADPH-cytochrome P450 reducatse, NADH-cytochrome b5 reductase, cytochrome P420, cytochrome b5), phase II (glutathione-S-transferase, DT diaphorase and γ-Glutamyl transpeptidase) and antioxidative enzymes (catalase, superoxide dismutase, ascorbate peroxidase, glutathione reductase, guiacol peroxidase and lactate dehydrogenase). Furthermore, other oxidative stress parameters (thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes and reduced glutathione) and liver marker enzymes (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and alkaline phosphatase) were also studied. The DMBA induced significant changes in activity of hepatic enzymes that was reversed by treatment with three dose levels of EAF. Conclusion It is concluded that EAF affords hepato-protection against DMBA in rats through modulation of phase I, II and anti-oxidative enzymes. PMID:24587216

  14. Suppression of silent information regulator 1 activity in noncancerous tissues of hepatocellular carcinoma: Possible association with non-B non-C hepatitis pathogenesis

    PubMed Central

    Konishi, Hideyuki; Shirabe, Ken; Nakagawara, Hidekazu; Harimoto, Norifumi; Yamashita, Yo-Ichi; Ikegami, Toru; Yoshizumi, Tomoharu; Soejima, Yuji; Oda, Yoshinao; Maehara, Yoshihiko

    2015-01-01

    Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase. In mice, mSirt1 deficiency causes the onset of fatty liver via regulation of the hepatic nutrient metabolism pathway. In this study, we demonstrate SIRT1 expression, activity and NAD+ regulation using noncancerous liver tissue specimens from hepatocellular carcinoma patients with non-B non-C (NBNC) hepatitis. SIRT1 expression levels were higher in NBNC patients than in healthy donors, while SIRT1 histone H3K9 deacetylation activity was suppressed in NBNC patients. In the liver of hepatitis patients, decreased NAD+ amounts and its regulatory enzyme nicotinamide phosphoribosyltransferase expression levels were observed, and this led to inhibition of SIRT1 activity. SIRT1 expression was associated with HIF1 protein accumulation in both the NBNC liver and liver cancer cell lines. These results may indicate that the NBNC hepatitis liver is exposed to hypoxic conditions. In HepG2 cells, hypoxia induced inflammatory chemokines, such as CXCL10 and MCP-1. These inductions were suppressed in rich NAD+ condition, and by SIRT1 activator treatment. In conclusion, hepatic SIRT1 activity was repressed in NBNC patients, and normalization of NAD+ amounts and activation of SIRT1 could improve the inflammatory condition in the liver of NBNC hepatitis patients. PMID:25736100

  15. Antidiabetic activity of Sedum dendroideum: metabolic enzymes as putative targets for the bioactive flavonoid kaempferitrin.

    PubMed

    Da Silva, Daniel; Casanova, Livia Marques; Marcondes, Mariah Celestino; Espindola-Netto, Jair Machado; Paixão, Larissa Pereira; De Melo, Giany Oliveira; Zancan, Patricia; Sola-Penna, Mauro; Costa, Sônia Soares

    2014-05-01

    The aim of this study was to evaluate the antidiabetic potential of a leaf extract and flavonoids from Sedum dendroideum (SD). Additionally, our goals were to establish a possible structure/activity relationship between these flavonoids and to assess the most active flavonoid on the glycolytic enzyme 6-phosphofructo-1-kinase (PFK). SD juice (LJ), a flavonoid-rich fraction (BF), and separately five flavonoids were evaluated intraperitoneally for their acute hypoglycemic activity in normal and streptozotocin-induced diabetic mice. First, the major flavonoids kaempferol 3,7-dirhamnoside or kaempferitrin (1), kaempferol 3-glucoside-7-rhamnoside (2), and kaempferol 3-neohesperidoside-7-rhamnoside (3) were tested. Then, the monoglycosides kaempferol 7-rhamnoside (5) and kaempferol 3-rhamnoside (6) were assayed to establish their structure/activity relationship. The effect of 1 on PFK was evaluated in skeletal muscle, liver, and adipose tissue from treated mice. LJ (400 mg/kg), BF (40 mg/kg), and flavonoid 1 (4 mg/kg) reduced glycemia in diabetic mice (120 min) by 52, 53, and 61%, respectively. Flavonoids 2, 3, 5, and 6 were inactive or showed little activity, suggesting that the two rhamnosyl moieties in kaempferitrin are important requirements. Kaempferitrin enhanced the PFK activity chiefly in hepatic tissue, suggesting that it is able to stimulate tissue glucose utilization. This result is confirmed testing kaempferitrin on C2C12 cell line, where it enhanced glucose consumption, lactate production, and the key regulatory glycolytic enzymes. The hypoglycemic activity of kaempferitrin depends on the presence of both rhamnosyl residues in the flavonoid structure when intraperitoneally administered. Our findings show for the first time that a flavonoid is capable of stimulating PFK in a model of diabetes and that kaempferitrin stimulates glucose-metabolizing enzymes. This study contributes to the knowledge of the mechanisms by which this flavonoid exerts its hypoglycemic

  16. Interdomain communications in bifunctional enzymes: how are different activities coordinated?

    PubMed

    Nagradova, Natalya

    2003-08-01

    Although bifunctional enzymes containing two different active centers located within separate domains are quite common in living systems, the significance of this bifunctionality is not always clear, and the molecular mechanisms of site-site interactions in such complex systems have come under the scrutiny of science only in recent years. This review summarizes recent data on the mechanisms of communication between active centers in bifunctional enzymes. Three types of enzymes are considered: (1) those catalyzing consecutive reactions of a metabolic pathway and exhibiting substrate channeling (glutamate synthase and imidazole glycerol phosphate synthase), (2) those catalyzing consecutive reactions without substrate channeling (lysine-ketoglutarate reductase/saccharopine dehydrogenase), and (3) those catalyzing opposed reactions (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase). The functional role of interdomain communications is briefly discussed. PMID:14609201

  17. A 19F NMR Study of Enzyme Activity

    NASA Astrophysics Data System (ADS)

    Peterman, Keith E.; Lentz, Kevin; Duncan, Jeffery

    1998-10-01

    This basic enzyme activity laboratory experiment demonstrates how 19F NMR can be used in biochemical studies and presents the advantages of 19F NMR over 1H NMR for studies of this nature. N-Trifluoroacetylglycine was selected as a commercially available model fluorine-tagged substrate that readily undergoes acylase I-catalyzed hydrolysis to produce trifluoroacetic acid and glycine. Progress of the reaction was monitored by following conversion of the trifluoroacetyl moiety peak of N-trifluoroacetylglycine to trifluoroacetic acid. The extent of hydrolysis was determined by comparing integrated ratios of the two 19F NMR peaks. A plot of percent hydrolysis versus enzyme concentration was used to calculate unit activity of the enzyme. This is a viable laboratory experiment for junior/senior-level courses in instrumental analytical chemistry, biochemistry, molecular biology, or spectroscopy.

  18. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity

    PubMed Central

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-01-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe2+ chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods. PMID:26451354

  19. Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay.

    PubMed Central

    Oberhaus, S M; Newbold, J E

    1993-01-01

    Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host. Images PMID:8411359

  20. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    PubMed

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying; Naleway, John Joseph

    2016-01-01

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

  1. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

    PubMed Central

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying

    2016-01-01

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

  2. Effects of adding some dietary fibers to a cystine diet on the activities of liver antioxidant enzymes and serum enzymes in rats.

    PubMed

    He, Guochun; Aoyama, Yoritaka

    2003-03-01

    This study investigates whether some dietary fibers can the toxicity due to cystine added to the diet. Wistar rats were investigated for the effects of adding pectin, sugar beet fiber or konjac mannan to a cystine diet on the growth rate and on the activities of liver antioxidant enzymes and serum enzymes. The addition of pectin, sugar beet fiber or konjac mannan to the cystine diet resulted in a significant increase in both the food intake and body weight gain. Feeding the cystine diet caused lower activities of total and Cu,Zn-superoxide dismutase, and of catalase in the liver. The addition of pectin to the cystine diet counteracted the activities of the total and Cu,Zn-superoxide dismutase, and of catalase in liver. Of the dietary fibers tested, konjac mannan prevented the elevation of the two enzyme activities in the serum induced by feeding the cystine diet, indicating that this fiber might have the ability to alleviate hepatic damage due to dietary cystine. PMID:12723612

  3. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

    Background A common assumption about enzyme active sites is that their structures are highly conserved to specifically distinguish between closely similar compounds. However, with the discovery of distinct enzymes with similar reaction chemistries, more and more studies discussing the structural flexibility of the active site have been conducted. Results Most of the existing works on the flexibility of active sites focuses on a set of pre-selected active sites that were already known to be flexible. This study, on the other hand, proposes an analysis framework composed of a new data collecting strategy, a local structure alignment tool and several physicochemical measures derived from the alignments. The method proposed to identify flexible active sites is highly automated and robust so that more extensive studies will be feasible in the future. The experimental results show the proposed method is (a) consistent with previous works based on manually identified flexible active sites and (b) capable of identifying potentially new flexible active sites. Conclusions This proposed analysis framework and the former analyses on flexibility have their own advantages and disadvantage, depending on the cause of the flexibility. In this regard, this study proposes an alternative that complements previous studies and helps to construct a more comprehensive view of the flexibility of enzyme active sites. PMID:21342563

  4. Enzyme activities by indicator of quality in organic soil

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  5. MICROBIAL COMMUNITY STRUCTURE AND ENZYME ACTIVITIES IN SEMIARID AGRICULTURAL SOILS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of management on the microbial community structure and enzyme activities of three semiarid soils from Southern High Plains of Texas were investigated. The soils (sandy clay loam, fine sandy loam and loam) were under continuous cotton (Gossypium hirsutum L.) or in cotton -peanut (Arachis h...

  6. Carbohydrate active enzymes revealed in Coptotermes formosanus transcriptome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A normalized cDNA library of Coptotermes formosanus was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. Sequencing of this library generated 131,637 EST and 25,939 unigenes were assembled. Carbohydrate active enzymes (CAZymes) revealed in this library we...

  7. Variation in Soil Enzyme Activities in a Temperate Agroforestry Watershed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Integration of agroforestry and grass buffers into row crop watersheds improves overall environmental quality, including soil quality. The objective of this study was to examine management and landscape effects on soil carbon, soil nitrogen, microbial diversity, enzyme activity, and DNA concentrati...

  8. MICROBIAL SUCCESSION AND INTESTINAL ENZYME ACTIVITIES IN THE DEVELOPING RAT

    EPA Science Inventory

    The succession of gastrointestinal flora in the developing rat was studied, concomitant with studies of intestinal enzyme activity. Aerobes and anaerobes were identified as members of 4 major bacterial groups, i.e., Lactobacilli spp., Gram positive enterococci, Gram negative rods...

  9. Hepatoprotective activity of Symplocos racemosa bark on carbon tetrachloride-induced hepatic damage in rats

    PubMed Central

    Wakchaure, Dhananjay; Jain, Dilpesh; Singhai, Abhay Kumar; Somani, Rahul

    2011-01-01

    The present study aims to evaluate the hepatoprotective activity of ethanol extract of Symplocos racemosa (EESR) bark on carbon tetrachloride (CCl4)-induced hepatic damage in rats. CCl4 with olive oil (1 : 1) (0.2 ml/kg, i.p.) was administered for ten days to induce hepatotoxicity. EESR (200 and 400 mg/kg, p.o.) and silymarin (100 mg/kg p.o.) were administered concomitantly for fourteen days. The degree of hepatoprotection was measured using serum transaminases (AST and ALT), alkaline phosphatase, bilirubin, albumin, and total protein levels. Metabolic function of the liver was evaluated by thiopentone-induced sleeping time. Antioxidant activity was assessed by measuring liver malondialdehyde, glutathione, catalase, and superoxide dismutase levels. Histopathological changes of liver sample were also observed. Significant hepatotoxicity was induced by CCl4 in experimental animals. EESR treatment showed significant dose-dependent restoration of serum enzymes, bilirubin, albumin, total proteins, and antioxidant levels. Improvements in hepatoprotection and morphological and histopathological changes were also observed in the EESR treated rats. It was therefore concluded that EESR bark is an effective hepatoprotective agent in CCl4-induced hepatic damage, and has potential clinical applications for treatment of liver diseases. PMID:22022156

  10. Potential enzyme activities in cryoturbated organic matter of arctic soils

    NASA Astrophysics Data System (ADS)

    Schnecker, J.; Wild, B.; Rusalimova, O.; Mikutta, R.; Guggenberger, G.; Richter, A.

    2012-12-01

    An estimated 581 Gt organic carbon is stored in arctic soils that are affected by cryoturbtion, more than in today's atmosphere (450 Gt). The high amount of organic carbon is, amongst other factors, due to topsoil organic matter (OM) that has been subducted by freeze-thaw processes. This cryoturbated OM is usually hundreds to thousands of years old, while the chemical composition remains largely unaltered. It has therefore been suggested, that the retarded decomposition rates cannot be explained by unfavourable abiotic conditions in deeper soil layers alone. Since decomposition of soil organic material is dependent on extracellular enzymes, we measured potential and actual extracellular enzyme activities in organic topsoil, mineral subsoil and cryoturbated material from three different tundra sites, in Zackenberg (Greenland) and Cherskii (North-East Siberia). In addition we analysed the microbial community structure by PLFAs. Hydrolytic enzyme activities, calculated on a per gram dry mass basis, were higher in organic topsoil horizons than in cryoturbated horizons, which in turn were higher than in mineral horizons. When calculated on per gram carbon basis, the activity of the carbon acquiring enzyme exoglucanase was not significantly different between cryoturbated and topsoil organic horizons in any of the three sites. Oxidative enzymes, i.e. phenoloxidase and peroxidase, responsible for degradation of complex organic substances, showed higher activities in topsoil organic and cryoturbated horizons than in mineral horizons, when calculated per gram dry mass. Specific activities (per g C) however were highest in mineral horizons. We also measured actual cellulase activities (by inhibiting microbial uptake of products and without substrate addition): calculated per g C, the activities were up to ten times as high in organic topsoil compared to cryoturbated and mineral horizons, the latter not being significantly different. The total amount of PLFAs, as a proxy for

  11. A Metal-Based Inhibitor of NEDD8-Activating Enzyme

    PubMed Central

    Chan, Daniel Shiu-Hin; Leung, Chung-Hang; Wang, Hui-Min; Ma, Dik-Lung

    2012-01-01

    A cyclometallated rhodium(III) complex [Rh(ppy)2(dppz)]+ (1) (where ppy = 2-phenylpyridine and dppz = dipyrido[3,2-a:2′,3′-c]phenazine dipyridophenazine) has been prepared and identified as an inhibitor of NEDD8-activating enzyme (NAE). The complex inhibited NAE activity in cell-free and cell-based assays, and suppressed the CRL-regulated substrate degradation and NF-κB activation in human cancer cells with potency comparable to known NAE inhibitor MLN4924. Molecular modeling analysis suggested that the overall binding mode of 1 within the binding pocket of the APPBP1/UBA3 heterodimer resembled that for MLN4924. Complex 1 is the first metal complex reported to suppress the NEDDylation pathway via inhibition of the NEDD8-activating enzyme. PMID:23185368

  12. Hepatoprotective and anti-hepatitis C viral activity of Platycodon grandiflorum extract on carbon tetrachloride-induced acute hepatic injury in mice.

    PubMed

    Kim, Tae-Won; Lim, Jong-Hwan; Song, In-Bae; Park, Sang-Jin; Yang, Jae-Won; Shin, Jung Cheul; Suh, Joo-Won; Son, Hwa-Young; Cho, Eun-Sang; Kim, Myoung-Seok; Lee, Sang-Wook; Kim, Jong-Woo; Yun, Hyo-In

    2012-01-01

    The present study aims to evaluate the anti-HCV activity of hotwater extract from Platycodon grandiflorum (BC703) with HCV genotype 1b subgenomic replicon system and investigate its hepatoprotective activity on carbon tetrachloride (CCl(4))-induced acute liver damage in mice. BC703 produced significant hepatoprotective effects against CCl(4)-induced acute hepatic injury by decreasing the activities of serum enzymes, nitric oxide and lipid peroxidation. Histopathological studies further substantiated the protective effect of BC703. Furthermore, BC703 inhibited the HCV RNA replication with an EC(50) value and selective index (CC(50)/EC(50)) of 2.82 µg/mL and above 35.46, respectively. However, digested BC703 using a simulated gastric juice showed poor protective effect against CCl(4)-induced hepatotoxicity in mice and decreased anti-HCV activity as compared to the intact BC703. Although further studies are necessary, BC703 may be a beneficial agent for the management of acute hepatic injury and chronic HCV infection. PMID:22878389

  13. Influence of liver disease and environmental factors on hepatic monooxygenase activity in vitro.

    PubMed

    Brodie, M J; Boobis, A R; Bulpitt, C J; Davies, D S

    1981-01-01

    The effects of liver disease and environmental factors on hepatic microsomal cytochrome P-450 content, NADPH-cytochrome c reductase (reductase) activity and aryl hydrocarbon hydroxylase (AHH) activity have been simultaneously investigated in 70 patients undergoing diagnostic liver biopsy. The activity of reductase was not significantly affected by the presence of liver disease or any of the environmental factors studied. Cytochrome P-450 content decreased with increasing severity of liver disease whereas AHH activity was only significantly reduced in biopsies showing hepatocellular destruction. None of the parameters of monooxygenase activity varied significantly with the age or sex of the patients. Alcohol excess was associated with decreased cytochrome P-450 content and AHH activity and this effect was independent of the histological status of the biopsy. Both high caffeine intake and cigarette smoking increased AHH activity in the absence of any change in cytochrome P-450 content. There was a positive correlation between the number of meat meals eaten per week and cytochrome P-450 content. Chronic treatment with enzyme-inducing anticonvulsants appeared to increase both cytochrome P-450 content and AHH activity. Despite differential effects of liver disease and environmental influences on cytochrome P-450 content and AHH activity there was a highly significant correlation between the two parameters. The results of the present study correlate well with the known effects of disease and environment on drug metabolism in vivo. PMID:7308271

  14. Hepatic Enzyme's Reference Intervals and Their Modulating Factors in Western Indian Population.

    PubMed

    Maksane, Shalini N; Dandekar, Sucheta P; Shukla, Akash; Bhatia, Shobna

    2016-03-01

    The reference intervals (RIs) of serum aminotransferases and Gamma-glutamyl transferase (GGT) have been established many years ago. Recent RIs are not available. The prospective study was conducted to re-evaluate the RIs of liver enzymes and the effect of demographic and anthropometric variables on them in western Indian population. A total of 1059 blood donors comprised the study population. Anthropometry and serum liver enzymes levels were measured. Subjects were categorized into normal weight and overweight by using body mass index (BMI) and waist circumference (WC). For RI determination, non-parametric methodology recommended by IFCC/CLSI was adopted. Mann-Whitney test and Spearman's rank correlation were used for statistical analysis. Upper limit of normal reference value of liver enzymes were lower in female compared to male. (ALT-23.55 F vs 36.00 M, GGT-34.58 F vs 36.20 M) When RI of liver enzymes were calculated according to body mass index, the upper limit of normal of ALT and GGT were higher in overweight group compared to normal weight group. (ALT-38.00 vs 27.00 IU/L and GGT-37.59 vs 35.26 IU/L). In both male and female, liver enzymes correlated significantly with age. WC and BMI were positively correlated with AST, ALT and GGT in both subgroups and the correlation was stronger in male. Demographic factors should be considered for making liver enzyme tests more clinically relevant. Gender based partitioning should be adopted for serum alanine aminotransferase (ALT) and GGT reference values for Western Indian population. PMID:26855497

  15. Functional Integrity of the Chimeric (Humanized) Mouse Liver: Enzyme Zonation, Physiologic Spaces, and Hepatic Enzymes and Transporters.

    PubMed

    Chow, Edwin C Y; Wang, Jason Z Ya; Quach, Holly P; Tang, Hui; Evans, David C; Li, Albert P; Silva, Jose; Pang, K Sandy

    2016-09-01

    Chimeric mouse liver models are useful in vivo tools for human drug metabolism studies; however, liver integrity and the microcirculation remain largely uninvestigated. Hence, we conducted liver perfusion studies to examine these attributes in FRGN [Fah(-/-), Rag2(-/-), and Il2rg(-/-), NOD strain] livers (control) and chimeric livers repopulated with mouse (mFRGN) or human (hFRGN) hepatocytes. In single-pass perfusion studies (2.5 ml/min), outflow dilution profiles of noneliminated reference indicators ((51)Cr-RBC, (125)I-albumin, (14)C-sucrose, and (3)H-water) revealed preservation of flow-limited distribution and reduced water and albumin spaces in hFRGN livers compared with FRGN livers, a view supported microscopically by tightly packed sinusoids. With prograde and retrograde perfusion of harmol (50 µM) in FRGN livers, an anterior sulfation (Sult1a1) over the posterior distribution of glucuronidation (Ugt1a1) activity was preserved, evidenced by the 42% lower sulfation-to-glucuronidation ratio (HS/HG) and 14% higher harmol extraction ratio (E) upon switching from prograde to retrograde flow. By contrast, zonation was lost in mFRGN and hFRGN livers, with HS/HG and E for both flows remaining unchanged. Remnant mouse genes persisted in hFRGN livers (10%-300% those of FRGN). When hFRGN livers were compared with human liver tissue, higher UGT1A1 and MRP2, lower MRP3, and unchanged SULT1A1 and MRP4 mRNA expression were observed. Total Sult1a1/SULT1A1 protein expression in hFRGN livers was higher than that of FRGN livers, consistent with higher harmol sulfate formation. The composite data on humanized livers suggest a loss of zonation, lack of complete liver humanization, and persistence of murine hepatocyte activities leading to higher sulfation. PMID:27342868

  16. CES1 genetic variation affects the activation of angiotensin-converting enzyme inhibitors.

    PubMed

    Wang, X; Wang, G; Shi, J; Aa, J; Comas, R; Liang, Y; Zhu, H-J

    2016-06-01

    The aim of the study was to determine the effect of carboxylesterase 1 (CES1) genetic variation on the activation of angiotensin-converting enzyme inhibitor (ACEI) prodrugs. In vitro incubation study of human liver, intestine and kidney s9 fractions demonstrated that the ACEI prodrugs enalapril, ramipril, perindopril, moexipril and fosinopril are selectively activated by CES1 in the liver. The impact of CES1/CES1VAR and CES1P1/CES1P1VAR genotypes and diplotypes on CES1 expression and activity on enalapril activation was investigated in 102 normal human liver samples. Neither the genotypes nor the diplotypes affected hepatic CES1 expression and activity. Moreover, among several CES1 nonsynonymous variants studied in transfected cell lines, the G143E (rs71647871) was a loss-of-function variant for the activation of all ACEIs tested. The CES1 activity on enalapril activation in human livers with the 143G/E genotype was approximately one-third of that carrying the 143G/G. Thus, some functional CES1 genetic variants (for example, G143E) may impair ACEI activation, and consequently affect therapeutic outcomes of ACEI prodrugs. PMID:26076923

  17. The Flavone Luteolin Suppresses SREBP-2 Expression and Post-Translational Activation in Hepatic Cells

    PubMed Central

    Wong, Tsz Yan; Lin, Shu-mei; Leung, Lai K.

    2015-01-01

    High blood cholesterol has been associated with cardiovascular diseases. The enzyme HMG CoA reductase (HMGCR) is responsible for cholesterol synthesis, and inhibitors of this enzyme (statins) have been used clinically to control blood cholesterol. Sterol regulatory element binding protein (SREBP) -2 is a key transcription factor in cholesterol metabolism, and HMGCR is a target gene of SREBP-2. Attenuating SREBP-2 activity could potentially minimize the expression of HMGCR. Luteolin is a flavone that is commonly detected in plant foods. In the present study, Luteolin suppressed the expression of SREBP-2 at concentrations as low as 1 μM in the hepatic cell lines WRL and HepG2. This flavone also prevented the nuclear translocation of SREBP-2. Post-translational processing of SREBP-2 protein was required for nuclear translocation. Luteolin partially blocked this activation route through increased AMP kinase (AMPK) activation. At the transcriptional level, the mRNA and protein expression of SREBP-2 were reduced through luteolin. A reporter gene assay also verified that the transcription of SREBF2 was weakened in response to this flavone. The reduced expression and protein processing of SREBP-2 resulted in decreased nuclear translocation. Thus, the transcription of HMGCR was also decreased after luteolin treatment. In summary, the results of the present study showed that luteolin modulates HMGCR transcription by decreasing the expression and nuclear translocation of SREBP-2. PMID:26302339

  18. Vanadium chemoprevention of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis: probable involvement of representative hepatic phase I and II xenobiotic metabolizing enzymes.

    PubMed

    Bishayee, A; Oinam, S; Basu, M; Chatterjee, M

    2000-09-01

    Vanadium, a non-platinum group metal and dietary micronutrient, is now proving to act as a promising antitumor agent. The present study was conducted to ascertain its antineoplastic potential against an experimental mammary carcinogenesis. Female Sprague-Dawley rats, at 50 days of age, were treated with 7,12-dimethylbenz(a)anthracene (DMBA) (0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Vanadium (ammonium monovanadate) at the concentration of 0.5 ppm was supplemented in drinking water and given ad libitum to the experimental group immediately after the carcinogen treatment and it continued until the termination of the study (24 weeks for histological and biochemical observations and 35 weeks for morphological findings). It was found that vanadium treatment brought about a substantial protection against DMBA-induced mammary carcinogenesis. This was evident from histological findings that showed no sign of hyperplasia or abnormality after vanadium treatment. There was a significant reduction in incidence (P < 0.05), total number, multiplicity (P < 0.01) and size of palpable mammary tumors and delay in mean latency period of tumor appearance (P < 0.001) following vanadium supplementation compared to DMBA control. From the cumulative results of various hepatic biochemical indices namely, lipid peroxidation, reduced glutathione level, superoxide dismutase activity, cytochrome P450 content and glutathione S-transferase activity, the anticarcinogenic potential of vanadium was well reflected through stabilization of these parameters. Results of the study indicate that the anticarcinogenic activity of vanadium during DMBA-initiated mammary carcinogenesis is mediated through alteration of hepatic antioxidant status as well as modulation of phase I and II drug metabolizing enzymes. On the basis of the observed results, vanadium can be considered as a readily available, promising and novel cancer chemopreventive agent. PMID:11097089

  19. Effect of standardized cranberry extract on the activity and expression of selected biotransformation enzymes in rat liver and intestine.

    PubMed

    Bártíková, Hana; Boušová, Iva; Jedličková, Pavla; Lněničková, Kateřina; Skálová, Lenka; Szotáková, Barbora

    2014-01-01

    The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious. PMID:25237750

  20. Micropollutant degradation via extracted native enzymes from activated sludge.

    PubMed

    Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

    2016-05-15

    A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

  1. [Activity of hydrogen sulfide production enzymes in kidneys of rats].

    PubMed

    Mel'nyk, A V; Pentiuk, O O

    2009-01-01

    An experimental research of activity and kinetic descriptions of enzymes participating in formation of hydrogen sulfide in the kidney of rats has been carried out. It was established that cystein, homocystein and thiosulphate are the basic substrates for hydrogen sulfide synthesis. The higest activity for hydrogen sulfide production belongs to thiosulfate-dithiolsulfurtransferase and cysteine aminotransferase, less activity is characteristic of cystathionine beta-synthase and cystathio-nine gamma-lyase. The highest affinity to substrate is registered for thiosulfate-dithiolsulfurtransferase and cystathionine gamma-lyase. It is discovered that the substrate inhibition is typical of all hydrogen sulfide formation enzymes, although this characteristic is the most expressed thiosulfat-dithiolsulfurtransferase. PMID:20387629

  2. A biologically active surface enzyme assembly that attenuates thrombus formation

    PubMed Central

    Qu, Zheng; Muthukrishnan, Sharmila; Urlam, Murali K.; Haller, Carolyn A.; Jordan, Sumanas W.; Kumar, Vivek A.; Marzec, Ulla M.; Elkasabi, Yaseen; Lahann, Joerg; Hanson, Stephen R.

    2013-01-01

    Activation of hemostatic pathways by blood-contacting materials remains a major hurdle in the development of clinically durable artificial organs and implantable devices. We postulate that surface-induced thrombosis may be attenuated by the reconstitution onto blood contacting surfaces of bioactive enzymes that regulate the production of thrombin, a central mediator of both clotting and platelet activation cascades. Thrombomodulin (TM), a transmembrane protein expressed by endothelial cells, is an established negative regulator of thrombin generation in the circulatory system. Traditional techniques to covalently immobilize enzymes on solid supports may modify residues contained within or near the catalytic site, thus reducing the bioactivity of surface enzyme assemblies. In this report, we present a molecular engineering and bioorthogonal chemistry approach to site-specifically immobilize a biologically active recombinant human TM fragment onto the luminal surface of small diameter prosthetic vascular grafts. Bioactivity and biostability of TM modified grafts is confirmed in vitro and the capacity of modified grafts to reduce platelet activation is demonstrated using a non-human primate model. These studies indicate that molecularly engineered interfaces that display TM actively limit surface-induced thrombus formation. PMID:23532366

  3. Microbial Community Structure and Enzyme Activities in Semiarid Agricultural Soils

    NASA Astrophysics Data System (ADS)

    Acosta-Martinez, V. A.; Zobeck, T. M.; Gill, T. E.; Kennedy, A. C.

    2002-12-01

    The effect of agricultural management practices on the microbial community structure and enzyme activities of semiarid soils of different textures in the Southern High Plains of Texas were investigated. The soils (sandy clay loam, fine sandy loam and loam) were under continuous cotton (Gossypium hirsutum L.) or in rotations with peanut (Arachis hypogaea L.), sorghum (Sorghum bicolor L.) or wheat (Triticum aestivum L.), and had different water management (irrigated or dryland) and tillage (conservation or conventional). Microbial community structure was investigated using fatty acid methyl ester (FAME) analysis by gas chromatography and enzyme activities, involved in C, N, P and S cycling of soils, were measured (mg product released per kg soil per h). The activities of b-glucosidase, b-glucosaminidase, alkaline phosphatase, and arylsulfatase were significantly (P<0.05) increased in soils under cotton rotated with sorghum or wheat, and due to conservation tillage in comparison to continuous cotton under conventional tillage. Principal component analysis showed FAME profiles of these soils separated distinctly along PC1 (20 %) and PC2 (13 %) due to their differences in soil texture and management. No significant differences were detected in FAME profiles due to management practices for the same soils in this sampling period. Enzyme activities provide early indications of the benefits in microbial populations and activities and soil organic matter under crop rotations and conservation tillage in comparison to the typical practices in semiarid regions of continuous cotton and conventional tillage.

  4. Increased serum cortisol binding in chronic active hepatitis

    SciTech Connect

    Orbach, O.; Schussler, G.C.

    1989-01-01

    A high serum cortisol concentration, apparently due to increased cortisol-binding globulin (CBG), was found in a patient (index case) with chronic active hepatitis (CAH). We therefore performed further studies to determine whether increased cortisol binding is generally associated with CAH. Serum samples were obtained from 15 hospitalized patients with long-term liver function test elevations but no evidence of cirrhosis, 15 normal subjects without a history of hepatitis, four healthy pregnant women, and 10 alcoholic patients with stigmata of cirrhosis. Serum cortisol binding was measured by an adaptation of a previously described charcoal uptake method. Thyroxine-binding globulin (TBG) and sex hormone-binding globulin were determined by radioimmunoassays. Charcoal uptake of 125I cortisol from sera of normal subjects and additional patients with CAH revealed that increased serum cortisol binding by a saturable site, presumably CBG, was associated with CAH. Cortisol binding was significantly correlated with immunoassayable TBG, suggesting that in CAH, similar mechanisms may be responsible for increasing the serum concentrations of CBG and TBG.

  5. Influence of environmental static electric field on antioxidant enzymes activities in hepatocytes of mice.

    PubMed

    Wu, S X; Xu, Y Q; Di, G Q; Jiang, J H; Xin, L; Wu, T Y

    2016-01-01

    With the increasing voltage of direct current transmission line, the intensity of the environmental static electric field has also increased. Thus, whether static electric fields cause biological injury is an important question. In this study, the effects of chronic exposure to environmental static electric fields on some antioxidant enzymes activities in the hepatocytes of mice were investigated. Male Institute of Cancer Research mice were exposed for 35 days to environmental static electric fields of different electric field intensities of 9.2-21.85 kV/m (experiment group I, EG-I), 2.3-15.4 kV/m (experiment group II, EG-II), and 0 kV/m (control group, CG). On days 7, 14, 21, and 35 of the exposure cycle, liver homogenates were obtained and the activities of antioxidant enzymes like superoxide dismutase, glutathione S-transferase, and glutathione peroxidase were determined, as well as the concentration of malonaldehyde. The results revealed a significant increase in superoxide dismutase activity in both EG-I and EG-II on the 7th (P < 0.05) and 35th days (P < 0.01) of the exposure cycle compared to that in the control group. However, the other test indices such as glutathione S-transferase, glutathione peroxidase, and malonaldehyde showed only minimal changes during the exposure cycle. These results revealed a weak relationship between the exposure to environmental static electric fields and hepatic oxidative stress in living organisms. PMID:27525865

  6. 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...

  7. Stoichiometry of soil enzyme activity at global scale.

    PubMed

    Sinsabaugh, Robert L; Lauber, Christian L; Weintraub, Michael N; Ahmed, Bony; Allison, Steven D; Crenshaw, Chelsea; Contosta, Alexandra R; Cusack, Daniela; Frey, Serita; Gallo, Marcy E; Gartner, Tracy B; Hobbie, Sarah E; Holland, Keri; Keeler, Bonnie L; Powers, Jennifer S; Stursova, Martina; Takacs-Vesbach, Cristina; Waldrop, Mark P; Wallenstein, Matthew D; Zak, Donald R; Zeglin, Lydia H

    2008-11-01

    Extracellular enzymes are the proximate agents of organic matter decomposition and measures of these activities can be used as indicators of microbial nutrient demand. We conducted a global-scale meta-analysis of the seven-most widely measured soil enzyme activities, using data from 40 ecosystems. The activities of beta-1,4-glucosidase, cellobiohydrolase, beta-1,4-N-acetylglucosaminidase and phosphatase g(-1) soil increased with organic matter concentration; leucine aminopeptidase, phenol oxidase and peroxidase activities showed no relationship. All activities were significantly related to soil pH. Specific activities, i.e. activity g(-1) soil organic matter, also varied in relation to soil pH for all enzymes. Relationships with mean annual temperature (MAT) and precipitation (MAP) were generally weak. For hydrolases, ratios of specific C, N and P acquisition activities converged on 1 : 1 : 1 but across ecosystems, the ratio of C : P acquisition was inversely related to MAP and MAT while the ratio of C : N acquisition increased with MAP. Oxidative activities were more variable than hydrolytic activities and increased with soil pH. Our analyses indicate that the enzymatic potential for hydrolyzing the labile components of soil organic matter is tied to substrate availability, soil pH and the stoichiometry of microbial nutrient demand. The enzymatic potential for oxidizing the recalcitrant fractions of soil organic material, which is a proximate control on soil organic matter accumulation, is most strongly related to soil pH. These trends provide insight into the biogeochemical processes that create global patterns in ecological stoichiometry and organic matter storage. PMID:18823393

  8. [A Case of Severe Chronic Active Epstein-Barr Virus Infection with Aplastic Anemia and Hepatitis].

    PubMed

    Lee, Ja In; Lee, Sung Won; Han, Nam Ik; Ro, Sang Mi; Noh, Yong-Sun; Jang, Jeong Won; Bae, Si Hyun; Choi, Jong Young; Yoon, Seung Kew

    2016-01-25

    Epstein-Barr virus (EBV) causes various acute and chronic diseases. Chronic active EBV infection (CAEBV) is characterized by infectious mononucleosis-like symptoms that persist for more than 6 months with high viral loads in peripheral blood and/or an unusual pattern of anti-EBV antibodies. Severe CAEBV is associated with poor prognosis with severe symptoms, an extremely high EBV-related antibody titer, and hematologic complications that often include hemophagocytic lymphohistiocytosis. However, CAEBV which led to the development of aplastic anemia (AA) has not been reported yet. A 73-year-old woman was admitted to our hospital with intermittent fever, general weakness and elevated liver enzymes. In the serologic test, EBV-related antibody titer was elevated, and real-time quantitative-PCR in peripheral blood showed viral loads exceeding 10(4) copies/μg DNA. Liver biopsy showed characteristic histopathological changes of EBV hepatitis and in situ hybridization with EBV-encoded RNA-1 was positive for EBV. Pancytopenia was detected in peripheral blood, and the bone marrow aspiration biopsy showed hypocellularity with replacement by adipocytes. AA progressed and the patient was treated with prednisolone but deceased 8 months after the diagnosis due to multiple organ failure and opportunistic infection. Herein, we report a rare case of severe CAEBV in an adult patient accompanied by AA and persistent hepatitis. PMID:26809631

  9. Hepatic 5'-monodeiodinase activity in teleosts in vitro: A survey of thirty-three species.

    PubMed

    Leatherland, J F; Reddy, P K; Yong, A N; Leatherland, A; Lam, T J

    1990-01-01

    The in vitro hepatic 5'-monodeiodination of thyroxine (T4) to triiodothyronine (T3) in Oreochromis mossambicus, Channa striata, Clarias batrachus, Cyprinus carpio and Oxyeleotris marmorata was found to be time, pH and temperature dependent, and related to the amount of substrate (T4) and homogenate introduced into the reaction vessel, in a manner which was consistent with Menton-Michaelis kinetics, and thus indicative of an enzyme-regulated process. Dithiothreitol introduced into the reaction vessel stimulated T3 production in a dose-related manner.Hepatic 5'-monodeiodinase activity was also detected in a further 28 species of teleosts suggesting that the peripheral monodeiodination of T4, which is well-documented in salmonids, is also widespread amongst other teleost fishes. All species examined exhibited evidence of enzymatic deiodination, but there were marked differences in Km and Vmax values between the species. There was no apparent phylogenetic or environmental relationships to explain the widely divergent Km and/or Vmax values, nor was there a correlation between Km and Vmax when the species were considered together. PMID:24221892

  10. Activities of N-mineralization enzymes associated with soil aggregates in three different tillage systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil enzymes released by microorganisms play a significant role in N mineralization process that determines N availability for plant growth. Soil aggregates of different sizes provide diverse microhabitats for microorganisms and therefore influence soil enzyme activities. We hypothesize that enzyme ...

  11. Altered Erythrocyte Glycolytic Enzyme Activities in Type-II Diabetes.

    PubMed

    Mali, Aniket V; Bhise, Sunita S; Hegde, Mahabaleshwar V; Katyare, Surendra S

    2016-07-01

    The activity of enzymes of glycolysis has been studied in erythrocytes from type-II diabetic patients in comparison with control. RBC lysate was the source of enzymes. In the diabetics the hexokinase (HK) activity increased 50 % while activities of phosphoglucoisomerase (PGI), phosphofructokinase (PFK) and aldolase (ALD) decreased by 37, 75 and 64 % respectively but were still several folds higher than that of HK. Hence, it is possible that in the diabetic erythrocytes the process of glycolysis could proceed in an unimpaired or in fact may be augmented due to increased levels of G6P. The lactate dehydrogenase (LDH) activity was comparatively high in both the groups; the diabetic group showed 85 % increase. In control group the HK, PFK and ALD activities showed strong positive correlation with blood sugar level while PGI activity did not show any correlation. In the diabetic group only PFK activity showed positive correlation. The LDH activity only in the control group showed positive correlation with marginal increase with increasing concentrations of glucose. PMID:27382204

  12. Hepatitis B virus: DNA polymerase activity of deletion mutants.

    PubMed

    Kim, Y; Hong, Y B; Jung, G

    1999-02-01

    The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain. PMID:10205676

  13. Analysis of enzyme activity regulation by non-denaturing electrophoresis and application of this regulation for enzyme reactor production.

    PubMed

    Shimazaki, Youji; Miki, Shizuka

    2013-10-01

    Non-denaturing electrophoresis can be used to screen enzymes that self-regulate their activities by using a combination of enzymes and their inhibitors. Furthermore, this technique can be applied to develop enzyme reactors that self-regulate their activities. After separation of proteins from mouse liver cytosol by non-denaturing isoelectric focusing, lactate dehydrogense (LDH) and esterase activities were qualitatively and quantitatively examined using a combination of two-dimensional electrophoresis (2-DE) and non-denaturing stacking gel electrophoresis. Activities of mouse liver-derived LDH and carboxylesterase were reversibly inhibited by oxamate and 6,9-diamino-2-ethoxyacridine (acrinol), respectively, in the stacking gels and recovered when the enzymes migrated towards the separation gels. After separation and immobilization of the enzymes, their activities were inhibited by inhibitors and recovered after inhibitor removal. These results indicate that non-denaturing electrophoresis can be applied to select enzymes that self-regulate their activities and subsequently aid in the development of enzyme reactors that can control the enzyme activities. PMID:22803677

  14. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  15. Activation of PPARγ is required for hydroxysafflor yellow A of Carthamus tinctorius to attenuate hepatic fibrosis induced by oxidative stress.

    PubMed

    Wang, C Y; Liu, Q; Huang, Q X; Liu, J T; He, Y H; Lu, J J; Bai, X Y

    2013-05-15

    Oxidative stress caused hepatic fibrosis by activating hepatic stellate cells (HSCs), which were implemented by depressing PPARγ activation. Hydroxysafflor yellow A (HSYA) as a nature active ingredient with antioxidant capacity was able to effectively attenuate oxidative stress mediated injury. So it will be very interesting to study effect of HSYA on HSCs activation and liver fibrosis, and reveal the role of PPARγ·CCl4 and H2O2 were used to mimic oxidative stress mediated hepatic injury in vitro and in vivo respectively. The anti-fibrosis effects of HSYA were evaluated and its mechanisms were disclosed by applying western blot, histopathological analysis, flow cytometry, RT-PCR and ELISA. Our results showed that HSCs activation and proliferation could be induced by oxidative stress, and the expressive levels of TGF-β1 and TIMP-1, the serum levels of ALT, AST, HA, LN, III-C and IV-C were also enhanced by oxidative stress, which is correlated with liver fibrosis (p<0.05 or p<0.01). HSYA was able to effectively inhibit oxidative stress mediated hepatic injury by increasing the activities of antioxidant enzymes, up regulating the expression of PPARγ and MMP-2, and down regulating the expression of TGF-β1 and TIMP-1, and reducing α-SMA level. The protective effect of HSYA can be significantly attenuated by GW9662 via blocking PPARγ (p<0.05 or p<0.01). Taken together, these results demonstrate that HSYA is able to significantly protect the liver from oxidative stress, which requires for HSYA to stimulate PPARγ activity, reduce cell proliferation and suppress ECM synthesis. PMID:23523101

  16. Depsides: Lichen Metabolites Active against Hepatitis C Virus

    PubMed Central

    Vu, Thi Huyen; Le Lamer, Anne-Cécile; Lalli, Claudia; Boustie, Joël; Samson, Michel

    2015-01-01

    A thorough phytochemical study of Stereocaulon evolutum was conducted, for the isolation of structurally related atranorin derivatives. Indeed, pilot experiments suggested that atranorin (1), the main metabolite of this lichen, would interfere with the lifecycle of hepatitis C virus (HCV). Eight compounds, including one reported for the first time (2), were isolated and characterized. Two analogs (5, 6) were also synthesized, to enlarge the panel of atranorin-related structures. Most of these compounds were active against HCV, with a half-maximal inhibitory concentration of about 10 to 70 µM, with depsides more potent than monoaromatic phenols. The most effective inhibitors (1, 5 and 6) were then added at different steps of the HCV lifecycle. Interestingly, atranorin (1), bearing an aldehyde function at C-3, inhibited only viral entry, whereas the synthetic compounds 5 and 6, bearing a hydroxymethyl and a methyl function, respectively, at C-3 interfered with viral replication. PMID:25793970

  17. Controlling the enzymatic activity of a restriction enzyme by light

    PubMed Central

    Schierling, Benno; Noël, Ann-Josée; Wende, Wolfgang; Hien, Le Thi; Volkov, Eugeny; Kubareva, Elena; Oretskaya, Tatiana; Kokkinidis, Michael; Römpp, Andreas; Spengler, Bernhard; Pingoud, Alfred

    2010-01-01

    For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis- or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when cross-linked show the largest “photoswitch effect,” i.e., difference in activity when illuminated with UV vs. blue light, > 30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V max rather than K m. PMID:20080559

  18. Chemoproteomic profiling of host and pathogen enzymes active in cholera.

    PubMed

    Hatzios, Stavroula K; Abel, Sören; Martell, Julianne; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A; Qadri, Firdausi; Ryan, Edward T; Davis, Brigid M; Weerapana, Eranthie; Waldor, Matthew K

    2016-04-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human choleric stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, and genetic disruption of all four proteases increased the abundance of intelectin, an intestinal lectin, and its binding to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting that it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialog in an animal model of infection. PMID:26900865

  19. Exploring the sheep rumen microbiome for carbohydrate-active enzymes.

    PubMed

    Lopes, Lucas Dantas; de Souza Lima, André Oliveira; Taketani, Rodrigo Gouvêa; Darias, Phillip; da Silva, Lília Raquel Fé; Romagnoli, Emiliana Manesco; Louvandini, Helder; Abdalla, Adibe Luiz; Mendes, Rodrigo

    2015-07-01

    The rumen is a complex ecosystem enriched for microorganisms able to degrade biomass during the animal's digestion process. The recovery of new enzymes from naturally evolved biomass-degrading microbial communities is a promising strategy to overcome the inefficient enzymatic plant destruction in industrial production of biofuels. In this context, this study aimed to describe the bacterial composition and functions in the sheep rumen microbiome, focusing on carbohydrate-active enzymes (CAE). Here, we used phylogenetic profiling analysis (inventory of 16S rRNA genes) combined with metagenomics to access the rumen microbiome of four sheep and explore its potential to identify fibrolytic enzymes. The bacterial community was dominated by Bacteroidetes and Firmicutes, followed by Proteobacteria. As observed for other ruminants, Prevotella was the dominant genus in the microbiome, comprising more than 30 % of the total bacterial community. Multivariate analysis of the phylogenetic profiling data and chemical parameters showed a positive correlation between the abundance of Prevotellaceae (Bacteroidetes phylum) and organic matter degradability. A negative correlation was observed between Succinivibrionaceae (Proteobacteria phylum) and methane production. An average of 2 % of the shotgun metagenomic reads was assigned to putative CAE when considering nine protein databases. In addition, assembled contigs allowed recognition of 67 putative partial CAE (NCBI-Refseq) representing 12 glycosyl hydrolase families (Pfam database). Overall, we identified a total of 28 lignocellulases, 22 amylases and 9 other putative CAE, showing the sheep rumen microbiome as a promising source of new fibrolytic enzymes. PMID:25900454

  20. Intestinal detoxification limits the activation of hepatic pregnane X receptor by lithocholic acid.

    PubMed

    Owen, Bryn M; Milona, Alexandra; van Mil, Saskia; Clements, Peter; Holder, Julie; Boudjelal, Mohamed; Cairns, William; Parker, Malcolm; White, Roger; Williamson, Catherine

    2010-01-01

    The intestinal-derived secondary bile acid (BA) lithocholic acid (LCA) is hepatotoxic and is implicated in the pathogenesis of cholestatic diseases. LCA is an endogenous ligand of the xenobiotic nuclear receptor pregnane X receptor (PXR), but there is currently no consensus on the respective roles of hepatic and intestinal PXR in mediating protection against LCA in vivo. Under the conditions reported here, we show that mice lacking Pxr are resistant to LCA-mediated hepatotoxicity. This unexpected phenotype is found in association with enhanced urinary BA excretion and elevated basal expression of drug metabolism enzymes and the hepatic sulfate donor synthesis enzyme Papss2 in Pxr(-/-) mice. By subsequently comparing molecular responses to dietary and intraperitoneal administration of LCA, we made two other significant observations: 1) LCA feeding induces intestinal, but not hepatic, drug-metabolizing enzymes in a largely Pxr-independent manner; and 2) in contrast to LCA feeding, bypassing first-pass gut transit by intraperitoneal administration of LCA did induce hepatic detoxification machinery and in a Pxr-dependent manner. These data reconcile important discrepancies in the reported molecular responses to this BA and suggest that Pxr plays only a limited role in mediating responses to gut-derived LCA. Furthermore, the route of administration must be considered in the future planning and interpretation of experiments designed to assess hepatic responses to BAs, orally administered pharmaceuticals, and dietary toxins. PMID:19797606

  1. Evaluation of pancreatin stability through enzyme activity determination.

    PubMed

    Terra, Gleysson De Paula; Vinícius De Farias, Marcus; Trevisan, Marcello Garcia; Garcia, Jerusa Simone

    2016-09-01

    Pancreatin is a biotechnological product containing an enzyme complex, obtained from porcine pancreas, that is employed in treating pancreatic diseases. Experiments regarding the stability of the pharmaceutical formulation containing pancreatin were performed using standard binary mixtures with 6 excipients in a 1:1 ratio (m/m) and a commercial formulation. To accomplish these goals, samples were stored for 1, 3 and 6 months at 40 ± 1 °C and 75 ± 5 % relative humidity (RH) and 40 ± 1 °C and 0 % RH. Stress testing was also performed. All samples were analyzed to evaluate the α-amylase, lipase and protease activities through UV/Vis spectrophotometry. The results revealed that the excipient proprieties and the storage conditions affected enzyme stability. Humidity was a strong influencing factor in the reduction of α-amylase and protease activities. Stress testing indicated that pH 9.0 and UV light did not induce substantial alterations in enzyme activity. PMID:27383890

  2. In vivo enzyme activity in inborn errors of metabolism

    SciTech Connect

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

    1990-08-01

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

  3. Digestive enzyme activities in larvae of sharpsnout seabream (Diplodus puntazzo).

    PubMed

    Suzer, Cüneyt; Aktülün, Sevim; Coban, Deniz; Okan Kamaci, H; Saka, Sahin; Firat, Kürşat; Alpbaz, Atilla

    2007-10-01

    The ontogenesis and specific activities of pancreatic and intestinal enzymes were investigated in sharpsnout sea bream, Diplodus puntazzo, during larval development until the end of weaning on day 50. The green-water technique was carried out for larval rearing in triplicate. Trypsin was first detected as early as hatching and sharply increased related to age and exogenous feeding until day 25, but a sharp decrease was observed towards the end of the experiment. Amylase was determined 2 days after hatching (DAH) and sharply increased to 10 DAH. Afterwards, slight decreases were found between 10 and 20 DAH and then slow alterations were continued until end of the experiment. Lipase was measured for the first time on day 4, and then slight increase was found to 25 DAH. After this date, slow variations were maintained until end of the experiment. Pepsin was firstly assayed 32 DAH related with stomach formation and sharply increased to 40 DAH. Then it was fluctuated until end of the experiment. Enzymes of brush border membranes, alkaline phosphatase and aminopeptidase N, showed similar pattern on specific activities during the first 10 days. Thereafter, while specific activity of alkaline phosphatase slightly decreased to 15 DAH and fluctuated until 20 DAH, aminopeptidase N activity slowly declined to 20 DAH. Afterwards, activity of alkaline phosphatase and aminopeptidase N were sharply increased to 30 DAH, showing maturation of the intestinal digestive process and also these activities continued to slight increase until end of the experiment. The specific activity of cytosolic peptidase, leucine-alanine peptidase sharply increased to on day 8, then suddenly declined to 12 DAH and further decreased until 20 DAH. After this date, in contrast to enzymes of brush border membranes, it sharply decreased to 25 DAH and continued to gradually decline until the end of the experiment. These converse expressions were indicative of a maturation of enterocytes and the transition to

  4. Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity[S

    PubMed Central

    Jaishy, Bharat; Zhang, Quanjiang; Chung, Heaseung S.; Riehle, Christian; Soto, Jamie; Jenkins, Stephen; Abel, Patrick; Cowart, L. Ashley; Van Eyk, Jennifer E.; Abel, E. Dale

    2015-01-01

    Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs. PMID:25529920

  5. Effects of cigarette smoke and dietary vitamin E levels on selected lung and hepatic biotransformation enzymes in mice

    SciTech Connect

    Graziano, M.J.; Gairola, C.; Dorough, H.W.

    1985-01-01

    Young male C57BL mice were exposed nose-only to cigarette smoke 20 min/day for 8 weeks while maintained on diets containing 0, 5, and 100 ppm of vitamin E. Smoking had no effect on hepatic aryl hydrocarbon hydroxylase (AHH), UDP-glucuronyltransferase, glutathione S-transferase, parathion desulfurase, or parathion esterase activity. Lung AHH activity was increased in all smoke-exposed mice, although the increase was significantly less in animals maintained on the vitamin E-free diet. All mice on the vitamin E-free diet showed reduced lung AHH activity and increased hepatic lipid peroxidation. No other biotransformations tested were significantly altered by varying vitamin E concentrations alone or in combination with cigarette smoke. For all vitamin E diets, both the smoke-exposed and sham-treated mice gained significantly less weight than the control animals. This effect was attributed to stress induced by restraint of the animals within the smoking apparatus. The results of these experiments show that both cigarette smoke and vitamin E-deficient diets may affect xenobiotic metabolism but that the combination does not appear to alter markedly their individual effects or to induce ones not previously observed.

  6. Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.

    PubMed

    Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

    2013-08-01

    Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals. PMID:23761660

  7. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-01-01

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach. PMID:22374998

  8. Elevated Levels of Endocannabinoids in Chronic Hepatitis C May Modulate Cellular Immune Response and Hepatic Stellate Cell Activation

    PubMed Central

    Patsenker, Eleonora; Sachse, Philip; Chicca, Andrea; Gachet, María Salomé; Schneider, Vreni; Mattsson, Johan; Lanz, Christian; Worni, Mathias; de Gottardi, Andrea; Semmo, Mariam; Hampe, Jochen; Schafmayer, Clemens; Brenneisen, Rudolf; Gertsch, Jürg; Stickel, Felix; Semmo, Nasser

    2015-01-01

    The endocannabinoid (EC) system is implicated in many chronic liver diseases, including hepatitis C viral (HCV) infection. Cannabis consumption is associated with fibrosis progression in patients with chronic hepatitis C (CHC), however, the role of ECs in the development of CHC has never been explored. To study this question, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were quantified in samples of HCV patients and healthy controls by gas and liquid chromatography mass spectrometry. Fatty acid amide hydrolase (FAAH) and monoaclyglycerol lipase (MAGL) activity was assessed by [3H]AEA and [3H]2-AG hydrolysis, respectively. Gene expression and cytokine release were assayed by TaqMan PCR and ELISpot, respectively. AEA and 2-AG levels were increased in plasma of HCV patients, but not in liver tissues. Hepatic FAAH and MAGL activity was not changed. In peripheral blood mononuclear cells (PBMC), ECs inhibited IFN-γ, TNF-α, and IL-2 secretion. Inhibition of IL-2 by endogenous AEA was stronger in PBMC from HCV patients. In hepatocytes, 2-AG induced the expression of IL-6, -17A, -32 and COX-2, and enhanced activation of hepatic stellate cells (HSC) co-cultivated with PBMC from subjects with CHC. In conclusion, ECs are increased in plasma of patients with CHC and might reveal immunosuppressive and profibrogenic effects. PMID:25826533

  9. Dimethylnitrosamine metabolism: I. In vitro activation of dimethylnitrosamine to mutagenic substance(s) by hepatic and renal tissues from three inbred strains of mice.

    PubMed

    Ampy, F R; Williams, A O

    1986-09-01

    The potential of hepatic and renal homogenates from three inbred strains of mice (BALB/c, C57BL and DBA) to activate dimethylnitrosamine (DMN) was investigated. Microsomal enzyme (S-9) preparations of liver and kidney from mature and immature mice were used in the Ames Salmonella mutagenicity assay. No age or sex-related differences in the formation of active mutagenic DMN Metabolites by liver microsomal enzymes were observed within any of the three inbred strains. In contrast, mature male kidney S-9 fractions from all three strains had a significantly greater potential to activate DMN than mature female and immature animals. Testosterone treatment resulted in no apparent changes in the ability of hepatic tissue to biotransform DMN to its mutagenic metabolites among age and sex classes. However, after testosterone treatment, renal microsomal fractions from mature female mice of all three strains did not differ significantly from their male counterparts in their ability to transform DMN to mutagenic metabolites. PMID:3747715

  10. Hepatic enzyme changes in bovine hepatogenous photosensitivity caused by water-damaged alfalfa hay.

    PubMed

    Putnam, M R; Qualls, C W; Rice, L E; Dawson, L J; Edwards, W C

    1986-07-01

    In the winter of 1983, practitioners reported extensive photosensitization in 7 herds of cattle. All herds had a history of having been fed water-damaged alfalfa hay. A cow from one herd was referred to the veterinary teaching hospital at Oklahoma State University. In this herd of approximately 40 adult Polled Herefords, all cattle had had some degree of clinical involvement over the past 4 to 6 weeks. Clinical signs included scaling and erythema of sparsely haired skin, muzzle, and teats, as well as icterus, anorexia, and weight loss. One cow died, and the remaining cattle recovered over an 8- to 10-week period after removal of the hay from the ration. In the referred cow, values for total and conjugated bilirubin, BUN, creatinine, sorbitol dehydrogenase, serum alkaline phosphatase, serum aspartate transaminase, and serum gamma-glutamyl transferase were higher than normal. In the herd of origin, extremely high serum gamma-glutamyl transferase values (180 to 1,400 IU/L) persisted (normal, 2 to 35 IU/L). Feeding the same alfalfa hay to 2 clinically normal cows reproduced the syndrome. The characteristic hepatic lesion was bile duct necrosis, with secondary bile duct hyperplasia. PMID:2874123

  11. Substrate-competitive activity-based profiling of ester prodrug activating enzymes

    PubMed Central

    Xu, Hao; Majmudar, Jaimeen D.; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H.; Carlson, Heather A.; Showalter, Hollis D.; Martin, Brent R.; Amidon, Gordon L.

    2015-01-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating pre-clinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a 4-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse, but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design and preclinical

  12. Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats

    PubMed Central

    Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A.; Correig, Xavier; Arola, Lluís; Bladé, Cinta

    2016-01-01

    Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD+) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD+ precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD+. Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD+ availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD+ levels. PMID:27102823

  13. Extracellular enzyme activities and nutrient availability during artificial groundwater recharge.

    PubMed

    Kolehmainen, Reija E; Korpela, Jaana P; Münster, Uwe; Puhakka, Jaakko A; Tuovinen, Olli H

    2009-02-01

    Natural organic matter (NOM) removal is the main objective of artificial groundwater recharge (AGR) for drinking water production and biodegradation plays a substantial role in this process. This study focused on the biodegradation of NOM and nutrient availability for microorganisms in AGR by the determination of extracellular enzyme activities (EEAs) and nutrient concentrations along a flow path in an AGR aquifer (Tuusula Water Works, Finland). Natural groundwater in the same area but outside the influence of recharge was used as a reference. Determination of the specific alpha-d-glucosidase (alpha-Glu), beta-d-glucosidase (beta-Glu), phosphomonoesterase (PME), leucine aminopeptidase (LAP) and acetate esterase (AEST) activities by fluorogenic model substrates revealed major increases in the enzymatic hydrolysis rates in the aquifer within a 10m distance from the basin. The changes in the EEAs along the flow path occurred simultaneously with decreases in nutrient concentrations. The results support the assumption that the synthesis of extracellular enzymes in aquatic environments is up and down regulated by nutrient availability. The EEAs in the basin sediment and pore water samples (down to 10cm) were in the same order of magnitude as in the basin water, suggesting similar nutritional conditions. Phosphorus was likely to be the limiting nutrient at this particular AGR site. Furthermore, the extracellular enzymes functioned in a synergistic and cooperative way. PMID:19028394

  14. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    PubMed

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized. PMID:26139824

  15. Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

    USGS Publications Warehouse

    Buhler, Donald R.; Benville, P.

    1969-01-01

    The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

  16. Hepatic injury induces contrasting response in liver and kidney to chemicals that are metabolically activated: Role of male sex hormone

    SciTech Connect

    Kim, Young C. Yim, Hye K.; Jung, Young S.; Park, Jae H.; Kim, Sung Y.

    2007-08-15

    Injury to liver, resulting in loss of its normal physiological/biochemical functions, may adversely affect a secondary organ. We examined the response of the liver and kidney to chemical substances that require metabolic activation for their toxicities in mice with a preceding liver injury. Carbon tetrachloride treatment 24 h prior to a challenging dose of carbon tetrachloride or acetaminophen decreased the resulting hepatotoxicity both in male and female mice as determined by histopathological examination and increases in serum enzyme activities. In contrast, the renal toxicity of the challenging toxicants was elevated markedly in male, but not in female mice. Partial hepatectomy also induced similar changes in the hepatotoxicity and nephrotoxicity of a challenging toxicant, suggesting that the contrasting response of male liver and kidney was associated with the reduction of the hepatic metabolizing capacity. Carbon tetrachloride pretreatment or partial hepatectomy decreased the hepatic xenobiotic-metabolizing enzyme activities in both sexes but elevated the renal p-nitrophenol hydroxylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase activities significantly only in male mice. Increases in Cyp2e1 and Cyp2b expression were also evident in male kidney. Castration of males or testosterone administration to females diminished the sex-related differences in the renal response to an acute liver injury. The results indicate that reduction of the hepatic metabolizing capacity induced by liver injury may render secondary target organs susceptible to chemical substances activated in these organs. This effect may be sex-specific. It is also suggested that an integrated approach should be taken for proper assessment of chemical hazards.

  17. The ameliorating effects of vitamin E on hepatic antioxidant system and xenobiotic-metabolizing enzymes in fenvalerate-exposed iodine-deficient rats.

    PubMed

    Kocer-Gumusel, Belma; Erkekoglu, Pinar; Caglayan, Aydan; Hincal, Filiz

    2016-07-01

    This study investigated the effects of vitamin E (VE) on hepatic antioxidant system and drug-metabolizing enzymes in fenvalerate (FEN)-exposed iodine-deficient (ID) Wistar rats. ID was produced by perchlorate containing drinking water. VE was introduced by a loading dose of 100 mg/kg/d, i.g. for the first three days in the last week of feeding period; then with a single maintenance dose of 40 mg/kg on the 4th day. During last week, FEN groups (F) received 100 mg/kg/d, i.p. FEN. VE alone did not significantly affect thyroid hormones and antioxidant parameters; however, significantly increased total cytochrome P450 (38%) and cytochrome b5 levels (36%). In all ID groups, plasma thyroid-stimulating hormone (TSH) levels increased markedly, but remained at control level in vitamin E plus FEN receiving iodine-deficient group (IDVF) group. Glutathione peroxidase activity showed marked increases in F (19%) and FEN-exposed iodine-deficient group (IDF, 48%) groups. FEN treatment significantly increased total cytochrome P450 (28%) and thiobarbituric acid reactive substance levels (36%), as well as 7-ethoxyresorufin O-deethylase (120%), 7-penthoxyresorufin O-deethylase (139%) and glutathione S-transferase (15%) activities and decreased total glutathione concentrations (28%) versus control. Overall results suggest that vitamin E has ameliorating effects on the measured parameters in ID and/or FEN exposure. PMID:26446907

  18. Redox regulation of apurinic/apyrimidinic endonuclease 1 activity in Long-Evans Cinnamon rats during spontaneous hepatitis.

    PubMed

    Karmahapatra, Soumendra Krishna; Saha, Tapas; Adhikari, Sanjay; Woodrick, Jordan; Roy, Rabindra

    2014-03-01

    The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson's disease. This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma. Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long-Evans Agouti rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3-phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA. PMID:24337968

  19. Prolidase Enzyme Activity in Conjunctiva and Pterygium Tissues

    PubMed Central

    Yıldırım, Yıldıray; Kaya, Abdullah; Kar, Taner; Muftuoglu, Tuba; Ayata, Ali

    2015-01-01

    Background The aim of this study was to determine prolidase activity in conjunctival tissue and its relationship with pterygium. Material/Methods Prolidase activity was measured in 23 pterygium and 25 healthy conjunctival tissues and the 2 groups were compared. Results Prolidase enzyme activity could not be measured in either the healthy conjunctival or in pterygium tissues. The mean serum prolidase levels of the control and pterygium groups were 967.46±353.64 and 858.29±301.83, respectively. Statistically, there was no significant difference between the groups with regard to serum prolidase levels (p>0.05). Conclusions In conclusion, absence of prolidase activity in pterygium tissue indicates that there is no collagen turnover in this tissue. We may explain this finding with the elastin-rich structure of the conjunctiva. PMID:26509313

  20. Plasma lysosomal enzyme activity in acute myocardial infarction.

    PubMed

    Welman, E; Selwyn, A P; Peters, T J; Colbeck, J F; Fox, K M

    1978-02-01

    N-acetyl-beta-glucosaminidase (EC 3.2.1.30, recommended name beta-N-Acetylglucosaminidase) was found to be a constituent of human cardiac lysosomes. beta-glucuronidase was also found in this tissue, while lysozyme, an enzyme present in leucocyte lysosomes, was not detectable in the heart. The activities of both N-acetyl-beta-glucosaminidase and beta-glucuronidase were elevated in plasma during the first 24 h after the onset of chest pain in patients with acute myocardial infarction and the peak levels of N-acetyl-beta-glucosaminidase correlated well with those of creatine kinase. N-acetyl-beta-glucosaminidase showed a further rise in plasma activity which gave a peak at 72 h after the onset of chest pain and this was accompanied by a rise in lysozyme activity. It is suggested that lysosome disruption caused by myocardial cell necrosis was responsible for the initial rise in plasma lysosomal enzyme activity and that the subsequent inflammatory reaction gave rise to the second peak. PMID:647716

  1. Two azole fungicides (carcinogenic triadimefon and non-carcinogenic myclobutanil) exhibit different hepatic cytochrome P450 activities in medaka fish.

    PubMed

    Lin, Chun-Hung; Chou, Pei-Hsin; Chen, Pei-Jen

    2014-07-30

    Conazoles are a class of imidazole- or triazole-containing drugs commonly used as fungicides in agriculture and medicine. The broad application of azole drugs has led to the contamination of surface aquifers receiving the effluent of municipal or hospital wastewater or agricultural runoff. Several triazoles are rodent carcinogens; azole pollution is a concern to environmental safety and human health. However, the carcinogenic mechanisms associated with cytochrome P450 enzymes (CYPs) of conazoles remain unclear. We exposed adult medaka fish (Oryzias latipes) to continuous aqueous solutions of carcinogenic triadimefon and non-carcinogenic myclobutanil for 7 to 20 days at sub-lethal or environmentally relevant concentrations and assessed hepatic CYP activity and gene expression associated with CYP-mediated toxicity. Both triadimefon and myclobutanil induced hepatic CYP3A activity, but only triadimefon enhanced CYP1A activity. The gene expression of cyp3a38, cyp3a40, pregnane x receptor (pxr), cyp26b, retinoid acid receptor γ1 (rarγ1) and p53 was higher with triadimefon than myclobutanil. As well, yeast-based reporter gene assay revealed that 4 tested conazoles were weak agonists of aryl hydrocarbon receptor (AhR). We reveal differential CYP gene expression with carcinogenic and non-carcinogenic conazoles in a lower vertebrate, medaka fish. Liver CYP-enzyme induction may be a key event in conazole-induced tumorigenesis. This information is essential to evaluate the potential threat of conazoles to human health and fish populations in the aquatic environment. PMID:24962053

  2. Characterization of the endogenous protein kinase activity of the hepatitis B virus.

    PubMed

    Kann, M; Thomssen, R; Köchel, H G; Gerlich, W H

    1993-01-01

    During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles. PMID:8260877

  3. Feed-drug interaction of orally applied butyrate and phenobarbital on hepatic cytochrome P450 activity in chickens.

    PubMed

    Mátis, G; Kulcsár, A; Petrilla, J; Hermándy-Berencz, K; Neogrády, Zs

    2016-08-01

    The expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes may be affected by several nutrition-derived compounds, such as by the commonly applied feed additive butyrate, possibly leading to feed-drug interactions. The aim of this study was to provide some evidence if butyrate can alter the activity of hepatic CYPs in chickens exposed to CYP-inducing xenobiotics, monitoring for the first time the possibility of such interaction. Ross 308 chickens in the grower phase were treated with daily intracoelomal phenobarbital (PB) injection (80 mg/kg BW), applied as a non-specific CYP-inducer, simultaneously with two different doses of intra-ingluvial sodium butyrate boluses (0.25 and 1.25 g/kg BW) for 5 days. Activity of CYP2H and CYP3A subfamilies was assessed by specific enzyme assays from isolated liver microsomes. According to our results, the lower dose of orally administered butyrate significantly attenuated the PB-triggered elevation of both hepatic CYP2H and CYP3A activities, which might be in association with the partly common signalling pathways of butyrate and CYP-inducing drugs, such as that of PB. Based on these data, butyrate may take part in pharmacoepigenetic interactions with simultaneously applied drugs or other CYP-inducing xenobiotics, with possible consequences for food safety and pharmacotherapy. Butyrate was found to be capable to maintain physiological CYP activity by attenuating CYP induction, underlining the safety of butyrate application in poultry nutrition. PMID:26614344

  4. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    PubMed

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs. PMID:27256897

  5. Tissue enzyme activities in the loggerhead sea turtle (Caretta caretta).

    PubMed

    Anderson, Eric T; Socha, Victoria L; Gardner, Jennifer; Byrd, Lynne; Manire, Charles A

    2013-03-01

    The loggerhead sea turtle, Caretta caretta, one of the seven species of threatened or endangered sea turtles worldwide, is one of the most commonly encountered marine turtles off the eastern coast of the United States and Gulf of Mexico. Although biochemical reference ranges have been evaluated for several species of sea turtles, tissue specificity of the commonly used plasma enzymes is lacking. This study evaluated the tissue specificity of eight enzymes, including amylase, lipase, creatine kinase (CK), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in 30 tissues from five stranded loggerhead sea turtles with no evidence of infectious disease. Amylase and lipase showed the greatest tissue specificity, with activity found only in pancreatic samples. Creatine kinase had high levels present in skeletal and cardiac muscle, and moderate levels in central nervous system and gastrointestinal samples. Gamma-glutamyl transferase was found in kidney samples, but only in very low levels. Creatine kinase, ALP, AST, and LDH were found in all tissues evaluated and ALT was found in most, indicating low tissue specificity for these enzymes in the loggerhead. PMID:23505704

  6. The carbohydrate-active enzymes database (CAZy) in 2013.

    PubMed

    Lombard, Vincent; Golaconda Ramulu, Hemalatha; Drula, Elodie; Coutinho, Pedro M; Henrissat, Bernard

    2014-01-01

    The Carbohydrate-Active Enzymes database (CAZy; http://www.cazy.org) provides online and continuously updated access to a sequence-based family classification linking the sequence to the specificity and 3D structure of the enzymes that assemble, modify and breakdown oligo- and polysaccharides. Functional and 3D structural information is added and curated on a regular basis based on the available literature. In addition to the use of the database by enzymologists seeking curated information on CAZymes, the dissemination of a stable nomenclature for these enzymes is probably a major contribution of CAZy. The past few years have seen the expansion of the CAZy classification scheme to new families, the development of subfamilies in several families and the power of CAZy for the analysis of genomes and metagenomes. This article outlines the changes that have occurred in CAZy during the past 5 years and presents our novel effort to display the resolution and the carbohydrate ligands in crystallographic complexes of CAZymes. PMID:24270786

  7. Characterization of cytidylyltransferase enzyme activity through high performance liquid chromatography.

    PubMed

    Brault, James P; Friesen, Jon A

    2016-10-01

    The cytidylyltransferases are a family of enzymes that utilize cytidine 5'-triphosphate (CTP) to synthesize molecules that are typically precursors to membrane phospholipids. The most extensively studied cytidylyltransferase is CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes conversion of phosphocholine and CTP to cytidine diphosphocholine (CDP-choline), a step critical for synthesis of the membrane phospholipid phosphatidylcholine (PC). The current method used to determine catalytic activity of CCT measures production of radiolabeled CDP-choline from (14)C-labeled phosphocholine. The goal of this research was to develop a CCT enzyme assay that employed separation of non-radioactive CDP-choline from CTP. A C18 reverse phase column with a mobile phase of 0.1 M ammonium bicarbonate (98%) and acetonitrile (2%) (pH 7.4) resulted in separation of solutions of the substrate CTP from the product CDP-choline. A previously characterized truncated version of rat CCTα (denoted CCTα236) was used to test the HPLC enzyme assay by measuring CDP-choline product formation. The Vmax for CCTα236 was 3850 nmol/min/mg and K0.5 values for CTP and phosphocholine were 4.07 mM and 2.49 mM, respectively. The HPLC method was applied to glycerol 3-phosphate cytidylyltransferase (GCT) and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively. PMID:27443959

  8. A Computational Methodology to Screen Activities of Enzyme Variants

    PubMed Central

    Hediger, Martin R.; De Vico, Luca; Svendsen, Allan; Besenmatter, Werner; Jensen, Jan H.

    2012-01-01

    We present a fast computational method to efficiently screen enzyme activity. In the presented method, the effect of mutations on the barrier height of an enzyme-catalysed reaction can be computed within 24 hours on roughly 10 processors. The methodology is based on the PM6 and MOZYME methods as implemented in MOPAC2009, and is tested on the first step of the amide hydrolysis reaction catalyzed by the Candida Antarctica lipase B (CalB) enzyme. The barrier heights are estimated using adiabatic mapping and shown to give barrier heights to within 3 kcal/mol of B3LYP/6-31G(d)//RHF/3-21G results for a small model system. Relatively strict convergence criteria (0.5 kcal/(molÅ)), long NDDO cutoff distances within the MOZYME method (15 Å) and single point evaluations using conventional PM6 are needed for reliable results. The generation of mutant structures and subsequent setup of the semiempirical calculations are automated so that the effect on barrier heights can be estimated for hundreds of mutants in a matter of weeks using high performance computing. PMID:23284627

  9. Similarities and Differences in the Expression of Drug-Metabolizing Enzymes between Human Hepatic Cell Lines and Primary Human HepatocytesS⃞

    PubMed Central

    Guo, Lei; Dial, Stacey; Shi, Leming; Branham, William; Liu, Jie; Fang, Jia-Long; Green, Bridgett; Deng, Helen; Kaput, Jim

    2011-01-01

    In addition to primary human hepatocytes, hepatoma cell lines, and transfected nonhepatoma, hepatic cell lines have been used for pharmacological and toxicological studies. However, a systematic evaluation and a general report of the gene expression spectra of drug-metabolizing enzymes and transporters (DMETs) in these in vitro systems are not currently available. To fill this information gap and to provide references for future studies, we systematically characterized the basal gene expression profiles of 251 drug-metabolizing enzymes in untreated primary human hepatocytes from six donors, four commonly used hepatoma cell lines (HepG2, Huh7, SK-Hep-1, and Hep3B), and one transfected human liver epithelial cell line. A large variation in DMET expression spectra was observed between hepatic cell lines and primary hepatocytes, with the complete absence or much lower abundance of certain DMETs in hepatic cell lines. Furthermore, the basal DMET expression spectra of five hepatic cell lines are summarized, providing references for researchers to choose carefully appropriate in vitro models for their studies of drug metabolism and toxicity, especially for studies with drugs in which toxicities are mediated through the formation of reactive metabolites. PMID:21149542

  10. Activity of enzyme immobilized on silanized Co-Cr-Mo.

    PubMed

    Puleo, D A

    1995-08-01

    The surface of an orthopedic biomaterial was modified by the covalent immobilization of biomolecules. Derivatization of Co-Cr-Mo samples with organic and aqueous solutions of gamma-aminopropyltriethoxysilane (APS) resulted in a concentration-dependent number of reactive NH2 groups on the surface available for coupling to protein. The enzyme trypsin was used as a model biomolecule to investigate the effect of immobilization on proteolytic activity. Trypsin was coupled to the silanized samples by formation of Schiff's base linkages via glutaraldehyde. The nature of the interaction between trypsin and biomaterial was then probed by treatment with concentrated guanidine hydrochloride (GuHCl) and urea. Residual activity (following treatment with chaotropic agents) of trypsin immobilized on silanized Co-Cr-Mo was dependent both on the nature of the silane solution and on the type of chaotropic agent. Organic silanization with APS required a minimum density of approximately 49 NH2 per nm2 of nominal surface area (> 0.021 M APS) for residual activity of immobilized trypsin. For aqueous silanization, approximately 5.4 NH2/nm2 (0.51 M APS) resulted in maximal residual trypsin activity. Treatment with GuHCl removed more trypsin activity from Co-Cr-Mo samples silanized with organic solutions of APS than did treatment with urea. On the contrary, with aqueous silanization the samples possessed greater residual activity following treatment with GuHCl than following urea. Compared to simple adsorption with protein onto Co-Cr-Mo, both methods of silanization with APS resulted in superior residual immobilized enzyme activity. PMID:7593038

  11. Global Profiling of Carbohydrate Active Enzymes in Human Gut Microbiome

    PubMed Central

    Mande, Sharmila S.

    2015-01-01

    Motivation Carbohydrate Active enzyme (CAZyme) families, encoded by human gut microflora, play a crucial role in breakdown of complex dietary carbohydrates into components that can be absorbed by our intestinal epithelium. Since nutritional wellbeing of an individual is dependent on the nutrient harvesting capability of the gut microbiome, it is important to understand how CAZyme repertoire in the gut is influenced by factors like age, geography and food habits. Results This study reports a comprehensive in-silico analysis of CAZyme profiles in the gut microbiomes of 448 individuals belonging to different geographies, using similarity searches of the corresponding gut metagenomic contigs against the carbohydrate active enzymes database. The study identifies a core group of 89 CAZyme families that are present across 85% of the gut microbiomes. The study detects several geography/age-specific trends in gut CAZyme repertoires of the individuals. Notably, a group of CAZymes having a positive correlation with BMI has been identified. Further this group of BMI-associated CAZymes is observed to be specifically abundant in the Firmicutes phyla. One of the major findings from this study is identification of three distinct groups of individuals, referred to as 'CAZotypes', having similar CAZyme profiles. Distinct taxonomic drivers for these CAZotypes as well as the probable dietary basis for such trends have also been elucidated. The results of this study provide a global view of CAZyme profiles across individuals of various geographies and age-groups. These results re-iterate the need of a more precise understanding of the role of carbohydrate active enzymes in human nutrition. PMID:26544883

  12. Neferine inhibits cultured hepatic stellate cell activation and facilitates apoptosis: A possible molecular mechanism.

    PubMed

    Ding, Hui; Shi, Jinghong; Wang, Ying; Guo, Jia; Zhao, Juhui; Dong, Lei

    2011-01-10

    Neferine is a major alkaloid component of "Lian Zi Xin", embryos of the seeds of Nelumbo nucifera Gaertner, Nymphaeaceae. Previous studies have shown that neferine has an inhibitory effect on pulmonary fibrosis through its anti-inflammatory and anti-oxidative activities and inhibition of cytokines and NF-κB. However, it is unknown whether neferine also has an inhibitory effect on liver fibrosis through inhibition of TGF-β1 and collagen I and facilitation of apoptosis of hepatic stellate cells. This study examined the effects of neferine on cultured hepatic stellate (HSC-T6) cells and explored its possible action mechanisms by means of MTT assay, enzyme-linked immunosorbent assay, flow-cytometric annexin V-PI assay and Hoechst 33258 staining, as well as real-time PCR and western blotting. The results showed that neferine administration (2, 4, 6, 8 and 10μmol/l) significantly decreased the TGF-β1 and collagen I produced in HSC-T6 cells, and increased the HSC-T6 cell apoptosis in a dose-dependent manner. Neferine treatment for 48h at concentrations of 6 and 10μmol/l significantly increased Bax and caspase 3 mRNAs and proteins, and reduced Bcl2 and alpha-smooth muscle actin (α-SMA) mRNAs and proteins. Our data indicate that neferine efficiently inhibits cultured HSC-T6 cell activation and induces apoptosis by increasing Bax and caspase 3 expression via the mitochondrial pathway. PMID:20969858

  13. Inhibitory effect of oestradiol on activation of rat hepatic stellate cells in vivo and in vitro

    PubMed Central

    Shimizu, I; Mizobuchi, Y; Yasuda, M; Shiba, M; Ma, Y; Horie, T; Liu, F; Ito, S

    1999-01-01

    Background—Hepatic stellate cells play a key role in the pathogenesis of hepatic fibrosis. 
Aims—To examine the inhibitory effect of oestradiol on stellate cell activation. 
Methods—In vivo, hepatic fibrosis was induced in rats by dimethylnitrosamine or pig serum. In vitro, rat stellate cells were activated by contact with plastic dishes resulting in their transformation into myofibroblast-like cells. 
Results—In the dimethylnitrosamine and pig serum models, treatment with oestradiol at gestation related doses resulted in a dose dependent suppression of hepatic fibrosis with restored content of hepatic retinyl palmitate, reduced collagen content, lower areas of stellate cells which express α smooth muscle actin (α-SMA) and desmin, and lower procollagen type I and III mRNA levels in the liver. In cultured stellate cells, oestradiol inhibited type I collagen production, α-SMA expression, and cell proliferation. These findings suggest that oestradiol is a potent inhibitor of stellate cell transformation. 
Conclusion—The antifibrogenic role of oestradiol in the liver may contribute to the sex associated differences in the progression from hepatic fibrosis to cirrhosis. 

 Keywords: hepatic stellate cells; hepatic fibrosis; oestradiol; α smooth muscle actin; retinyl palmitate PMID:9862839

  14. Activation of Hepatic STAT3 Maintains Pulmonary Defense during Endotoxemia

    PubMed Central

    Hilliard, Kristie L.; Allen, Eri; Traber, Katrina E.; Kim, Yuri; Wasserman, Gregory A.; Jones, Matthew R.; Mizgerd, Joseph P.

    2015-01-01

    Pneumonia and infection-induced sepsis are worldwide public health concerns. Both pathologies elicit systemic inflammation and induce a robust acute-phase response (APR). Although APR activation is well regarded as a hallmark of infection, the direct contributions of liver activation to pulmonary defense during sepsis remain unclear. By targeting STAT3-dependent acute-phase changes in the liver, we evaluated the role of liver STAT3 activity in promoting host defense in the context of sepsis and pneumonia. We employed a two-hit endotoxemia/pneumonia model, whereby administration of 18 h of intraperitoneal lipopolysaccharide (LPS; 5 mg/kg of body weight) was followed by intratracheal Escherichia coli (106 CFU) in wild-type mice or those lacking hepatocyte STAT3 (hepSTAT3−/−). Pneumonia alone (without endotoxemia) was effectively controlled in the absence of liver STAT3. Following endotoxemia and pneumonia, however, hepSTAT3−/− mice, with significantly reduced levels of circulating and airspace acute-phase proteins, exhibited significantly elevated lung and blood bacterial burdens and mortality. These data suggested that STAT3-dependent liver responses are necessary to promote host defense. While neither recruited airspace neutrophils nor lung injury was altered in endotoxemic hepSTAT3−/− mice, alveolar macrophage reactive oxygen species generation was significantly decreased. Additionally, bronchoalveolar lavage fluid from this group of hepSTAT3−/− mice allowed greater bacterial growth ex vivo. These results suggest that hepatic STAT3 activation promotes both cellular and humoral lung defenses. Taken together, induction of liver STAT3-dependent gene expression programs is essential to countering the deleterious consequences of sepsis on pneumonia susceptibility. PMID:26216424

  15. Activation of Hepatic STAT3 Maintains Pulmonary Defense during Endotoxemia.

    PubMed

    Hilliard, Kristie L; Allen, Eri; Traber, Katrina E; Kim, Yuri; Wasserman, Gregory A; Jones, Matthew R; Mizgerd, Joseph P; Quinton, Lee J

    2015-10-01

    Pneumonia and infection-induced sepsis are worldwide public health concerns. Both pathologies elicit systemic inflammation and induce a robust acute-phase response (APR). Although APR activation is well regarded as a hallmark of infection, the direct contributions of liver activation to pulmonary defense during sepsis remain unclear. By targeting STAT3-dependent acute-phase changes in the liver, we evaluated the role of liver STAT3 activity in promoting host defense in the context of sepsis and pneumonia. We employed a two-hit endotoxemia/pneumonia model, whereby administration of 18 h of intraperitoneal lipopolysaccharide (LPS; 5 mg/kg of body weight) was followed by intratracheal Escherichia coli (10(6) CFU) in wild-type mice or those lacking hepatocyte STAT3 (hepSTAT3(-/-)). Pneumonia alone (without endotoxemia) was effectively controlled in the absence of liver STAT3. Following endotoxemia and pneumonia, however, hepSTAT3(-/-) mice, with significantly reduced levels of circulating and airspace acute-phase proteins, exhibited significantly elevated lung and blood bacterial burdens and mortality. These data suggested that STAT3-dependent liver responses are necessary to promote host defense. While neither recruited airspace neutrophils nor lung injury was altered in endotoxemic hepSTAT3(-/-) mice, alveolar macrophage reactive oxygen species generation was significantly decreased. Additionally, bronchoalveolar lavage fluid from this group of hepSTAT3(-/-) mice allowed greater bacterial growth ex vivo. These results suggest that hepatic STAT3 activation promotes both cellular and humoral lung defenses. Taken together, induction of liver STAT3-dependent gene expression programs is essential to countering the deleterious consequences of sepsis on pneumonia susceptibility. PMID:26216424

  16. Early activated hepatic stellate cell-derived molecules reverse acute hepatic injury

    PubMed Central

    Chang, Wen-Ju; Song, Lu-Jun; Yi, Tuo; Shen, Kun-Tang; Wang, Hong-Shan; Gao, Xiao-Dong; Li, Min; Xu, Jian-Min; Niu, Wei-Xin; Qin, Xin-Yu

    2015-01-01

    AIM: To test whether hepatic stellate cells (HSCs) at different activation stages play different roles in acetaminophen (APAP)-induced acute liver injury (ALI). METHODS: HSCs were isolated from mouse liver and cultured in vitro. Morphological changes of initiation HSCs [HSCs (5d)] and perpetuation HSCs [HSCs (p3)] were observed by immunofluorescence and transmission electron microscopy. The protective effects of HSC-derived molecules, cell lysates and HSC-conditioned medium (HSC-CM) were tested in vivo by survival and histopathological analyses. Liver injury was determined by measuring aminotransferase levels in the serum and by histologic examination of tissue sections under a light microscope. Additionally, to determine the molecular mediators of the observed protective effects of initiation HSCs, we examined HSC-CM using a high-density protein array. RESULTS: HSCs (5d) and HSCs (p3) had different morphological and phenotypic traits. HSCs (5d) presented a star-shaped appearance with expressing α-SMA at non-uniform levels between cells. However, HSCs (p3) evolved into myofibroblast-like cells without lipid droplets and expressed a uniform and higher level of α-SMA. HSC-CM (5d), but not HSC-CM (p3), provided a significant survival benefit and showed a dramatic reduction of hepatocellular necrosis and panlobular leukocyte infiltrates in mice exposed to APAP. However, this protective effect was abrogated at higher cell masses, indicating a therapeutic window of effectiveness. Furthermore, the protein array screen revealed that HSC-CM (5d) was composed of many chemokines and growth factors that correlated with inflammatory inhibition and therapeutic activity. When compared with HSC-CM (p3), higher levels of monocyte chemoattractant protein-1, macrophage inflammatory protein-1γ, hepatocyte growth factor, interleukin-10, and matrix metalloproteinase-2, but lower levels of stem cell factor and Fas-Ligand were observed in HSC-CM (5d). CONCLUSION: These data indicated

  17. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  18. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, E.E.; Roessler, P.G.

    1999-07-27

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

  19. Glucocerebrosidase enzyme activity in GBA mutation Parkinson's disease.

    PubMed

    Ortega, Roberto A; Torres, Paola A; Swan, Matthew; Nichols, William; Boschung, Sarah; Raymond, Deborah; Barrett, Matthew J; Johannes, Brooke A; Severt, Lawrence; Shanker, Vicki; Hunt, Ann L; Bressman, Susan; Pastores, Gregory M; Saunders-Pullman, Rachel

    2016-06-01

    Mutations in the glucocerebrosidase (GBA1) gene, the most common genetic contributor to Parkinson's disease (PD), are associated with an increased risk of PD in heterozygous and homozygous carriers. While glucocerebrosidase enzyme (GCase) activity is consistently low in Gaucher disease, there is a range of leukocyte GCase activity in healthy heterozygous GBA1 mutation carriers. To determine whether GCase activity may be a marker for PD with heterozygous GBA1 mutations (GBA1 mutation PD, GBA PD), GBA PD patients (n=15) were compared to PD patients without heterozygous GBA1 mutations (idiopathic PD; n=8), heterozygous GBA1 carriers without PD (asymptomatic carriers; n=4), and biallelic mutation carriers with PD (Gaucher disease with PD, GD1 PD; n=3) in a pilot study. GCase activity (nmol/mg protein/hour) in GD1 PD (median [interquartile range]; minimum-maximum: 6.4 [5.7]; 5.3-11) was lower than that of GBA PD (16.0 [7.0]; 11-40) (p=0.01), while GCase activity in GBA PD was lower than idiopathic PD (28.5 [15.0]; 16-56) (p=0.01) and asymptomatic carriers (25.5 [2.5]; 23-27) (p=0.04). Therefore, GCase activity appears to be a possible marker of heterozygous GBA1 mutation PD, and larger studies are warranted. Prospective studies are also necessary to determine whether lower GCase activity precedes development of PD. PMID:26857292

  20. Engineering Enzymes in Energy Crops: Conditionally Activated Enzymes Expressed in Cellulosic Energy Crops

    SciTech Connect

    2010-01-15

    Broad Funding Opportunity Announcement Project: Enzymes are required to break plant biomass down into the fermentable sugars that are used to create biofuel. Currently, costly enzymes must be added to the biofuel production process. Engineering crops to already contain these enzymes will reduce costs and produce biomass that is more easily digested. In fact, enzyme costs alone account for $0.50-$0.75/gallon of the cost of a biomass-derived biofuel like ethanol. Agrivida is genetically engineering plants to contain high concentrations of enzymes that break down cell walls. These enzymes can be “switched on” after harvest so they won’t damage the plant while it’s growing.

  1. Stable Colloidal Drug Aggregates Catch and Release Active Enzymes.

    PubMed

    McLaughlin, Christopher K; Duan, Da; Ganesh, Ahil N; Torosyan, Hayarpi; Shoichet, Brian K; Shoichet, Molly S

    2016-04-15

    Small molecule aggregates are considered nuisance compounds in drug discovery, but their unusual properties as colloids could be exploited to form stable vehicles to preserve protein activity. We investigated the coaggregation of seven molecules chosen because they had been previously intensely studied as colloidal aggregators, coformulating them with bis-azo dyes. The coformulation reduced colloid sizes to <100 nm and improved uniformity of the particle size distribution. The new colloid formulations are more stable than previous aggregator particles. Specifically, coaggregation of Congo Red with sorafenib, tetraiodophenolphthalein (TIPT), or vemurafenib produced particles that are stable in solutions of high ionic strength and high protein concentrations. Like traditional, single compound colloidal aggregates, the stabilized colloids adsorbed and inhibited enzymes like β-lactamase, malate dehydrogenase, and trypsin. Unlike traditional aggregates, the coformulated colloid-protein particles could be centrifuged and resuspended multiple times, and from resuspended particles, active trypsin could be released up to 72 h after adsorption. Unexpectedly, the stable colloidal formulations can sequester, stabilize, and isolate enzymes by spin-down, resuspension, and release. PMID:26741163

  2. Activity of extracellular enzymes on the marine beach differing in the level of antropopressure.

    PubMed

    Perliński, P; Mudryk, Z J

    2016-03-01

    The level of activity of extracellular enzymes was determined on two transects characterised by different anthropic pressure on a sandy beach in Ustka, the southern coast of the Baltic Sea. Generally, the level of activity of the studied enzymes was higher on the transect characterised by high anthropic pressure. The ranking order of the mean enzyme activity rates in the sand was as follows: lipase > phosphatase > aminopeptidase > β-glucosidase > α-glucosidase > chitinase. Each enzyme had its characteristic horizontal profile of activity. The levels of activity of the studied enzymes were slightly higher in the surface than subsurface sand layer. Extracellular enzymatic activities were strongly influenced by the season. PMID:26911592

  3. Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-α converting enzyme as potential therapeutics for hepatitis.

    PubMed

    Wu, Dan; Gu, Qiuhong; Zhao, Ning; Xia, Fei; Li, Zhiwei

    2015-12-01

    The human tumor necrosis factor-α converting enzyme (TACE) has recently been raised as a new and promising therapeutic target of hepatitis and other inflammatory diseases. Here, we reported a successful application of the solved crystal structure of TACE complex with a peptide-like ligand INN for rational design of novel peptide hydroxamic acid inhibitors with high potency and selectivity to target and inhibit TACE. First, the intermolecular interactions between TACE catalytic domain and INN were characterized through an integrated bioinformatics approach, with which the key substructures of INN that dominate ligand binding were identified. Subsequently, the INN molecular structure was simplified to a chemical sketch of peptide hydroxamic acid compound, which can be regarded as a linear tripeptide capped by a N-terminal carboxybenzyl group (chemically protective group) and a C-terminal hydroxamate moiety (coordinated to the Zn(2+) at TACE active site). Based on the sketch, a virtual combinatorial library containing 180 peptide hydroxamic acids was generated, from which seven samples were identified as promising candidates by using a knowledge-based protein-peptide affinity predictor and were then tested in vitro with a standard TACE activity assay protocol. Consequently, three designed peptide hydroxamic acids, i.e. Cbz-Pro-Ile-Gln-hydroxamic acid, Cbz-Leu-Ile-Val-hydroxamic acid and Cbz-Phe-Val-Met-hydroxamic acid, exhibited moderate or high inhibitory activity against TACE, with inhibition constants Ki of 36 ± 5, 510 ± 46 and 320 ± 26 nM, respectively. We also examined the structural basis and non-bonded profile of TACE interaction with a designed peptide hydroxamic acid inhibitor, and found that the inhibitor ligand is tightly buried in the active pocket of TACE, forming a number of hydrogen bonds, hydrophobic forces and van der Waals contacts at the interaction interface, conferring both stability and specificity for TACE-inhibitor complex

  4. Evolution of an Antibiotic Resistance Enzyme Constrained by Stability and Activity Trade-offs

    SciTech Connect

    Wang, Xiaojun; Minasov, George; Shoichet, Brian K.

    2010-03-08

    Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the {beta}-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182 {yields} Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.

  5. Effect of sulfite intake on intestinal enzyme activity in rats.

    PubMed

    Rodriguez Vieytes, M; Martinez-Sapiña, J; Taboada Montero, C; Lamas Aneiros, M

    1994-01-01

    Sulfites are usually added to food, beverages and pharmaceuticals as preservative antioxidants, bleaching agents, and dough conditioning agents. Ingestion of foods containing sulfites can cause abdominal pain, diarrhoea, seizures and death. Sulfite can react with cellular components and can cause toxicity. Changes in mucosal disaccharidases and phosphatase alkaline after sodium metabisulfite administration were investigated in the small intestine of rats. Female Wistar rats were given a diet supplemented with 0.25 or 2.5% sodium metabisulfite for 5 weeks. Sucrase, maltase, lactase and alkaline phosphatase were assayed in intestinal homogenates and in brush border membrane fractions. The intake of only 2.5% sulfite induced an increase in the specific activities of sucrase, maltase, and alkaline phosphatase compared to control levels (P < 0.05). Lactase levels were affected in a variable manner. The origin of such altered enzyme activities is still unknown. PMID:7958644

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

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the "hot-spot" within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

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

    PubMed Central

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

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

    NASA Astrophysics Data System (ADS)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

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

  9. Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis

    PubMed Central

    Li, Jian-ping; Gao, Yan; Chu, Shi-feng; Zhang, Zhao; Xia, Cong-yuan; Mou, Zheng; Song, Xiu-yun; He, Wen-bin; Guo, Xiao-feng; Chen, Nai-hong

    2014-01-01

    Aim: To investigate the anti-fibrosis effects of ginsenoside Rg1 on alcohol- and CCl4-induced hepatic fibrosis in rats and to explore the mechanisms of the effects. Methods: Rats were given 6% alcohol in water and injected with CCl4 (2 mL/kg, sc) twice a week for 8 weeks. Rg1 (10, 20 and 40 mg/kg per day, po) was administered in the last 2 weeks. Hepatic fibrosis was determined by measuring serum biochemical parameters, HE staining, Masson's trichromic staining, and hydroxyproline and α-SMA immunohistochemical staining of liver tissues. The activities of antioxidant enzymes, lipid peroxidation, and Nrf2 signaling pathway-related proteins (Nrf2, Ho-1 and Nqo1) in liver tissues were analyzed. Cultured hepatic stellate cells (HSCs) of rats were prepared for in vitro studies. Results: In the alcohol- and CCl4-treated rats, Rg1 administration dose-dependently suppressed the marked increases of serum ALT, AST, LDH and ALP levels, inhibited liver inflammation and HSC activation and reduced liver fibrosis scores. Rg1 significantly increased the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and reduced MDA levels in liver tissues. Furthermore, Rg1 significantly increased the expression and nuclear translocation of Nrf2 that regulated the expression of many antioxidant enzymes. Treatment of the cultured HSCs with Rg1 (1 μmol/L) induced Nrf2 translocation, and suppressed CCl4-induced cell proliferation, reversed CCl4- induced changes in MDA, GPX, PCIII and HA contents in the supernatant fluid and α-SMA expression in the cells. Knockdown of Nrf2 gene diminished these actions of Rg1 in CCl4-treated HSCs in vitro. Conclusion: Rg1 exerts protective effects in a rat model of alcohol- and CCl4-induced hepatic fibrosis via promoting the nuclear translocation of Nrf2 and expression of antioxidant enzymes. PMID:24976156

  10. Multiple mechanisms regulate circadian expression of the gene for cholesterol 7alpha-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis.

    PubMed

    Noshiro, Mitsuhide; Usui, Emiko; Kawamoto, Takeshi; Kubo, Hiroshi; Fujimoto, Katsumi; Furukawa, Masae; Honma, Sato; Makishima, Makoto; Honma, Ken-ichi; Kato, Yukio

    2007-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A) and sterol 12alpha-hydroxylase (CYP8B) in bile acid biosynthesis and 3-hydroxyl-3-methylglutaryl CoA reductase (HMGCR) in cholesterol biosynthesis are the key enzymes in hepatic metabolic pathways, and their transcripts exhibit circadian expression profiles in rodent liver. The authors determined transcript levels of these enzymes and the regulatory factors for Cyp7a--including Dbp, Dec2, E4bp4, Hnf4alpha, Pparalpha, Lxralpha, Rev-erbalpha, and Rev-erbbeta--in the liver of wild-type and homozygous Clock mutant mice (Clock/Clock) and examined the effects of these transcription factors on the transcription activities of Cyp7a. The expression profile of the Cyp7a transcript in wild-type mice showed a strong circadian rhythm in both the 12L:12D light-dark cycle and constant darkness, and that in Clock/Clock also exhibited a circadian rhythm at an enhanced level with a lower amplitude, although its protein level became arrhythmic at a high level. The expression profile of Cyp8b mRNA in wild-type mice showed a shifted circadian rhythm from that of Cyp7a, becoming arrhythmic in Clock/Clock at an expression level comparable to that of wild-type mice. The expression profile of Hmgcr mRNA also lost its strong circadian rhythm in Clock/Clock , showing an expression level comparable to that of wild-type mice. The expressions of Dbp, Dec2, Rev-erbalpha, and Rev-erb beta--potent regulators for Cyp7a expression--were abolished or became arrhythmic in Clock/Clock, while other regulators for Cyp7a-Lxralpha, Hnf4alpha, Pparalpha, and E4bp4--had either less affected or enhanced expression in Clock/Clock. In luciferase reporter assays, REV-ERBalpha/beta, DBP, LXRalpha, and HNF4alpha increased the promoter activity of Cyp7a, whereas DEC2 abolished the transcription from the Cyp7a promoter: E4BP4 and PPARalpha were moderate negative regulators. Furthermore, knockdown of REV-ERBalpha/beta with siRNA suppressed Cyp7a transcript levels, and in the

  11. Hepatic drug-oxidizing enzyme systems and urinary D-glucaric acid excretion in patients with congestive heart failure.

    PubMed Central

    Tokola, O; Pelkonen, O; Karki, N T; Luoma, P

    1975-01-01

    Drug-oxidizing enzyme systems in liver biopsy samples and the urinary excretion of D-glucaric acid were studied in two different groups of patients with cardiac insufficiency. 2. In one group of six patients, the activities of drug-metabolizing enzymes had decreased considerably as compared with the control values, but in four liver samples from patients treated with oral hypoglycaemic agents for their diabetes, activities were higher than in control samples from ten patients. 3. In the other group of seven patients, the urinary excretion of D-glucaric acid (isolated by ion-exchange chromatography) was 60% lower than in the control group of nine humans, whereas in four patients taking antiepileptic agents excretion rate was higher than control values. 4. Because the age distribution was markedly different between cardiac insufficiency and control groups, it is difficult to conclude, if the impairment of drug metabolism was a consequence of the old age or of the disease process. However, drug-oxidizing enzyme systems seem to be inducible also in old age. 5. The results support further the opinion that the urinary excretion of D-glucaric acid may be one useful index in assessing an individual's capacity to metabolize foreign compounds especially in the patients with lowered drug metabolizing capacity. PMID:786355

  12. Inhibition of hepatic microsomal carboxylesterase activity by paraoxon.

    PubMed

    Castle, M C

    1988-01-01

    A large number of therapeutic agents are esters of carboxylic acids and are thus substrates for microsomal carboxylesterase enzymes. These studies characterized the effects of the organophosphate compound, paraoxon, on the hydrolysis of several drug esters (procaine, chloramphenicol succinate, prednisolone succinate, lidocaine, procainamide and methylparaben) by microsomal preparations from guinea-pigs. These investigations demonstrate that carboxylesterase activity toward several drug esters is present in liver, lung and kidney. The liver is by far the major site of hydrolysis of these ester compounds. Since no hydrolysis was observed with the two amide esters, the hydrolysis of carboxylesters and amide esters appears to be mediated by different enzymes in the guinea-pig. At the substrate concentrations studied, the hydrolysis of methylparaben followed zero-order kinetics. When added to isolated microsomal preparations, paraoxon produced a dose-dependent inhibition of hydrolysis of all substrates. Administration of paraoxon to guinea-pigs prior to isolation of microsomes did not produce consistent effects with any substrate. Inhibition of ester hydrolysis was observed with some pretreatments, while either no change or increased hydrolysis was observed with other pretreatment regimens. PMID:3245748

  13. County-Scale Spatial Distribution of Soil Enzyme Activities and Enzyme Activity Indices in Agricultural Land: Implications for Soil Quality Assessment

    PubMed Central

    Xie, Baoni; Wang, Junxing; He, Wenxiang; Wang, Xudong; Wei, Gehong

    2014-01-01

    Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km2) scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI) and the geometric mean of enzyme activities (GME). At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality. PMID:25610908

  14. Hepatitis C Virus Translation Preferentially Depends on Active RNA Replication

    PubMed Central

    Liu, Helene Minyi; Aizaki, Hideki; Machida, Keigo; Ou, J.-H. James; Lai, Michael M. C.

    2012-01-01

    Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome. PMID:22937067

  15. Effect of Aerobic and Resistance Exercise Training on Liver Enzymes and Hepatic Fat in Iranian Men With Nonalcoholic Fatty Liver Disease

    PubMed Central

    Shamsoddini, Alireza; Sobhani, Vahid; Ghamar Chehreh, Mohammad Ebrahim; Alavian, Seyed Moayed; Zaree, Ali

    2015-01-01

    Background: Nonalcoholic fatty liver disease (NAFLD) has different prevalence rates in various parts of the world and is a risk factor for diabetes and cardiovascular disease that could progress to nonalcoholic steatohepatitis, cirrhosis, and liver failure. Objectives: The current study aimed to investigate the effect of Aerobic Training (AT) and resistance training (RT) on hepatic fat content and liver enzyme levels in Iranian men. Patients and Methods: In a randomized clinical trial study, 30 men with clinically defined NAFLD were allocated into three groups (aerobic, resistance and control). An aerobic group program consisted of 45 minutes of aerobic exercise at 60% - 75% maximum heart rate intensity, a resistance group performed seven resistance exercises at intensity of 50% - 70% of 1 repetition maximum (1RM ) and the control group had no exercise training program during the study. Before and after training, anthropometry, insulin sensitivity, liver enzymes and hepatic fat were elevated. Results: After training, hepatic fat content was markedly reduced, to a similar extent, in both the aerobic and resistance exercise training groups (P ≤ 0.05). In the two exercise training groups, alanine amino transferase and aspartate amino transferase serum levels were significantly decreased compared to the control group (P = 0.002) and (P = 0.02), respectively. Moreover, body fat (%), fat mass (kg), homeostasis model assessment insulin resistance (HOMI-IR) were all improved in the AT and RT. These changes in the AT group were independent of weight loss. Conclusions: This study demonstrated that RT and AT are equally effective in reducing hepatic fat content and liver enzyme levels among patients with NAFLD. However, aerobic exercise specifically improves NAFLD independent of any change in body weight. PMID:26587039

  16. The Hepatoselective Glucokinase Activator PF-04991532 Ameliorates Hyperglycemia without Causing Hepatic Steatosis in Diabetic Rats

    PubMed Central

    Erion, Derek M.; Lapworth, Amanda; Amor, Paul A.; Bai, Guoyun; Vera, Nicholas B.; Clark, Ronald W.; Yan, Qingyun; Zhu, Yimin; Ross, Trenton T.; Purkal, Julie; Gorgoglione, Matthew; Zhang, Guodong; Bonato, Vinicius; Baker, Levenia; Barucci, Nicole; D’Aquila, Theresa; Robertson, Alan; Aiello, Robert J.; Yan, Jiangli; Trimmer, Jeff; Rolph, Timothy P.; Pfefferkorn, Jeffrey A.

    2014-01-01

    Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase

  17. ARGINASE 2 DEFICIENCY RESULTS IN SPONTANEOUS STEATOHEPATITIS: A NOVEL LINK BETWEEN INNATE IMMUNE ACTIVATION AND HEPATIC DE NOVO LIPOGENESIS

    PubMed Central

    Navarro, Laura A.; Wree, Alexander; Povero, Davide; Berk, Michael P.; Eguchi, Akiko; Ghosh, Sudakshina; Papouchado, Bettina G.; Erzurum, Serpil C.; Feldstein, Ariel E.

    2016-01-01

    BACKGROUND & AIMS Innate immune activation has been postulated as a central mechanism for disease progression from hepatic steatosis to steatohepatitis in obesity-related fatty liver disease. Arginase 2 competes with inducible nitric oxide synthase (iNOS) for its substrate and the balance between these two enzymes plays a crucial role in regulating immune responses and macrophage activation. Our aim was to test the hypothesis that arginase 2 deficiency in mice favors progression from isolated hepatic steatosis, induced by high fat feeding to steatohepatitis. METHODS Arginase 2-knockout (Arg2−/−) mice were studied for changes in liver histology and metabolic phenotype at baseline and after a short term course (7 week) feeding with a high fat (HFAT) diet. In additional experiments, Arg2−/− mice received tail vein injections of liposome-encapsulated clodronate (CLOD) over a three-week period to selectively deplete liver macrophages. RESULTS Unexpectedly, Arg2−/− mice showed profound changes in their livers at baseline characterized by significant steatosis as demonstrated with histological and biochemical analysis. These changes were independent of systemic metabolic parameters and associated with marked increase mRNA levels of genes involved in hepatic de novo lipogenesis. Liver injury and inflammation were present with elevated serum ALT, marked infiltration of F4/80 positive cells, and increased mRNA levels of inflammatory genes. HFAT feeding exacerbated these changes. Macrophage depletion after CLOD injection significantly attenuated lipid deposition and normalized lipogenic mRNA profile of livers from Arg2−/− mice. CONCLUSIONS This study identifies arginase 2 as novel link between innate immune responses, hepatic lipid deposition, and liver injury. PMID:25234945

  18. PP2A inhibition results in hepatic insulin resistance despite Akt2 activation.

    PubMed

    Galbo, Thomas; Perry, Rachel J; Nishimura, Erica; Samuel, Varman T; Quistorff, Bjørn; Shulman, Gerald I

    2013-10-01

    In the liver, insulin suppresses hepatic gluconeogenesis by activating Akt, which inactivates the key gluconeogenic transcription factor FoxO1 (Forkhead Box O1). Recent studies have implicated hyperactivity of the Akt phosphatase Protein Phosphatase 2A (PP2A) and impaired Akt signaling as a molecular defect underlying insulin resistance. We therefore hypothesized that PP2A inhibition would enhance insulin-stimulated Akt activity and decrease glucose production. PP2A inhibitors increased hepatic Akt phosphorylation and inhibited FoxO1in vitro and in vivo, and suppressed gluconeogenesis in hepatocytes. Paradoxically, PP2A inhibition exacerbated insulin resistance in vivo. This was explained by phosphorylation of both hepatic glycogen synthase (GS) (inactivation) and phosphorylase (activation) resulting in impairment of glycogen storage. Our findings underline the significance of GS and Phosphorylase as hepatic PP2A substrates and importance of glycogen metabolism in acute plasma glucose regulation. PMID:24150286

  19. Assays for Hepatitis B Virus DNA-and RNA-Dependent DNA Polymerase Activities.

    PubMed

    Shaw, T; Locarnini, S A

    2000-01-01

    Genomes of the hepatitis B viruses (HBVs) consist of approx 3.2 kb of partly double-stranded DNA containing three or four overlapping open reading frames, the largest of which encodes the viral polymerase (Pol) protein. After entry into the cell and uncoating, the viral genome is transported to the nucleus where it is converted into a covalently closed circular (CCC) or supercoiled molecule by cellular repair mechanisms. The viral CCC DNA is transcribed, presumably by host cell RNA polymerase II, into unspliced, capped polyadenylated mRNA species from which viral proteins are transcribed. In addition, terminally redundant 3.5-kb RNA transcripts, which function as pregenomes, are produced and exported to the cytoplasm where they are packaged into viral core particles in which reverse transcription, pregenome degradation, and duplication occurs, reproducing the partly double-stranded HBV genome (for recent review, see ref. 1). Besides its essential role in HBV genome replication, HBV Pol is also involved in virus assembly, and because hepadnaviruses do not encode enzymes functionally equivalent to deoxynucleoside kinases (2), functions associated with HBV Pol are probably the only virus-specific targets for antiviral activity of nucleoside analogs. In vitro assays for inhibition of HBV Pol functions by deoxynucleoside triphosphate (dNTP) analogs are useful indicators but, because of restrictions imposed by hepatocyte enzymology, provide no guarantee of potential anti-HBV activity of the parent (deoxy)nucleoside analogs in intact cells (2). PMID:21331902

  20. Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy.

    PubMed

    Guo, Qing; He, Yufan; Lu, H Peter

    2015-11-10

    Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure-function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2-amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme-substrate interactions, enzyme-substrate active complex formation, and protein folding-binding interactions. PMID:26512103

  1. Fisetin improves glucose homeostasis through the inhibition of gluconeogenic enzymes in hepatic tissues of streptozotocin induced diabetic rats.

    PubMed

    Prasath, Gopalan Sriram; Pillai, Subramanian Iyyam; Subramanian, Sorimuthu Pillai

    2014-10-01

    Liver plays a vital role in blood glucose homeostasis. Recent studies have provided considerable evidence that hepatic glucose production (HGP) plays an important role in the development of fasting hyperglycemia in diabetes. From this perspective, diminution of HGP has certainly been considered for the treatment of diabetes. In the present study, we have analyzed the modulatory effects of fisetin, a flavonoid of strawberries, on the expression of key enzymes of carbohydrate metabolism in STZ induced experimental diabetic rats. The physiological criterions such as food and fluid intake were regularly monitored. The levels of blood glucose, plasma insulin, hemoglobin and glycosylated hemoglobin were analyzed. The mRNA and protein expression levels of gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were determined by immunoblot as well as PCR analysis. Diabetic group of rats showed significant increase in food and water intake when compared with control group of rats. Upon oral administration of fisetin as well as gliclazide to diabetic group of rats, the levels were found to be decreased. Oral administration of fisetin (10 mg/kg body weight) to diabetic rats for 30 days established a significant decline in blood glucose and glycosylated hemoglobin levels and a significant increase in plasma insulin level. The mRNA and protein expression levels of gluconeogenic genes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), were decreased in liver tissues upon treatment with fisetin. The results of the present study suggest that fisetin improves glucose homeostasis by direct inhibition of gluconeogenesis in liver. PMID:25064342

  2. Diurnal Variations of Mouse Plasma and Hepatic Bile Acid Concentrations as well as Expression of Biosynthetic Enzymes and Transporters

    PubMed Central

    Zhang, Yu-Kun Jennifer; Guo, Grace L.; Klaassen, Curtis D.

    2011-01-01

    Background Diurnal fluctuation of bile acid (BA) concentrations in the enterohepatic system of mammals has been known for a long time. Recently, BAs have been recognized as signaling molecules beyond their well-established roles in dietary lipid absorption and cholesterol homeostasis. Methods and Results The current study depicted diurnal variations of individual BAs detected by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) in serum and livers collected from C57BL/6 mice fed a regular chow or a chow containing cholestyramine (resin). Circadian rhythms of mRNA of vital BA-related nuclear receptors, enzymes, and transporters in livers and ilea were determined in control- and resin-fed mice, as well as in farnesoid X receptor (FXR) null mice. The circadian profiles of BAs showed enhanced bacterial dehydroxylation during the fasting phase and efficient hepatic reconjugation of BAs in the fed phase. The resin removed more than 90% of BAs with β-hydroxy groups, such as muricholic acids and ursodeoxycholic acid, from serum and livers, but did not exert as significant influence on CA and CDCA in both compartments. Both resin-fed and FXR-null mouse models indicate that BAs regulate their own biosynthesis through the FXR-regulated ileal fibroblast growth factor 15. BA flux also influences the daily mRNA levels of multiple BA transporters. Conclusion BA concentration and composition exhibit circadian variations in mouse liver and serum, which influences the circadian rhythms of BA metabolizing genes in liver and ileum. The diurnal variations of BAs appear to serve as a signal that coordinates daily nutrient metabolism in mammals. PMID:21346810

  3. Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children.

    PubMed Central

    Yaginuma, K; Kobayashi, H; Kobayashi, M; Morishima, T; Matsuyama, K; Koike, K

    1987-01-01

    Attention was directed to hepatitis B virus (HBV) integration in tissues obtained from an hepatocellular carcinoma (HCC) of an 11-year-old boy and from the liver of his 6-year-old brother, who had chronic active hepatitis. Multiple HBV DNA integration sites were demonstrated in both tissues. Cell population(s) in the HCC and liver from the patient with chronic active hepatitis were assumed to be heterogeneous with regard to HBV integration. The integrated forms in the two tissues showed similar genetic organization without gross rearrangement. The location of one of the virus-chromosomal junctions was restricted to the 5'-end region of the minus-strand DNA of HBV. The experimental results support our previous model for the mechanism of HBV integration, in which minus-strand replicative intermediates integrate into chromosomal DNA. The integrated HBV DNAs were conserved in the same region of the viral genome, spanning from the C gene through the S gene to the X gene, which contains intrinsic promoter-enhancer sequences. Images PMID:3033312

  4. Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity

    PubMed Central

    Kellogg, Joshua; Grace, Mary H.; Lila, Mary Ann

    2014-01-01

    Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose (IC50 of 112.0 and 137.8 μg/mL, respectively). The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS) was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia. PMID:25341030

  5. Bacterial communities and enzyme activities of PAHs polluted soils.

    PubMed

    Andreoni, V; Cavalca, L; Rao, M A; Nocerino, G; Bernasconi, S; Dell'Amico, E; Colombo, M; Gianfreda, L

    2004-11-01

    Three soils (i.e. a Belgian soil, B-BT, a German soil, G, and an Italian agricultural soil, I-BT) with different properties and hydrocarbon-pollution history with regard to their potential to degrade phenanthrene were investigated. A chemical and microbiological evaluation of soils was done using measurements of routine chemical properties, bacterial counts and several enzyme activities. The three soils showed different levels of polycyclic aromatic hydrocarbons (PAHs), being their contamination strictly associated to their pollution history. High values of enzyme activities and culturable heterotrophic bacteria were detected in the soil with no or negligible presence of organic pollutants. Genetic diversity of soil samples and enrichment cultures was measured as bands on denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil and enrichment community DNAs. When analysed by Shannon index (H'), the highest genetic biodiversity (H'=2.87) was found in the Belgian soil B-BT with a medium-term exposition to PAHs and the poorest biodiversity (H'=0.85) in the German soil with a long-term exposition to alkanes and PAHs and where absence, or lower levels of enzyme activities were measured. For the Italian agricultural soil I-BT, containing negligible amounts of organic pollutants but the highest Cu content, a Shannon index=2.13 was found. The enrichment of four mixed cultures capable of degrading solid phenanthrene in batch liquid systems was also studied. Phenanthrene degradation rates in batch systems were culture-dependent, and simple (one-slope) and complex (two-slope) kinetic behaviours were observed. The presence of common bands of microbial species in the cultures and in the native soil DNA indicated that those strains could be potential in situ phenanthrene degraders. Consistent with this assumption are the decrease of PAH and phenanthrene contents of Belgian soil B-BT and the isolation of phenanthrene-degrading bacteria. From

  6. [Polyclonal activation due to Epstein-Barr virus superinfection in a case with chronic hepatitis B].

    PubMed

    Bakir Ozbey, Saliha; Mistik, Reşit; Gürcüoğlu, Emel; Oral, Barbaros; Göral, Güher

    2007-10-01

    Primary infection with Epstein-Barr virus (EBV) often occurs subclinically during childhood, resulting in a latent infection of B lymphocytes. In this report, a chronic hepatitis B case who presented with a serologic profile mimicking acute hepatitis B virus (HBV) infection and exhibiting transient autoantibody positivities because of the polyclonal activation of B cells due to EBV reactivation has been presented. The test results of 56 years old male patient who suffered from fatigue and pain on the right upper quadrant, revealed high levels of liver enzymes (AST: 187 U/L, ALT: 569 U/L), positivity of HBsAg, anti-HBc IgG and anti-HBe, and negativity of anti-HBc IgM, HBeAg and anti-HBs. Since HBV-DNA level was found 405,974 copies/mL by quantitative real time polymerase chain reaction (PCR), the patient was taken into follow-up. At the 6th month AST and ALT levels further elevated (352 U/L and 609 U/L, respectively), and anti-HBc IgM and anti-HBs became positive in addition to the previous positive markers of HBV. With the suspicion of superinfection, further laboratory investigations yielded negative results in CMV-IgM and Paul Bunnel test, while positive results in EBV anti-VCA IgM and IgG, anti-EBNA IgM and IgG, anti-p22 IgM and IgG and anti-EA IgM. In the follow-up period high levels of autoantibody positivities [rheumatoid factor (42.200 U/ml), anti-nuclear antibody (1/100) and anti-Ro-52] together with increased levels of total IgG, IgM and IgA were detected. In the following months, the levels of transaminases, total immunoglobulins and HBV-DNA have distinctively decreased, and in the 20th month the previous HBV profile regained (HBsAg, anti-HBc IgG and anti-HBe positive, anti-HBc IgM and anti-HBs negative, HBV-DNA: 6984 copies/ml) and the other pathological test results returned to normal. As a result, ALT increases seen during the course of chronic hepatitis B should not always be considered as HBV manifestations and the unusual serologic patterns should

  7. Effect of clofibrate on the enzyme activity of rat liver plasma membranes.

    PubMed

    Renaud, G; Foliot, A; Marais, J; Infante, R

    1980-03-15

    The activity of 3 plasma membranes marker enzymes (5'-nucleotidase, Mg++-ATPase and alkaline phosphodiesterase-I) was determined in plasma membranes isolated from liver of control and of clofibrate-treated rats. A complete indentity of plasma membranes enzyme activity in the 2 groups of experimental animals was observed for the 3 enzymes studied. PMID:6102923

  8. Effects of Fertilization on Tomato Growth and Soil Enzyme Activity

    NASA Astrophysics Data System (ADS)

    Mu, Zhen; Hu, Xue-Feng; Cheng, Chang; Luo, Zhi-qing

    2015-04-01

    To study the effects of different fertilizer applications on soil enzyme activity, tomato plant growth and tomato yield and quality, a field experiment on tomato cultivation was carried out in the suburb of Shanghai. Three fertilizer treatments, chemical fertilizer (CF) (N, 260 g/kg; P, 25.71g/kg; K, 83.00g/kg), rapeseed cake manure (CM) (N, 37.4 g/kg; P, 9.0 g/kg; K, 8.46 g/kg), crop-leaf fermenting manure (FM) (N, 23.67 g/kg; P, 6.39 g/kg; K 44.32 g/kg), and a control without using any fertilizers (CK), were designed. The total amounts of fertilizer application to each plot for the CF, CM, FM and CK were 0.6 kg, 1.35 kg, 3.75 kg and 0 kg, respectively, 50% of which were applied as base fertilizer, and another 50% were applied after the first fruit picking as top dressing. Each experimental plot was 9 m2 (1 m × 9 m) in area. Each treatment was replicated for three times. No any pesticides and herbicides were applied during the entire period of tomato growth to prevent their disturbance to soil microbial activities. Soil enzyme activities at each plot were constantly tested during the growing period; the tomato fruit quality was also constantly analyzed and the tomato yield was calculated after the final harvesting. The results were as follows: (1) Urease activity in the soils treated with the CF, CM and FM increased quickly after applying base fertilizer. That with the CF reached the highest level. Sucrase activity was inhibited by the CF and CM to some extent, which was 32.4% and 11.2% lower than that with the CK, respectively; while that with the FM was 15.7% higher than that with the CK. Likewise, catalase activity with the CF increased by 12.3% - 28.6%; that with the CM increased by 87.8% - 95.1%; that with the FM increased by 86.4% - 93.0%. Phosphatase activity with the CF increased rapidly and reached a maximum 44 days after base fertilizer application, and then declined quickly. In comparison, that with the CM and FM increased slowly and reached a maximum

  9. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation.

    PubMed

    Dzamukova, Maria R; Naumenko, Ekaterina A; Lvov, Yuri M; Fakhrullin, Rawil F

    2015-01-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

  10. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    PubMed Central

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-01-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

  11. Mobilization of hepatic calcium pools by platelet activating factor

    SciTech Connect

    Lapointe, D.S.; Hanahan, D.J.; Olson, M.S.

    1987-03-24

    In the perfused rat liver, platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), infusion produces an extensive but transient glycogenolytic response which at low AGEPC concentrations is markedly dependent upon the perfusate calcium levels. The role of calcium in the glycogenolytic response of the liver to AGEPC was investigated by assessing the effect of AGEPC on various calcium pools in the intact liver. Livers from fed rats were equilibrated with /sup 45/Ca/sup 2 +/, and the kinetics of /sup 45/Ca/sup 2 +/ efflux were determined in control, AGEPC-stimulated, and phenylephrine-stimulated livers during steady-state washout of /sup 45/Ca/sup 2 +/. AGEPC treatment had only a slight if any effect on the pattern of steady-state calcium efflux from the liver, as opposed to major perturbations in the pattern of calcium efflux effected by the ..cap alpha..-adrenergic agonist phenylephrine. Infusion of short pulses of AGEPC during the washout of /sup 45/Ca/sup 2 +/ from labeled livers caused a transient release of /sup 45/Ca/sup 2 +/ which was not abolished at low calcium concentrations in the perfusate. Infusion of latex beads, which are removed by the reticuloendothelial cells, caused the release of hepatic /sup 45/Ca/sup 2 +/ in a fashion similar to the case with AGEPC. The findings indicate that AGEPC does not perturb a major pool of calcium within the liver as occurs upon ..cap alpha..-adrenergic stimulation; it is likely that AGEPC mobilizes calcium from a smaller yet very important pool, very possibly from nonparenchymal cells in the liver.

  12. Microbial enzyme activities of peatland soils in south central Alaska lowlands

    EPA Science Inventory

    Microbial enzyme activities related to carbon and nutrient acquisition were measured on Alaskan peatland soils as indicators of nutrient limitation and biochemical sustainability. Peat decomposition is mediated by microorganisms and enzymes that in turn are limited by various ph...

  13. A Review on the Effects of Supercritical Carbon Dioxide on Enzyme Activity

    PubMed Central

    Wimmer, Zdeněk; Zarevúcka, Marie

    2010-01-01

    Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO2. The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability. PMID:20162013

  14. Salacia oblonga root improves postprandial hyperlipidemia and hepatic steatosis in Zucker diabetic fatty rats: Activation of PPAR-{alpha}

    SciTech Connect

    Hsun-Wei Huang, Tom; Peng Gang; Qian Li, George; Yamahara, Johji; Roufogalis, Basil D.; Li Yuhao . E-mail: yuhao@pharm.usyd.edu.au

    2006-02-01

    Salacia oblonga (SO) root is an Ayurvedic medicine with anti-diabetic and anti-obese properties. Peroxisome proliferator-activated receptor (PPAR)-{alpha}, a nuclear receptor, plays an important role in maintaining the homeostasis of lipid metabolism. Here, we demonstrate that chronic oral administration of the water extract from the root of SO to Zucker diabetic fatty (ZDF) rats, a genetic model of type 2 diabetes and obesity, lowered plasma triglyceride and total cholesterol (TC) levels, increased plasma high-density lipoprotein levels and reduced the liver contents of triglyceride, non-esterified fatty acids (NEFA) and the ratio of fatty droplets to total tissue. By contrast, the extract had no effect on plasma triglyceride and TC levels in fasted ZDF rats. After olive oil administration to ZDF the extract also inhibited the increase in plasma triglyceride levels. These results suggest that SO extract improves postprandial hyperlipidemia and hepatic steatosis in ZDF rats. Additionally, SO treatment enhanced hepatic expression of PPAR-{alpha} mRNA and protein, and carnitine palmitoyltransferase-1 and acyl-CoA oxidase mRNAs in ZDF rats. In vitro, SO extract and its main component mangiferin activated PPAR-{alpha} luciferase activity in human embryonic kidney 293 cells and lipoprotein lipase mRNA expression and enzyme activity in THP-1 differentiated macrophages; these effects were completely suppressed by a selective PPAR-{alpha} antagonist MK-886. The findings from both in vivo and in vitro suggest that SO extract functions as a PPAR-{alpha} activator, providing a potential mechanism for improvement of postprandial hyperlipidemia and hepatic steatosis in diabetes and obesity.

  15. The inhibition of major human hepatic cytochrome P450 enzymes by 18 pesticides: comparison of the N-in-one and single substrate approaches.

    PubMed

    Abass, Khaled; Pelkonen, Olavi

    2013-08-01

    In the present study on human hepatic microsomes, the N-in-one assay with ten probe substrates for nine cytochrome-P450 enzymes (CYPs) was compared with the single substrate assays to investigate pesticides-CYP interactions. CYP inhibition was measured by liquid chromatography-tandem mass spectrometry (LC/MS-MS). As illustrated by the initial screening at 100 μM concentration of 18 pesticides, CYPs are more sensitive to organophosphates (OPs) than to other pesticide groups. Chlorpyrifos and fenitrothion were most effective in inhibiting CYP1A1/2, and CYP2B6. Profenofos was also inhibitory towards multiple CYPs. Pyrethroids, e.g. deltamethrin, fenvalerate and lambda-cyhalothrin, potently inhibited CYP2D6. CYP3A4 activity was moderately inhibited by fenvalerate and potently by alpha-cypermethrin. The correlations between IC50 values obtained from the N-in-one and single substrate approaches were highly significant for CYP2Cs (r(2)=0.94), CYP3A4, omeprazole-sulfoxidation, (r(2)=0.89), followed by CYP1A2 and CYP2B6 (r(2)=0.82), and CYP2D6 (r(2)=0.80). In contrast no correlation was observed with CYP2E1 and CYP3A4 (midazolam-1'-hydroxylation). The N-in-one screening assay seems useful and reliable for most CYP activities when a comprehensive and quick evaluation of potential interactions with CYPs is needed. However, at the present moment, it does not enable discrimination on the basis of mechanism of inhibition. A strict comparison between single and N-in-one assays is a prerequisite for more extensive routine use. PMID:22634058

  16. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

    PubMed Central

    Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

  17. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

    PubMed

    Tang, Yujin; Wang, Ruipu; Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

  18. Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis

    PubMed Central

    Song, Zhenyuan; Deaciuc, Ion; Zhou, Zhanxiang; Song, Ming; Chen, Theresa; Hill, Daniell; McClain, Craig J.

    2014-01-01

    Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate or prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 ± 0.5 g (means ± SE) were divided into four groups (8 mice per group) and started on one of four treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS), and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (wt/vol) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system. PMID:17702954

  19. Nifedipine lowers cocaine-induced brain and liver enzyme activity and cocaine urinary excretion in rats.

    PubMed

    Vitcheva, Vessela; Simeonova, Rumyana; Karova, Dima; Mitcheva, Mitka

    2011-06-01

    The aim of this study was to see how nifedipine counters the effects of cocaine on hepatic and brain enzymatic activity in rats and whether it affects urinary excretion of cocaine. Male Wistar rats were divided in four groups of six: control, nifedipine group (5 mg kg-1i.p. a day for five days); cocaine group (15 mg kg-1i.p. a day for five days), and the nifedipine+cocaine group. Twenty-four hours after the last administration, we measured neuronal nitric oxide synthase (nNOS) activity in the brain and cytochrome P450 quantity, ethylmorphine-N-demethylase, and anilinehydroxylase activity in the liver. Urine samples were collected 24 h after the last cocaine and cocaine+nifedipine administration. Urinary cocaine concentration was determined using the GC/MS method.Cocaine administration increased brain nNOS activity by 55 % (p<0.05) in respect to control, which indicates the development of tolerance and dependence. In the combination group, nifedipine decreased the nNOS activity in respect to the cocaine-only group.In the liver, cocaine significantly decreased and nifedipine significantly increased cytochrome P450, ethylmorphine-N-demethylase, and anilinehydroxylase in respect to control. In combination, nifedipine successfully countered cocaine effects on these enzymes.Urine cocaine excretion in the cocaine+nifedipine group significantly dropped (by 35 %) compared to the cocaine-only group.Our results have confirmed the effects of nifedipine against cocaine tolerance and development of dependence, most likely due to metabolic interactions between them. PMID:21705300

  20. Application of Activity-Based Protein Profiling to Study Enzyme Function in Adipocytes

    PubMed Central

    Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F.; Saez, Enrique

    2014-01-01

    Activity-Based Protein Profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes, and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. PMID:24529438

  1. Constitutive Activation of the Nlrc4 Inflammasome Prevents Hepatic Fibrosis and Promotes Hepatic Regeneration after Partial Hepatectomy

    PubMed Central

    DeSantis, David A.; Ko, Chih-Wei; Wang, Lan; Lee, Peter; Croniger, Colleen M.

    2015-01-01

    TThe molecular mechanisms responsible for the development of hepatic fibrosis are not fully understood. The Nlrc4 inflammasome detects cytosolic presence of bacterial components, activating inflammatory cytokines to facilitate clearance of pathogens and infected cells. We hypothesized that low-grade constitutive activation of the Nlrc4 inflammasome may lead to induced hepatocyte proliferation and prevent the development of hepatic fibrosis. The gene of Nlrc4 contains two single nucleotide polymorphisms (SNPs), one located within the Nlrc4 promoter and one contained within exon 5. These SNPs regulate Nlrc4 gene transcription and activation as measured through gene reporter assays and IL-1β secretion. The 17C-6 mice have increased IL-1β in plasma after chronic carbon tetrachloride (CCl4) administration compared to B6 mice. After two-thirds partial hepatectomy (2/3PH) 17C-6 mice have earlier restoration of liver mass with greater cyclin D1 protein and BrdU incorporation compared to B6 mice at several time points. These data reveal mild constitutive activation of the Nlrc4 inflammasome as the results of two SNPs, which leads to the stimulation of hepatocyte proliferation. The increased liver regeneration induces rapid liver mass recovery after hepatectomy and may prevent the development of hepatotoxin-induced liver fibrosis. PMID:26635450

  2. Correlation Among Soil Enzyme Activities, Root Enzyme Activities, and Contaminant Removal in Two-Stage In Situ Constructed Wetlands Purifying Domestic Wastewater.

    PubMed

    Ni, Lixiao; Xu, Jiajun; Chu, Xianglin; Li, Shiyin; Wang, Peifang; Li, Yiping; Li, Yong; Zhu, Liang; Wang, Chao

    2016-07-01

    Two-stage in situ wetlands (two vertical flow constructed wetlands in parallel and a horizontal flow constructed wetland) were constructed for studying domestic wastewater purification and the correlations between contaminant removal and plant and soil enzyme activities. Results indicated the removal efficiency of NH4 (+) and NO3 (-) were significantly correlated with both urease and protease activity, and the removal of total phosphorus was significantly correlated with phosphatase activity. Chemical oxygen demand removal was not correlated with enzyme activity in constructed wetlands. Plant root enzyme (urease, phosphatase, protease and cellulose) activity correlation was apparent with all contaminant removal in the two vertical flow constructed wetlands. However, the correlation between the plant root enzyme activity and contaminant removal was poor in horizontal flow constructed wetlands. Results indicated that plant roots clearly played a role in the removal of contaminants. PMID:27230025

  3. Effects of petroleum on adrenocortical activity and on hepatic naphthalene-metabolizing activity in mallard ducks

    USGS Publications Warehouse

    Gorsline, J.; Holmes, W.N.

    1981-01-01

    Unstressed mallard ducks (Anas platyrhychos), given uncontaminated food and maintained on a short photoperiod, show two daily maxima in plasma corticosterone concentration ([B]); one occurring early in the light phase and a second just before the onset of darkness. After one week of exposure to food containing 3% (v/w) South Louisiana crude oil, plasma [B] were significantly lowered throughout the day. Similar abrupt declines in plasma [B] also occurred during the first 10 days of exposure to food containing 1% and 0.5% crude oil. Although the plasma [B] in birds consuming food contaminated with 0.5% crude oil increased between 10 and 50 days of exposure, the concentration after 50 days was still lower than normal. During the same interval, normal plasma [B] were restored in birds consuming food containing 1% and 3% crude oil. Significant increases occurred in the naphthalene-metabolizing properties of hepatic microsomes prepared from birds acutely exposed to all levels of petroleum-contaminated food and elevated levels were sustained throughout the first 50 days of exposure. Birds given food containing 3% crude oil for more than 50 days, however, showed steady declines in hepatic naphthalene-metabolizing activity. After 500 days, the activity was similar to that found in contemporaneous controls. During the same interval, the plasma [B] increased until the levels were higher than normal after 500 days of exposure; at this time, an inverse relationship, similar to that seen during the first week of exposure to contaminated food, was once more established between plasma [B] and the concomitant hepatic naphthalene-metabolizing activity.

  4. Human hepatic N-acetylglutamate content and N-acetylglutamate synthase activity. Determination by stable isotope dilution.

    PubMed Central

    Tuchman, M; Holzknecht, R A

    1990-01-01

    N-Acetyl-L-glutamate (N-acetylglutamate) content and N-acetylglutamate synthase activity ranges were established in human liver tissue homogenates by stable isotope dilution. The methods employ N-[methyl-2H3]acetyl[15N]glutamate as internal standard, extraction of N-acetylglutamate by anion-exchange technique and its determination by g.l.c.-mass spectrometry by using selected ion monitoring. Hepatic N-acetylglutamate content in 16 different human livers, normal in structure and function, ranged from 6.8 to 59.7 nmol/g wet wt. (25.0 +/- 13.4 mean +/- S.D.) or from 64.6 to 497.6 nmol/g of protein (223.2 +/- 104.2 mean +/- S.D.). In vitro, N-acetylglutamate synthase activity in liver tissue homogenate ranged from 44.5 to 374.5 (132.0 +/- 90.6 mean +/- S.D.) nmol/min per g wet wt. or from 491.7 to 3416.9 (1159.6 +/- 751.1 mean +/- S.D.) nmol/min per g of protein. No correlation was found between hepatic N-acetylglutamate concentrations and the respective maximal enzymic activities in vitro of N-acetylglutamate synthase. The marked variability in this system among individual livers may reflect its regulatory role in ureagenesis. PMID:2241918

  5. Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

    PubMed Central

    2013-01-01

    Background For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity. Results During uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity. Conclusion Specific activity calculated from the level of

  6. Ultrasonic Monitoring of Enzyme Catalysis; Enzyme Activity in Formulations for Lactose-Intolerant Infants.

    PubMed

    Altas, Margarida C; Kudryashov, Evgeny; Buckin, Vitaly

    2016-05-01

    The paper introduces ultrasonic technology for real-time, nondestructive, precision monitoring of enzyme-catalyzed reactions in solutions and in complex opaque media. The capabilities of the technology are examined in a comprehensive analysis of the effects of a variety of diverse factors on the performance of enzyme β-galactosidase in formulations for reduction of levels of lactose in infant milks. These formulations are added to infant's milk bottles prior to feeding to overcome the frequently observed intolerance to lactose (a milk sugar), a serious issue in healthy development of infants. The results highlight important impediments in the development of these formulations and also illustrate the capability of the described ultrasonic tools in the assessment of the performance of enzymes in complex reaction media and in various environmental conditions. PMID:27018312

  7. Hepatitis C virus core protein triggers expansion and activation of CD4(+)CD25(+) regulatory T cells in chronic hepatitis C patients.

    PubMed

    Zhai, Naicui; Chi, Xiumei; Li, Tianyang; Song, Hongxiao; Li, Haijun; Jin, Xia; Crispe, Ian Nicholas; Su, Lishan; Niu, Junqi; Tu, Zhengkun

    2015-11-01

    CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) are increased in patients with chronic hepatitis C, which may contribute to the sustained suppression of hepatitis C virus (HCV)-specific T-cell responses and viral persistence in HCV-infected individuals. We postulated that HCV core protein (HCVc) directly contributes to the expansion of Tregs in HCV-infected patients, and we provide evidence to support this hypothesis in the report. Peripheral blood mononuclear cells (PBMCs) and sera were collected from 87 treatment-naïve chronic HCV-infected patients, CD4(+)CD25(+) Tregs were measured by flow cytometry, and HCV RNA and HCVc levels were detected using qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. CD4(+), CD8(+), CD4(+)CD25(+) and CD4(+)CD25(-) T cells were purified from healthy donors and cultured with recombinant HCVc and Toll-like receptor (TLR) ligands. Flow cytometry was used to analyze cell proliferation, and ELISA was performed to measure cytokine production. In the 87 chronic HCV-infected patients, HCVc showed a significant correlation with HCV RNA and CD4(+)CD25(+) Tregs. Mechanistic studies showed that HCVc, together with anti-CD3 antibody, augmented CD4(+)CD25(+) Treg proliferation, but inhibited CD4(+)CD25(-) T-cell proliferation and IFN-γ production, in a dose-dependent and Treg-dependent manner. Moreover, unlike the TLR3 ligand (poly I:C) and the TLR4 ligand (lipopolysaccharide, LPS), the TLR2 ligand (lipoteichoic acid, LTA) and HCVc both inhibited TCR-induced CD4(+) T-cell proliferation and IFN-γ secretion in a Treg-dependent manner. These data indicate that HCVc, like other TLR2 ligands, triggers CD4(+)CD25(+) Treg activation and expansion to inhibit host immune responses, which may play a critical role in viral persistence in HCV-infected patients. PMID:25531392

  8. Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

    SciTech Connect

    Agarwal, Pratul K

    2012-01-01

    Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted that mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.

  9. Activity, life time and effect of hydrolytic enzymes for enhanced biogas production from sludge anaerobic digestion.

    PubMed

    Odnell, Anna; Recktenwald, Michael; Stensén, Katarina; Jonsson, Bengt-Harald; Karlsson, Martin

    2016-10-15

    As an alternative to energy intensive physical methods, enzymatic treatment of sludge produced at wastewater treatment plants for increased hydrolysis and biogas production was investigated. Several hydrolytic enzymes were assessed with a focus on how enzyme activity and life time was influenced by sludge environments. It could be concluded that the activity life time of added enzymes was limited (<24 h) in both waste activated sludge and anaerobic digester sludge environments and that this was, for the majority of enzymes, due to endogenous protease activity. In biogas in situ experiments, subtilisin at a 1% mixture on basis of volatile solids, was the only enzyme providing a significantly increased biomethane production of 37%. However, even at this high concentration, subtilisin could not hydrolyze all available substrate within the life time of the enzyme. Thus, for large scale implementation, enzymes better suited to the sludge environments are needed. PMID:27498254

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. Sechium edule Shoot Extracts and Active Components Improve Obesity and a Fatty Liver That Involved Reducing Hepatic Lipogenesis and Adipogenesis in High-Fat-Diet-Fed Rats.

    PubMed

    Yang, Mon-Yuan; Chan, Kuei-Chuan; Lee, Yi-Ju; Chang, Xiao-Zong; Wu, Cheng-Hsun; Wang, Chau-Jong

    2015-05-13

    Excess fat accumulation in the liver increases the risk of developing progressive liver injuries ranging from a fatty liver to hepatocarcinoma. In a previous study, we demonstrated that the polyphenol components of Sechium edule shoots attenuated hepatic lipid accumulation in vitro. Therefore, we investigated the effects and mechanisms of the extract of S. edule shoots (SWE) to modulate fat accumulation in a high-fat-diet (HFD)-induced animal model. In this study, we found that the SWE can reduce the body weight, adipose tissue fat, and regulate hepatic lipid contents (e.g., triglyceride and cholesterol). Additionally, treatment of caffeic acid (CA) and hesperetin (HPT), the main ingredients of SWE, also inhibited oleic acid (OA)-induced lipid accumulation in HepG2 cells. SWE enhanced the activation of AMP-activating protein kinase (AMPK) and decreased numerous lipogenic-related enzymes, such as sterol regulator element-binding proteins (SREBPs), e.g., SREBP-1 and SREBP-2, and HMG-CoA reductase (HMGCoR) proteins, which are critical regulators of hepatic lipid metabolism. Taken together, the results demonstrated that SWE can prevent a fatty liver and attenuate adipose tissue fat by inhibiting lipogenic enzymes and stimulating lipolysis via upregulating AMPK. It was also demonstrated that the main activation components of SWE are both CA and HPT. PMID:25912298

  13. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    SciTech Connect

    Shlomai, Amir; Shaul, Yosef

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  14. Preparation of biocatalytic nanofibres with high activity and stability via enzyme aggregate coating on polymer nanofibres

    NASA Astrophysics Data System (ADS)

    Kim, Byoung Chan; Nair, Sujith; Kim, Jungbae; Kwak, Ja Hun; Grate, Jay W.; Kim, Seong H.; Gu, Man Bock

    2005-07-01

    We have developed a unique approach for the fabrication of enzyme aggregate coatings on the surfaces of electrospun polymer nanofibres. This approach employs covalent attachment of seed enzymes onto nanofibres consisting of a mixture of polystyrene and poly(styrene-co-maleic anhydride), followed by a glutaraldehyde (GA) treatment that cross-links additional enzyme molecules and aggregates from the solution onto the covalently attached seed enzyme molecules. These cross-linked enzyme aggregates, covalently attached to the nanofibres via the linkers of seed enzyme molecules, are expected to improve the enzyme activity due to increased enzyme loading, and also the enzyme stability. To demonstrate the principle, we coated α-chymotrypsin (CT) on nanofibres electrospun from a mixture of polystyrene and poly(styrene-co-maleic anhydride). The initial activity of CT-aggregate-coated nanofibres was nine times higher than nanofibres with just a layer of covalently attached CT molecules. The enzyme stability of CT-aggregate-coated nanofibres was greatly improved with essentially no measurable loss of activity over a month of observation under rigorous shaking conditions. This new approach of enzyme coating on nanofibres, yielding high activity and stability, creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, and biosensors.

  15. Angiotensin-Converting Enzyme Inhibitors and Active Tuberculosis

    PubMed Central

    Wu, Jiunn-Yih; Lee, Meng-Tse Gabriel; Lee, Si-Huei; Lee, Shih-Hao; Tsai, Yi-Wen; Hsu, Shou-Chien; Chang, Shy-Shin; Lee, Chien-Chang

    2016-01-01

    Abstract Numerous epidemiological data suggest that the use of angiotensin-converting enzyme inhibitors (ACEis) can improve the clinical outcomes of pneumonia. Tuberculosis (TB) is an airborne bacteria like pneumonia, and we aimed to find out whether the use of ACEis can decrease the risk of active TB. We conducted a nested case–control analysis by using a 1 million longitudinally followed cohort, from Taiwan national health insurance research database. The rate ratios (RRs) for TB were estimated by conditional logistic regression, and adjusted using a TB-specific disease risk score (DRS) with 71 TB-related covariates. From January, 1997 to December, 2011, a total of 75,536 users of ACEis, and 7720 cases of new active TB were identified. Current use (DRS adjusted RR, 0.87 [95% CI, 0.78–0.97]), but not recent and past use of ACEis, was associated with a decrease in risk of active TB. Interestingly, it was found that chronic use (>90 days) of ACEis was associated with a further decrease in the risk of TB (aRR, 0.74, [95% CI, 0.66–0.83]). There was also a duration response effect, correlating decrease in TB risk with longer duration of ACEis use. The decrease in TB risk was also consistent across all patient subgroups (age, sex, heart failure, cerebrovascular diseases, myocardial infraction, renal diseases, and diabetes) and patients receiving other cardiovascular medicine. In this large population-based study, we found that subjects with recent and chronic use of ACEis were associated with decrease in TB risk. PMID:27175655

  16. Enzyme catalysis: C-H activation is a Reiske business

    NASA Astrophysics Data System (ADS)

    Bruner, Steven D.

    2011-05-01

    Enzymes that selectively oxidize unactivated C-H bonds are capable of constructing complex molecules with high efficiency. A new member of this enzyme family is RedG, a Reiske-type oxygenase that catalyses chemically challenging cyclizations in the biosynthesis of prodiginine natural products.

  17. Light/dark modulation of enzyme activity in photosynthesis

    SciTech Connect

    Anderson, L.E.; Ashton, A.R.; Mohamed, A.H.; Scheibe, R.

    1982-02-01

    In photosynthetic species ranging from cyanobacteria to higher plants, many enzymes are light modulated. Most of the known light modulated enzymes are chloroplastic. Four mechanisms of modulation have been proposed. One function of light modulation is probably the autocatalytic build-up of reductive pentose phosphate cycle intermediates during the induction period of photosynthetic CO/sub 2/ fixation.

  18. Dynamic relationships between microbial biomass, respiration, inorganic nutrients and enzyme activities: informing enzyme-based decomposition models

    PubMed Central

    Moorhead, D. L.; Rinkes, Z. L.; Sinsabaugh, R. L.; Weintraub, M. N.

    2013-01-01

    We re-examined data from a recent litter decay study to determine if additional insights could be gained to inform decomposition modeling. Rinkes et al. (2013) conducted 14-day laboratory incubations of sugar maple (Acer saccharum) or white oak (Quercus alba) leaves, mixed with sand (0.4% organic C content) or loam (4.1% organic C). They measured microbial biomass C, carbon dioxide efflux, soil ammonium, nitrate, and phosphate concentrations, and β-glucosidase (BG), β-N-acetyl-glucosaminidase (NAG), and acid phosphatase (AP) activities on days 1, 3, and 14. Analyses of relationships among variables yielded different insights than original analyses of individual variables. For example, although respiration rates per g soil were higher for loam than sand, rates per g soil C were actually higher for sand than loam, and rates per g microbial C showed little difference between treatments. Microbial biomass C peaked on day 3 when biomass-specific activities of enzymes were lowest, suggesting uptake of litter C without extracellular hydrolysis. This result refuted a common model assumption that all enzyme production is constitutive and thus proportional to biomass, and/or indicated that part of litter decay is independent of enzyme activity. The length and angle of vectors defined by ratios of enzyme activities (BG/NAG vs. BG/AP) represent relative microbial investments in C (length), and N and P (angle) acquiring enzymes. Shorter lengths on day 3 suggested low C limitation, whereas greater lengths on day 14 suggested an increase in C limitation with decay. The soils and litter in this study generally had stronger P limitation (angles >45°). Reductions in vector angles to <45° for sand by day 14 suggested a shift to N limitation. These relational variables inform enzyme-based models, and are usually much less ambiguous when obtained from a single study in which measurements were made on the same samples than when extrapolated from separate studies. PMID:23964272

  19. A krill oil supplemented diet reduces the activities of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes in rats.

    PubMed

    Ferramosca, A; Conte, L; Zara, V

    2012-04-01

    The mitochondrial tricarboxylate carrier supplies cytosol with the carbon units necessary for hepatic lipogenesis. The activities of cytosolic acetyl-CoA carboxylase and fatty acid synthetase are therefore strictly connected to the function of mitochondrial tricarboxylate carrier. Dietary polyunsaturated fatty acids (PUFA) are potent modulators of hepatic lipogenesis. In rats fed with a diet enriched with 2.5% krill oil (KO), a novel source of dietary n-3 PUFA, a time-dependent decrease in the activities of the mitochondrial tricarboxylate carrier and of the lipogenic enzymes was found. The KO induced inhibition of hepatic lipogenesis was more pronounced than that found in fish oil (FO)-fed rats, at least at short feeding times. The decrease in the activity of the mitochondrial tricarboxylate carrier caused by KO was due to a reduced expression of the protein. Furthermore, in the KO-fed animals a greater reduction in the levels of hepatic triglycerides and cholesterol was found in comparison to FO-fed rats. PMID:21429045

  20. Detection of Sulfatase Enzyme Activity with a CatalyCEST MRI Contrast Agent.

    PubMed

    Sinharay, Sanhita; Fernández-Cuervo, Gabriela; Acfalle, Jasmine P; Pagel, Mark D

    2016-05-01

    A chemical exchange saturation transfer (CEST) MRI contrast agent has been developed that detects sulfatase enzyme activity. The agent produces a CEST signal at δ=5.0 ppm before enzyme activity, and a second CEST signal appears at δ=9.0 ppm after the enzyme cleaves a sulfate group from the agent. The comparison of the two signals improved detection of sulfatase activity. PMID:26956002

  1. Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation.

    PubMed Central

    Aoufouchi, S; Shall, S

    1997-01-01

    Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation. PMID:9230139

  2. Mice expressing reduced levels of hepatic glucose-6-phosphatase-α activity do not develop age-related insulin resistance or obesity.

    PubMed

    Kim, Goo-Young; Lee, Young Mok; Cho, Jun-Ho; Pan, Chi-Jiunn; Jun, Hyun Sik; Springer, Danielle A; Mansfield, Brian C; Chou, Janice Y

    2015-09-15

    Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes. PMID:26089201

  3. Carbohydrate-active enzymes exemplify entropic principles in metabolism

    PubMed Central

    Kartal, Önder; Mahlow, Sebastian; Skupin, Alexander; Ebenhöh, Oliver

    2011-01-01

    Glycans comprise ubiquitous and essential biopolymers, which usually occur as highly diverse mixtures. The myriad different structures are generated by a limited number of carbohydrate-active enzymes (CAZymes), which are unusual in that they catalyze multiple reactions by being relatively unspecific with respect to substrate size. Existing experimental and theoretical descriptions of CAZyme-mediated reaction systems neither comprehensively explain observed action patterns nor suggest biological functions of polydisperse pools in metabolism. Here, we overcome these limitations with a novel theoretical description of this important class of biological systems in which the mixing entropy of polydisperse pools emerges as an important system variable. In vitro assays of three CAZymes essential for central carbon metabolism confirm the power of our approach to predict equilibrium distributions and non-equilibrium dynamics. A computational study of the turnover of the soluble heteroglycan pool exemplifies how entropy-driven reactions establish a metabolic buffer in vivo that attenuates fluctuations in carbohydrate availability. We argue that this interplay between energy- and entropy-driven processes represents an important regulatory design principle of metabolic systems. PMID:22027553

  4. Cross-linked enzyme aggregates (CLEAs) of Pencilluim notatum lipase enzyme with improved activity, stability and reusability characteristics.

    PubMed

    Rehman, Saima; Bhatti, Haq Nawaz; Bilal, Muhammad; Asgher, Muhammad

    2016-10-01

    Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. In this study, Pencillium notatum lipase (PNL) was immobilized as carrier free cross-linked enzyme aggregates using glutaraldehyde (GLA) and Ethylene glycol-bis [succinic acid N-hydroxysuccinimide] (EG-NHS) as cross-linking agents. The optimal conditions for the synthesis of an efficient lipase CLEAs such as precipitant type, the nature and amount of cross-linking reagent, and cross-linking time were optimized. The recovered activities of CLEAs were considerably dependent on the concentration of GLA; however, the activity recovery was not severely affected by EG-NHS as a mild cross-linker. The EG-NHS aggregates displayed superior hydrolytic (52.08±2.52%) and esterification (64.42%) activities as compared to GLA aggregates which showed 23.8±1.86 and 34.54% of hydrolytic and esterification activity, respectively. Morphological analysis by fluorescence and scanning electron microscope revealed that EG-NHS aggregates were smaller in size with larger surface area compared to GLA aggregates. The pH optima of both types of CLEAs were displaced to slightly alkaline region and higher temperature as compared to native enzyme. Highest enzyme activity of CLEAs was achieved at the pH of 9.0 and 42°C temperature. Moreover, a significant improvement in the thermal resistance was also recorded after immobilization. After ten reusability cycles in aqueous medium, GLA and EG-NHS cross-linked lipase CLEAs preserved 63.62% and 70.9% of their original activities, respectively. The results suggest that this novel CLEA-lipase is potentially usable in many industrial applications. PMID:27365121

  5. Optimization of collective enzyme activity via spatial localization

    NASA Astrophysics Data System (ADS)

    Buchner, Alexander; Tostevin, Filipe; Hinzpeter, Florian; Gerland, Ulrich

    2013-10-01

    The spatial organization of enzymes often plays a crucial role in the functionality and efficiency of enzymatic pathways. To fully understand the design and operation of enzymatic pathways, it is therefore crucial to understand how the relative arrangement of enzymes affects pathway function. Here we investigate the effect of enzyme localization on the flux of a minimal two-enzyme pathway within a reaction-diffusion model. We consider different reaction kinetics, spatial dimensions, and loss mechanisms for intermediate substrate molecules. Our systematic analysis of the different regimes of this model reveals both universal features and distinct characteristics in the phenomenology of these different systems. In particular, the distribution of the second pathway enzyme that maximizes the reaction flux undergoes a generic transition from co-localization with the first enzyme when the catalytic efficiency of the second enzyme is low, to an extended profile when the catalytic efficiency is high. However, the critical transition point and the shape of the extended optimal profile is significantly affected by specific features of the model. We explain the behavior of these different systems in terms of the underlying stochastic reaction and diffusion processes of single substrate molecules.

  6. Enzyme activities in plasma, kidney, liver, and muscle of five avian species

    USGS Publications Warehouse

    Franson, J.C.; Murray, H.C.; Bunck, C.

    1985-01-01

    Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH) were determined in plasma, kidney, liver, and muscle from five species of captive birds. Few differences occurred in plasma activities between sexes but considerable differences occurred between species. All five enzymes were detected in each of the tissues sampled. Relative enzyme activities in liver, kidney, and muscle were similar for each species. CPK activity was much higher in muscle than in liver or kidney and, of the five enzymes studied, may be the best indicator of muscle damage. Most of the other enzymes were more evenly distributed among the three tissues, and no organ-specific enzyme could be identified for liver or kidney. Because of interspecific variations in plasma enzyme activities, it is important to establish baseline values for each species to ensure accurate interpretation of results.

  7. Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

    PubMed Central

    Guo, Qing; He, Yufan; Lu, H. Peter

    2015-01-01

    Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions. PMID:26512103

  8. Enzyme induction, mutagen activation and carcinogen testing in yeast

    SciTech Connect

    Wiseman, A.

    1987-01-01

    This book documents the scientific basis for using yeasts to detect mutagenic chemicals likely to cause cancer in humans, a phenomenon explained by the presence of the enzyme cytochrome P-450 in some tissues. Explains the nature and roles of this enzyme in detail, and explores a range of related topics, including the genetic features of yeast, the mitochondrial DNA system and petite mutants, the molecular biology of transcription of genes in yeast, and enzyme induction. Also examined is DNA repair and how mutagenesis in yeast and other microorganisms relates to the practical detection of mutagens.

  9. Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator.

    PubMed Central

    Kato, N; Lan, K H; Ono-Nita, S K; Shiratori, Y; Omata, M

    1997-01-01

    The hepatitis C virus (HCV) nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids and fused to the DNA-binding domain of GAL4, strongly activates transcription in yeast and human hepatoma cells. Transcriptional activation by the HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis. PMID:9343247

  10. The effect of insulin on plasma glucose concentrations, expression of hepatic glucose transporters and key gluconeogenic enzymes during the perinatal period in broiler chickens.

    PubMed

    Franssens, Lies; Lesuisse, Jens; Wang, Yufeng; Willems, Els; Willemsen, Hilke; Koppenol, Astrid; Guo, Xiaoquan; Buyse, Johan; Decuypere, Eddy; Everaert, Nadia

    2016-06-01

    Chickens have blood glucose concentrations that are twofold higher than those observed in mammals. Moreover, the insulin sensitivity seems to decrease with postnatal age in both broiler and layer chickens. However, little is known about the response of insulin on plasma glucose concentrations and mRNA abundance of hepatic glucose transporters 1, 2, 3, 8, 9 and 12 (GLUT1, 2, 3, 8, 9 and 12) and three regulatory enzymes of the gluconeogenesis, phosphoenolpyruvate carboxykinase 1 and 2 (PCK1 and 2) or fructose-1,6-biphosphatase 1 (FBP1) in chicks during the perinatal period. In the present study, broiler embryos on embryonic day (ED)16, ED18 or newly-hatched broiler chicks were injected intravenously with bovine insulin (1μg/g body weight (BW)) to examine plasma glucose response and changes in hepatic mRNA abundance of the GLUTs, PCK1 and 2 and FBP1. Results were compared with a non-treated control group and a saline-injected sham group. Plasma glucose levels of insulin-treated ED18 embryos recovered faster from their minimum level than those of insulin-treated ED16 embryos or newly-hatched chicks. In addition, at the minimum plasma glucose level seven hours post-injection (PI), hepatic GLUT2, FBP1 and PCK2 mRNA abundance was decreased in insulin-injected embryos, compared to sham and control groups, being most pronounced when insulin injection occurred on ED16. PMID:26723190

  11. The pathogenesis of arthritis associated with acute hepatitis-B surface antigen-positive hepatitis. Complement activation and characterization of circulating immune complexes.

    PubMed Central

    Wands, J R; Mann, E; Alpert, E; Isselbacher, K J

    1975-01-01

    Circulating immune complexes were identified in cryoproteins isolated from serial samples of serum from six patients with acute viral hepatitis with and without arthritic symptoms. Cryoprecipitates were analyzed for the presence of hepatitis-B surface antigen (HBsAg) and hepatitis-B surface antibody (anti-HBs) by hemagglutination inhibition and hemagglutination. Complement components were detected by counter electrophoresis, and immunoglobulins were detected by gel diffusion. HBsAg, IgG, and IgM were identified in cryoprecipitates from all hepatitis patients, but were higher in concentration in patients with arthritis. Only cryoprecipitates from hepatitis patients with arthritis contained IgA and complement components C3, C4, and C5 as well as IgG and IgM, which disappear with resolution of the arthritis. The subtypes of IgG in these cryoprecipitates were predominantly the complement-fixing IgG1 and IgG3, HBsAg and anti-HBs were concentrated several-fold in the cryoprecipitates when compared to the serum concentration. Sequential studies in two patients demonstrated that the initial appearance of anti-HBs in the cryoprotein complex was associated with the detection in the complex of IgM suggesting a primary immune response to HBsAg. The C3 activator fragment (C3A) of the properdin complex was found in fresh serum obtained from three hepatitis patients with arthritis and not in uncomplicated hepatitis. The cryoprecipitable immune complexes from patients with arthritis converted C3PA in fresh normal sera to C3A in vitro whereas cryoprotein isolated from patients with uncomplicated hepatitis had no such effect. Thus, the transient appearance of circulating complement-fixing immune complexes in patients with the arthritis of acute hepatitis is associated with activation of both classical and alternate complement pathways and suggests that they play an important role in the pathogenesis of these serum sickness-like extrahepatic symptoms. Images PMID:1123429

  12. Also with a restrictive transfusion policy, screening with second-generation anti-hepatitis C virus enzyme-linked immunosorbent assay would have reduced post-transfusion hepatitis C after open-heart surgery.

    PubMed

    Mathiesen, U L; Karlsson, E; Foberg, U; Frydén, A; Franzén, L; Widell, A; Bodemar, G

    1993-07-01

    The incidence of post-transfusion hepatitis non-A, non-B (PTH-NANB) was prospectively assessed among open-heart surgery patients from the southeast region of Sweden before the introduction of antihepatitis C virus (HCV) blood donor screening. Blood samples for alanine aminotransferase analysis were drawn before and 2, 3, and 4 months after transfusion. Surgery was performed in four centres. Of 190 transfused and followed-up patients 2 (1.1%) contracted PTH-NANB, both operated on at the centre with significantly fewer transfusions than the other centres. One patient had antibodies to HCV detected by first-generation (C100-3) and later by second-generation anti-HCV enzyme-linked immunosorbent assay (ELISA-2) and by positive second-generation recombinant immunoblot assay (4-RIBA). The other patient, although negative by first-generation anti-HCV ELISA, was positive by second-generation ELISA and by 4-RIBA. Both patients were hepatitis C-viremic by polymerase chain reaction (PCR). All the six donors implicated in the two hepatitis cases were first-generation anti-HCV-negative, but two, one for each patient, were positive by second-generation anti-HCV ELISA. This finding was confirmed by positive 4-RIBA in only 1 donor, the other being 'indeterminate'. However, in both donors hepatitis C viremia was found by PCR. This study shows that the second-generation anti-HCV ELISA will further reduce the risk for PTH-NANB/C and draws attention to the problem of evaluation of confirmatory tests. PMID:7689744

  13. Differential regulation of detoxification enzymes in hepatic and mammary tissue by hops (Humulus lupulus) in vitro and in vivo

    PubMed Central

    Dietz, Birgit M.; Hagos, Ghenet K.; Eskra, Jillian N.; Wijewickrama, Gihani T.; Anderson, Jeffrey R.; Nikolic, Dejan; Guo, Jian; Wright, Brian; Chen, Shao-Nong; Pauli, Guido F.; van Breemen, Richard B.; Bolton, Judy L.

    2013-01-01

    Scope Hops contain the phytoestrogen, 8-prenylnaringenin, and the cytoprotective compound, xanthohumol (XH). XH induces the detoxification enzyme, NAD(P)H-quinone oxidoreductase (NQO1) in vitro; however, the tissue distribution of XH and 8-prenylnaringenin and their tissue specific activity have not been analyzed. Methods and results A standardized hop extract (p.o.) and XH (s.c.) were administered to Sprague-Dawley rats over four days. LC-MS-MS analysis of plasma, liver and mammary gland revealed that XH accumulated in liver and mammary glands. Compared with the low level in the original extract, 8-prenylnaringenin was enriched in the tissues. Hops and XH induced NQO1 in the liver, while only hops reduced NQO1 activity in the mammary gland. Mechanistic studies revealed that hops modulated NQO1 through three mechanisms. In liver cells, 1) XH modified Keap1 leading to Nrf2 translocation and antioxidant response element (ARE) activation; 2) hop-mediated ARE induction was partially mediated through phosphorylation of Nrf2 by PKC; 3) in breast cells, 8-prenylnaringenin reduced NQO1 likely through binding to ERα, recruiting Nrf2, and downregulating ARE-regulated genes. Conclusions XH and 8-prenylnaringenin in dietary hops are bioavailable to the target tissues. While hops and XH might be cytoprotective in the liver, 8-prenylnaringenin seems responsible for hop-mediated NQO1 reduction in the mammary gland. PMID:23512484

  14. Inconsistencies and ambiguities in calculating enzyme activity: The case of laccase.

    PubMed

    Baltierra-Trejo, Eduardo; Márquez-Benavides, Liliana; Sánchez-Yáñez, Juan Manuel

    2015-12-01

    Laccase is a key enzyme in the degradation of lignin by fungi. Reports indicate that the activity of this enzyme ranges from 3.5 to 484,000 U L(-1). Our aim was to analyze how laccase activity is calculated in the literature, and to determine statistically whether variations in activity are due to biological properties or to inconsistencies in calculation. We found a general lack of consensus on the definition of enzyme activity, and enzymes are sometimes characterized in terms of reaction rate and specific activity. Moreover, enzyme activity is calculated using at least seven different equations. Therefore, it is critical to standardize the calculation of laccase activity in order to compare results directly. PMID:26459230

  15. Effects of Fresh Yellow Onion Consumption on CEA, CA125 and Hepatic Enzymes in Breast Cancer Patients: A Double- Blind Randomized Controlled Clinical Trial.

    PubMed

    Jafarpour-Sadegh, Farnaz; Montazeri, Vahid; Adili, Ali; Esfehani, Ali; Rashidi, Mohammad-Reza; Mesgari, Mehran; Pirouzpanah, Saeed

    2015-01-01

    Onion (Allium cepa) consumption has been remarked in folk medicine which has not been noted to be administered so far as an adjunct to conventional doxorubicin-based chemotherapy in breast cancer patients. To our knowledge, this is the first study aimed to investigate the effects of consuming fresh yellow onions on hepatic enzymes and cancer specific antigens compared with a low-onion containing diet among breast cancer (BC) participants treated with doxorubicin. This parallel design randomized controlled clinical trial was conducted on 56 BC patients whose malignancy was confirmed with histopathological examination. Subjects were assigned in a stratified-random allocation into either group received body mass index dependent 100-160 g/d of onion as high onion group (HO; n=28) or 30-40 g/d small onion in low onion group (LO; n=28) for eight weeks intervention. Participants, care givers and laboratory assessor were blinded to the assignments (IRCT registry no: IRCT2012103111335N1). The compliance of participants in the analysis was appropriate (87.9%). Comparing changes throughout pre- and post-dose treatments indicated significant controls on carcinoembryonic antigen, cancer antigen-125 and alkaline phosphatase levels in the HO group (P<0.05). Our findings for the first time showed that regular onion administration could be effective for hepatic enzyme conveying adjuvant chemotherapy relevant toxicity and reducing the tumor markers in BC during doxorubicin-based chemotherapy. PMID:26625755

  16. Dynamically Achieved Active Site Precision in Enzyme Catalysis

    PubMed Central

    2015-01-01

    Conspectus The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes’ enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme–substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed. PMID:25539048

  17. Influence of dietary zinc on hepatic collagen and prolyl hydroxylase activity in alcoholic rats.

    PubMed

    Giménez, A; Caballería, J; Parés, A; Alié, S; Deulofeu, R; Andreu, H; Rodés, J

    1992-09-01

    The effects of dietary zinc on hepatic collagen and prolyl hydroxylase activity in normal and alcoholic rats has been investigated in four groups of pair-fed male Wistar rats given either liquid ethanol or a control diet for 12 wk. Each group of pair-fed animals received a diet with a different zinc concentration (standard diet, 7.6 mg/L; low-zinc diet, 3.4 mg/L; zinc-supplemented diet, 76 mg/L; and zinc-extrasupplemented, 300 mg/L. There were no significant differences in hepatic collagen concentration and prolyl hydroxylase activity between alcoholic and normal rats receiving a standard diet (collagen, 77 +/- 5 and 73 +/- 6 micrograms/mg protein; and prolyl hydroxylase; 37 +/- 26 and 36 +/- 22 cpm/mg protein). Alcoholic rats fed a low-zinc diet showed increased prolyl hydroxylase activity (75 +/- 10 cpm/mg protein, p less than 0.05), although no changes in hepatic collagen (77 +/- 10 micrograms/mg protein) were observed in comparison with rats fed a standard alcoholic diet. By contrast, hepatic collagen was significantly lower in alcoholic rats fed a zinc-supplemented diet (66 +/- 4 and 63 +/- 3 micrograms/mg protein, p less than 0.05 and p less than 0.01, respectively), and hepatic prolyl hydroxylase activity was particularly lower in rats receiving zinc 300 mg/L (18 +/- 20 cpm/mg protein). Similar effects were observed in normal rats. We conclude that dietary zinc influences hepatic prolyl hydroxylase activity and collagen deposition in alcoholic rats, and in consequence, the control of dietary zinc is necessary to assess the effects of alcohol on collagen metabolism in rats. PMID:1324218

  18. Preparation of biocatalytic nanofibers with high activity and stability via enzyme aggregate coating on polymer nanofibers

    SciTech Connect

    Kim, Byoung Chan; Nair, Sujith; Kim, Jungbae; Kwak, Ja Hun; Grate, Jay W.; Kim, Seong H.; Gu, Man Bock

    2005-04-01

    We have developed a unique approach for the fabrication of enzyme coating on the surface of electrospun polymer nanofibers. This approach employs covalent attachment of seed enzymes onto nanofibers, followed by the glutaraldehyde treatment that crosslinks additional enzymes onto the seed enzyme molecules. These crosslinked enzyme aggregates, covalently attached to the nanofibers via seed enzyme linker, would improve not only the enzyme activity due to increased enzyme loading, but also the enzyme stability. To demonstrate the principle of concept, we fabricated the coating of alpha-chymotrypsin (CT) on the nanofibers electrospun from a mixture of polystyrene and poly(styrene-co-maleic anhydride). The addition of poly(styrene-co-maleic anhydride) makes it much easier to attach the seed enzyme molecules onto electrospun nanofibers without any rigorous functionalization of nanofibers for the attachment of enzymes. The initial activity of final CT coating was 17 and 9 times higher than those of simply-adsorbed CT and covalently-attached CT, respectively. While adsorbed and covalently-attached CT resulted in a serious enzyme leaching during initial incubation in a shaking condition, the CT coating did not show any leaching from the beginning of incubation in the same condition. As a result, the enzyme stability of CT coating was impressively improved with a half-life of 686 days under rigorous shaking while the half-life of covalently-attached CT was only 21 hours. This new approach of enzyme coating with high stability and activity will make a great impact in various applications of enzymes such as bioconversion, bioremediation, and biosensors.

  19. [Activity of proteolytic and nucleolytic enzymes from the gonades of hydrobionts].

    PubMed

    Pozdniakova, Iu M; Pivnenko, T N; Epshteĭn, L M

    2004-01-01

    The activity of nucleolytic and proteolytic enzymes in milt of nine kinds of fishes belonging to various families and of three kinds invertebrates is determined. There is carried out electrophoreses division of preparations DNA, received from milts by various methods; there are determined structure and molecular weights of oligonucleotides. The influence of activity tissue enzymes on a destruction degree of DNA is established at addition enzymes of exogenic origin. PMID:19621738

  20. The biochemistry of fatty liver and kidney syndrome. Biotin-mediated restoration of hepatic gluconeogenesis in vitro and its relationship to pyruvate carboxylase activity.

    PubMed Central

    Bannister, D W

    1976-01-01

    Liver slices from chicks affected by the fatty liver and kidney syndrome display an extremely low extent of hepatic gluconeogenesis which is associated with decreased activities of certain rate-limiting gluconeogenic enzymes. Pyruvate carboxylase activity is particularly severely affected, being less than 4% of control values. Incubation of affected slices in a biotin-containing nutrient medium restores both gluconeogenesis and pyruvate carboxylase actiivity (the latter to approx. 35% of the control valve). Activities of the other enzymes studied were not greatly affected by this treatment. Restoration of gluconeogenesis did not occur if biotin was excluded from the nutrient medium, nor was it prevented by protein-synthesis inhibitors. It is concluded that the syndrome involves the lack of available biotin in the liver rather than suppression of apocarboxylase synthesis. PMID:182141

  1. Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity

    SciTech Connect

    Pols, Thijs W.H.; Ottenhoff, Roelof; Vos, Mariska; Levels, Johannes H.M.; Quax, Paul H.A.; Meijers, Joost C.M.; Pannekoek, Hans; Groen, Albert K.; Vries, Carlie J.M. de

    2008-02-22

    NR4A nuclear receptors are induced in the liver upon fasting and regulate hepatic gluconeogenesis. Here, we studied the role of nuclear receptor Nur77 (NR4A1) in hepatic lipid metabolism. We generated mice expressing hepatic Nur77 using adenoviral vectors, and demonstrate that these mice exhibit a modulation of the plasma lipid profile and a reduction in hepatic triglyceride. Expression analysis of >25 key genes involved in lipid metabolism revealed that Nur77 inhibits SREBP1c expression. This results in decreased SREBP1c activity as is illustrated by reduced expression of its target genes stearoyl-coA desaturase-1, mitochondrial glycerol-3-phosphate acyltransferase, fatty acid synthase and the LDL receptor, and provides a mechanism for the physiological changes observed in response to Nur77. Expression of LXR target genes Abcg5 and Abcg8 is reduced by Nur77, and may suggest involvement of LXR in the inhibitory action of Nur77 on SREBP1c expression. Taken together, our study demonstrates that Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity.

  2. Neuroinflammation contributes to hypokinesia in rats with hepatic encephalopathy: ibuprofen restores its motor activity.

    PubMed

    Cauli, Omar; Rodrigo, Regina; Piedrafita, Blanca; Llansola, Marta; Mansouri, Mohammad T; Felipo, Vicente

    2009-05-01

    Patients with hepatic encephalopathy show altered motor function, psychomotor slowing, and hypokinesia, which are reproduced in rats with portacaval shunts (PCS). Increased extracellular glutamate in substantia nigra pars reticulata (SNr) is responsible for hypokinesia in PCS rats. The mechanisms by which liver failure leads to increased extracellular glutamate in SNr remain unclear. Inflammation seems to act synergistically with hyperammonemia to induce neurological alterations in hepatic encephalopathy. It is therefore possible that inflammation-associated alterations may contribute to motor alterations in hepatic encephalopathy. The aim of this work was to assess whether treatment with an antiinflammatory, ibuprofen, is able to normalize extracellular glutamate in SNr and/or to improve hypokinesia in PCS rats. The amounts of the glutamate transporters GLT-1 and EAAC-1 are reduced by 26% and 32%, respectively, in SNr of PCS rats. This reduction is associated with a tenfold increase in extracellular glutamate in SNr and a reduction in motor activity. Chronic treatment with 30 mg/kg ibuprofen completely normalizes the amount of GLT-1 and EAAC-1 and significantly reduces (by 53%) extracellular glutamate in SNr of PCS rats. Moreover, ibuprofen, at 15 or 30 (but not at 5) mg/kg/day, completely eliminates hypokinesia, restoring normal motor activity. This supports the idea that inflammation is a main contributor to the induction of hypokinesia in hepatic encephalopathy. Moreover, these data point to the possible therapeutic utility of decreasing inflammation, by safe procedures, in the treatment of the motor deficits in patients with hepatic encephalopathy. PMID:19025766

  3. Hepatic SRC-1 Activity Orchestrates Transcriptional Circuitries of Amino Acid Pathways with Potential Relevance for Human Metabolic Pathogenesis

    PubMed Central

    Tannour-Louet, Mounia; York, Brian; Tang, Ke; Stashi, Erin; Bouguerra, Hichem; Zhou, Suoling; Yu, Hui; Wong, Lee-Jun C.; Stevens, Robert D.; Xu, Jianming; Newgard, Christopher B.; O'Malley, Bert W.

    2014-01-01

    Disturbances in amino acid metabolism are increasingly recognized as being associated with, and serving as prognostic markers for chronic human diseases, such as cancer or type 2 diabetes. In the current study, a quantitative metabolomics profiling strategy revealed global impairment in amino acid metabolism in mice deleted for the transcriptional coactivator steroid receptor coactivator (SRC)-1. Aberrations were hepatic in origin, because selective reexpression of SRC-1 in the liver of SRC-1 null mice largely restored amino acids concentrations to normal levels. Cistromic analysis of SRC-1 binding sites in hepatic tissues confirmed a prominent influence of this coregulator on transcriptional programs regulating amino acid metabolism. More specifically, SRC-1 markedly impacted tyrosine levels and was found to regulate the transcriptional activity of the tyrosine aminotransferase (TAT) gene, which encodes the rate-limiting enzyme of tyrosine catabolism. Consequently, SRC-1 null mice displayed low TAT expression and presented with hypertyrosinemia and corneal alterations, 2 clinical features observed in the human syndrome of TAT deficiency. A heterozygous missense variant of SRC-1 (p.P1272S) that is known to alter its coactivation potential, was found in patients harboring idiopathic tyrosinemia-like disorders and may therefore represent one risk factor for their clinical symptoms. Hence, we reinforce the concept that SRC-1 is a central factor in the fine orchestration of multiple pathways of intermediary metabolism, suggesting it as a potential therapeutic target that may be exploitable in human metabolic diseases and cancer. PMID:25148457

  4. Changes in the spectrum and rates of extracellular enzyme activities in seawater following aggregate formation

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; Steen, A. D.; Arnosti, C.

    2010-03-01

    Marine snow aggregates are heavily colonized by heterotrophic microorganisms that express high levels of hydrolytic activities, making aggregates hotspots for carbon remineralization in the ocean. To assess how aggregate formation influences the ability of seawater microbial communities to access organic carbon, we compared hydrolysis rates of six polysaccharides in coastal seawater after aggregates had been formed (via incubation on a roller table) with hydrolysis rates in seawater from the same site that had not incubated on a roller table (referred to as whole seawater). Hydrolysis rates in the aggregates themselves were up to three orders of magnitude higher on a volume basis than in whole seawater. The enhancement of enzyme activity in aggregates relative to whole seawater differed by substrate, suggesting that the enhancement was under cellular control, rather than due to factors such as lysis or grazing. A comparison of hydrolysis rates in whole seawater with those in aggregate-free seawater, i.e. the fraction of water from the roller bottles that did not contain aggregates, demonstrated a nuanced microbial response to aggregate formation. Activities of laminarinase and xylanase enzymes in aggregate-free seawater were higher than in whole seawater, while activities of chondroitin, fucoidan, and arabinogalactan hydrolyzing enzymes were lower than in whole seawater. These data suggest that aggregate formation enhanced production of laminarinase and xylanase enzymes, and the enhancement also affected the surrounding seawater. Decreased activities of chondroitin, fucoidan, and arabinoglactan-hydrolyzing enzymes in aggregate-free seawaters relative to whole seawater are likely due to shifts in enzyme production by the aggregate-associated community, coupled with the effects of enzyme degradation. Enhanced activities of laminarin- and xylan-hydrolyzing enzymes in aggregate-free seawater were due at least in part to cell-free enzymes. Measurements of enzyme

  5. Changes in enzyme activities in tissues of rats exposed to hypoxia (Short Communication)

    PubMed Central

    Cryer, Anthony; Bartley, Walter

    1973-01-01

    Rats were exposed to various degrees of hypoxia and enzyme activities in their tissues were determined. In general, oxidative metabolism was not increased in response to hypoxia, nor was anaerobic metabolism. Physiological and anatomical changes were concluded to be more important than changes in cellular enzyme activities in the overall adaptation to acute hypoxia. PMID:4357712

  6. Macromolecular (pro)drugs with concurrent direct activity against the hepatitis C virus and inflammation.

    PubMed

    Wohl, Benjamin M; Smith, Anton A A; Jensen, Bettina E B; Zelikin, Alexander N

    2014-12-28

    Macromolecular prodrugs (MPs) are a powerful tool to alleviate side-effects and improve the efficacy of the broad-spectrum antiviral agent ribavirin. In this work, we sought an understanding of what makes an optimal formulation within the macromolecular parameter space--nature of the polymer carrier, average molar mass, drug loading, or a good combination thereof. A panel of MPs based on biocompatible synthetic vinylic and (meth)acrylic polymers was tested in an anti-inflammatory assay with relevance to alleviating inflammation in the liver during hepatitis C infection. Pristine polymer carriers proved to have a pronounced anti-inflammatory activity, a notion which may prove significant in developing MPs for antiviral and anticancer treatments. With conjugated ribavirin, MPs revealed enhanced activity but also higher toxicity. Therapeutic windows and therapeutic indices were determined and discussed to reveal the most potent formulation and those with optimized safety. Polymers were also tested as inhibitors of replication of the hepatitis C viral RNA using a subgenomic viral replicon system. For the first time, negatively charged polymers are revealed to have an intracellular activity against hepatitis C virus replication. Concerted activity of the polymer and ribavirin afforded MPs which significantly increased the therapeutic index of ribavirin-based treatment. Taken together, the systematic investigation of the macromolecular space identified lead candidates with high efficacy and concurrent direct activity against the hepatitis C virus and inflammation. PMID:25451544

  7. EPAC activation inhibits acetaldehyde-induced activation and proliferation of hepatic stellate cell via Rap1.

    PubMed

    Yang, Yan; Yang, Feng; Wu, Xiaojuan; Lv, Xiongwen; Li, Jun

    2016-05-01

    Hepatic stellate cells (HSCs) activation represents an essential event during alcoholic liver fibrosis (ALF). Previous studies have demonstrated that the rat HSCs could be significantly activated after exposure to 200 μmol/L acetaldehyde for 48 h, and the cAMP/PKA signaling pathways were also dramatically upregulated in activated HSCs isolated from alcoholic fibrotic rat liver. Exchange protein activated by cAMP (EPAC) is a family of guanine nucleotide exchange factors (GEFs) for the small Ras-like GTPases Rap, and is being considered as a vital mediator of cAMP signaling in parallel with the principal cAMP target protein kinase A (PKA). Our data showed that both cAMP/PKA and cAMP/EPAC signaling pathways were involved in acetaldehyde-induced HSCs. Acetaldehyde could reduce the expression of EPAC1 while enhancing the expression of EPAC2. The cAMP analog Me-cAMP, which stimulates the EPAC/Rap1 pathway, could significantly decrease the proliferation and collagen synthesis of acetaldehyde-induced HSCs. Furthermore, depletion of EPAC2, but not EPAC1, prevented the activation of HSC measured as the production of α-SMA and collagen type I and III, indicating that EPAC1 appears to have protective effects on acetaldehyde-induced HSCs. Curiously, activation of PKA or EPAC perhaps has opposite effects on the synthesis of collagen and α-SMA: EPAC activation by Me-cAMP increased the levels of GTP-bound (activated) Rap1 while PKA activation by Phe-cAMP had no significant effects on such binding. These results suggested that EPAC activation could inhibit the activation and proliferation of acetaldehyde-induced HSCs via Rap1. PMID:26854595

  8. Enzyme Activity Profiles during Fruit Development in Tomato Cultivars and Solanum pennellii1[W][OA

    PubMed Central

    Steinhauser, Marie-Caroline; Steinhauser, Dirk; Koehl, Karin; Carrari, Fernando; Gibon, Yves; Fernie, Alisdair R.; Stitt, Mark

    2010-01-01

    Enzymes interact to generate metabolic networks. The activities of more than 22 enzymes from central metabolism were profiled during the development of fruit of the modern tomato cultivar Solanum lycopersicum ‘M82’ and its wild relative Solanum pennellii (LA0716). In S. pennellii, the mature fruit remains green and contains lower sugar and higher organic acid levels. These genotypes are the parents of a widely used near introgression line population. Enzymes were also profiled in a second cultivar, S. lycopersicum ‘Moneymaker’, for which data sets for the developmental changes of metabolites and transcripts are available. Whereas most enzyme activities declined during fruit development in the modern S. lycopersicum cultivars, they remained high or even increased in S. pennellii, especially enzymes required for organic acid synthesis. The enzyme profiles were sufficiently characteristic to allow stages of development and cultivars and the wild species to be distinguished by principal component analysis and clustering. Many enzymes showed coordinated changes during fruit development of a given genotype. Comparison of the correlation matrices revealed a large overlap between the two modern cultivars and considerable overlap with S. pennellii, indicating that despite the very different development responses, some basic modules are retained. Comparison of enzyme activity, metabolite profiles, and transcript profiles in S. lycopersicum ‘Moneymaker’ revealed remarkably little connectivity between the developmental changes of transcripts and enzymes and even less between enzymes and metabolites. We discuss the concept that the metabolite profile is an emergent property that is generated by complex network interactions. PMID:20335402

  9. Extracellular enzyme activity in anaerobic bacterial cultures: evidence of pullulanase activity among mesophilic marine bacteria.

    PubMed

    Arnosti, C; Repeta, D J

    1994-03-01

    The extracellular enzymatic activity of a mixed culture of anaerobic marine bacteria enriched on pullulan [alpha(1,6)-linked maltotriose units] was directly assessed with a combination of gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR). Hydrolysis products of pullulan were separated by GPC into three fractions with molecular weights of > or = 10,000, approximately 5,000, and < or = 1,200. NMR spectra of these fractions demonstrated that pullulan was rapidly and specifically hydrolyzed at alpha(1,6) linkages by pullulanase enzymes, most likely type II pullulanase. Although isolated pullulanase enzymes have been shown to hydrolyze pullulan completely to maltotriose (S. H. Brown, H. R. Costantino, and R. M. Kelly, Appl. Environ. Microbiol. 56:1985-1991, 1990; M. Klingeberg, H. Hippe, and G. Antranikian, FEMS Microbiol. Lett. 69:145-152, 1990; R. Koch, P. Zablowski, A. Spreinat, and G. Antranikian, FEMS Microbiol. Lett. 71:21-26, 1990), the smallest carbohydrate detected in the bacterial cultures consisted of two maltotriose units linked through one alpha(1,6) linkage. Either the final hydrolysis step was closely linked to substrate uptake, or specialized porins similar to maltoporin might permit direct transport of large oligosaccharides into the bacterial cell. This is the first report of pullulanase activity among mesophilic marine bacteria. The combination of GPC and NMR could easily be used to assess other types of extracellular enzyme activity in bacterial cultures. PMID:8161177

  10. A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.

    PubMed

    Arnold, Laurence H; Kunzelmann, Simone; Webb, Martin R; Taylor, Ian A

    2015-01-01

    The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents. PMID:25331707

  11. Carbohydrate-active enzymes: sequences, shapes, contortions and cells.

    PubMed

    Davies, Gideon J; Williams, Spencer J

    2016-02-01

    The enzyme-catalysed degradation of oligo and polysaccharides is of considerable interest in many fields ranging from the fundamental-understanding the intrinsic chemical beauty-through to the applied, including diverse practical applications in medicine and biotechnology. Carbohydrates are the most stereochemically-complex biopolymer, and myriad different natural polysaccharides have led to evolution of multifaceted enzyme consortia for their degradation. The glycosidic bonds that link sugar monomers are among the most chemically-stable, yet enzymatically-labile, bonds in the biosphere. That glycoside hydrolases can achieve a rate enhancement (kcat/kuncat) >10(17)-fold provides testament to their remarkable proficiency and the sophistication of their catalysis reaction mechanisms. The last two decades have seen significant advances in the discovery of new glycosidase sequences, sequence-based classification into families and clans, 3D structures and reaction mechanisms, providing new insights into enzymatic catalysis. New impetus to these studies has been provided by the challenges inherent in plant and microbial polysaccharide degradation, both in the context of environmentally-sustainable routes to foods and biofuels, and increasingly in human nutrition. Study of the reaction mechanism of glycoside hydrolases has also inspired the development of enzyme inhibitors, both as mechanistic probes and increasingly as therapeutic agents. We are on the cusp of a new era where we are learning how to dovetail powerful computational techniques with structural and kinetic data to provide an unprecedented view of conformational details of enzyme action. PMID:26862192

  12. Mapping Metabolic Brain Activity in Three Models of Hepatic Encephalopathy

    PubMed Central

    Méndez, Marta; Fidalgo, Camino; Aller, María Ángeles; Arias, Jaime; Arias, Jorge L.

    2013-01-01

    Cirrhosis is a common disease in Western countries. Liver failure, hyperammonemia, and portal hypertension are the main factors that contribute to human cirrhosis that frequently leads to a neuropsychiatric disorder known as hepatic encephalopathy (HE). In this study, we examined the differential contribution of these leading factors to the oxidative metabolism of diverse brain limbic system regions frequently involved in memory process by histochemical labelling of cytochrome oxidase (COx). We have analyzed cortical structures such as the infralimbic and prelimbic cotices, subcortical structures such as hippocampus and ventral striatum, at thalamic level like the anterodorsal, anteroventral, and mediodorsal thalamus, and, finally, the hypothalamus, where the mammillary nuclei (medial and lateral) were measured. The severest alteration is found in the model that mimics intoxication by ammonia, followed by the thioacetamide-treated group and the portal hypertension group. No changes were found at the mammillary bodies for any of the experimental groups. PMID:23573412

  13. Functional activity of sphingomyelin cycle in rat liver in chronic toxic hepatitis.

    PubMed

    Serebrov, V Yu; Kuzmenko, D I; Burov, P G; Novitsky, S V

    2008-12-01

    Activities of sphingomyelinase and ceramidase decreased in the liver in chronic toxic hepatitis and the balance between the levels of proapoptotic ceramide and antiapoptotic sphyngosine-1-phosphate shifts towards the latter substance. Pronounced changes in the qualitative and quantitative composition of fatty acids in the sphingomyelin cycle effector molecules were revealed. PMID:19513367

  14. DOES IRON OR HEME CONTROL RAT HEPATIC DELTA-AMINOLEVULINIC ACID SYNTHETASE ACTIVITY

    EPA Science Inventory

    Disodium ethylenediamine tetraacetic acid and/or allylisopropylacetamide administration to rat pups did not evoke a premature induction of hepatic d-aminolevulinic acid synthetase. Administration of iron to adult rats did not alter d-aminolevulinic acid synthetase activity and ha...

  15. Spatial distribution of enzyme activities along the root and in the rhizosphere of different plants

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

    Extracellular enzymes are important for decomposition of many biological macromolecules abundant in soil such as cellulose, hemicelluloses and proteins. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. So far acquisition of in situ data about local activity of different enzymes in soil has been challenged. That is why there is an urgent need in spatially explicit methods such as 2-D zymography to determine the variation of enzymes along the roots in different plants. Here, we developed further the zymography technique in order to quantitatively visualize the enzyme activities (Spohn and Kuzyakov, 2013), with a better spatial resolution We grew Maize (Zea mays L.) and Lentil (Lens culinaris) in rhizoboxes under optimum conditions for 21 days to study spatial distribution of enzyme activity in soil and along roots. We visualized the 2D distribution of the activity of three enzymes:β-glucosidase, leucine amino peptidase and phosphatase, using fluorogenically labelled substrates. Spatial resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography shows different pattern of spatial distribution of enzyme activity along roots and soil of different plants. We observed a uniform distribution of enzyme activities along the root system of Lentil. However, root system of Maize demonstrated inhomogeneity of enzyme activities. The apical part of an individual root (root tip) in maize showed the highest activity. The activity of all enzymes was the highest at vicinity of the roots and it decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify

  16. Uronic Acid products release from enzymically active cell wall from tomato fruit and its dependency on enzyme quantity and distribution.

    PubMed

    Huber, D J; Lee, J H

    1988-07-01

    Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed. PMID:16666191

  17. Enzyme markers

    MedlinePlus

    ... or defects passed down through families (inherited) can affect how enzymes work. Some enzymes are affected by several genes. Test results are usually reported as a percentage of normal enzyme activity.

  18. A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

    PubMed Central

    Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  19. A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

    PubMed

    Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

    2015-04-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  20. NADPH-dependent generation of a cytosolic dithiol which activates hepatic iodothyronine 5'-deiodinase. Demonstration by alkylation with iodoacetamide.

    PubMed Central

    Das, A K; Hummel, B C; Walfish, P G

    1986-01-01

    We have assessed a previously proposed mechanism mediating 5'-deiodinase activation involving enzymic reduction of disulphides to thiols in non-glutathione cytosolic components of Mr approx. 13,000 (Fraction B) catalysed by NADPH in the presence of other cytosolic components of Mr greater than 60,000 (Fraction A). The extent of Fraction B reduction under various experimental conditions was monitored by determining the amount of 14C incorporated into chromatographically isolated Fractions B and A after their alkylation with iodo[14C]acetamide. Incorporation of 14C into B was found to require the simultaneous presence of NADPH and A, to be directly proportional to the concentration of NADPH added, and to be unaffected by either propylthiouracil or iopanoate. Activation of 5'-deiodinase attainable using B after its partial reduction by various concentrations of NADPH and subsequent alkylation with non-radioactive iodoacetamide was inversely proportional to the previously added concentration of NADPH. Fraction B was stable at 100 degrees C for 5 min, while similar heat treatment of Fraction A or omission of NADPH resulted in a complete loss of 14C incorporation. A greater than 90% reduction in iodo[14C]acetamide incorporation was revealed when 0.2 mM-sodium arsenite was added after enzymic reduction of B, as well as when NADPH was replaced by NADH. Fraction B could be labelled more extensively after reduction non-specifically, with dithiothreitol or NaBH4, but not by GSH. These observations provide strong evidence for the presence in vivo of a cytosolic disulphide (DFBS2) in Fraction B which can be reduced enzymically to a dithiol [DFB(SH)2] by NADPH and cytosolic components in Fraction A. The degree of activation of hepatic 5'-deiodinase correlated with the amount of available (unalkylated) Fraction B. PMID:3814095

  1. Heating of vegetable oils influences the activity of enzymes participating in arachidonic acid formation in Wistar rats.

    PubMed

    Stawarska, Agnieszka; Białek, Agnieszka; Tokarz, Andrzej

    2015-10-01

    Dietary intake of lipids and their fatty acids profile influence many aspects of health. Thermal processing changes the properties of edible oils and can also modify their metabolism, for example, eicosanoids formation. The aim of our study was to verify whether the activity of desaturases can be modified by lipids intake, especially by the fatty acids content. The experimental diets contained rapeseed oil, sunflower oil, and olive oil, both unheated and heated (for 10 minutes at 200 °C each time before administration), and influenced the fatty acids composition in serum and the activity of enzymes participating in arachidonic acid (AA) formation. The activity of desaturases was determined by measuring the amounts of AA formed in vitro derived from linoleic acid as determined in liver microsomes of Wistar rats. In addition, the indices of ∆(6)-desaturase (D6D) and ∆(5)-desaturase (D5D) have been determined. To realize this aim, the method of high-performance liquid chromatography has been used with ultraviolet-visible spectrophotometry detection. Diet supplementation with the oils rich in polyunsaturated fatty acids affects the fatty acids profile in blood serum and the activity of D6D and ∆(5)-desaturase in rat liver microsomes, the above activities being dependent on the kind of oil applied. Diet supplementation with heated oils has been found to increase the amount of AA produced in hepatic microsomes; and in the case of rapeseed oil and sunflower oil, it has also increased D6D activity. PMID:26094213

  2. Changes in the spectrum and rates of extracellular enzyme activities in seawater following aggregate formation

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; Steen, A. D.; Arnosti, C.

    2009-12-01

    Marine snow aggregates are heavily colonized by heterotrophic microorganisms that express high levels of hydrolytic activities, making aggregates hotspots for carbon remineralization in the ocean. To assess how aggregate formation influences the ability of seawater microbial communities to access organic carbon, we compared hydrolysis rates of six polysaccharides in coastal seawater after aggregates had been formed (via incubation on a roller table) with hydrolysis rates in seawater from the same site that had not incubated on a roller table (referred to as whole seawater). Hydrolysis rates in the aggregates themselves were up to three orders of magnitude higher on a volume basis than in whole seawater. The enhancement of enzyme activity in aggregates relative to whole seawater differed by substrate, suggesting that the enhancement was under cellular control, rather than due to factors such as lysis or grazing. A comparison of hydrolysis rates in whole seawater with those in aggregate-free seawater, i.e. the fraction of water from the roller bottles that did not contain aggregates, demonstrated a nuanced microbial response to aggregate formation. Activities of laminarinase and xylanase enzymes in aggregate-free seawater were higher than in whole seawater, while activities of chondroitin, fucoidan, and arabinogalactan hydrolyzing enzymes were lower than in whole seawater. These data suggest that aggregate formation enhanced production of laminarinase and xylanase enzymes, and the enhancement also affected the surrounding seawater. Decreased activities of chondroitin, fucoidan, and arabinoglactan-hydrolyzing enzymes in aggregate-free seawater relative to whole seawater are likely due to shifts in enzyme production by the aggregate-associated community, coupled with the effects of enzyme degradation. Enhanced activities of laminarin- and xylan-hydrolyzing enzymes in aggregate-free seawater were due at least in part to cell-free enzymes. Measurements of enzyme lifetime

  3. Effects of orally applied butyrate bolus on histone acetylation and cytochrome P450 enzyme activity in the liver of chicken – a randomized controlled trial

    PubMed Central

    2013-01-01

    Background Butyrate is known as histone deacetylase inhibitor, inducing histone hyperacetylation in vitro and playing a predominant role in the epigenetic regulation of gene expression and cell function. We hypothesized that butyrate, endogenously produced by intestinal microbial fermentation or applied as a nutritional supplement, might cause similar in vivo modifications in the chromatin structure of the hepatocytes, influencing the expression of certain genes and therefore modifying the activity of hepatic microsomal drug-metabolizing cytochrome P450 (CYP) enzymes. Methods An animal study was carried out in chicken as a model to investigate the molecular mechanisms of butyrate’s epigenetic actions in the liver. Broiler chicks in the early post-hatch period were treated once daily with orally administered bolus of butyrate following overnight starvation with two different doses (0.25 or 1.25 g/kg body weight per day) for five days. After slaughtering, cell nucleus and microsomal fractions were separated by differential centrifugation from the livers. Histones were isolated from cell nuclei and acetylation of hepatic core histones was screened by western blotting. The activity of CYP2H and CYP3A37, enzymes involved in biotransformation in chicken, was detected by aminopyrine N-demethylation and aniline-hydroxylation assays from the microsomal suspensions. Results Orally added butyrate, applied in bolus, had a remarkable impact on nucleosome structure of hepatocytes: independently of the dose, butyrate caused hyperacetylation of histone H2A, but no changes were monitored in the acetylation state of H2B. Intensive hyperacetylation of H3 was induced by the higher administered dose, while the lower dose tended to increase acetylation ratio of H4. In spite of the observed modification in histone acetylation, no significant changes were observed in the hepatic microsomal CYP2H and CYP3A37 activity. Conclusion Orally added butyrate in bolus could cause in vivo

  4. APPL1-mediated activation of STAT3 contributes to inhibitory effect of adiponectin on hepatic gluconeogenesis.

    PubMed

    Ding, Youming; Zhang, Deling; Wang, Bin; Zhang, Yemin; Wang, Lei; Chen, Xiaoyan; Li, Mingxin; Tang, Zhao; Wang, Changhua

    2016-09-15

    Adiponectin has been shown to suppress hepatic gluconeogenesis. However, the signaling pathways underlying its action remain ill-defined. The purpose of this study was to examine the potential role of APPL1 in mediating anti-gluconeogenic ability of adiponectin. Primary hepatocytes were isolated from male C57BL/6 mice. Western blot and RT-PCR were performed to detect protein expression and mRNA level, respectively. The protein-protein association was determined by immunoprecipitation and GST pull-down assay. We found that APPL1 protein levels were negatively associated with expressions of proteins and mRNAs of gluconeogenesis enzymes under stimulation with adiponectin. In addition, adiponectin-stimulated STAT3 phosphorylation and acetylation were positively regulated by APPL1 and negative regulated by SirT1. Pharmacological and genetic inhibition of STAT3 mitigated impact of adiponectin on hepatic gluconeogenesis. Furthermore, adiponectin administration facilitated the binding of APPL1 to SirT1 and suppressed the association of SirT1 with STAT3. Taken together, our study showed that APPL1-SirT1-STAT3 pathway mediated adiponectin signaling in primary hepatocytes. This new finding provides a novel mechanism by which adiponectin suppresses hepatic gluconeogenesis. PMID:27246173

  5. Serum and urinary enzyme activities in renal artery embolism.

    PubMed

    Donadio, C; Auner, I; Giordani, R; Lucchetti, A; Pentimone, F

    1986-10-31

    Renal artery embolism is not a rare occurrence, especially in patients with valvular heart disease, but the early diagnosis of this condition is infrequently accomplished. We report the clinical and laboratory data of 2 patients with valvular heart disease who presented with unilateral renal artery embolization. The usefulness of the determination of serum and urinary enzymes and renal function tests is discussed. We propose that these parameters support an earlier and more accurate diagnosis of renal artery embolism. PMID:2877758

  6. Quantitative Packaging of Active Enzymes into a Protein Cage.

    PubMed

    Azuma, Yusuke; Zschoche, Reinhard; Tinzl, Matthias; Hilvert, Donald

    2016-01-22

    Genetic fusion of cargo proteins to a positively supercharged variant of green fluorescent protein enables their quantitative encapsulation by engineered lumazine synthase capsids possessing a negatively charged lumenal surface. This simple tagging system provides a robust and versatile means of creating hierarchically ordered protein assemblies for use as nanoreactors. The generality of the encapsulation strategy and its effect on enzyme function were investigated with eight structurally and mechanistically distinct catalysts. PMID:26695342

  7. Photoregulation of Biological Activity by Photochromic Reagents, IV. A Model for Diurnal Variation of Enzymic Activity*

    PubMed Central

    Bieth, Joseph; Wassermann, Norbert; Vratsanos, Spyros M.; Erlanger, Bernard F.

    1970-01-01

    Levels of acetylcholinesterase activity can be made to vary in response to the presence or absence of sunlight in a system that can be considered as a model for photoperiodic processes found in nature. The enzyme is rendered photosensitive by the presence of a photochromic inhibitor, N-p-phenylazophenylcarbamyl choline, which changes from a trans to a cis isomer under the influence of the light of the sun and reverts back to the trans isomer in the dark. The two isomers differ in their ability acetylcholinesterase, thus rendering the enzyme system responsive to sunlight. The relationship of this system to photoresponsive processes in nature is discussed, and a possible role in photoregulation is suggested for naturally occurring carotenoids. PMID:5269248

  8. Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity.

    PubMed

    Federico, R; Angelini, R; Cesta, A; Pini, C

    1985-09-01

    A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified (125)I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor. PMID:16664402

  9. Antifouling activity of enzyme-functionalized silica nanobeads.

    PubMed

    Zanoni, Michele; Habimana, Olivier; Amadio, Jessica; Casey, Eoin

    2016-03-01

    The amelioration of biofouling in industrial processing equipment is critical for performance and reliability. While conventional biocides are effective in biofouling control, they are potentially hazardous to the environment and in some cases corrosive to materials. Enzymatic approaches have been shown to be effective and can overcome the disadvantages of traditional biocides, however they are typically uneconomic for routine biofouling control. The aim of this study was to design a robust and reusable enzyme-functionalized nano-bead system having biofilm dispersion properties. This work describes the biochemical covalent functionalization of silica-based nanobeads (hereafter referred to as Si-NanoB) with Proteinase K (PK). Results showed that PK-functionalized Si-NanoB are effective in dispersing both protein-based model biofilms and structurally altering Pseudomonas fluorescens biofilms, with significant decreases in surface coverage and thickness of 30.1% and 38.85%, respectively, while increasing surface roughness by 19 % following 24 h treatments on bacterial biofilms. This study shows that enzyme-functionalized nanobeads may potentially be an environmentally friendly and cost effective alternative to pure enzyme and chemical treatments. PMID:26370186

  10. Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity.

    PubMed Central

    Bauer, P I; Buki, K G; Hakam, A; Kun, E

    1990-01-01

    The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was

  11. High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

    PubMed Central

    Bell, Colin W.; Fricks, Barbara E.; Rocca, Jennifer D.; Steinweg, Jessica M.; McMahon, Shawna K.; Wallenstein, Matthew D.

    2013-01-01

    Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample). Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil

  12. Histidine Augments the Suppression of Hepatic Glucose Production by Central Insulin Action

    PubMed Central

    Kimura, Kumi; Nakamura, Yusuke; Inaba, Yuka; Matsumoto, Michihiro; Kido, Yoshiaki; Asahara, Shun-ichiro; Matsuda, Tomokazu; Watanabe, Hiroshi; Maeda, Akifumi; Inagaki, Fuyuhiko; Mukai, Chisato; Takeda, Kiyoshi; Akira, Shizuo; Ota, Tsuguhito; Nakabayashi, Hajime; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2013-01-01

    Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes. PMID:23474485

  13. Periphytic photosynthetic stimulation of extracellular enzyme activity in aquatic microbial communities associated with decaying typha litter.

    PubMed

    Francoeur, Steven N; Schaecher, Mark; Neely, Robert K; Kuehn, Kevin A

    2006-11-01

    We examined the effect of light on extracellular enzyme activities of periphytic/endogenous microbial assemblages associated with decomposing litter of an emergent macrophyte Typha angustifolia within a small inland wetland in southeastern Michigan. Standing-dead Typha leaf litter was collected, placed into floating wire mesh litter baskets, and submerged in a wetland pool. Enzyme saturation assays were conducted on three occasions following litter submergence (days 9, 28, and 44) to generate saturation curves for the individual enzymes tested and to examine potential differences in enzyme saturation kinetics during microbial colonization and development. Experimental light manipulations were conducted on two occasions during microbial development (days 10 and 29). Short-term (30 min) light exposure significantly increased extracellular beta-glucosidase activity of litter-associated microbial communities. Activities of beta-xylosidase and leucine-aminopeptidase were not stimulated, and stimulation of phosphatase activity was variable. The exact mechanism for increased enzyme activity remains unknown, but it may have been increased pH arising from periphytic algal photosynthesis. These results suggest that extracellular enzyme activity in microbial communities colonizing natural organic substrata may be influenced by light/photosynthesis, as has previously been demonstrated for periphyton communities grown on artificial, inert substrata. Thus, light/photosynthetic mediated stimulation of extracellular enzyme activities may be a common occurrence in microbial communities associated with natural decaying plant litter in wetlands and might engender diurnal patterns in other microbial decay processes (e.g., production, organic matter decomposition, and mineralization). PMID:17082997

  14. Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells.

    PubMed

    Lněničková, Kateřina; Procházková, Eliška; Skálová, Lenka; Matoušková, Petra; Bártíková, Hana; Souček, Pavel; Szotáková, Barbora

    2016-01-01

    The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE) and epigallocatechin gallate (EGCG) using both proliferating (P) and differentiated (D) caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes' activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST) activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT) activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well. PMID:27617982

  15. Functional heterogeneity of UDP-glucuronosyltransferase as indicated by its differential development and inducibility by glucocorticoids. Demonstration of two groups within the enzyme's activity towards twelve substrates.

    PubMed Central

    Wishart, G J

    1978-01-01

    1. UDP-glucuronosyltransferase activity towards 12 substrates has been assessed in rat liver during the perinatal period. 2. Between days 16 and 20 of gestation, enzyme activities towards the substrates 2-aminophenol, 2-aminobenzoate, 4-nitrophenol, 1-naphthol, 4-methylumbelliferone and 5-hydroxytryptamine (the 'late foetal' group) surge to reach adult values, while activities towards bilirubin, testosterone, beta-oestradiol, morphine, phenolphthalein, and chloramphenicol (the 'neonatal' group) remain negligible or at less than 10% of adult values. 3. By the second postnatal day, enzyme activities towards the neonatal group have attained, or approached adult values. 4. Dexamethasone precociously stimulates in 17-day foetal liver in utero transferase activities in the late foetal, but not the neonatal group. A similar inductive pattern is found for 15-day foetal liver in organ culture. 5. It is suggested that foetal glucocorticoids, whose synthesis markedly increases between days 16 and 20 of gestation, are responsibile for triggering the simultaneous surge of all the hepatic UDP-glucuronosyltransferase activities in the late foetal group. The neonatal group of activities apparently require a different or additional stimulus for their appearance. 6. The relationship of these two groups of transferase activities to other similar groups observed during induction by xenobiotics and enzyme purification is discussed. PMID:101211

  16. Ionone Derivatives from the Mycelium of Phellinus linteus and the Inhibitory Effect on Activated Rat Hepatic Stellate Cells

    PubMed Central

    Huang, Shiow-Chyn; Kuo, Ping-Chung; Hung, Hsin-Yi; Pan, Tai-Long; Chen, Fu-An; Wu, Tian-Shung

    2016-01-01

    Three new γ-ionylideneacetic acid derivatives, phellinulins A–C (1–3), were characterized from the mycelium extract of Phellinus linteus. The chemical structures were established based on the spectroscopic analysis. In addition, phellinulin A (1) was subjected to the examination of effects on activated rat hepatic stellate cells and exhibited significant inhibition of hepatic fibrosis. PMID:27164091

  17. Zinc might prevent heat-induced hepatic injury by activating the Nrf2-antioxidant in mice.

    PubMed

    Wang, F; Li, Y; Cao, Y; Li, C

    2015-05-01

    Zinc (Zn) is generally known to be an essential trace element with growth-promoting and antioxidant activities. The present study was performed to clarify the role of Zn in the livers of heat-treated mice. Eight-week-old male mice were divided into control (Con), heat treatment (HT) and heat treatment plus zinc groups (HT + Zn) and were fed diets containing 60, 60, or 300 mg/kg Zn (zinc sulfate), respectively. After 30 days of feeding on their respective diets, the control group was maintained at a controlled temperature (25 °C), whereas the HT and HT + Zn groups were exposed to an elevated ambient temperature (40-42 °C) for 2 h each day. After heat exposure for seven consecutive days, sera and liver tissues were collected. The mice in the HT group exhibited reduced liver weights and lower hepatosomatic indices. Histological findings revealed that the hepatocytes of the HT group were subjected to serious damage and exhibited irregular arrangements and nuclear pyknosis. Moreover, in the HT group, the hepatic malondialdehyde levels were significantly increased, while the serum alkaline phosphatase levels, hepatic copper/zinc-superoxide dismutase (CuZn-SOD) and glutathione peroxidase activities were significantly reduced compared to those of the control group. However, in the HT + Zn group, the histomorphology of the liver was restored, the serum aspartate aminotransferase (AST) level was significantly decreased, and the hepatic CuZn-SOD activity was significantly increased compared to the HT group. Furthermore, expressions of the hepatic Nrf2 protein and Nrf2, Keap1, and NQO1 genes in the HT + Zn group were not only higher than the HT group but also higher than the control group. Zn might alleviate heat-induced hepatic injury as revealed by restored histomorphology and AST level. Our results further suggest that Zn might exert its protective effects via the activation of the Nrf2-antioxidant pathway. PMID:25586622

  18. Thrombin activation and liver inflammation in advanced hepatitis C virus infection

    PubMed Central

    González-Reimers, Emilio; Quintero-Platt, Geraldine; Martín-González, Candelaria; Pérez-Hernández, Onán; Romero-Acevedo, Lucía; Santolaria-Fernández, Francisco

    2016-01-01

    Hepatitis C virus (HCV) infection is associated with increased thrombotic risk. Several mechanisms are involved including direct endothelial damage by the HCV virus, with activation of tissue factor, altered fibrinolysis and increased platelet aggregation and activation. In advanced stages, chronic HCV infection may evolve to liver cirrhosis, a condition in which alterations in the portal microcirculation may also ultimately lead to thrombin activation, platelet aggregation, and clot formation. Therefore in advanced HCV liver disease there is an increased prevalence of thrombotic phenomena in portal vein radicles. Increased thrombin formation may activate hepatic stellate cells and promote liver fibrosis. In addition, ischemic changes derived from vascular occlusion by microthrombi favor the so called parenchymal extinction, a process that promotes collapse of hepatocytes and the formation of gross fibrous tracts. These reasons may explain why advanced HCV infection may evolve more rapidly to end-stage liver disease than other forms of cirrhosis. PMID:27182154

  19. Thrombin activation and liver inflammation in advanced hepatitis C virus infection.

    PubMed

    González-Reimers, Emilio; Quintero-Platt, Geraldine; Martín-González, Candelaria; Pérez-Hernández, Onán; Romero-Acevedo, Lucía; Santolaria-Fernández, Francisco

    2016-05-14

    Hepatitis C virus (HCV) infection is associated with increased thrombotic risk. Several mechanisms are involved including direct endothelial damage by the HCV virus, with activation of tissue factor, altered fibrinolysis and increased platelet aggregation and activation. In advanced stages, chronic HCV infection may evolve to liver cirrhosis, a condition in which alterations in the portal microcirculation may also ultimately lead to thrombin activation, platelet aggregation, and clot formation. Therefore in advanced HCV liver disease there is an increased prevalence of thrombotic phenomena in portal vein radicles. Increased thrombin formation may activate hepatic stellate cells and promote liver fibrosis. In addition, ischemic changes derived from vascular occlusion by microthrombi favor the so called parenchymal extinction, a process that promotes collapse of hepatocytes and the formation of gross fibrous tracts. These reasons may explain why advanced HCV infection may evolve more rapidly to end-stage liver disease than other forms of cirrhosis. PMID:27182154

  20. Stabilization of glucose-6-phosphatase activity by a 21 000-dalton hepatic microsomal protein.

    PubMed Central

    Burchell, A; Burchell, B; Monaco, M; Walls, H E; Arion, W J

    1985-01-01

    Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2996501

  1. Retaining and recovering enzyme activity during degradation of TCE by methanotrophs

    SciTech Connect

    Palumbo, A.V.; Strong-Gunderson, J.M.; Carroll, S.

    1997-12-31

    To determine if compounds added during trichloroethylene (TCE) degradation could reduce the loss of enzyme activity or increase enzyme recovery, different compounds serving as energy and carbon sources, pH buffers, or free radical scavengers were tested. Formate and formic acid (reducing power and a carbon source), as well as ascorbic acid and citric acid (free radical scavengers) were added during TCE degradation at a concentration of 2 mM. A saturated solution of calcium carbonate was also tested to address pH concerns. In the presence of formate and methane, only calcium carbonate and formic acid had a beneficial effect on enzyme recovery. The calcium carbonate and formic acid both reduced the loss of enzyme activity and resulted in the highest levels of enzyme activity after recovery. 19 refs., 3 figs.

  2. Hepatitis C virus infection in sexually active homosexual men.

    PubMed

    Buchbinder, S P; Katz, M H; Hessol, N A; Liu, J; O'Malley, P M; Alter, M J

    1994-11-01

    While hepatitis C virus (HCV) is known to be transmitted parenterally, the role of sexual transmission remains unclear. In order to examine the association of sexual risk factors with HCV seroprevalence at a time when unprotected sexual practices were still quite common, 435 homosexual men recruited from a municipal sexually transmitted disease clinic with behavioural data and serologic specimens from 1983-1984 were evaluated. Overall, 25% of men reporting injecting drug use (IDU) and 5% of men with no IDU were anti-HCV positive; the rate in the non-IDU was significantly higher than age-matched rates in blood donors (summary odds ratio 3.5, 95% confidence interval 2.8-4.2). In addition to IDU, amphetamine and phencyclidine use were also associated with anti-HCV positivity on univariate analysis. Sexual risk factors for anti-HCV positivity included anal receptive intercourse, 'fisting', having an IDU sexual partner, a self-reported history of genital herpes and HIV seropositivity. On multivariate analysis, only IDU was significantly associated with anti-HCV positivity. Thus, sexual practices appear to play a minor role in transmission of HCV. PMID:7884219

  3. The Enterococcus hirae Mur-2 enzyme displays N-acetylglucosaminidase activity.

    PubMed

    Eckert, Catherine; Magnet, Sophie; Mesnage, Stéphane

    2007-02-20

    Enterococcus hirae produces two autolytic enzymes named Mur-1 and Mur-2, both previously described as N-acetylmuramidases. We used tandem mass spectrometry to show that Mur-2 in fact displays N-acetylglucosaminidase activity. This result reveals that Mur-2 and its counterparts studied to date, which are members of glycosyl hydrolase family 73 from the CAZy (Carbohydrate-Active enZyme) database, display the same catalytic activity. PMID:17258207

  4. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes. PMID:25449264

  5. Enzymes of Glycerol and Glyceraldehyde Metabolism in Mouse Liver: Effects of Caloric Restriction and Age on Activities

    PubMed Central

    Hagopian, Kevork; Ramsey, Jon J.; Weindruch, Richard

    2008-01-01

    Synopsis The influence of caloric restriction on hepatic glyceraldehyde and glycerol metabolizing enzyme activities of young and old mice were studied. Glycerol kinase and cytoplasmic glycerol-3-phosphate dehydrogenase activities were increased in both young and old CR mice when compared to controls, while triokinase increased only in old CR mice. Aldehyde dehydrogenase and aldehyde reductase activities in both young and old CR were unchanged by CR. Mitochondrial glycerol-3-phosphate dehydrogenase showed a trend towards an increased activity in old CR mice, while a trend towards a decreased activity in alcohol dehydrogenase was observed in both young and old CR mice. Serum glycerol levels decreased in young and old CR mice. Therefore, increases in glycerol kinase and glycerol-3-phosphate dehydrogenase were associated with a decrease in fasting blood glycerol levels in CR animals. A prominent role for triokinase in glyceraldehyde metabolism with CR was also observed. The results indicate that long-term CR induces sustained increases in the capacity for gluconeogenesis from glycerol. PMID:18429748

  6. Enzyme activity in terrestrial soil in relation to exploration of the Martian surface

    NASA Technical Reports Server (NTRS)

    Mclaren, A. D.

    1974-01-01

    Sensitive tests for the detection of extracellular enzyme activity in Martian soil was investigated using simulated Martian soil. Enzyme action at solid-liquid water interfaces and at low humidity were studied, and a kinetic scheme was devised and tested based on the growth of microorganisms and the oxidation of ammonium nitrite.

  7. Genome-level and biochemical diversity of the acyl-activating enzyme superfamily in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In higher plants, the superfamily of carboxyl-CoA ligases and related proteins, collectively called acyl activating enzymes (AAEs), has evolved to provide enzymes for many pathways of primary and secondary metabolism and for the conjugation of hormones to amino acids. Across the superfamily there is...

  8. Quantitation of Lipase Activity from a Bee: An Introductory Enzyme Experiment.

    ERIC Educational Resources Information Center

    Farley, Kathleen A.; Jones, Marjorie A.

    1989-01-01

    This four-hour experiment uses a bee as a source of the enzyme which is reacted with a radioactive substrate to determine the specific activity of the enzyme. Uses thin layer chromatography, visible spectrophotometry, and liquid scintillation spectrometry (if not available a Geiger-Muller counter can be substituted). (MVL)

  9. Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the effects of a biochar made from switchgrass on four soil enzymes (ß- glucosidase, ß-N-acetylglucosaminidase, lipase, and leucine aminopeptidase) to determine if biochar would consistently modify soil biological activities. Inconsistent results from enzyme assays of char-amended soils s...

  10. Open-mouthed hybrid microcapsules with elevated enzyme loading and enhanced catalytic activity.

    PubMed

    Shi, Jiafu; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi

    2014-10-25

    Open-mouthed hybrid microcapsules (HMCs) are synthesized through a hard-templating method. When utilized for enzyme immobilization and enzymatic catalysis, the open-mouthed HMCs show high enzyme loading capability, enhanced catalytic activity and desirable recycling stability, due to their fully exposed outer and inner surfaces. PMID:25189769

  11. Measuring potential denitrification enzyme activity rates using the membrane inlet mass spectrometer

    EPA Science Inventory

    The denitrification enzyme activity (DEA) assay, provides a quantitative assessment of the multi enzyme, biological process of reactive nitrogen removal via the reduction of N03 to N2. Measured in soil, usually under non limiting carbon and nitrate concentrations, this short ter...

  12. Soil Enzyme Activities as Affected by Manure Types, Application Rates and Management Practices

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The application of manure can restore soil ecosystem services related to nutrient cycling and soil organic matter (SOM) dynamics through biochemical transformations mediated by soil enzymes. Enzyme activities are very crucial in soil metabolic functioning as they drive the decomposition of organic r...

  13. Illustrating the Effect of pH on Enzyme Activity Using Gibbs Energy Profiles

    ERIC Educational Resources Information Center

    Bearne, Stephen L.

    2014-01-01

    Gibbs energy profiles provide students with a visual representation of the energy changes that occur during enzyme catalysis, making such profiles useful as teaching and learning tools. Traditional kinetic topics, such as the effect of pH on enzyme activity, are often not discussed in terms of Gibbs energy profiles. Herein, the symbolism of Gibbs…

  14. Purification and Properties of Two Proteolytic Enzymes with Carboxypeptidase Activity in Germinated Wheat 1

    PubMed Central

    Preston, Ken R.; Kruger, James E.

    1976-01-01

    Two proteolytic enzymes with carboxypeptidase activity have been isolated from a germinated wheat extract and partially characterized. Both enzymes rapidly released amino acids from hemoglobin and gluten and hydrolyzed carbobenzoxy-phenylalanylalanine. The enzymes were inhibited by diisopropylphosphofluoridate, but unaffected by salts, ethylenediaminetetraacetate, and sulfhydryl reagents at lower concentrations, and had molecular weights of approximately 55,000 and 61,000. Analysis of the hydrolysis products of hemoglobin and gluten indicated that both enzymes had broad specificities, including the ability to release proline. Images PMID:16659708

  15. In vitro enzyme inhibition activities of crude ethanolic extracts derived from medicinal plants of Pakistan.

    PubMed

    Khattak, Somia; Saeed-Ur-Rehman; Shah, Hameed Ullah; Khan, Taous; Ahmad, Manzoor

    2005-09-01

    Twenty two crude ethanolic extracts from 14 indigenous medicinal plants were subjected to enzyme inhibition screening against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and lipoxygenase enzymes (LO). Three extracts showed activity against AChE, nine extracts were found to be active against BChE and four extracts inhibited the enzyme LO. The most significant inhibition activities (> or =50%) were found in extracts derived from Aloe vera (leaves), Alpinia galanga (rhizome), Curcuma longa (rhizome), Cymbopogon citratus (leaves), Ocimum americanum (leaves), Ocimum americanum (stem) and Withania somnifera (roots). PMID:16010821

  16. Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

    NASA Astrophysics Data System (ADS)

    Liu, Jianguo; Zhang, Xiaoli; Sun, Yanhong; Lin, Wei

    2010-01-01

    The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O{2/-}). The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H. pluvialis during exposure to reactive oxygen species (ROS) such as O{2/-}. Astaxanthin reacted with ROS much faster than did the protective enzymes, and had the strongest antioxidative capacity to protect against lipid peroxidation. The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells. Astaxanthin-enriched red cells had the strongest antioxidative capacity, followed by brown cells, and astaxanthin-deficient green cells. Although there was no significant increase in expression of protective enzymes, the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin, which quenched O{2/-} before the protective enzymes could act. In green cells, astaxanthin is very low or absent; therefore, scavenging of ROS is inevitably reliant on antioxidative enzymes. Accordingly, in green cells, these enzymes play the leading role in scavenging ROS, and the expression of these enzymes is rapidly increased to reduce excessive ROS. However, because ROS were constantly increased in this study, the enhance enzyme activity in the green cells was not able to repair the ROS damage, leading to elevated MDA content. Of the four defensive enzymes measured in astaxanthin-deficient green cells, SOD eliminates O{2/-}, POD eliminates H2O2, which is a by-product of SOD activity, and APX and CAT are then initiated to scavenge excessive ROS.

  17. Experimental strategy to discover microbes with gluten-degrading enzyme activities

    NASA Astrophysics Data System (ADS)

    Helmerhorst, Eva J.; Wei, Guoxian

    2014-06-01

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  18. Activation of NLRP3 and AIM2 inflammasomes in Kupffer cells in hepatic ischemia/reperfusion.

    PubMed

    Kim, Hyo-Yeon; Kim, Seok-Joo; Lee, Sun-Mee

    2015-01-01

    Inflammasome activation by danger signals in ischemia/reperfusion (I/R) injury is responsible for the sterile inflammatory response. Signals triggering formation and activation of the inflammasome involve the generation of oxidative stress. The aim of this study was to examine the molecular mechanisms of inflammasome activation and the involvement of reactive oxygen species in hepatic I/R. I/R induced the formation of nucleotide-binding domain leucine-rich repeat containing family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes and the subsequent serum release of interleukin 1β. Pannexin-1 inhibitor and anti-cathepsin B antibody attenuated I/R-induced inflammasome activation and hepatic injury. The expression of the thioredoxin-interacting protein gene and the interaction between NLRP3 and the thioredoxin-interacting protein increased after I/R. Treatment with the antioxidant N-acetylcysteine significantly attenuated protein conversion of interleukin 1β after hepatic I/R. Moreover, pannexin-1 protein expression and cathepsin B release were strongly attenuated by N-acetylcysteine. The depletion of Kupffer cells with gadolinium chloride markedly decreased NLRP3 and AIM2 inflammasome expression and activation of their signaling pathways, and also reduced the level of caspase-1 protein in F4/80-positive cells. Our findings suggest that reactive-oxygen-species-mediated activation of NLRP3 and AIM2 inflammasomes leads to I/R-induced inflammatory responses in which Kupffer cells play a crucial role. PMID:25327779

  19. Eastern region represents a worrying cluster of active hepatitis C in Algeria in 2012.

    PubMed

    Bensalem, Aïcha; Selmani, Karima; Hihi, Narjes; Bencherifa, Nesrine; Mostefaoui, Fatma; Kerioui, Cherif; Pineau, Pascal; Debzi, Nabil; Berkane, Saadi

    2016-08-01

    Algeria is the largest country of Africa, peopled with populations living a range of traditional/rural and modern/urban lifestyles. The variations of prevalence of chronic active hepatitis care poorly known on the Algerian territory. We conducted a retrospective survey on all patients (n = 998) referred to our institution in 2012 and confirmed by us for an active hepatitis C. Half of the hepatitis C virus (HCV) isolates were genotyped. Forty Algerian regions out of the 48 were represented in our study. Three geographical clusters (Aïn-Temouchent/SidiBelAbbes, Algiers, and a large Eastern region) with an excess of active hepatitis C were observed. Patients coming from the Eastern cluster (Batna, Khenchela, Oum el Bouaghi, and Tebessa) were strongly over-represented (49% of cases, OR = 14.5, P < 0.0001). The hallmarks of Eastern region were an excess of women (65% vs. 46% in the remaining population, P < 0.0001) and the almost exclusive presence of HCV genotype 1 (93% vs. 63%, P = 0.0001). The core of the epidemics was apparently located in Khenchela (odds ratio = 24.6, P < 0.0001). This situation is plausibly connected with nosocomial transmission or traditional practices as scarification (Hijama), piercing or tattooing, very lively in this region. Distinct hepatitis C epidemics are currently affecting Algerian population. The most worrying situation is observed in rural regions located east of Algeria. J. Med. Virol. 88:1394-1403, 2016. © 2016 Wiley Periodicals, Inc. PMID:26856380

  20. [Effect of space flight on the Kosmos-1129 biosatellite on enzyme activity of the rat liver].

    PubMed

    Nemeth, S; Tigranian, R A

    1983-01-01

    After the 18.5 day Cosmos-1129 flight the activity of 7 glucocorticoid-stimulated enzymes of the rat liver was measured. Immediately postflight the activity of tyrosine aminotransferase, tryptophan pyrolase and serine dehydrogenase increased. These enzymes rapidly (within several hours) react to increased glucocorticoids. The activity of aspartate and alanine aminotransferases also increased. These enzymes require many days of a continuous effect of glucocorticoids. The glycogen concentration in the rat liver also grew. At R + 6 the activity of tryptophan pyrolase and serine dehydrogenase decreased and that of the other enzymes returned to normal. The immobilization stress applied postflight led to an increased activity of tyrosine aminotransferase and tryptophan pyrolase. This study gives evidence that after space flight rats are in an acute stress state, evidently, produced by the biosatellite recovery. PMID:6620954

  1. PNPLA3 148M Carriers with Inflammatory Bowel Diseases Have Higher Susceptibility to Hepatic Steatosis and Higher Liver Enzymes

    PubMed Central

    Mancina, Rosellina Margherita; Spagnuolo, Rocco; Milano, Marta; Brogneri, Simona; Morrone, Attilio; Cosco, Cristina; Lazzaro, Veronica; Russo, Cristina; Ferro, Yvelise; Pingitore, Piero; Pujia, Arturo; Montalcini, Tiziana; Doldo, Patrizia; Garieri, Pietro; Piodi, Luca; Caprioli, Flavio; Valenti, Luca

    2015-01-01

    Background: Inflammatory bowel diseases (IBD) are characterized by chronic relapsing inflammation of the gastrointestinal tract and encompass Crohn's disease and ulcerative colitis. IBD are often associated with extraintestinal manifestations affecting multiple organs including the liver. Increased levels of serum aminotransferases, possibly related to nonalcoholic fatty liver disease, constitute one of the most frequently described IBD-related liver diseases. The PNPLA3 I148M substitution is a major common genetic determinant of hepatic fat content and progression to chronic liver disease. The aim of this study was to investigate whether carriers of PNPLA3 148M allele with IBD have higher risk of liver steatosis and increase in transaminases levels. Methods: The PNPLA3 I148M (rs738409) genotype was performed by Taqman assays in 158 individuals from Southern Italy (namely, Catanzaro cohort) and in 207 individuals from Northern Italy (namely, Milan cohort) with a definite diagnosis of IBD. Demographic and clinical data and also alanine transaminase levels were collected for both cohorts. The Catanzaro cohort underwent liver evaluation by sonography and liver stiffness and controlled attenuation parameter measurements by transient elastography. Results: Here, we show for the first time that carriers of the PNPLA3 148M allele with IBD have a greater risk of hepatic steatosis (odds ratio, 2.9, and confidence interval, 1.1–7.8), higher controlled attenuation parameter values (P = 0.029), and increased circulating alanine transaminase (P = 0.035) in the Catanzaro cohort. We further confirm the higher alanine transaminase levels in the Milan cohort (P < 0.001). Conclusions: Our results show that PNPLA3 148M carriers with IBD have higher susceptibility to hepatic steatosis and liver damage. PMID:26355465

  2. Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity

    PubMed Central

    Ma, Lei-lei; Wu, Zhi-tao; Wang, Le; Zhang, Xue-feng; Wang, Jing; Chen, Chen; Ni, Xuan; Lin, Yun-fei; Cao, Yi-yi; Luan, Yang; Pan, Guo-yu

    2016-01-01

    Aim: Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo. Methods: The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg·kg−1·d−1, ig) for 4 weeks, their blood samples were analyzed. Results: A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 μmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 μmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg−1·d−1) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg−1·d−1, all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats. Conclusion: Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major

  3. Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

    NASA Astrophysics Data System (ADS)

    Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

    2015-04-01

    Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse

  4. Bioengineering of stainless steel surface by covalent immobilization of enzymes. Physical characterization and interfacial enzymatic activity.

    PubMed

    Caro, Anne; Humblot, Vincent; Méthivier, Christophe; Minier, Michel; Barbes, Lucica; Li, Joachim; Salmain, Michèle; Pradier, Claire-Marie

    2010-09-01

    Two hydrolytic enzymes, namely lysozyme and trypsin, were covalently immobilized onto stainless steel surfaces using wet chemistry processes. The immobilization strategy took advantage of the spontaneous physisorption of the polymer poly(ethylene imine) (PEI) onto stainless steel to yield a firmly attached, thin organic layer containing a high density of primary amine functions. Both enzymes were then covalently grafted to the surface via a glutaraldehyde cross-linker. Alternatively, a thicker underlayer of PEI was chemisorbed by cross-linking two PEI layers by glutaraldehyde. The effective presence of both enzymes on the stainless steel surfaces and their relative amount were assessed by immunochemical assays employing specific anti-enzyme antibodies. Eventually, the hydrolytic activity of the immobilized enzymes was evaluated by local enzymatic tests with suitable substrates. This work demonstrates that, although the amount of enzymes did not vary significantly with the underlayer thickness, their hydrolytic activity could be much improved by increasing the distance from the oxide surface and, likely, by favoring their accessibility. Our data suggest that the immobilization of enzymes on solid oxide surfaces is feasible and efficient, and that the enzymes retain catalytic activity. It may thus provide a promising route towards biofilm-resistant materials. PMID:20566201

  5. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    PubMed

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. PMID:26257292

  6. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes.

    PubMed Central

    Khan, A. R.; James, M. N.

    1998-01-01

    Proteolytic enzymes are synthesized as inactive precursors, or "zymogens," to prevent unwanted protein degradation, and to enable spatial and temporal regulation of proteolytic activity. Upon sorting or appropriate compartmentalization, zymogen conversion to the active enzyme typically involves limited proteolysis and removal of an "activation segment." The sizes of activation segments range from dipeptide units to independently folding domains comprising more than 100 residues. A common form of the activation segment is an N-terminal extension of the mature enzyme, or "prosegment," that sterically blocks the active site, and thereby prevents binding of substrates. In addition to their inhibitory role, prosegments are frequently important for the folding, stability, and/or intracellular sorting of the zymogen. The mechanisms of conversion to active enzymes are diverse in nature, ranging from enzymatic or nonenzymatic cofactors that trigger activation, to a simple change in pH that results in conversion by an autocatalytic mechanism. Recent X-ray crystallographic studies of zymogens and comparisons with their active counterparts have identified the structural changes that accompany conversion. This review will focus upon the structural basis for inhibition by activation segments, as well as the molecular events that lead to the conversion of zymogens to active enzymes. PMID:9568890

  7. Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum.

    PubMed

    Saari, L L; Pope, M R; Murrell, S A; Ludden, P W

    1986-04-15

    Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity. PMID:3082874

  8. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    NASA Astrophysics Data System (ADS)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  9. Chronic Kidney Disease Alters Vitamin A Homeostasis via Effects on Hepatic RBP4 Protein Expression and Metabolic Enzymes.

    PubMed

    Jing, J; Isoherranen, N; Robinson-Cohen, C; Petrie, I; Kestenbaum, B R; Yeung, C K

    2016-08-01

    Vitamin A, via retinoic acid (RA), is a critical micronutrient. Normally, plasma concentrations are tightly regulated. Concentrations of vitamin A metabolites (13cis-RA, atRA) and relationships between RBP4 and retinoids have never been fully evaluated in adult patients with CKD. We measured retinoid and RBP4 concentrations in plasma and urine from 55 adult patients with CKD and 21 matched healthy subjects. RBP4 and retinol levels were increased approximately twofold in patients with CKD, with a negative correlation between plasma retinol and eGFR (p = 0.006) and plasma RBP4 and eGFR (p = 0.0007). RBP4 renal clearance was higher in patients with CKD than healthy subjects but not associated with eGFR. Circulating concentrations of atRA increased and concentrations of 13cis-RA decreased in subjects with CKD with no change in RA-to-retinol ratio. Increases in circulating retinol, RBP4, and atRA may be due to increased hepatic RBP4 synthesis, retinyl ester hydrolysis, and/or hepatic secretion of RBP4-retinol. PMID:27277845

  10. SOME EFFECTS OF AGE, SPECIES DIFFERENCE, ANTIBIOTICS AND TOXICANT EXPOSURE ON INTESTINAL ENZYME ACTIVITY AND GENOTOXICITY

    EPA Science Inventory

    Altered intestinal enzyme activity significantly affects the biotransformation and toxicity of many xenobiotics. his review summarizes research, supported by the Air Force Bioenvironmental Hazards Research Program, that employs a novel gas-liquid chromatographic assay to investig...

  11. Modeling in situ soil enzyme activity using continuous field soil moisture and temperature data

    NASA Astrophysics Data System (ADS)

    Steinweg, J. M.; Wallenstein, M. D.

    2010-12-01

    Moisture and temperature are key drivers of soil organic matter decomposition, but there is little consensus on how climate change will affect the degradation of specific soil compounds under field conditions. Soil enzyme activities are a useful metric of soil community microbial function because they are they are the direct agents of decomposition for specific substrates in soil. However, current standard enzyme assays are conducted under optimized conditions in the laboratory and do not accurately reflect in situ enzyme activity, where diffusion and substrate availability may limit reaction rates. The Arrhenius equation, k= A*e(-Ea/RT), can be used to predict enzyme activity (k), collision frequency (A) or activation energy (Ea), but is difficult to parameterize when activities are measured under artificial conditions without diffusion or substrate limitation. We developed a modifed equation to estimate collision frequency and activation energy based on soil moisture to model in-situ enzyme activites. Our model was parameterized using data we collected from the Boston Area Climate Experiment (BACE) in Massachusetts; a multi-factor climate change experiment that provides an opportunity to assess how changes in moisture availability and temperature may impact enzyme activity. Soils were collected from three precipitation treatments and four temperature treatments arranged in a full-factorial design at the BACE site in June 2008, August 2008, January 2009 and June 2009. Enzyme assays were performed at four temperatures (4, 15, 25 and 35°C) to calculate temperature sensitivity and activation energy over the different treatments and seasons. Enzymes activities were measured for six common enzymes involved in carbon (β-glucosidase, cellobiohydrolase, xylosidase), phosphorus (phosphatase) and nitrogen cycling (N-acetyl glucosaminidase, and leucine amino peptidase). Potential enzyme activity was not significantly affected by precipitation, warming or the interaction of

  12. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  13. Regulation of Hepatic Triacylglycerol Metabolism by CGI-58 Does Not Require ATGL Co-activation.

    PubMed

    Lord, Caleb C; Ferguson, Daniel; Thomas, Gwynneth; Brown, Amanda L; Schugar, Rebecca C; Burrows, Amy; Gromovsky, Anthony D; Betters, Jenna; Neumann, Chase; Sacks, Jessica; Marshall, Stephanie; Watts, Russell; Schweiger, Martina; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Lathia, Justin D; Sakaguchi, Takuya F; Lehner, Richard; Haemmerle, Guenter; Zechner, Rudolf; Brown, J Mark

    2016-07-26

    Adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) are critical regulators of triacylglycerol (TAG) turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG) and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes. PMID:27396333

  14. Attenuated viral hepatitis in Trem1-/- mice is associated with reduced inflammatory activity of neutrophils.

    PubMed

    Kozik, Jan-Hendrik; Trautmann, Tanja; Carambia, Antonella; Preti, Max; Lütgehetmann, Marc; Krech, Till; Wiegard, Christiane; Heeren, Joerg; Herkel, Johannes

    2016-01-01

    TREM1 (Triggering Receptor Expressed on Myeloid Cells 1) is a pro-inflammatory receptor expressed by phagocytes, which can also be released as a soluble molecule (sTREM1). The roles of TREM1 and sTREM1 in liver infection and inflammation are not clear. Here we show that patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection manifest elevated serum levels of sTREM1. In mice, experimental viral hepatitis induced by infection with Lymphocytic Choriomeningitis Virus (LCMV)-WE was likewise associated with increased sTREM1 in serum and urine, and with increased TREM1 and its associated adapter molecule DAP12 in the liver. Trem1-/- mice showed accelerated clearance of LCMV-WE and manifested attenuated liver inflammation and injury. TREM1 expression in the liver of wild-type mice was mostly confined to infiltrating neutrophils, which responded to LCMV by secretion of CCL2 and TNF-α, and release of sTREM1. Accordingly, the production of CCL2 and TNF-α was decreased in the livers of LCMV-infected Trem1-/- mice, as compared to LCMV-infected wildtype mice. These findings indicate that TREM1 plays a role in viral hepatitis, in which it seems to aggravate the immunopathology associated with viral clearance, mainly by increasing the inflammatory activity of neutrophils. PMID:27328755

  15. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

    PubMed Central

    Feng, Qin; Gou, Xiao-jun; Meng, Sheng-xi; Huang, Cheng; Zhang, Yu-quan; Tang, Ya-jun; Wang, Wen-jing; Xu, Lin; Peng, Jing-hua; Hu, Yi-yang

    2013-01-01

    Qushi Huayu Decoction (QHD), a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD) in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK) in vivo and in vitro. Nonalcoholic fatty liver (NAFL) model was duplicated with high-fat diet in rats and with free fatty acid (FFA) in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC) and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1) and carbohydrate-responsive element-binding protein (ChREBP) in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro. PMID:23573117

  16. Capsaicin modulates proliferation, migration, and activation of hepatic stellate cells.

    PubMed

    Bitencourt, Shanna; Mesquita, Fernanda; Basso, Bruno; Schmid, Júlia; Ferreira, Gabriela; Rizzo, Lucas; Bauer, Moises; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis; Mannaerts, Inge; van Grunsven, Leo Adrianus; Oliveira, Jarbas

    2014-03-01

    Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro. PMID:23955514

  17. Quantum dot based enzyme activity sensors present deviations from Michaelis-Menten kinetic model

    NASA Astrophysics Data System (ADS)

    Díaz, Sebastián. A.; Brown, Carl W.; Malanoski, Anthony P.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

    2016-03-01

    Nanosensors employing quantum dots (QDs) and enzyme substrates with fluorescent moieties offer tremendous promise for disease surveillance/diagnostics and as high-throughput co-factor assays. Advantages of QDs over other nanoscaffolds include their small size and inherent photochemical properties such as size tunable fluorescence, ease in attaching functional moieties, and resistance to photobleaching. These properties make QDs excellent Förster Resonance Energy Transfer (FRET) donors; well-suited for rapid, optical measurement applications. We report enzyme sensors designed with a single FRET donor, the QD donor acting as a scaffold to multiple substrates or acceptors. The QD-sensor follows the concrete activity of the enzyme, as compared to the most common methodologies that quantify the enzyme amount or its mRNA precursor. As the sensor reports on the enzyme activity in real-time we can actively follow the kinetics of the enzyme. Though classic Michaelis-Menten (MM) parameters can be obtained to describe the activity. In the course of these experiments deviations, both decreasing and increasing the kinetics, from the common MM model were observed upon close examinations. From these observations additional experiments were undertaken to understand the varying mechanisms. Different enzymes can present different deviations depending on the chosen target, e.g. trypsin appears to present a positive hopping mechanism while collagenase demonstrates a QD caused reversible inhibition.

  18. Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs.

    PubMed

    Busk, Peter Kamp; Lange, Lene

    2013-06-01

    Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision. PMID:23524681

  19. Studies on antioxidant activity of teasaponins after hydrolyzed by enzyme

    NASA Astrophysics Data System (ADS)

    Tian, Jing; Zhao, Sen; Xu, Longquan; Fei, Xu; Wang, Xiuying; Wang, Yi

    The biological activity of teasaponins and their molecular structure are closely related, and the activity of saponins may be increased with the change of their molecular structure. In this report, teasaponins were hydrolyzed by Aspergillus niger for increasing the antioxidant activity. The antioxidant activity of teasaponins before and after hydrolyzed was tested by DPPH, and the result showed four new teasaponins were produced after hydrolysis, and their antioxidant activity was increased significantly than the original teasaponins before hydrolysis, the radical scavenging capacity (RSC) was partly up to 95 %.

  20. In vitro effects of the citrus flavonoids diosmin, naringenin and naringin on the hepatic drug-metabolizing CYP3A enzyme in human, pig, mouse and fish.

    PubMed

    Burkina, Viktoriia; Zlabek, Vladimir; Halsne, Ruth; Ropstad, Erik; Zamaratskaia, Galia

    2016-06-15

    Flavonoids are known to have effects on cytochrome P450 enzymatic activity. However, little effort has been made to examine species differences and the relevance of studies on mammalian and fish microsomes so that extrapolations can be made to humans. Therefore, the effects of several naturally occurring flavonoids on the activity of CYP3A-dependent 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD) were evaluated in human, pig, mouse, and juvenile rainbow trout sources of hepatic microsomes. Each was exposed to three concentrations (1, 10, and 100μM) of diosmin, naringin, and naringenin. Naringenin competitively inhibited BFCOD activity (Ki values were 24.6μM in human, 15.6μM in pig, and 19.6μM in mouse microsomes). In fish, BFCOD activity was inhibited in a noncompetitive manner (Ki=7μM). Neither diosmin nor naringenin affected BFCOD activity in hepatic microsomes from the studied model organisms. These results suggest that dietary flavonoids potentially inhibit the metabolism of clinical drugs. PMID:27107807

  1. A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis

    PubMed Central

    Gutschow, Patrick; Schmidt, Paul J.; Han, Huiling; Ostland, Vaughn; Bartnikas, Thomas B.; Pettiglio, Michael A.; Herrera, Carolina; Butler, James S.; Nemeth, Elizabeta; Ganz, Tomas; Fleming, Mark D.; Westerman, Mark

    2015-01-01

    Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbbth3/+), hereditary hemochromatosis (Hfe−/−, Hjv−/−, and Tfr2Y245X/Y245X), hypotransferrinemia (Trfhpx/hpx), heterozygous transferrin receptor 1 deficiency (Tfrc+/−) and iron refractory iron deficiency anemia (Tmprss6−/− and Tmprss6hem8/hem8). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups. PMID:25425686

  2. Increased physical activity decreases hepatic free fatty acid uptake: a study in human monozygotic twins.

    PubMed

    Hannukainen, Jarna C; Nuutila, Pirjo; Borra, Ronald; Ronald, Borra; Kaprio, Jaakko; Kujala, Urho M; Janatuinen, Tuula; Heinonen, Olli J; Kapanen, Jukka; Viljanen, Tapio; Haaparanta, Merja; Rönnemaa, Tapani; Parkkola, Riitta; Knuuti, Juhani; Kalliokoski, Kari K

    2007-01-01

    Exercise is considered to be beneficial for free fatty acid (FFA) metabolism, although reports of the effects of increased physical activity on FFA uptake and oxidation in different tissues in vivo in humans have been inconsistent. To investigate the heredity-independent effects of physical activity and fitness on FFA uptake in skeletal muscle, the myocardium, and liver we used positron emission tomography (PET) in nine healthy young male monozygotic twin pairs discordant for physical activity and fitness. The cotwins with higher physical activity constituting the more active group had a similar body mass index but less body fat and 18 +/- 10% higher (P < 0.001) compared to the less active brothers with lower physical activity. Low-intensity knee-extension exercise increased skeletal muscle FFA and oxygen uptake six to 10 times compared to resting values but no differences were observed between the groups at rest or during exercise. At rest the more active group had lower hepatic FFA uptake compared to the less active group (5.5 +/- 4.3 versus 9.0 +/- 6.1 micromol (100 ml)(-1) min(-1), P = 0.04). Hepatic FFA uptake associated significantly with body fat percentage (P = 0.05). Myocardial FFA uptake was similar between the groups. In conclusion, in the absence of the confounding effects of genetic factors, moderately increased physical activity and aerobic fitness decrease body adiposity even in normal-weighted healthy young adult men. Further, increased physical activity together with decreased intra-abdominal adiposity seems to decrease hepatic FFA uptake but has no effects on skeletal muscle or myocardial FFA uptake. PMID:17053033

  3. Serum Amyloid A Induces Inflammation, Proliferation and Cell Death in Activated Hepatic Stellate Cells

    PubMed Central

    Siegmund, Sören V.; Schlosser, Monika; Schildberg, Frank A.; Seki, Ekihiro; De Minicis, Samuele; Uchinami, Hiroshi; Kuntzen, Christian; Knolle, Percy A.; Strassburg, Christian P.; Schwabe, Robert F.

    2016-01-01

    Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs. PMID:26937641

  4. Serum Amyloid A Induces Inflammation, Proliferation and Cell Death in Activated Hepatic Stellate Cells.

    PubMed

    Siegmund, Sören V; Schlosser, Monika; Schildberg, Frank A; Seki, Ekihiro; De Minicis, Samuele; Uchinami, Hiroshi; Kuntzen, Christian; Knolle, Percy A; Strassburg, Christian P; Schwabe, Robert F

    2016-01-01

    Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs. PMID:26937641

  5. Hepatoprotective Activity of Methanolic Extract of Bauhinia purpurea Leaves against Paracetamol-Induced Hepatic Damage in Rats

    PubMed Central

    Yahya, F.; Mamat, S. S.; Kamarolzaman, M. F. F.; Seyedan, A. A.; Jakius, K. F.; Mahmood, N. D.; Shahril, M. S.; Suhaili, Z.; Mohtarrudin, N.; Susanti, D.; Somchit, M. N.; Teh, L. K.; Salleh, M. Z.; Zakaria, Z. A.

    2013-01-01

    In an attempt to further establish the pharmacological properties of Bauhinia purpurea (Fabaceae), hepatoprotective potential of methanol extract of B. purpurea leaves (MEBP) was investigated using the paracetamol- (PCM-) induced liver toxicity in rats. Five groups of rats (n = 6) were used and administered orally once daily with 10