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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. Activities of the enzymes of hepatic gluconeogenesis in periparturient dairy cows with induced fatty liver.

    PubMed

    Murondoti, Absolom; Jorritsma, Ruurd; Beynen, Anton C; Wensing, Theo; Geelen, Math J H

    2004-05-01

    The objective was to measure the activities of all the enzymes essential for hepatic gluconeogenesis in dairy cows with induced fatty liver. We aimed to induce severe fatty liver in ten experimental cows by overfeeding them during the dry period while seven control cows were maintained on a restricted diet. To induce a marked negative energy balance, the experimental cows were deprived of feed for 8 h immediately after parturition. In addition, the experimental cows were given a restricted amount of diet during the first 5 d of lactation. Liver samples were collected 1 week before and 1, 2 and 4 weeks after parturition. Before parturition, liver triacylglycerol concentrations did not differ between the two groups. After parturition, the experimental cows developed marked fatty liver as indicated by a higher level of triacylglycerols in the liver compared with the control cows. Before parturition, all gluconeogenic enzymes in the liver were lower in experimental cows than in control cows. Phosphoenolpyruvate carboxykinase, pyruvate carboxylase and propionyl-CoA carboxylase were significantly lower and fructose 1,6-bisphosphatase and glucose 6-phosphatase tended to be lower in the experimental cows. The activities of two crucial enzymes for gluconeogenesis in ruminants, i.e., phosphoenolpyruvate carboxykinase and propionyl-CoA carboxylase, remained low throughout the sampling period post partum. Activities of pyruvate carboxylase and glucose 6-phosphatase in the experimental cows post partum were upgraded to values similar to those of the control cows. The results showed that the capacity for hepatic gluconeogenesis before parturition was lower in cows with induced fatty liver than in control cows. After parturition, the low activities of crucial gluconeogenic enzymes indicated insufficient production of glucose. It is suggested that the low gluconeogenic capacity leads successively to low blood glucose concentrations, low insulin levels and high rates of

  3. Effects of Chinese, Japanese and Western tea on hepatic P450 enzyme activities in rats.

    PubMed

    Niwattisaiwong, N; Luo, X X; Coville, P F; Wanwimolruk, S

    2004-01-01

    Previous studies have reported that green tea effectively protects against cancers caused by various dietary carcinogens. As P450 enzymes are the major system responsible for the metabolism of many carcinogens, we hypothesise that tea consumption may alter the catalytic activities of P450 enzymes. We conducted this study to screen the effects of four different teas on the activities of P450 enzymes. Tea solutions (2.5%) were prepared by adding boiling water to tea leaves and filtering. Female Wistar rats were divided into five groups (n = 4 each); each had free access to tea solutions while the control group was supplied with water for 4 weeks. Animals were sacrificed and livers were removed for preparation of microsomes. Enzyme activities were determined by incubation of liver microsomes with the appropriate CYP substrate. The activity of CYP1A1 in livers from rats receiving Oolong (Chinese) tea (185 +/- 63 pmol/mg/min), Japanese green tea (197 +/- 22 pmol/mg/min) and Earl Grey tea (228 +/- 40 pmol/mg/min) was significantly higher (p < 0.05) than in the control group (94 +/- 34 pmol/mg/min), whereas no change was observed in the activity of CYP1A2 in any of tested animals. The hepatic activity of CYP2D6 was greater only in rats drinking Earl Grey tea compared to the controls (235 +/- 37 vs 161 +/- 41 pmol/mg/min, p < 0.05). There were also significant increases (p < 0.05) in the activity of CYP3A in livers of animals given Oolong tea (653 +/- 174 vs 382 +/- 114 pmol/mg/min) and Earl Grey tea (751 +/- 202 pmol/mg/min), while Jasmine and Japanese green tea had no significant effect. These results indicate that not all types of tea cause alterations in liver CYP enzymes as some elevated activities and some did not. Further studies are needed to determine whether there is a relationship between the effect of tea on CYP activities and anti-carcinogenesis.

  4. Circulating lysosomal enzymes and acute hepatic necrosis.

    PubMed Central

    Gove, C D; Wardle, E N; Williams, R

    1981-01-01

    The activities of the lysosomal enzymes acid and neutral protease, N-acetylglucosaminidase, and acid phosphatase were measured in the serum of patients with fulminant hepatic failure. Acid protease (cathepsin D) activity was increased about tenfold in patients who died and nearly fourfold in those who survived fulminant hepatic failure after paracetamol overdose, whereas activities were increased equally in patients with fulminant hepatic failure due to viral hepatitis whether or not they survived. A correlation was found between serum acid protease activity and prothrombin time, and the increase in cathepsin D activity was sustained over several days compared with aspartate aminotransferase, which showed a sharp early peak and then a fall. Circulating lysosomal proteases can damage other organs, and measurement of their activity may therefore be of added value in assessing prognosis in this condition. PMID:7007443

  5. Di2-ethylhexyl phthalate disrupts thyroid hormone homeostasis through activating the Ras/Akt/TRHr pathway and inducing hepatic enzymes

    PubMed Central

    Ye, Hanfeng; Ha, Mei; Yang, Min; Yue, Ping; Xie, Zhengyuan; Liu, Changjiang

    2017-01-01

    Di(2-ethylhexyl) phthalate (DEHP), as a widespread environmental pollutant and an endocrine disruptor, can disturb the homeostasis of thyroid hormones (THs). In order to elucidate roles of the MAPK and PI3K/Akt pathways and hepatic enzymes in thyroid-disrupting effects of DEHP, Sprague-Dawley rats were dosed with DEHP by gavage for 30 consecutive days; Nthy-ori 3-1 cells were treated with DEHP with NAC, k-Ras siRNA or inhibitors (U0126 and wortmannin). Results showed that DEHP led to histopathologic changes in rat thyroid and liver, such as the decrease in thyroid follicular cavity diameter, hepatocyte edema. Triiodothyronine (T3), thyroxine (T4) and thyrotropin releasing hormone (TRH) were reduced. DEHP caused ROS production, oxidative stress and k-Ras upregulation, thereby activating the ERK and Akt pathways in vivo and in vitro. Moreover, TRH receptor (TRHr) level was elevated after the activation of the Akt pathway and was downregulated after the inhibition of the Akt pathway. However, TRHr was not modulated by the ERK pathway. Additionally, hepatic enzymes, including Ugt1a1, CYP2b1, Sult1e1, and Sult2b1, were significantly induced after DEHP exposure. Taken together, DEHP can perturb TH homeostasis and reduce TH levels. The activated Ras/Akt/TRHr pathway and induced hepatic enzymes play vital roles in thyroid-disrupting effects of DEHP. PMID:28065941

  6. Di2-ethylhexyl phthalate disrupts thyroid hormone homeostasis through activating the Ras/Akt/TRHr pathway and inducing hepatic enzymes.

    PubMed

    Ye, Hanfeng; Ha, Mei; Yang, Min; Yue, Ping; Xie, Zhengyuan; Liu, Changjiang

    2017-01-09

    Di(2-ethylhexyl) phthalate (DEHP), as a widespread environmental pollutant and an endocrine disruptor, can disturb the homeostasis of thyroid hormones (THs). In order to elucidate roles of the MAPK and PI3K/Akt pathways and hepatic enzymes in thyroid-disrupting effects of DEHP, Sprague-Dawley rats were dosed with DEHP by gavage for 30 consecutive days; Nthy-ori 3-1 cells were treated with DEHP with NAC, k-Ras siRNA or inhibitors (U0126 and wortmannin). Results showed that DEHP led to histopathologic changes in rat thyroid and liver, such as the decrease in thyroid follicular cavity diameter, hepatocyte edema. Triiodothyronine (T3), thyroxine (T4) and thyrotropin releasing hormone (TRH) were reduced. DEHP caused ROS production, oxidative stress and k-Ras upregulation, thereby activating the ERK and Akt pathways in vivo and in vitro. Moreover, TRH receptor (TRHr) level was elevated after the activation of the Akt pathway and was downregulated after the inhibition of the Akt pathway. However, TRHr was not modulated by the ERK pathway. Additionally, hepatic enzymes, including Ugt1a1, CYP2b1, Sult1e1, and Sult2b1, were significantly induced after DEHP exposure. Taken together, DEHP can perturb TH homeostasis and reduce TH levels. The activated Ras/Akt/TRHr pathway and induced hepatic enzymes play vital roles in thyroid-disrupting effects of DEHP.

  7. Activity and mRNA Levels of Enzymes Involved in Hepatic Fatty Acid Synthesis in Rats Fed Naringenin.

    PubMed

    Hashimoto, Toru; Ide, Takashi

    2015-11-04

    We investigated the physiological activity of naringenin in affecting hepatic lipogenesis and serum and liver lipid levels in rats. Rats were fed diets containing 0, 1, or 2.5 g/kg naringenin for 15 d. Naringenin at a dietary level of 2.5 g/kg significantly decreased the activities and the mRNA levels of various lipogenic enzymes and sterol regulatory element binding protein-1c (SREBP-1c) mRNA level. The activities and the mRNA levels were also 9-22% and 12-38% lower, respectively, in rats fed a 1 g/kg naringenin diet than in the animals fed a naringenin-free diet, although the differences were not significant in many cases. Naringenin at 2.5 g/kg significantly lowered serum triacylglycerol, cholesterol, and phospholipid and hepatic triacylglycerol and cholesterol. This flavonoid at 1.0 g/kg also significantly lowered these parameters except for serum triacylglycerol. Naringenin levels in serum and liver dose-dependently increased, and hepatic concentrations reached levels that can affect various signaling pathways.

  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. 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

  10. Physical properties, lipid composition and enzyme activities of hepatic subcellular membranes from chick embryo after ethanol treatment

    SciTech Connect

    Sanchez-Amate, M.C.; Marco, C.; Segovia, J.L. )

    1992-01-01

    Exposure of chick embryos to ethanol resulted in significant alterations to the lipid composition of various different hepatic subcellular membranes. A marked decrease in cholesterol levels and an increase in the phospholipid content of microsomes and mitochondria was observed. Ethanol also affected the fatty acid profiles, mainly by decreasing the percentage of oleic acid in phosphatidylcholine and phosphatidylethanolamine in the mitochondria and phosphatidylethanolamine in the microsomes. In spite of these changes ethanol only induced alterations in the fluidity of the mitochondrial membranes, which showed a more rigid core, in contrast to the phospholipid-head region, which was not affected. In accordance with the changes observed in the physical state of the membrane, the enzymes involved in the microsomal electron-transport systems were not modified by ethanol, while cytochrome oxidase activity decreased by 50% compared to the activity in the mitochondria from control chick embryos.

  11. 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.

  12. [Activity of the marker liver enzymes under the conditions of toxic hepatitis and alimentary deprivation of protein].

    PubMed

    Voloshchuk, O N; Kopyl'chuk, G P; Buchkovskaia, I M

    2014-01-01

    The activity of the sorbitoldehydrogenase (SDH), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in the blood serum of rats with acetaminophen-induced hepatitis under the conditions of alimentary deprivation of protein was studied. The animals were divided into 3 groups: 1--rats with acute acetaminophen-induced hepatitis, maintained on the full ration; 2--rats with acute acetaminophen-induced hepatitis, maintained under the conditions of alimentary deprivation of protein; 3--control. The activity of the sorbitol dehydrogenase in blood serum was determined by the kinetic method, activity of the alanine aminotransferase and alkaline phosphatase - photometrically. It is shown, that in animals with the model hepatitis the activity of sorbitol dehydrogenase in blood serum increases 20-fold, wherein statistical significance between animals with hepatitis maintained under the conditions of full ration and those of low-protein diet is not established. In the group of animals with acetaminophen-induced hepatitis the preservation on the control level of the alkaline phosphatase activity on the base of the increase of alanine aminotransferase by 2.2 times and ratio ALT/ALP>5 testifies about hepatocellular liver injury. In the group of animals with drug-induced hepatitis and alimentary deprivation of protein, the increase of the alkaline phosphatase and alanine aminotransferase activity is observed, herewith the ratio ALT/ALP ranges from 2 to 5 and testifies about mixed liver injury. The conclusion was made, that alimentary deprivation of protein is the critical factor for the development of the disturbances of functional and structural liver integrity, and the therapeutic approaches to the correction of the drug-induced liver injury should be different depending on the value of protein ration in the anamnesis, taking into account the different types of liver injury.

  13. Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol.

    PubMed Central

    Spady, D K; Cuthbert, J A; Willard, M N; Meidell, R S

    1995-01-01

    Clinical interventions that accelerate conversion of cholesterol to bile acids reduce circulating low density lipoprotein (LDL) cholesterol concentrations. The initial and rate-limiting step in the bile acid biosynthetic pathway is catalyzed by hepatic cholesterol 7 alpha-hydroxylase. To examine the effects of transient primary overexpression of this enzyme on sterol metabolism and lipoprotein transport, we constructed a recombinant adenovirus in which a cDNA encoding rat 7 alpha-hydroxylase is expressed from the human cytomegalovirus immediate-early promoter (AdCMV7 alpha). Syrian hamsters administered AdCMV7 alpha intravenously accumulated transgene-specific mRNA in the liver and demonstrated a dose-dependent increase in hepatic microsomal 7 alpha-hydroxylase activity. The increased conversion of cholesterol to bile acids resulted in a compensatory increase in hepatic cholesterol synthesis. In addition, overexpression of 7 alpha-hydroxylase reduced the rate of LDL cholesterol entry into the plasma space and, in animals maintained on a Western-type diet, restored hepatic LDL receptor expression. As a consequence, plasma LDL concentrations fell by approximately 60% in animals maintained on control diet and by approximately 75% in animals consuming a Western-type diet. Plasma high density lipoprotein cholesterol levels were reduced to a lesser degree. These results demonstrate that transient upregulation of bile acid synthesis by direct transfer of a 7 alpha-hydroxylase gene favorably alters circulating lipoprotein profiles and suggest one potential molecular target for genetic strategies aimed at reducing cardiovascular risk. Images PMID:7635963

  14. 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.

  15. 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...

  16. Temperature modulates hepatic carbohydrate metabolic enzyme activity and gene expression in juvenile GIFT tilapia (Oreochromis niloticus) fed a carbohydrate-enriched diet.

    PubMed

    Qiang, J; He, J; Yang, H; Wang, H; Kpundeh, M D; Xu, P; Zhu, Z X

    2014-02-01

    The effects of rearing temperature on hepatic glucokinase (GK), glucose-6-phosphatase (G6Pase) and Glucose-6-phosphate dehydrogenase (G6PD) activity and gene expression were studied in GIFT (genetically improved farmed tilapia) tilapia fed a high carbohydrate diet containing 28% crude protein, 5% crude lipid and 40% wheat starch. Triplicate groups of fish (11.28 g initial body weight) were fed the diet for 45 days at 22 °C, 28 °C or 34 °C. At the end of the trial, final body weight of juvenile at 28 °C (59.12 g) was higher than that of the fish reared at 22 °C (27.13 g) and 34 °C (43.17 g). Feed intake, feed efficiency and protein efficiency ratio were also better at 28 °C. Liver glycogen levels were higher at 28 °C, while plasma glucose levels were higher in the 22 °C group. Significant (P<0.05) effects of water temperature on enzymes activities and gene expression were observed. Hepatic GK activity and mRNA level were higher at 28 °C than at 34 °C. Higher G6Pase and G6PD activity and gene expression were observed at 22 °C. Overall, the data show that juveniles reared at 28 °C exhibited enhanced liver glycolytic capacity. In contrast, hepatic gluconeogenesis and lipogenesis were increased by low temperature (22 °C).

  17. 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)...

  18. The changes of serum testosterone level and hepatic microsome enzyme activity of crucian carp (Carassius carassius) exposed to a sublethal dosage of pentachlorophenol.

    PubMed

    Zhang, Min; Yin, Daqiang; Kong, Fanxiang

    2008-10-01

    Pentachlorophenol (PCP), which was reported to be a typical persistent organic pollutant and environmental endocrine disruptor, may cause threat to aquatic species. In this study, serum testosterone concentration, activity of liver microsome ethoxyresorufin O-deethylase (EROD) and glutathione S-transferases (GST) of crucian carp (Carassius carassius) exposed to PCP for 7 and 15 d, respectively, were examined. The results showed that testosterone concentration was induced remarkably after 7 d (P<0.05), and the testosterone concentrations in 15 d treatment crucian carp were higher than those in 7 d treatment. It was found that there were significant effects on activities of EROD and GST after crucian carp were exposed to PCP for 7 and 15 d (P<0.05), compared to the controls. EROD and GST activities increased with increase in PCP concentration and also with increase in time on exposure. The results indicated that serum testosterone, EROD and GST were sensitive endpoints to PCP. PCP may have endocrine disrupting activities and may affect the reproductive success of this species. It is possible that the changes of hepatic microsome enzyme activities may result in alterations of serum testosterone levels in crucian carp.

  19. 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.

  20. Dietary D-psicose, a C-3 epimer of D-fructose, suppresses the activity of hepatic lipogenic enzymes in rats.

    PubMed

    Matsuo, T; Baba, Y; Hashiguchi, M; Takeshita, K; Izumori, K; Suzuki, H

    2001-01-01

    D-Psicose (D-ribo-2-hexulose), a C-3 epimer of D-fructose, is present in small quantities in commercial carbohydrate complexes or agricultural products. Wistar male rats were fed experimental diets which consisted of 5% D-psicose, cellulose, D-fructose or D-glucose for 28 days. Abdominal adipose tissue weight was significantly lower (P < 0.05) in rats fed the D-psicose diet than in rats fed a D-fructose and D-glucose diets, even though the four dietary groups were offered the same amount throughout the experimental period. Fatty acid synthase and glucose 6-phosphate dehydrogenase activities in the liver were significantly lower (P < 0.05) in rats fed the D-psicose diet than in rats fed the D-fructose and D-glucose diets. However, lipoprotein lipase activities in the heart, soleus muscle and perirenal adipose tissue were the same. These results suggest that a supplement of D-psicose in the diet suppresses hepatic lipogenic enzyme activities. The lower abdominal fat accumulation in rats fed a D-psicose diet might result from lower lipogenesis in the liver.

  1. 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

  2. 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

    2016-05-13

    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.

  3. Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus).

    PubMed

    Li, Hongxia; Boon, Jan P; Lewis, Wilma E; van den Berg, Martin; Nyman, Madeleine; Letcher, Robert J

    2003-03-01

    Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccumulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho-Cl substituents (CB-15 [4,4'-Cl2] and CB-77 [3,3',4,4'-Cl4]), vicinal meta and para-H atoms (CB-52 [2,2',5,5'-Cl4], and -101 [2,2',4,5,5'-Cl5]) or with both characteristics in combination with either only one ortho-Cl (CB-26 [2,3',5-Cl3], CB-31 [2,4',5-Cl3]) or two ortho-Cl substituents (CB-44 [2,2',3,5'-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations < or = 1.0 microM because these inhibitors became decreasingly selective at higher concentrations. At < 1.0 microM, ellipticine (CYPIAI/2 inhibitor) selectively inhibited CB-15, -26, -31, and -77 metabolism, with no significant inhibition of CB-44, -52, and -101 metabolism. Inhibition of CB-52 and -101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 microM and maximized at about 100% at 10 microM. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, -31, and -44 metabolism relative to CB-15, -77, and -52 at concentrations < or = 1.0 microM. Major testosterone metabolites formed in vitro were 2beta-(CYP3A), 6beta- (CYP3A, CYPIA), and 16beta- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory

  4. Mutagenic activation and detoxification of benzo[a]pyrene in vitro by hepatic cytochrome P450 1A1 and phase II enzymes in three meat-producing animals.

    PubMed

    Darwish, W; Ikenaka, Y; Eldaly, E; Ishizuka, M

    2010-01-01

    The mutagenic activation activity of hepatic microsomes from three meat-producing animals (cattle, deer and horses) was compared with those of rats as a reference species. In the Ames Salmonella typhimurium TA98 assay, the liver microsomes of all examined animals mutagenically activated benzo[a]pyrene, an ideal promutagens, in terms of production of histidine-independent revertant colonies. The microsomes of horses had the highest ability to produce revertant colonies of the examined animals under both low and high substrate concentrations. Inhibition of this mutagenic activity using alpha-naphthoflavone, anti-rat CYP1A1, CYP3A2 and CYP2E1 antibodies suggests that this activity was mainly because of CYP1A1 in these animals as well as in rats. The addition of co-factors for two phase II enzymes, microsomal UDP glucoronosyl transferase and cytosolic glutathione-S-transferase, reduced the production of the revertant colonies in a concentration-dependent manner. Interestingly, horses had the highest reduction rate among the examined animals, suggesting that phase II enzymes play a great role in producing a state of balance between the bioactivation and detoxification of xenobiotics in these meat-producing animals. This report is the first to investigate the mutagenic activation activity of the hepatic microsomes and the role of phase II enzymes against this activity in meat-producing animals.

  5. 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

  6. Hepatic Xenobiotic Metabolizing Enzyme Gene Expression ...

    EPA Pesticide Factsheets

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

  7. Grapefruit and oroblanco enhance hepatic detoxification enzymes in rats: possible role in protection against chemical carcinogenesis.

    PubMed

    Hahn-Obercyger, Michal; Stark, Aliza H; Madar, Zecharia

    2005-03-09

    Citrus fruits are considered to be functional foods that promote good health. This study was carried out to assess the effect of oroblanco and grapefruit consumption on hepatic detoxification enzymes. Male Sprague-Dawley rats were provided with either regular drinking water (control) or experimental treatments of oroblanco juice, grapefruit juice, or a sugar mix for 6 weeks. After 1 week of treatment, half the animals in each group were injected with the procarcinogen 1,2-dimethylhydrazine. Grapefruit juice significantly increased activity and expression of the hepatic phase I enzyme, cytochrome P450 CYP1A1, with a marked trend toward enhanced NAD(P)H:quinone reductase (QR) activity. Oroblanco juice significantly increased glutathione S-transferase phase II enzyme activity along with CYP1A1 expression and a notable trend toward increased activity of both CYP1A1 and QR. These results suggest that these citrus fruits are bifunctional inducers, modulating both phase I and phase II drug-metabolizing enzymes to enhance hepatic detoxification.

  8. Ameliorating effect of betanin, a natural chromoalkaloid by modulating hepatic carbohydrate metabolic enzyme activities and glycogen content in streptozotocin - nicotinamide induced experimental rats.

    PubMed

    Dhananjayan, Indumathi; Kathiroli, Sujithra; Subramani, Srinivasan; Veerasamy, Vinothkumar

    2017-04-01

    Betanin, a chromoalkaloid of beetroot, has shown significant biological effects of antioxidants, anti-inflammatory and anticarcinogenic activities. So, we attempted to determine whether betanin (a natural pigment) would be protective against hyperglycemia in streptozotocin (STZ) - nicotinamide (NA) induced diabetic rats. Rats were injected with STZ (40mg/kgb.w.) 15 mins after the administration of NA (110mg/kgb.w.) by intraperitonially (i.p.) 30days for the induction of experimental diabetes mellitus. After 72h diabetic rats were treated with betanin orally at a doses of 10, 20 and 40mg/kg b.w., respectively in a dose dependent manner and glibenclamide (600μg/kgb.w.). The promising character of betanin against diabetic rats was evaluated by performing the various biochemical parameters and histomorphological changes in liver and pancreas. Among the three doses, 20mg/kgb.w. of betanin was able to positively regulate plasma glucose, insulin, glycosylated hemoglobin (HbA1c) and hemoglobin (Hb) levels by significantly increasing the activity of glycolytic enzyme (glucokinase and pyruvate kinase), glucose-6-phosphate dehydrogenase and significantly decreasing the activity of gluconeogenic enzymes (glucose-6-phosphatase and fructose-1,6-bisphosphatase) thereby increasing the glycogen content in the liver. We put forward that betanin could significantly restore the levels of carbohydrate metabolic key enzymes to near normal in diabetic rat. Immunohistochemical observation of pancreas revealed that betanin treated diabetic rats showed increased insulin immunoreactive β-cells, which confirmed the biochemical findings. Taken together, present study suggests that betanin modulates the carbohydrate metabolism and has beneficial effects in glucose homeostasis.

  9. Role of hepatitis B virus X repression of C/EBPbeta activity in the down-regulation of glutathione S-transferase A2 gene: implications in other phase II detoxifying enzyme expression.

    PubMed

    Cho, I J; Ki, S H; Brooks, C; Kim, S G

    2009-02-01

    1. A genome-wide in silico screening rendered the genes of phase II enzymes in the rat genome whose promoters contain the putative DNA elements interacting with CCAAT/enhancer binding protein (C/EBP) and NF-E2-related factor (Nrf2). The hepatitis B virus X (HBx) protein strongly modulates the transactivation and/or the repression of genes regulated by some bZIP transcription factors. 2. This study investigated the effects of HBx on the induction of phase II enzymes with the aim of elucidating the role of HBx interaction with C/EBPbeta or Nrf2 bZIP transcription factors in hepatocyte-derived cells. 3. Immunoblot and reporter gene analyses revealed that transfection of HBx interfered with the constitutive and inducible GSTA2 transactivation promoted by oltipraz (C/EBPbeta activator), but not that by tert-butylhydroquinone (t-BHQ, Nrf2 activator). Moreover, HBx transfection completely inhibited GSTA2 reporter gene activity induced by C/EBPbeta, but failed to inhibit that by Nrf2. 4. Gel shift assays identified that HBx inhibited the increase in C/EBPbeta-DNA complex formation by oltipraz, but not the increase in Nrf2-DNA complex by t-BHQ. Immunoprecipitation and immunoblot assays verified the direct interaction between HBx and C/EBPbeta. Moreover, chromatin immunoprecipitation assays confirmed HBx inhibition of C/EBPbeta binding to its binding site in the GSTA2 gene promoter. HBx repressed the induction of other phase II enzymes including GSTP, UDP-glucuronyltransferase 1A, microsomal epoxide hydrolase, GSTM1, GSTM2, and gamma-glutamylcysteine synthase. 5. These results demonstrate that HBx inhibits the induction of phase II detoxifying enzymes, which is mediated by its interaction with C/EBPbeta, but not Nrf2, substantiating the specific role of HBx in phase II detoxifying capacity.

  10. Effect of Helicteres isora bark extract on blood glucose and hepatic enzymes in experimental diabetes.

    PubMed

    Kumar, G; Murugesan, A G; Rajasekara Pandian, M

    2006-04-01

    The effect of oral administration of an aqueous extract of the bark of Helicteres isora was investigated on blood glucose and plasma antioxidant status in streptozotocin (STZ) induced diabetic rats. The study was also undertaken to evaluate the role of hepatic enzymes in experimental diabetes. Oral administration of a bark extract of Helicteres isora (100, 200 mg/kg) in STZ diabetic rats caused a significant increase in body weight, hepatic hexokinase activity and significant decrease in hepatic glucose-6-phosphatase, serum acid phosphatase (ACP), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). Based on these findings, we suggest that Helicteres isora possesses hypoglycemic and hepatoprotective activity and is able to ameliorate biochemical damage in STZ induced diabetic rats.

  11. 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.

  12. 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

  13. 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

  14. Catalase ameliorates hepatic fibrosis by inhibition of hepatic stellate cells activation.

    PubMed

    Dong, Yuwei; Qu, Ying; Xu, Mingyi; Wang, Xingpeng; Lu, Lungen

    2014-01-01

    Catalase, an endogenous antioxidant enzyme, is thought to have rescue effects on hepatic fibrosis. In this study, the regulation of catalase in CCl₄-induced hepatic fibrogenesis was investigated. Our results indicated that catalase expression was decreased upon CCl₄ treatment in a time-dependent manner, while the expression of several profibrosis and proangiogenic factors, including transforming growth factor (TGF)-beta 1, vascular endothelial growth factor (VEGF), and angiopoietin 1 were significantly increased. To assess the role of catalase in hepatic fibrosis, catalase was overexpressed in HSC-T6 cells. This overexpression resulted in the inhibition of cell proliferation, migratory activity, and alpha-smooth muscle actin (alpha-SMA) expression, key features that characterize activation of hepatic stellate cells (HSC). Overexpression of catalase led to a decrease in the secretion of collagen type 1 and angiopoietin 1. These results indicate that loss of catalase activity is involved in the pathogenesis of hepatic fibrosis caused by the activation of HSCs.

  15. Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase.

    PubMed

    Shin, Hye-Jun; Park, Young-Ho; Kim, Sun-Uk; Moon, Hyung-Bae; Park, Do Sim; Han, Ying-Hao; Lee, Chul-Ho; Lee, Dong-Seok; Song, In-Sung; Lee, Dae Ho; Kim, Minhye; Kim, Nam-Soon; Kim, Dae-Ghon; Kim, Jin-Man; Kim, Sang-Keun; Kim, Yo Na; Kim, Su Sung; Choi, Cheol Soo; Kim, Young-Bum; Yu, Dae-Yeul

    2011-08-26

    Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

  16. 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

  17. Effect of peppermint oil on serum lipid peroxidation and hepatic enzymes after immobility stress in mice.

    PubMed

    Marjani, Abdoljalal; Rahmati, Reza; Mansourian, Azad Reza; Veghary, Gholamreza

    2012-01-01

    This study was undertaken to determine the influences of various doses of peppermint oil on the hepatic en-zymes, alanine transaminase, apartate tranaminase, alkaline phosphotase and gamma glutamyl transferase and the level of malondialdehyde in the serum of mice with and without immobility stress. The mice exposed to drink water, 0.9, 27 and 60 mg/kg peppermint oil from the days 1 to 5 for a period of 4 h before and after immobility stress. Serum MDA in-creased in treatment group II, III and IV after immobility stress. There was a significant decrease in ALT in treatment group III and IV after immobility stress. There were also significant decreases in ALP and GGT in treatment group IV af-ter immobility stress. This result may suggest that, MDA level is higher in immobilization stress group than in the un-immobilized animals in serum and this results show that enzyme activities decreased after immobilization stress.

  18. Tissue-specific contaminant accumulation and associated effects on hepatic serum analytes and cytochrome P450 enzyme activities in hooded seals (Cystophora cristata) from the Gulf of St. Lawrence.

    PubMed

    Wolkers, Hans; Boily, France; Fink-Gremmels, Johanna; van Bavel, Bert; Hammill, Mike O; Primicerio, Raul

    2009-02-01

    The current study aims to assess contaminant levels and tissue burdens in hooded seal (Cystophora cristata) blubber, liver, and blood in association with cytochrome P450 (CYP) enzymes (CYP1A and -3A) and serum analytes (hepatic enzymes like alanine aminotransferase [ALT], aspartate aminotransferase, alkaline phosphatase [AP], and gamma-glutamyltransferase [GGT], serum proteins, and creatine kinase). Contaminant accumulation levels and patterns of polychlorinated biphenyls, chlorinated pesticides, and polybrominated diphenyl ethers (PBDEs) differed between tissues and seal groups, with the highest levels in liver. Pups showed higher liver contaminant levels, especially for PBDEs, than adults. These high levels might be associated with the ingestion of large amounts of contaminated milk and subsequent accumulation in the liver. Adult males and females mainly differed in PBDE levels, which were higher in females, possibly due to a sex-specific diet. The association between blubber contaminant burdens and the diagnostic enzymes ALT, GGT, and AP, and serum albumin, was inconclusive. In contrast, several CYP isoenzymes showed a clear positive relationship with the overall blubber contaminant burden, indicating enzyme induction following exposure to polyhalogenated hydrocarbons. Therefore, liver CYP isoenzymes may serve as a sensitive biomarker for long-term exposure to polyhalogenated hydrocarbons.

  19. Activation of thiamin diphosphate in enzymes.

    PubMed

    Hübner, G; Tittmann, K; Killenberg-Jabs, M; Schäffner, J; Spinka, M; Neef, H; Kern, D; Kern, G; Schneider, G; Wikner, C; Ghisla, S

    1998-06-29

    Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

  20. The effects of fenvalerate on hepatic and cerebral xenobiotic metabolizing enzymes in selenium and/or iodine deficient rats

    PubMed Central

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

    2016-01-01

    Objective(s): Particularly in developing countries, selenium and/or iodine deficiencies are encountered and use of pesticides in agriculture are not well-controlled. Fenvalerate is a pyrethroid insectide used in agriculture and has applications against a wide range of pests. This study was designed to evaluate the effects of fenvalerate on hepatic and cerebral xenobiotic metabolizing enzyme activities in the presence of iodine and/or selenium deficiency on a rat model. Materials and Methods: Iodine and/or selenium deficiency was induced by feeding three-week-old Wistar rats with a diet containing <0.005 mg selenium kg-1, and/or administering 1% sodium perchlorate in drinking water for 7 weeks. Test groups received fenvalerate (100 mg kg-1 BW IP) for the last 7 days. Hepatic and cerebral microsomal aniline hydroxylase (CYP2E1) and cytosolic glutathione S-transferase (GST) activities were determined. Besides, hepatic NADPH-cytochrome P450 reductase (P450R), ethoxyresorufin O-deethylase (EROD, CYP1A1/1A2) and penthoxyresorufin O-depenthylase (PROD, CYP2B1/2B2), activities were also measured. Results: Fenvalerate had a general inductive effect on the hepatic and cerebral xenobiotic metabolizing enzyme activities. Moreover, enzyme activities were also altered by iodine and/or selenium deficiency, but the effects seemed to be enzyme- and tissue-specific. Conclusion: The inductive effect of fenvalerate, particularly in high dose exposures, may change the metabolism of several xenobiotics, including drugs, as well as endogenous substrates. The effects may vary depending on the selenium and/or iodine status of individual. PMID:27872699

  1. Schisandra Chinensis Baillon regulates the gene expression of phase II antioxidant/detoxifying enzymes in hepatic damage induced rats

    PubMed Central

    Jang, Han I; Do, Gyeong-Min; Lee, Hye Min; Ok, Hyang Mok; Shin, Jae-Ho

    2014-01-01

    BACKGROUND/OBJECTIVES This study investigated the antioxidant activities and hepatoprotective effects of Schisandra chinensis Baillon extract (SCE) against tert-butyl hydroperoxide (t-BHP)-induced oxidative hepatic damage in rats. MATERIALS/METHODS Sprague-Dawley (SD) rats were pretreated with SCE (300, 600, and 1,200 mg/kg BW) or saline once daily for 14 consecutive days. On day 14, each animal, except those belonging to the normal control group, were injected with t-BHP (0.8 mmol/kg BW/i.p.), and all of the rats were sacrificed 16 h after t-BHP injection. RESULTS Although no significant differences in AST and ALT levels were observed among the TC and SCE groups, the high-dose SCE group showed a decreasing tendency compared to the TC group. However, erythrocyte SOD activity showed a significant increase in the low-dose SCE group compared with the TC group. On the other hand, no significant differences in hepatic total glutathione (GSH) level, glutathione reductase (GR), and glutathione peroxidase (GSH-Px) activities were observed among the TC and SCE groups. Hepatic histopathological evaluation revealed that pretreatment with SCE resulted in reduced t-BHP-induced incidence of lesions, such as neutrophil infiltration, swelling of liver cells, and necrosis. In particular, treatment with a high dose of SCE resulted in induction of phase II antioxidant/detoxifying enzyme expression, such as glutathione S-transferase (GST) and glutamate-cysteine ligase catalytic subunit (GCLC). CONCLUSIONS Based on these results, we conclude that SCE exerts protective effects against t-BHP induced oxidative hepatic damage through the reduction of neutrophil infiltration, swelling of liver cells, and necrosis. In addition, SCE regulates the gene expression of phase II antioxidant/detoxifying enzymes independent of hepatic antioxidant enzyme activity. PMID:24944771

  2. 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.

  3. Hepatic xenobiotic metabolizing enzymes in two species of benthic fish showing different prevalences of contaminant-associated liver neoplasms

    SciTech Connect

    Collier, T.K.; Singh, S.V.; Awasthi, Y.C.; Varanasi, U. )

    1992-04-01

    English sole (Parophrys vetulus) and starry flounder (Platichthys stellatus) are closely related benthic fish which show substantial differences in prevalences of contaminant-associated hepatic neoplasms and putatively preneoplastic foci of cellular alteration when captured from estuaries containing a variety of organic chemical contaminants, including polycyclic aromatic hydrocarbons (PAH) and chlorinated hydrocarbons. Because PAH are strongly implicated as causative agents in the etiology of these lesions, several of the hepatic enzymes involved in activation and detoxication of PAH were studied in these two species. Hepatic aryl hydrocarbon hydroxylase (AHH), epoxide hydrolase (EH), and glutathione S-transferase (GST) activities were measured in animals sampled from both contaminated and reference areas. English sole, the species showing higher prevalences of contaminant-associated hepatic lesions, had higher (1- to 2-fold) hepatic activities of AHH and lower activities of EH (0.8-fold) and GST (1.8-fold) than those of starry flounder, regardless of site of capture. These results are largely consistent with increased activation and decreased detoxication of PAH by English sole in comparison to starry flounder. Both laboratory and field data suggested that the observed species differences in enzyme activities were constitutive and not related to differential exposure to contaminants. There were also substantial differences between these species with respect to expression of GST isoenzymes, in that starry flounder expressed two highly anionic GST isoenzymes which did not correspond to any GST isoenzymes expressed in English sole liver; a previous study in an elasmobranch fish showed that an anionic GST was most active toward PAH oxides.

  4. 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)…

  5. (+)-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.

  6. Sex difference in induction of hepatic CYP2B and CYP3A subfamily enzymes by nicardipine and nifedipine in rats.

    PubMed

    Konno, Yoshihiro; Sekimoto, Masashi; Nemoto, Kiyomitsu; Degawa, Masakuni

    2004-04-01

    Male and female of F344 rats were treated per os with nicardipine (Nic) and nifedipine (Nif), and changes in the levels of mRNA and protein of hepatic cytochrome P450 (P450) enzymes, CYP2B1, CYP2B2, CYP3A1, CYP3A2, CYP3A9, and CYP3A18 were examined. Furthermore, hepatic microsomal activities for pentoxyresorufin O-dealkylation (PROD) and nifedipine oxidation, which are mainly mediated by CYP2B and CYP3A subfamily enzymes, respectively, were measured. Analyses of RT-PCR and Western blotting revealed that Nic and Nif induced predominantly CYP3A and CYP2B enzymes, respectively. As for the gene activation of CYP2B enzymes, especially CYP2B1, Nif showed high capacity in both sexes of rats, whereas Nic did a definite capacity in the males but little in the females. Gene activations of CYP3A1, CYP3A2, and CYP3A18 by Nic occurred in both sexes of rats, although that of CYP3A9 did only in the male rats. Although gene activations of CYP3A1 and CYP3A2 by Nif were observed in both sexes of rats, a slight activation of the CYP3A9 gene occurred only in female rats, and the CYP3A18 gene activation, in neither male nor female rats. Thus, changes in levels of the mRNA or protein of CYP2B and CYP3A enzymes, especially CYP2B1 and CYP3A2, were closely correlated with those in hepatic PROD and nifedipine oxidation activities, respectively. The present findings demonstrate for the first time the sex difference in the Nic- and Nif-mediated induction of hepatic P450 enzymes in rats and further indicate that Nic and Nif show different specificities and sex dependencies in the induction of hepatic P450 enzymes.

  7. 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)

  8. 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.

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

    PubMed Central

    Yang, Peng; Subbaiah, Papasani V.

    2015-01-01

    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

  10. Citrus unshiu peel extract ameliorates hyperglycemia and hepatic steatosis by altering inflammation and hepatic glucose- and lipid-regulating enzymes in db/db mice.

    PubMed

    Park, Hae-Jin; Jung, Un Ju; Cho, Su-Jung; Jung, Hee-Kyung; Shim, Sangphil; Choi, Myung-Sook

    2013-02-01

    Insulin resistance in Type 2 diabetes leads to hepatic steatosis that can accompanied by progressive inflammation of the liver. Citrus unshiu peel is a rich source of citrus flavonoids that possess anti-inflammatory, anti-diabetic and lipid-lowering effects. However, the ability of citrus unshiu peel ethanol extract (CPE) to improve hyperglycemia, adiposity and hepatic steatosis in Type 2 diabetes is unknown. Thus, we evaluated the effects of CPE on markers for glucose, lipid metabolism and inflammation in Type 2 diabetic mice. Male C57BL/KsJ-db/db mice were fed a normal diet with CPE (2 g/100 g diet) or rosiglitazone (0.001 g/100 g diet) for 6 weeks. Mice supplemented with the CPE showed a significant decrease in body weight gain, body fat mass and blood glucose level. The antihyperglycemic effect of CPE appeared to be partially mediated through the inhibition of hepatic gluconeogenic phosphoenolpyruvate carboxykinase mRNA expression and its activity and through the induction of insulin/glucagon secretion. CPE also ameliorated hepatic steatosis and hypertriglyceridemia via the inhibition of gene expression and activities of the lipogenic enzymes and the activation of fatty acid oxidation in the liver. These beneficial effects of CPE may be related to increased levels of anti-inflammatory adiponectin and interleukin (IL)-10, and decreased levels of pro-inflammatory markers (IL-6, monocyte chemotactic protein-1, interferon-γ and tumor necrosis factor-α) in the plasma or liver. Taken together, we suggest that CPE has the potential to improve both hyperglycemia and hepatic steatosis in Type 2 diabetes.

  11. 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)

  12. Urinary D-glucaric acid and serum hepatic enzyme levels in chronic alcoholics.

    PubMed

    Tutor, J C; Alvarez-Prechous, A; Bernabeu, F; Pardiñas, M C; Paz, J M; Lareu, V

    1988-06-01

    Urinary D-glucaric acid (DGA) and the activities of gamma-glutamyl transferase (GGT) and other hepatic enzymes in serum were determined in 33 noncirrhotic male alcoholics who had continued to consume alcohol until at least 24 h prior to the taking of samples. DGA excretion was significantly greater in them than in a group of 30 healthy controls (p less than 0.001), exceeding the upper reference level in 38% of the alcoholic cases (as compared with 88% for GGT). In the alcoholic patients, there was highly significant correlation between urinary DGA and serum GGT (r = 0.613, p less than 0.001), suggesting that in both cases the increased levels are due to enzyme induction. None of the biochemical variables studied were significantly correlated with estimated daily alcohol consumption. Urinary DGA levels fell off rapidly with abstinence, and in 31 alcoholic patients who had consumed no alcohol for 5 days, there was no statistically significant correlation between DGA excretion and serum GGT (r = 0.158, p congruent to 0.4).

  13. 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.

  14. 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.

  15. How thiamine diphosphate is activated in enzymes.

    PubMed

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

    1997-01-03

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

  16. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  17. 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.

  18. Central neural regulation by adrenergic nerves of the daily rhythm in hepatic tyrosine transaminase activity

    PubMed Central

    Black, Ira B.; Reis, Donald J.

    1971-01-01

    1. In adrenalectomized fasted rats transection of the spinal cord at C7-C8 or placement of bilateral electrolytic lesions in the lateral hypothalamus when performed in the morning interrupted the daily rhythm of hepatic tyrosine transaminase by elevating low (AM) enzyme activities to high (PM) levels; lesions placed in PM did not affect the late afternoon rise in enzyme activity. 2. Bilateral thalamic lesions had no affect on enzyme activity. 3. The activity of hepatic catechol-O-methyl transferase was unaffected by hypothalamic lesions. 4. The lesion-evoked rise of tyrosine transaminase activity was abolished by exogenously administered norepinephrine. 5. Cycloheximide blocked the rise of tyrosine transaminase activity caused by hypothalamic lesions. 6. The results suggest that rhythmic activity of sympathetic nerves governed by lateral hypothalamus contribute to regulation of the daily rhythm in tyrosine transaminase by regulating the release of norepinephrine peripherally; norepinephrine may block the daily rise of enzyme by interfering with protein synthesis, possibly of new enzyme, by competing with pyridoxal co-factor. 7. It is proposed that alternating activity of sympathetic-adrenergic and vagal-cholinergic nerves to liver, controlled by the C.N.S., contribute to rhythmic activity of hepatic tyrosine transaminase. ImagesFig. 2 PMID:4400586

  19. [Hepatic manifestation of a macrophage activation syndrome (MAS)].

    PubMed

    Nagel, Michael; Schwarting, Andreas; Straub, Beate K; Galle, Peter R; Zimmermann, Tim

    2017-04-04

    Background Elevated liver values are the most common pathological laboratory result in Germany. Frequent findings, especially in younger patients, are nutritive- or medicamentous- toxic reasons, viral or autoimmune hepatitis. A macrophage activation syndrome (MAS) may manifest like a viral infectious disease with fever, hepatosplenomegaly and pancytopenia and is associated with a high mortality. It is based on an enhanced activation of macrophages with increased cytokine release, leading to organ damage and multi-organ failure. In addition to genetic causes, MAS is commonly associated with infections and rheumatic diseases. We report the case of a 26-year-old female patient suffering from MAS as a rare cause of elevated liver enzymes. Methods Patient characteristics, laboratory values, liver histology, bone marrow and radiological imaging were documented and analyzed. Case Report After an ordinary upper airway infection with bronchitis, a rheumatic arthritis appeared and was treated with leflunomide und methotrexate. In the further course of the disease, the patient developed an acute hepatitis with fever, pancytopenia and massive hyperferritinemia. Immunohistochemistry of the liver biopsy revealed hemophagocytosis and activation of CD68-positive macrophages. In the radiological and histological diagnostics of the liver and bone marrow, an MAS was diagnosed as underlying disease of the acute hepatitis. Under therapy with prednisolone, the fever disappeared and transaminases and ferritin rapidly normalized. Conclusion Aside from the frequent causes of elevated liver values in younger patients, such as nutritive toxic, drug induced liver injury, viral or autoimmune hepatitis, especially in case of massive hyperferritinemia, a MAS should be considered as a rare cause of acute liver disease.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Activity assessment of microbial fibrinolytic enzymes.

    PubMed

    Kotb, Essam

    2013-08-01

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

  6. Effect of Laser Irradiation on Enzyme Activity

    NASA Astrophysics Data System (ADS)

    Murakami, Satoshi; Kashii, Masafumi; Kitano, Hiroshi; Adachi, Hiroaki; Takano, Kazufumi; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Mori, Yusuke; Doi, Masaaki; Sugamoto, Kazuomi; Yoshikawa, Hideki; Sasaki, Takatomo

    2005-11-01

    We previously developed a protein crystallization technique using a femtosecond laser and protein crystal processing and detaching techniques using a pulsed UV laser. In this study, we examine the effect of laser irradiation on protein integrity. After several kinds of laser were irradiated on part of a solution of glycerol-6-phosphate dehydrogenase from Leuconostoc mesenteroides, we measured the enzyme activity. Femtosecond and deep-UV laser irradiations have little influence on the whole enzyme activity, whereas the enzyme lost its activity upon high-power near-infrared laser irradiation at a wavelength of 1547 nm. These results suggest that suitable laser irradiation has no remarkable destructive influence on protein crystallization or crystal processing.

  7. Evaluation of the synergistic effect of Allium sativum, Eugenia jambolana, Momordica charantia, Ocimum sanctum, and Psidium guajava on hepatic and intestinal drug metabolizing enzymes in rats

    PubMed Central

    Kumar, Devendra; Trivedi, Neerja; Dixit, Rakesh K.

    2016-01-01

    Aims/Background: This study was to investigated the synergistic effect of polyherbal formulations (PHF) of Allium sativum L., Eugenia jambolana Lam., Momordica charantia L., Ocimum sanctum Linn., and Psidium guajava L. in the inhibition/induction of hepatic and intestinal cytochrome P450 (CYPs) and Phase-II conjugated drug metabolizing enzymes (DMEs). Consumption of these herbal remedy has been extensively documented for diabetes treatment in Ayurveda. Methodology: PHF of these five herbs was prepared, and different doses were orally administered to Sprague–Dawley rats of different groups except control group. Expression of mRNA and activity of DMEs were examined by real-time polymerase chain reaction and high performance liquid chromatography in isolated liver and intestine microsomes in PHF pretreated rats. Results: The activities of hepatic and intestinal Phase-II enzyme levels increased along with mRNA levels except CYP3A mRNA level. PHF administration increases the activity of hepatic and intestinal UDP-glucuronyltransferase and glutathione S-transferase in response to dose and time; however, the activity of hepatic sulfotransferase increased at higher doses. Conclusions: CYPs and Phase-II conjugated enzymes levels can be modulated in dose and time dependent manner. Observations suggest that polyherbal formulation might be a possible cause of herb-drug interaction, due to changes in pharmacokinetic of crucial CYPs and Phase-II substrate drug. PMID:27757267

  8. 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...

  9. Hepatic cannabinoid receptor type 1 mediates alcohol-induced regulation of bile acid enzyme genes expression via CREBH.

    PubMed

    Chanda, Dipanjan; Kim, Yong-Hoon; Li, Tiangang; Misra, Jagannath; Kim, Don-Kyu; Kim, Jung Ran; Kwon, Joseph; Jeong, Won-Il; Ahn, Sung-Hoon; Park, Tae-Sik; Koo, Seung-Hoi; Chiang, John Y L; Lee, Chul-Ho; Choi, Hueng-Sik

    2013-01-01

    Bile acids concentration in liver is tightly regulated to prevent cell damage. Previous studies have demonstrated that deregulation of bile acid homeostasis can lead to cholestatic liver disease. Recently, we have shown that ER-bound transcription factor Crebh is a downstream effector of hepatic Cb1r signaling pathway. In this study, we have investigated the effect of alcohol exposure on hepatic bile acid homeostasis and elucidated the mediatory roles of Cb1r and Crebh in this process. We found that alcohol exposure or Cb1r-agonist 2-AG treatment increases hepatic bile acid synthesis and serum ALT, AST levels in vivo alongwith significant increase in Crebh gene expression and activation. Alcohol exposure activated Cb1r, Crebh, and perturbed bile acid homeostasis. Overexpression of Crebh increased the expression of key bile acid synthesis enzyme genes via direct binding of Crebh to their promoters, whereas Cb1r knockout and Crebh-knockdown mice were protected against alcohol-induced perturbation of bile acid homeostasis. Interestingly, insulin treatment protected against Cb1r-mediated Crebh-induced disruption of bile acid homeostasis. Furthermore, Crebh expression and activation was found to be markedly increased in insulin resistance conditions and Crebh knockdown in diabetic mice model (db/db) significantly reversed alcohol-induced disruption of bile acid homeostasis. Overall, our study demonstrates a novel regulatory mechanism of hepatic bile acid metabolism by alcohol via Cb1r-mediated activation of Crebh, and suggests that targeting Crebh can be of therapeutic potential in ameliorating alcohol-induced perturbation of bile acid homeostasis.

  10. Hepatic cytochrome P450 enzymes belonging to the CYP2C subfamily from an Australian marsupial, the koala (Phascolarctos cinereus).

    PubMed

    Jones, Brett R; El-Merhibi, Adaweyah; Ngo, Suong N T; Stupans, Ieva; McKinnon, Ross A

    2008-09-01

    Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. We have previously reported that the obligate Eucalyptus feeder koala (Phascolarctos cinereus) exhibits a higher hepatic CYP2C activity as compared to non-Eucalyptus feeders human or rat, with stimulation of CYP2C activity by cineole. In the present study, we examine CYP2C expression by immunohistochemistry and describe the identification and cloning of koala CYP2Cs. Utilising anti-rat CYP2C6 antibody, the expression of CYP2C was found to be uniform across the hepatic sections, being consistent with that observed in human and rat. Two 1647 and 1638 bp koala liver CYP2C complete cDNAs, designated CYP2C47 and CYP2C48 respectively, were cloned by cDNA library screening. The koala CYP2C cDNAs encode a protein of 495 amino acids. Three additional partial CYP2C sequences were also identified from the koala, indicating the multiplicity of the CYP2C subfamily in this unique marsupial species. The results of this study demonstrate the presence of koala hepatic CYP2Cs that share several common features with other published CYP2Cs; however CYP2C47 and CYP2C48 contain four extra amino acid residues at the NH2-terminal, a transmembrane anchor which was reported being a fundamentally conserved structure core of all eukaryote CYP enzymes.

  11. 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

  12. Effect of Traumatic Brain Injury, Erythropoietin, and Anakinra on Hepatic Metabolizing Enzymes and Transporters in an Experimental Rat Model.

    PubMed

    Anderson, Gail D; Peterson, Todd C; Vonder Haar, Cole; Farin, Fred M; Bammler, Theo K; MacDonald, James W; Kantor, Eric D; Hoane, Michael R

    2015-09-01

    In contrast to considerable data demonstrating a decrease in cytochrome P450 (CYP) activity in inflammation and infection, clinically, traumatic brain injury (TBI) results in an increase in CYP and UDP glucuronosyltransferase (UGT) activity. The objective of this study was to determine the effects of TBI alone and with treatment with erythropoietin (EPO) or anakinra on the gene expression of hepatic inflammatory proteins, drug-metabolizing enzymes, and transporters in a cortical contusion impact (CCI) injury model. Microarray-based transcriptional profiling was used to determine the effect on gene expression at 24 h, 72 h, and 7 days post-CCI. Plasma cytokine and liver protein concentrations of CYP2D4, CYP3A1, EPHX1, and UGT2B7 were determined. There was no effect of TBI, TBI + EPO, or TBI + anakinra on gene expression of the inflammatory factors shown to be associated with decreased expression of hepatic metabolic enzymes in models of infection and inflammation. IL-6 plasma concentrations were increased in TBI animals and decreased with EPO and anakinra treatment. There was no significant effect of TBI and/or anakinra on gene expression of enzymes or transporters known to be involved in drug disposition. TBI + EPO treatment decreased the gene expression of Cyp2d4 at 72 h with a corresponding decrease in CYP2D4 protein at 72 h and 7 days. CYP3A1 protein was decreased at 24 h. In conclusion, EPO treatment may result in a significant decrease in the metabolism of Cyp-metabolized drugs. In contrast to clinical TBI, there was not a significant effect of experimental TBI on CYP or UGT metabolic enzymes.

  13. 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)

  14. Can chronic maternal drug therapy alter the nursing infant's hepatic drug metabolizing enzyme pattern?

    PubMed

    Toddywalla, V S; Patel, S B; Betrabet, S S; Kulkarni, R D; Kombo, I; Saxena, B N

    1995-10-01

    This study was carried out to investigate whether minute quantities of maternal drugs ingested over an extended period of time by a breast-feeding infant can alter the activity pattern of the infant's hepatic drug metabolizing enzyme (HDME). The HDME activity patterns of 12 breast-fed infants whose mothers were not on drug therapy were compared with those of 11 infants whose mothers had been taking 30 micrograms levo-norgesterel daily for 90 to 195 days (oral contraceptives group) and of 10 infants whose mothers had been taking ethambutol and isoniazid daily since pregnancy (tuberculosis group). As 6 beta hydroxycortisol in urine is considered to be a good and acceptable reflector of HDME activity, it was estimated from the infants' urine using enzyme-linked immunosorbent assay (ELISA) technique. A comparison of the patterns between 90 days of age and 195 days of age of the infants in the control group and the two study groups indicated an increase from 36.6 ng/mL to 58.4 ng/mL at 195 days in the control group. An initial decrease from 36.6 ng/mL to 26.2 ng/mL was noted with commencement of maternal levo-norgesterel therapy, followed by a slow and steady rise to 47.8 ng/mL at 195 days of age, with a shift in the peak from 120 to 135 days of infants age in the oral contraceptive group. A suppressed pattern with decreased levels of 6 beta hydroxycortisol ranging from 19.3 ng/mL to 26.5 ng/mL at 195 days was found in the tuberculosis group. The data were analyzed by two-way analysis of variance (ANOVA) coupled with Duncan's Multiple range test. Both treatment group showed significant differences from the control group at the 0.050 level. The HDME plays an important role in determining the final outcome of any drug in humans, as it controls the metabolism of drugs. Hence, alterations in its activity caused by the transfer of maternal drugs over a prolonged period of time could pose a serious problem to nurslings when they require drugs for their own benefit.

  15. Naturalistic observation on the hepatic enzyme changes in patients treated with either risperidone or olanzapine alone.

    PubMed

    Pae, Chi-Un; Lim, Hyun-Kook; Kim, Tae-Suk; Kim, Jung-Jin; Lee, Chang-Uk; Lee, Soo-Jung; Lee, Chul; Paik, In-Ho

    2005-05-01

    This retrospective study aimed to compare differences in hepatic enzyme elevation during treatment with either risperidone or olanzapine alone in patients with psychotic disorders. The charts were reviewed for six hundred and sixty-seven (667) inpatients with psychotic disorders who were treated with either risperidone (n=289) or olanzapine (n=145) alone at a university-affiliated hospital between 1998 and 2002. Frequencies of elevation greater than the reference level in any enzyme among aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphotase (ALP) were higher in the olanzapine-treated group (26.9%) than in the risperidone-treated group (14.2%) [odds ratio (OR)=2.225, 95% confidence interval (CI)=1.362-3.638, P=0.002]. Frequencies of elevation greater than the reference level in ALT were higher in the olanzapine-treated group than in the risperidone-treated group (OR=2.182, P=0.004), as were frequencies with two-fold (OR=3.064, P=0.017) and three-fold (OR=2.883, P=0.039) elevation. Recovery time was longer in the olanzapine-treated group than in the risperidone-treated group (P=0.0059), as was latency time (P=0.0044). These results suggest that there are potential differences in antipsychotic-associated hepatic enzyme alterations between risperidone and olanzapine treatment. Controlled, prospective studies should be conducted to identify the risk factors associated with an alteration in hepatic enzymes related to treatment with risperidone and olanzapine.

  16. Hepatic antioxidant enzymes SOD and CAT of Nile tilapia (Oreochromis niloticus) in response to pesticide methomyl and recovery pattern.

    PubMed

    Meng, Shun Long; Chen, Jia Zhang; Xu, Pao; Qu, Jian Hong; Fan, Li Min; Song, Chao; Qiu, Li Ping

    2014-04-01

    Hepatic antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) of Nile tilapia in response to pesticide methomyl and recovery pattern were researched by exposing tilapia to sub-lethal methomyl concentrations of 0, 0.2, 2, 20 and 200 μg/L for 30 days, and then transferred to methomyl-free water for 18 days. Hepatic SOD and CAT were measured at 10 min (day 0), 6, 12, 18, 24 and 30 days after starting the experiment and at 18 days after transferring to methomyl-free water. The results showed hepatic SOD and CAT activities in 2, 20 and 200 μg/L groups were affected significantly, however, that in 0.2 μg/L group didn't change significantly compared to control during 30-day exposure period. Thus it would appear the 0.2 μg/L methomyl might be considered the no observed adverse effect level. Recovery data showed that, for SOD, the effects produced by lower concentration of methomyl 2 μg/L were reversible but not at concentrations higher than 20 μg/L, however, for CAT, the effects produced by all the concentrations were reversible.

  17. 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

  18. Hepatitis

    MedlinePlus

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Hepatitis KidsHealth > For Teens > Hepatitis Print A A A ... to a liver condition called hepatitis . What Is Hepatitis? The liver is one of the body's powerhouses. ...

  19. Hepatitis

    MedlinePlus

    ... de los dientes Video: Getting an X-ray 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- ...

  20. [Effects of Hg on soil enzyme activity].

    PubMed

    Yang, Chun-Lu; Sun, Tie-Heng; He, Wen-Xiang; Chen, Su

    2007-03-01

    With simulation test, this paper studied the effects of Hg on the activities of urease, invertase and neutral phosphotase in four soils. The results showed that Hg inhibited soil urease and invertase activities markedly, but its inhibitory effect differed with test soils. There was a significant logarithmic correlation between the concentration of HgCl2 and the activities of these two enzymes (P < 0.05). In test soils, the ED50 of urease activity was 87.99, 5.47, 24.05 and 19.88 mg x kg(-1), and that of invertase activity was 76.68, 727.49, 236.52 and 316.59 mg x kg(-1), respectively. Urease was more sensitive than invertase to Hg contamination, while organic matter had a protective effect on soil enzymes. Soil neutral phosphatase was not sensitive to Hg contamination, except that it was significantly activated by Hg in the meadow brown soil applied with plenty of organic fertilizer.

  1. The trans-10,cis-12 isomer of conjugated linoleic acid reduces hepatic triacylglycerol content without affecting lipogenic enzymes in hamsters.

    PubMed

    Zabala, Amaia; Churruca, Itziar; Macarulla, M Teresa; Rodríguez, Víctor M; Fernández-Quintela, Alfredo; Martínez, J Alfredo; Portillo, María P

    2004-09-01

    Conjugated linoleic acid (CLA) refers to the positional and geometric dienoic isomers of linoleic acid. The dietary intake of CLA has been associated with changes in lipid metabolism. The aim of the present work was to assess the effects of the two main isomers of CLA on sterol regulatory element binding protein (SREBP)-1a and SREBP-1c mRNA levels, as well as on mRNA levels and the activities of several lipogenic enzymes in liver. For this purpose hamsters were fed an atherogenic diet supplemented with 5 g linoleic acid, cis-9,trans-11 or trans-10,cis-12 CLA/kg diet for 6 weeks. The trans-10,cis-12 isomer intake produced significantly greater liver weight, but also significantly decreased liver fat accumulation. No changes in mRNA levels of SREBP-1a, SREBP-1c and lipogenic enzymes, or in the activities of these enzymes, were observed. There was no effect of feeding cis-9,trans-11 CLA. These results suggest that increased fat accumulation in liver does not occur on the basis of liver enlargement produced by feeding the trans-10,cis-12 isomer of CLA in hamsters. The reduction in hepatic triacylglycerol content induced by this isomer was not attributable to changes in lipogenesis.

  2. Iodothyronine deiodinase enzyme activities in bone.

    PubMed

    Williams, Allan J; Robson, Helen; Kester, Monique H A; van Leeuwen, Johannes P T M; Shalet, Stephen M; Visser, Theo J; Williams, Graham R

    2008-07-01

    Euthyroid status is essential for normal skeletal development and maintenance of the adult skeleton, but the mechanisms which control supply of thyroid hormone to bone cells are poorly understood. Thyroid hormones enter target cells via monocarboxylate transporter-8 (MCT8), which provides a functional link between thyroid hormone uptake and metabolism in the regulation of T3-action but has not been investigated in bone. Most circulating active thyroid hormone (T3) is derived from outer ring deiodination of thyroxine (T4) mediated by the type 1 deiodinase enzyme (D1). The D2 isozyme regulates intra-cellular T3 supply and determines saturation of the nuclear T3-receptor (TR), whereas a third enzyme (D3) inactivates T4 and T3 to prevent hormone availability and reduce TR-saturation. The aim of this study was to determine whether MCT8 is expressed in the skeleton and whether chondrocytes, osteoblasts and osteoclasts express functional deiodinases. Gene expression was analyzed by RT-PCR and D1, D2 and D3 function by sensitive and highly specific determination of enzyme activities. MCT8 mRNA was expressed in chondrocytes, osteoblasts and osteoclasts at all stages of cell differentiation. D1 activity was undetectable in all cell types, D2 activity was only present in mature osteoblasts whereas D3 activity was evident throughout chondrocyte, osteoblast and osteoclast differentiation in primary cell cultures. These data suggest that T3 availability especially during skeletal development may be limited by D3-mediated catabolism rather than by MCT8 mediated cellular uptake or D2-dependent T3 production.

  3. Effect of stress on hepatic 11beta-hydroxysteroid dehydrogenase activity and its influence on carbohydrate metabolism.

    PubMed

    Altuna, María Eugenia; Lelli, Sandra Marcela; San Martín de Viale, Leonor C; Damasco, María Cristina

    2006-10-01

    Stress activates the synthesis and secretion of catecholamines and adrenal glucocorticoids, increasing their circulating levels. In vivo, hepatic 11beta-hydroxysteroid dehydrogenase 1 (HSD1) stimulates the shift of 11-dehydrocorticosterone to corticosterone, enhancing active glucocorticoids at tissue level. We studied the effect of 3 types of stress, 1 induced by bucogastric overload with 200 mmol/L HCl causing metabolic acidosis (HCl), the second induced by bucogastric overload with 0.45% NaCl (NaCl), and the third induced by simulated overload (cannula), on the kinetics of hepatic HSD1 of rats and their influence on the activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase, glycemia, and glycogen deposition. Compared with unstressed controls, all types of stress significantly increased HSD1 activity (146% cannula, 130% NaCl, and 253% HCl), phosphoenolpyruvate carboxykinase activity (51% cannula, 48% NaCl, and 86% HCl), and glycemia (29% cannula, 30% NaCl, and 41% HCl), but decreased hepatic glycogen (68% cannula, 68% NaCl, and 78% HCl). Owing to these results, we suggest the following events occur when stress is induced: an increase in hepatic HSD1 activity, augmented active glucocorticoid levels, increased gluconeogenesis, and glycemia. Also involved are the multiple events indirectly related to glucocorticoids, which lead to the depletion of hepatic glycogen deposits, thereby contributing to increased glycemia. This new approach shows that stress increments the activity of hepatic HSD1 and suggests that this enzyme could be involved in the development of the Metabolic Syndrome.

  4. Lymphocyte activation and hepatic cellular infiltration in immunocompetent mice infected by dengue virus.

    PubMed

    Chen, Hsuen-Chin; Lai, Show-Yun; Sung, Jui-Min; Lee, Shu-Hwae; Lin, Yu-Chin; Wang, Wei-Kung; Chen, Yee-Chun; Kao, Chuan-Liang; King, Chwan-Chuen; Wu-Hsieh, Betty A

    2004-07-01

    Activation and expansion of dengue virus-specific T cells and abnormal liver functions in dengue patients have been documented. However, it remains to be determined whether T cells are involved in the pathogenic mechanism of dengue virus infection. In this study, immunocompetent C57BL/6 mice were employed to study dengue virus-induced T cell activation. Mice were inoculated with 10(8) PFU dengue virus serotype 2 strain 16681 by the intravenous route. Dengue viral core RNA was detected by RT-PCR in mouse serum, liver, spleen, and brain at different time points after infection. Splenic T cells were activated as evidenced by their expression of CD69 and O-glycosylated CD43 at as early as day 3 after infection. Splenic T cell expression of O-glycosylated CD43 and IFN-gamma production coordinately peaked at day 5. Coincided with the peak of splenic T cell activation was hepatic lymphocyte infiltration and elevation of liver enzymes. Flow cytometric analysis revealed the infiltrating CD8(+) T cell to CD4(+) T cell ratio was 5/3. After a second inoculation of dengue virus, hepatic T cell infiltration and liver enzyme levels increased sharply. The infiltrating hepatic CD8(+) T cell to CD4(+) T cell ratio increased to 5.8/1. A strong correlation was found between T cell activation and hepatic cellular infiltration in immunocompetent mice infected with dengue virus. The kinetics of liver enzyme elevation also correlated with that of T cell activation. These data suggest a relationship between T cell infiltration and elevation of liver enzymes.

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

    PubMed

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

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

  6. Characterization of human hepatic and extrahepatic UDP-glucuronosyltransferase enzymes involved in the metabolism of classic cannabinoids.

    PubMed

    Mazur, Anna; Lichti, Cheryl F; Prather, Paul L; Zielinska, Agnieszka K; Bratton, Stacie M; Gallus-Zawada, Anna; Finel, Moshe; Miller, Grover P; Radomińska-Pandya, Anna; Moran, Jeffery H

    2009-07-01

    Tetrahydrocannabinol (Delta(9)-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible for these cytochrome P450-dependent oxidations, but little work has been done to characterize phase II metabolic pathways. In this study, we test the hypothesis that there are specific human UGTs responsible for classic cannabinoid metabolism. The activities of 12 human recombinant UGTs toward classic cannabinoids [cannabinol (CBN), cannabidiol (CBD), (-)-Delta(8)-THC, (-)-Delta(9)-THC, (+/-)-11-hydroxy-Delta(9)-THC (THC-OH), and (-)-11-nor-9-carboxy-Delta(9)-THC (THC-COOH)] were evaluated using high-performance liquid chromatography-tandem mass spectrometry and labeling assays. Despite activity by UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7 toward CBN, CBD, THC-OH, and THC-COOH, only selected UGTs demonstrate sufficient activity for further characterization of steady-state kinetics. CBN was the most recognized substrate as evidenced by activities from hepatic UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect the introduction of an aromatic ring to Delta(9)-THC, leading to favorable pi stacking with phenylalanines in the UGT active site. Likewise, oxidation of Delta(9)-THC to THC-OH results in UGT1A9 and UGT1A10 activity toward the cannabinoid. Further oxidation to THC-COOH surprisingly leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of THC-COOH is the main metabolite found in urine, and thus these hepatic enzymes play a critical role in the metabolic clearance of cannabinoids. Taken together, glucuronidation of cannabinoids depends on upstream processing including enzymes such as CYP2C9 and CYP3A4.

  7. Characterization of Human Hepatic and Extrahepatic UDP-Glucuronosyltransferase Enzymes Involved in the Metabolism of Classic Cannabinoids

    PubMed Central

    Mazur, Anna; Lichti, Cheryl F.; Prather, Paul L.; Zielinska, Agnieszka K.; Bratton, Stacie M.; Gallus-Zawada, Anna; Finel, Moshe; Miller, Grover P.; Radomińska-Pandya, Anna; Moran, Jeffery H.

    2009-01-01

    Tetrahydrocannabinol (Δ9-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible for these cytochrome P450-dependent oxidations, but little work has been done to characterize phase II metabolic pathways. In this study, we test the hypothesis that there are specific human UGTs responsible for classic cannabinoid metabolism. The activities of 12 human recombinant UGTs toward classic cannabinoids [cannabinol (CBN), cannabidiol (CBD), (–)-Δ8-THC, (–)-Δ9-THC, (±)-11-hydroxy-Δ9-THC (THC-OH), and (–)-11-nor-9-carboxy-Δ9-THC (THC-COOH)] were evaluated using high-performance liquid chromatography-tandem mass spectrometry and labeling assays. Despite activity by UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7 toward CBN, CBD, THC-OH, and THC-COOH, only selected UGTs demonstrate sufficient activity for further characterization of steady-state kinetics. CBN was the most recognized substrate as evidenced by activities from hepatic UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect the introduction of an aromatic ring to Δ9-THC, leading to favorable π stacking with phenylalanines in the UGT active site. Likewise, oxidation of Δ9-THC to THC-OH results in UGT1A9 and UGT1A10 activity toward the cannabinoid. Further oxidation to THC-COOH surprisingly leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of THC-COOH is the main metabolite found in urine, and thus these hepatic enzymes play a critical role in the metabolic clearance of cannabinoids. Taken together, glucuronidation of cannabinoids depends on upstream processing including enzymes such as CYP2C9 and CYP3A4. PMID:19339377

  8. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Lu, Guoxin

    2007-01-01

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

  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. 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

  11. Hepatic SATB1 induces paracrine activation of hepatic stellate cells and is upregulated by HBx

    PubMed Central

    Gong, Jin; Tu, Wei; Han, Jian; He, Jiayi; Liu, Jingmei; Han, Ping; Wang, Yunwu; Li, Mengke; Liu, Mei; Liao, Jiazhi; Tian, Dean

    2016-01-01

    Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver diseases, but its involvement in hepatic fibrogenesis remains unclear. Special AT-rich binding protein 1 (SATB1) has been implicated in reprogramming chromatin organization and transcription profiles in many cancers and non-cancer-related conditions. We found that hepatic SATB1 expression was significantly up-regulated in fibrotic tissues from chronic hepatitis B virus (HBV)-infected patients and HBV transgenic (HBV-Tg) mouse model. Knockdown of SATB1 in the liver significantly alleviated CCl4-induced fibrosis in HBV-Tg mouse model. Moreover, we suggested HBV encoded x protein (HBx) induced SATB1 expression through activation of JNK and ERK pathways. Enforced expression of SATB1 in hepatocytes promoted the activation and proliferation of hepatic stellate cells (HSCs) by secretion of connective tissue growth factor (CTGF), Interleukin-6 (IL-6) and platelet derived growth factor-A (PDGF-AA). Our findings demonstrated that HBx upregulated hepatic SATB1 which exerted pro-fibrotic effects by paracrine activation of stellate cells in HBV-related fibrosis. PMID:27883059

  12. Hepatitis

    MedlinePlus

    ... clotting problems or chronic liver disease. previous continue Hepatitis B and Hepatitis C Although hep A is a ... does — through direct contact with infected body fluids. Hepatitis B and C are even more easily passed in ...

  13. Hepatitis

    MedlinePlus

    ... A if they've been vaccinated against it. Hepatitis B Hepatitis B is a more serious infection. It may lead ... of which cause severe illness and even death. Hepatitis B virus (HBV) is transmitted from person to person ...

  14. Hepatitis

    MedlinePlus

    ... Issues Listen Español Text Size Email Print Share Hepatitis Page Content Article Body Hepatitis means “inflammation of ... it has been associated with drinking contaminated water. Hepatitis Viruses Type Transmission Prognosis A Fecal-oral (stool ...

  15. [Hepatitis non-A, non-B: epidemiological significance in acute viral hepatitis and chronic active hepatitis of hepatological consultation].

    PubMed

    Jmelnitzky, A C; Basualdo, J A; Belloni, P O; Ponce de León, H H; García, C; Curciarello, J

    1987-01-01

    157 acute viral hepatitis and 60 chronic active ones have been analyzed focusing on NANB etiology. HAV was implicated in 36.3% of the hole acute viral hepatitis sample, HBV in 29.3%, and HNANBV was presumed as etiology in 31.2%, 5 patients (3.2%) had acute infection by HAV, on previous one by HBV, except for Epstein-Barr virus, no other test for viruses were determined (CMV, HSV, etc.). Male/female ratio was 1.4:1, 1.9:1, and 1.4:1 for HAV, HBV and HNANBV acute hepatitis respectively; HAV was the main etiology in the 0-9 age group (72.2%) although it only represents 11.5% of the sample; small occurrence of HAV hepatitis were found in patients over 40 (8.8%); HBV was clearly prevalent in patients over 50 (65.2%); the highest concentration of NANB etiology was found between 20-39 years old, but it was represented in all age-groups. Out of 49 NANB acute hepatitis, 12.2% had related transfusional antecedents, 12.2% belonged to health care worker group, and 4.1% had a close family NANB hepatitis contact; 71.5% had no reported antecedent. Viral source was presumably implicated in 75.0% of chronic active hepatitis, 25.0% attributable to HNANBV. Results seem not feasible to transfer to general population due to the facts that most patients were of specialized consult, and pediatric assistance is unusual to the authors practice.

  16. [Chronic active hepatitis: clinical, biochemical, and histopathologic correlation].

    PubMed

    Subauste, M C

    1989-01-01

    A retrospective study over 26 female patients with chronic active hepatitis was made. The mean age was 39 years old, the mean length of illness of 8 months; 5 patients had positive markers for hepatitis B. Patients were selected with the grade of histological activity: 8 patients had a mild form from disease (2A) and 16 with a severe one (2B). The predominant group was 2B. Severe inflammatory infiltration was the hallmark and multiobulillar necrosis, bridging, eosinophils and hiperplasia of kuppfer cells were found only in this group. Clinical features range from hepatic manifestations to systemic ones. Chronic active hepatitis may present with cholestasis, but the latter is not always related with the grade of activity. Group 2B had elevated aminotransferases and a low concentration for protrobine.

  17. Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines.

    PubMed

    Papageorgiou, Ioannis; Grepper, Susan; Unadkat, Jashvant D

    2013-02-01

    CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17β-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 1× total) and the 10× total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.

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

    PubMed

    Shisler, Krista A; Broderick, Joan B

    2014-03-15

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

  19. Influence of Piper betle on hepatic marker enzymes and tissue antioxidant status in D-galactosamine-induced hepatotoxic rats.

    PubMed

    Pushpavalli, Ganesan; Veeramani, Chinnadurai; Pugalendi, Kodukkur Viswanathan

    2008-01-01

    D-galactosamine is a well-established hepatotoxicant that induces a diffuse type of liver injury closely resembling human viral hepatitis. D-galactosamine by its property of generating free radicals causes severe damage to the membrane and affects almost all organs of the human body. The leaves of Piper betle L., a commonly used masticatory in Asian countries, possess several biological properties. Our aim is to investigate the in vivo antioxidant potential of P. betle leaf-extract against oxidative stress induced by D-galactosamine intoxication in male albino Wistar rats. Toxicity was induced by an intraperitoneal injection of D-galactosamine, 400 mg/kg body weight (BW) for 21 days. Rats were treated with P. betle extract (200 mg/kg BW) via intragastric intubations. We assessed the activities of liver marker enzymes (aspartate amino-transferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase) and levels of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides, superoxide dismutase, catalase, glutathione peroxidase, vitamin C, vitamin E, and reduced glutathione. The extract significantly improved the status of antioxidants and decreased TBARS, hydroperoxides, and liver marker enzymes when compared with the D-galactosamine treated group, demonstrating its hepatoprotective and antioxidant properties.

  20. Influence of Piper betle on hepatic marker enzymes and tissue antioxidant status in ethanol-treated Wistar rats.

    PubMed

    Saravanan, R; Prakasam, A; Ramesh, B; Pugalendi, K V

    2002-01-01

    Piper betle L. is a commonly used masticatory in Asia. This study was carried out to investigate the hepatoprotective and antioxidant properties of P. betle, using ethanol intoxication as a model of hepatotoxic and oxidative damage. Ethanol-treated rats exhibited elevation of hepatic marker enzymes and disturbances in antioxidant defense when compared with normal rats. Oral administration of P. betle extract (100, 200, or 300 mg/kg body weight) for 30 days significantly (P <.05) decreased aspartate aminotransferase (AST), alanine aminotransferase (ALT), thiobarbituric acid reactive substances (TBARS), and lipid hydroperoxides in ethanol treated rats. The extract also improved the tissue antioxidant status by increasing the levels of nonenzymatic antioxidants (reduced glutathione, vitamin C, and vitamin E) and the activities of free radical-detoxifying enzymes such as superoxide dismutase, catalase, and glutathione peroxidase in liver and kidney of ethanol-treated rats. The highest dose of P. betle extract (300 mg/kg body weight) was most effective. The results were comparable with the known hepatoprotective drug, silymarin. These results indicate that P. betle could afford a significant hepatoprotective and antioxidant effect.

  1. Temperature and the catalytic activity of enzymes: a fresh understanding.

    PubMed

    Daniel, Roy M; Danson, Michael J

    2013-09-02

    The discovery of an additional step in the progression of an enzyme from the active to inactive state under the influence of temperature has led to a better match with experimental data for all enzymes that follow Michaelis-Menten kinetics, and to an increased understanding of the process. The new model of the process, the Equilibrium Model, describes an additional mechanism by which temperature affects the activity of enzymes, with implications for ecological, metabolic, structural, and applied studies of enzymes.

  2. Investigation of antiaromatase activity using hepatic microsomes of Nile tilapia (Oreochromis niloticus).

    PubMed

    Sassa-Deepaeng, Tanongsak; Chaisri, Wasana; Pikulkaew, Surachai; Okonogi, Siriporn

    2017-03-19

    Microsomal aromatase enzymes of humans and rats have been used in antiaromatase assays, but enzyme activity is species-specific. The current study extracted hepatic microsomes of Nile tilapia (Oreochromis niloticus) to investigate and compare the antiaromatase activity of chrysin, quercetin, and quercitrin. This activity was evaluated using a dibenzylfluorescein (DBF) assay. Results revealed that the age and body weight of Nile tilapia affected the yield of extracted microsomes. Extraction of hepatic microsomes of Nile tilapia was most effective when using a reaction medium with a pH of 8.0. A DBF assay using Nile tilapia microsomes revealed significant differences in levels of antiaromatase activity for chrysin, quercetin, and quercitrin. Chrysin was the most potent aromatase inhibitor, with an IC50 of 0.25 mg/mL. In addition, chrysin is an aromatase inhibitor that also inhibits the proliferation of cancer cells. Hepatic microsomes of Nile tilapia can be used to investigate and compare the antiaromatase activity of different compounds.

  3. 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.

  4. 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).

  5. Individual variation in hepatic aldehyde oxidase activity.

    PubMed

    Al-Salmy, H S

    2001-04-01

    Aldehyde oxidase (AO) is a molybdo-flavo enzyme expressed predominantly in the liver, lung, and kidney. AO plays a major role in oxidation of aldehydes, as well as oxidation of various N-heterocyclic compounds of pharmacological and toxicological importance including antiviral (famciclovir), antimalarial (quinine), antitumour (methotrexate), and nicotine. The aim of this study was to investigate cytosolic aldehyde oxidase activity in human liver. Cytosolic AO was characterised using both the metabolism of N-[(2-dimethylamino)ethyl] acridine-4-carboxamide (DACA) and benzaldehyde to form DACA-9(10H)-acridone (quantified by HPLC with fluorescence detection) and benzoic acid (quantified spectrophotometrically). Thirteen livers (10 female, 3 male) were examined. The intrinsic clearance (Vmax/Km) of DACA varied 18-fold (0.03-0.50 m/min/mg). Vmax ranged from 0.20-3.10 nmol/ min/mg, and Km ranged from 3.5-14.2 microM. In the same specimens, the intrinsic clearance for benzaldehyde varied 5-fold (0.40-1.8 ml/min/mg). Vmax ranged from 3.60-12.6 nmol/min/mg and Km ranged from 3.6-14.6 microM. Furthermore, there were no differences in AO activity between male and female human livers, nor was there any relationship to age of donor (range 29-73 years), smoking status, or disease status. In conclusion, our results showed that there are variations in AO activity in human liver. These variations in aldehyde oxidase activity might reflect individual variations or they might be due to AO stability during processing and storage.

  6. 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

  7. 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.

  8. Lipoprotein electrostatic properties regulate hepatic lipase association and activity.

    PubMed

    Boucher, Jonathan G; Nguyen, Trang; Sparks, Daniel L

    2007-12-01

    The effect of lipoprotein electrostatic properties on the catalytic regulation of hepatic lipase (HL) was investigated. Enrichment of serum or very low density lipoprotein (VLDL) with oleic acid increased lipoprotein negative charge and stimulated lipid hydrolysis by HL. Similarly, enrichment of serum or isolated lipoproteins with the anionic phospholipids phosphatidylinositol (PI), phosphatidic acid, or phosphatidylserine also increased lipoprotein negative charge and stimulated hydrolysis by HL. Anionic lipids had a small effect on phospholipid hydrolysis, but significantly stimulated triacylglyceride (TG) hydrolysis. High density lipoprotein (HDL) charge appears to have a specific effect on lipolysis. Enrichment of HDL with PI significantly stimulated VLDL-TG hydrolysis by HL. To determine whether HDL charge affects the association of HL with HDL and VLDL, HL-lipoprotein interactions were probed immunochemically. Under normal circumstances, HL associates with HDL particles, and only small amounts bind to VLDL. PI enrichment of HDL blocked the binding of HL with HDL. These data indicate that increasing the negative charge of HDL stimulates VLDL-TG hydrolysis by reducing the association of HL with HDL. Therefore, HDL controls the hydrolysis of VLDL by affecting the interlipoprotein association of HL. Lipoprotein electrostatic properties regulate lipase association and are an important regulator of the binding and activity of lipolytic enzymes.

  9. 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.

  10. Effect of selenium-containing compounds on hepatic chemoprotective enzymes in mice.

    PubMed

    El-Sayed, Wael M; Aboul-Fadl, Tarek; Lamb, John G; Roberts, Jeanette C; Franklin, Michael R

    2006-03-15

    Selenite and organoselenium compounds have been examined at supranutritional levels for their ability to influence the activity and mRNA levels of chemoprotective enzymes in the livers of selenium-sufficient mice and the changes compared to those elicited by oltipraz. Compounds investigated included novel selenocysteine prodrugs that have previously been evaluated for their ability to reduce the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. Following seven daily doses (i.g.), all compounds except 2-methylselenazolidine-4(R)-carboxylic acid (MSCA) increased thioredoxin reductase activity (43-92%) but only for 2-oxoselenazolidine-4(R)-carboxylic acid (OSCA) was there an accompanying increase in mRNA. No compound enhanced glutathione peroxidase activity, although sodium selenite significantly elevated the mRNA of this enzyme. Oltipraz was an efficacious inducer of both thioredoxin reductase and glutathione peroxidase mRNAs. Sodium selenite, selenazolidine-4(R)-carboxylic acid (SCA), and OSCA elevated NAD(P)H-quinone oxidoreductase mRNA but only for OSCA was the elevation in mRNA accompanied by an increase in enzyme activity. L-Selenocystine significantly increased this activity without increasing mRNA levels. Sodium selenite, L-selenocystine, L-selenomethionine, and Se-methyl-L-selenocysteine all enhanced glutathione S-transferase activity. The increased activity with sodium selenite was accompanied by increases in mRNAs of Gst alpha, Gst mu and Gst pi classes, while for L-selenocystine and Se-methyl-L-selenocysteine, only an elevation in the mRNA for the Gst alpha class was observed. Gst alpha and Gst mu class mRNAs were elevated by OSCA without a significant elevation in enzyme activity. SCA and MSCA both elevated a Gst pi mRNA and MSCA elevated Gst mu in addition. By comparison, oltipraz only significantly elevated the mRNA of Gst mu, adding to the conclusion that across the entire study, no selenium compound appears to be acting

  11. Angiotensin Receptor Blockade Recovers Hepatic UCP2 Expression and Aconitase and SDH Activities and Ameliorates Hepatic Oxidative Damage in Insulin Resistant Rats

    PubMed Central

    Montez, Priscilla; Vázquez-Medina, José Pablo; Rodríguez, Rubén; Thorwald, Max A.; Viscarra, José A.; Lam, Lisa; Peti-Peterdi, Janos; Nakano, Daisuke; Nishiyama, Akira

    2012-01-01

    Metabolic syndrome (MetS) is commonly associated with elevated renin-angiotensin system, oxidative stress, and steatohepatitis with down-regulation of uncoupling proteins (UCPs). However, the mechanisms linking renin-angiotensin system, steatosis, and UCP2 to hepatic oxidative damage during insulin resistance are not described. To test the hypothesis that angiotensin receptor activation contributes to decreased hepatic UCP2 expression and aconitase activity and to increased oxidative damage after increased glucose intake in a model of MetS, lean and obese Long Evans rats (n = 10/group) were randomly assigned to the following groups: 1) untreated Long Evans Tokushima Otsuka (lean, strain control), 2) untreated Otsuka Long Evans Tokushima Fatty (OLETF) (MetS model), 3) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg·d × 6 wk), 4) OLETF + high glucose (HG) (5% in drinking water × 6 wk), and 5) OLETF + ARB + HG (ARB/HG × 6 wk). HG increased body mass (37%), plasma triglycerides (TGs) (35%), plasma glycerol (87%), plasma free fatty acids (28%), and hepatic nitrotyrosine (74%). ARB treatment in HG decreased body mass (12%), plasma TG (15%), plasma glycerol (23%), plasma free fatty acids (14%), and hepatic TG content (42%), suggesting that angiotensin receptor type 1 (AT1) activation and increased adiposity contribute to the development of obesity-related dyslipidemia. ARB in HG also decreased hepatic nitrotyrosine and increased hepatic UCP2 expression (59%) and aconitase activity (40%), as well as antioxidant enzyme activities (50-120%), suggesting that AT1 activation also contributes to protein oxidation, impaired lipid metabolism, and antioxidant metabolism in the liver. Thus, in addition to promoting obesity-related hypertension, AT1 activation may also impair lipid metabolism and antioxidant capacity, resulting in steatosis via decreased UCP2 and tricarboxylic acid cycle activity. PMID:23087176

  12. Angiotensin receptor blockade recovers hepatic UCP2 expression and aconitase and SDH activities and ameliorates hepatic oxidative damage in insulin resistant rats.

    PubMed

    Montez, Priscilla; Vázquez-Medina, José Pablo; Rodríguez, Rubén; Thorwald, Max A; Viscarra, José A; Lam, Lisa; Peti-Peterdi, Janos; Nakano, Daisuke; Nishiyama, Akira; Ortiz, Rudy M

    2012-12-01

    Metabolic syndrome (MetS) is commonly associated with elevated renin-angiotensin system, oxidative stress, and steatohepatitis with down-regulation of uncoupling proteins (UCPs). However, the mechanisms linking renin-angiotensin system, steatosis, and UCP2 to hepatic oxidative damage during insulin resistance are not described. To test the hypothesis that angiotensin receptor activation contributes to decreased hepatic UCP2 expression and aconitase activity and to increased oxidative damage after increased glucose intake in a model of MetS, lean and obese Long Evans rats (n = 10/group) were randomly assigned to the following groups: 1) untreated Long Evans Tokushima Otsuka (lean, strain control), 2) untreated Otsuka Long Evans Tokushima Fatty (OLETF) (MetS model), 3) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg·d × 6 wk), 4) OLETF + high glucose (HG) (5% in drinking water × 6 wk), and 5) OLETF + ARB + HG (ARB/HG × 6 wk). HG increased body mass (37%), plasma triglycerides (TGs) (35%), plasma glycerol (87%), plasma free fatty acids (28%), and hepatic nitrotyrosine (74%). ARB treatment in HG decreased body mass (12%), plasma TG (15%), plasma glycerol (23%), plasma free fatty acids (14%), and hepatic TG content (42%), suggesting that angiotensin receptor type 1 (AT1) activation and increased adiposity contribute to the development of obesity-related dyslipidemia. ARB in HG also decreased hepatic nitrotyrosine and increased hepatic UCP2 expression (59%) and aconitase activity (40%), as well as antioxidant enzyme activities (50-120%), suggesting that AT1 activation also contributes to protein oxidation, impaired lipid metabolism, and antioxidant metabolism in the liver. Thus, in addition to promoting obesity-related hypertension, AT1 activation may also impair lipid metabolism and antioxidant capacity, resulting in steatosis via decreased UCP2 and tricarboxylic acid cycle activity.

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

    PubMed Central

    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

  14. The importance of myeloperoxidase enzyme activity in the pathogenesis of Crimean-Congo haemorrhagic fever.

    PubMed

    Guven, F M K; Aydin, H; Yildiz, G; Engin, A; Celik, V K; Bakir, D; Deveci, K

    2013-03-01

    Crimean-Congo haemorrhagic fever (CCHF) is a disease with a severe course including acute viral haemorrhagic fever, ecchymosis, thrombocytopenia, hepatic function disorder and high mortality. Myeloperoxidase (MPO) is an enzyme located in neutrophil granulocytes and plays an important role in the destruction of phagocytosed micro-organisms. The aim of this study was to analyse MPO enzyme activity in CCHF cases compared with a control group. A total of 47 randomly selected CCHF patients admitted to the Department of Infectious Diseases of Cumhuriyet University Hospital in Sivas, Turkey, were studied, and as a control group, 41 age- and sex-matched individuals without any systemic disease were included in this study. MPO enzyme activity was measured in plasma and leukocytes for both groups by the ELISA method. MPO plasma and MPO leukocyte values were calculated as 57.62 ± 8.85 and 44.84 ± 9.71 in CCHF patients, and 0.79 ± 0.29 and 0.49 ± 0.11 in the controls, respectively. MPO enzyme activity was statistically significantly higher in patients with CCHF when compared to the control group. In conclusion, MPO enzyme activity is directly related to the activation of phagocytic leukocytes, and increases in both the plasma and leukocytes in CCHF patients. The increase of the MPO enzyme activity in leukocytes due to viral load leads to the destruction of the leukocyte. It is thought that MPO enzyme activity in plasma was higher in CCHF patients due to the destruction of leukocytes. MPO enzyme activity may be important in terms of the prognosis in patients with CCHF; however, more extensive studies are required on this subject.

  15. Phase I and II liver enzyme activities in juvenile alligators (Alligator mississippiensis) collected from three sites in the Kissimmee-Everglades drainage, Florida (USA).

    PubMed

    Gunderson, M P; Oberdörster, E; Guillette, L J

    2004-10-01

    We examined CYP1A (measured using hepatic EROD and MROD activities) and glutathione-S-transferase (GST) activities in juvenile alligators (Alligator mississippiensis) collected from three sites with varying contamination in the Kissimmee-Everglades drainage in south Florida. We hypothesized that contaminants present in areas with intermediate or higher contaminant concentrations would alter hepatic enzyme activities in juvenile alligators from those sites when compared to hepatic enzyme activity in animals from the area with the least contamination. EROD activity was found to be higher in animals from the site with lower reported levels of contamination relative to those from the site with the highest reported contamination suggesting an inhibition of CYP1A expression or activity. No differences among animals from the three sites were observed for hepatic MROD and GST activities. A significant negative relationship between EROD, MROD, and GST activities and body size was exhibited in alligators from the site with the lowest contamination. No relationship between body size and hepatic enzyme activity was found in animals from the sites with intermediate and higher contamination, suggesting that contaminants present at these sites act to alter this relationship. No correlation was observed in this study between plasma steroid concentrations (estradiol-17 beta or testosterone) and hepatic EROD, MROD, or GST activities.

  16. Short communication: Regulation of hepatic gluconeogenic enzymes by dietary glycerol in transition dairy cows.

    PubMed

    White, H M; Carvalho, E R; Koser, S L; Schmelz-Roberts, N S; Pezzanite, L M; Slabaugh, A C; Doane, P H; Donkin, S S

    2016-01-01

    Nutritional status and glucose precursors are known regulators of gluconeogenic gene expression. Glycerol can replace corn in diets fed to dairy cows and use of glycerol is linked to increased rumen propionate production. The effect of dietary glycerol on the regulation of gluconeogenic enzymes is unknown. The objective of this study was to examine the effect of glycerol on expression of pyruvate carboxylase (PC), cytosolic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M), and glucose-6-phosphatase. Twenty-six multiparous Holstein cows were fed either a control diet or a diet where high-moisture corn was replaced by glycerol from -28 through +56 d relative to calving (DRTC). Liver tissue was collected via percutaneous liver biopsy at -28, -14, +1, +14, +28, and +56 DRTC for RNA analysis. Expression of PC mRNA increased 6-fold at +1 and 4-fold at +14 DRTC relative to precalving levels. Dietary glycerol did not alter expression of PC mRNA expression. Expression of PEPCK-C increased 2.5-fold at +14 and 3-fold at +28 DRTC compared with +1 DRTC. Overall, dietary glycerol increased PEPCK-C expression compared with that of cows fed control diets. The ratio of PC to PEPCK-C was increased 6.3-fold at +1 DRTC compared with precalving and tended to be decreased in cows fed glycerol. We detected no effect of diet or DRTC on PEPCK-M or glucose-6-phosphatase mRNA, and there were no interactions of dietary treatment and DRTC for any transcript measured. Substituting corn with glycerol increased the expression of PEPCK-C mRNA during transition to lactation and suggests that dietary energy source alters hepatic expression. The observed increase in PEPCK-C expression with glycerol feeding may indicate regulation of hepatic gene expression by changes in rumen propionate production.

  17. 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

  18. Therapeutic insulin and hepatic glucose-6-phosphatase activity in preterm infants

    PubMed Central

    Burchell, A; McGeechan, A; Hume, R

    2000-01-01

    BACKGROUND—Hepatic glucose-6-phosphatase activity is low at birth, and in term infants rises rapidly to adult levels. In contrast, in most preterm infants, it remains low postnatally making them vulnerable to repeated hypoglycaemic episodes, resultant cerebral damage, or risk of sudden and unexpected death.
AIMS—To investigate the clinical features of preterm infants with low glucose-6-phosphatase enzyme activity to determine the influencing factors.
METHODS—Clinical data from 36 preterm infants were correlated by stepwise multiple regression analysis with Vmax of hepatic glucose-6-phosphatase as the dependent variable.
RESULTS—The most significant correlation was with the administration of insulin (units/kg/h postnatal life) with lesser effects of respiratory distress syndrome and dopamine administration. The Vmax changes reflected changes in the level of expression of the glucose-6-phosphatase protein.
CONCLUSION—In a variety of animal models, hepatic glucose-6-phosphatase levels have been shown to decrease in response to insulin, which also decreases transcription of the glucose-6-phosphatase gene. The association of insulin administration with high levels of hepatic glucose-6-phosphatase activity and protein expression was therefore most unexpected. Results from model systems, or adults, must be extrapolated to the metabolism of preterm infants with caution.

 PMID:10794792

  19. 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…

  20. JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

    PubMed Central

    Kochat, Veena; Equbal, Zaffar; Baligar, Prakash; Kumar, Vikash; Srivastava, Madhulika; Mukhopadhyay, Asok

    2017-01-01

    The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule

  1. 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

  2. Effects of Angiotensin Converting Enzyme Inhibitors on Liver Fibrosis in HIV and Hepatitis C Coinfection.

    PubMed

    Reese, Lindsey J; Tider, Diane S; Stivala, Alicia C; Fishbein, Dawn A

    2012-01-01

    Background. Liver fibrosis is accelerated in HIV and hepatitis C coinfection, mediated by profibrotic effects of angiotensin. The objective of this study was to determine if angiotensin converting enzyme inhibitors (ACE-Is) attenuate liver fibrosis in coinfection. Methods. A retrospective review of 156 coinfected subjects was conducted to analyze the association between exposure to ACE-Is and liver fibrosis. Noninvasive indices of liver fibrosis (APRI, FIB-4, Forns indices) were compared between subjects who had taken ACE-Is and controls who had not taken them. Linear regression was used to evaluate ACE-I use as an independent predictor of fibrosis. Results. Subjects taking ACE-Is for three years were no different than controls on the APRI and the FIB-4 but had significantly higher scores than controls on the Forns index, indicating more advanced fibrosis. The use of ACE-Is for three years remained independently associated with an elevated Forns score when adjusted for age, race, and HIV viral load (P < 0.001). There were significant associations between all of the indices and significant fibrosis, as determined clinically and radiologically. Conclusions. There was not a protective association between angiotensin inhibition and liver fibrosis in coinfection. These noninvasive indices may be useful for ruling out significant fibrosis in coinfection.

  3. Hepatitis B virus replication in steroid-treated severe HBsAg-positive chronic active hepatitis.

    PubMed

    Davis, G L; Czaja, A J; Taswell, H F; Ludwig, J; Go, V L

    1985-02-01

    To determine the effect of corticosteroids on the replication of hepatitis B virus and to assess the relationship between virus replication and prognosis, the behavior of serum and tissue HBcAg was evaluated in 16 patients with severe HBsAg-positive chronic active hepatitis who were treated with prednisone and followed for up to 10 years (mean +/- SEM, 66 +/- 9 months). Hepatitis B virus replication was assessed in serum by a solid-phase radioimmunoassay of Dane particle-associated HBcAg and in liver tissue by indirect immunoperoxidase staining for HBcAg. Despite the presence of severe inflammatory activity, only low levels of hepatitis B virus replication were demonstrated. Mean serum HBcAg levels were low at accession and remained essentially unchanged or gradually decreased during corticosteroid therapy. Serum HBcAg appeared in only one patient in whom no virus replication was detected prior to therapy. HBeAg was frequently detected at low titers by radioimmunoassay when serum HBcAg was undetectable. Loss of HBcAg preceded loss of HBeAg by radioimmunoassay, and disappearance of both markers was a prerequisite for sustained histologic remission. In eight patients, inflammation was present despite absence of serum or tissue HBcAg; in three of these, disease activity continued after loss of HBeAg. We conclude that low levels of hepatitis B virus replication may be associated with severe inflammatory activity, and these levels are not increased by long-term corticosteroid therapy. Inflammation can continue despite loss of HBeAg and absence of detectable virus replication.

  4. Cold-active enzymes studied by comparative molecular dynamics simulation.

    PubMed

    Spiwok, Vojtech; Lipovová, Petra; Skálová, Tereza; Dusková, Jarmila; Dohnálek, Jan; Hasek, Jindrich; Russell, Nicholas J; Králová, Blanka

    2007-04-01

    Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included alpha-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein.

  5. Ultrasound in Enzyme Activation and Inactivation

    NASA Astrophysics Data System (ADS)

    Mawson, Raymond; Gamage, Mala; Terefe, Netsanet Shiferaw; Knoerzer, Kai

    As discussed in previous chapters, most effects due to ultrasound arise from cavitation events, in particular, collapsing cavitation bubbles. These collapsing bubbles generate very high localized temperatures and pressure shockwaves along with micro-streaming that is associated with high shear forces. These effects can be used to accelerate the transport of substrates and reaction products to and from enzymes, and to enhance mass transfer in enzyme reactor systems, and thus improve efficiency. However, the high velocity streaming, together with the formation of hydroxy radicals and heat generation during collapsing of bubbles, may also potentially affect the biocatalyst stability, and this can be a limiting factor in combined ultrasound/enzymatic applications. Typically, enzymes can be readily denatured by slight changes in environmental conditions, including temperature, pressure, shear stress, pH and ionic strength.

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

    NASA Astrophysics Data System (ADS)

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

    2004-12-01

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

  7. Manganese enzymes with binuclear active sites

    SciTech Connect

    Dismukes, G.C.

    1996-11-01

    The purpose of this article is twofold. First, to review the recent literature dealing with the mechanisms of catalysis by binuclear manganese enzymes. Second, to summarize and illustrate the general principles of catalysis which distinguish binuclear metalloenzymes from monometallic centers. This review covers primarily the published literature from 1991 up to May 1996. A summary of the major structurally characterized dimanganese enzymes is given. These perform various reaction types including several redox reactions, (de)hydrations, isomerizations, (de)phosphorylation, and phosphoryl transfer. 114 refs.

  8. 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.

  9. 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,…

  10. Multicentric evaluation of new commercial enzyme immunoassays for the detection of immunoglobulin M and total antibodies against hepatitis A virus.

    PubMed

    Arcangeletti, M C; Dussaix, E; Ferraglia, F; Roque-Afonso, A M; Graube, A; Chezzi, C

    2011-08-01

    A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively).

  11. Multicentric Evaluation of New Commercial Enzyme Immunoassays for the Detection of Immunoglobulin M and Total Antibodies against Hepatitis A Virus▿

    PubMed Central

    Arcangeletti, M. C.; Dussaix, E.; Ferraglia, F.; Roque-Afonso, A. M.; Graube, A.; Chezzi, C.

    2011-01-01

    A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively). PMID:21653739

  12. [Gallbladder motor activity in patients with virus hepatitis B].

    PubMed

    Mamos, Arkadiusz; Wichan, Paweł; Chojnacki, Jan; Grzegorczyk, Krzysztof

    2003-12-01

    In acute stage of virus hepatitis B patients often complain of dyspeptic discomfort. They may be a consequence of alimentary tract motor activity disorders including these of gallbladder. Routine ultrasonography in an early phase of virus hepatitis often reveals gallbladder wall thickening what may confirm the above thesis. Thus, a group of 15 patients in an acute phase of virus hepatitis B was subjected to examinations. Gallbladder motor activity was assessed by ultrasonographic method determining its total volume and ejection fraction and volume after test meal stimulus. First examination was performed in the first week since the appearance of yellowing of the walls, successive in 4 and 8 week of the disease. Obtained results were compared to the values obtained in the group of 25 healthy volunteers. It was found out that gallbladder volume was significantly decreased and ejection fraction increased in the acute phase of virus hepatitis B than in the controls. This may speak for gallbladder hyperreactivity in patients in the course of virus hepatitis B. These disorders decreased during two-month observation but even in the 8 week the investigated parameters differed from those found in the control group.

  13. Puerarin ameliorates hepatic steatosis by activating the PPARα and AMPK signaling pathways in hepatocytes.

    PubMed

    Kang, Ok-Hwa; Kim, Sung-Bae; Mun, Su-Hyun; Seo, Yun-Soo; Hwang, Hyeong-Chil; Lee, Young-Mi; Lee, Ho-Seob; Kang, Dae-Gil; Kwon, Dong-Yeul

    2015-03-01

    Non-alcoholic fatty liver disease (NAFLD) is characterized by the hepatic manifestation of metabolic syndrome and is the leading cause of chronic liver disease. Steatohepatitis plays a critical role in the process resulting in liver fibrosis and cirrhosis. Puerarin is a herbal product widely used in Asia, and is believed to have therapeutic benefits for alleviating the symptoms of steatohepatitis. The present study was designed to investigate the effects and mechanisms of action of puerarin in reducing lipid accumulation in oleic acid (OA)-treated HepG2 cells. Hepatocytes were treated with OA with or without puerarin to observe lipid accumulation by Oil Red O staining. We also examined hepatic lipid contents (e.g., triacylglycerol and cholesterol) following treatment with puerarin. Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were used to measure sterol regulatory element binding protein (SREBP)-1, fatty acid synthase (FAS), peroxisome proliferator-activated receptor α (PPARα) and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) protein and mRNA expression, respectively. Our results revealed that puerarin suppressed OA-induced lipid accumulation, and reduced the triacylglycerol and cholesterol levels. Furthermore, puerarin decreased the expression levels of lipogenic enzymes, such as FAS and SREBPs, and increased the expression levels of PPARα, which are critical regulators of hepatic lipid metabolism through the AMPK signaling pathway. These results indicate that puerarin has the same ability to activate AMPK, and reduce SREBP-1 and FAS expression, thus inhibiting hepatic lipogenesis and increasing hepatic antioxidant activity. We found that puerarin exerted a regulatory effect on lipid accumulation by decreasing lipogenesis in hepatocytes. Therefore, puerarin extract may have therapeutic benefits in the treatment of fatty liver and lipid-related metabolic disorders.

  14. 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.

  15. 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...

  16. Gluconeogenic substrates and hepatic gluconeogenic enzymes in streptozotocin-diabetic rats: effect of mulberry (Morus indica L.) leaves.

    PubMed

    Andallu, B; Varadacharyulu, N C

    2007-03-01

    Mulberry (Morus indica L.) leaves, the sole food of the silk worm, were evaluated for antidiabetic effects in streptozotocin (STZ)-diabetic rats. Treatment with dried mulberry leaf powder at 25% of the diet for a period of 8 weeks was found to be remarkably beneficial to STZ-diabetic rats as evidenced by controlled hyperglycemia and glycosuria. In addition, mulberry leaves countered (reversed) the alterations in gluconeogenic substrates in STZ-diabetic rats as indicated by significant reduction in serum pyruvic and lactic acid levels, a significant increase in proteins and a significant decrease in free amino acid, urea, and creatinine levels in blood, and a decreased urinary excretion of urea and creatinine. Anomalies in the activities of hepatic gluconeogenic enzymes associated with impaired glucose homeostasis in STZ-diabetic rats were ameliorated by feeding the mulberry leaf-supplemented diet, indicating that control over hyperglycemia and associated complications in the diabetic state by mulberry leaves is by way of regulation of gluconeogenesis. With respect to all the parameters, mulberry leaves were more effective than the oral hypoglycemic drug glibenclamide.

  17. 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.

  18. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  19. 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.

  20. Effect of malonate and p-chlorophenoxy acetic acid on hepatic succinic dehydrogenase activity of ageing lizards.

    PubMed

    Jena, B S; Patnaik, B K

    1990-01-01

    The degree of inhibition of hepatic succinic dehydrogenase activity by malonate, a competitive inhibitor, did not differ between young and middle-aged lizards. On the other hand, the same parameter increased significantly between middle-aged and old lizards. The percent inhibition of enzyme activity by p-chlorophenoxy acetic acid was also age-dependent, being higher in middle-aged and old than in young lizards.

  1. Enzyme

    MedlinePlus

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

  2. 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

  3. Metformin regulates hepatic lipid metabolism through activating AMP-activated protein kinase and inducing ATGL in laying hens.

    PubMed

    Chen, Wei-Lu; Wei, Hen-Wei; Chiu, Wen-Zan; Kang, Ching-Hui; Lin, Ting-Han; Hung, Chien-Ching; Chen, Ming-Chun; Shieh, Ming-Song; Lee, Chin-Cheng; Lee, Horng-Mo

    2011-12-05

    Although many clinical trials have showed that metformin improves non-alcoholic fatty liver disease, which is a common liver disease associated with hepatic enzyme abnormalities, an animal model is required to investigate the effects of altered gene expression and post-translational processing (proteins) in mediating the observed responses. Laying hens appear to develop fatty livers, as in the case in human beings, when ingesting energy in excess of maintenance, and they can be used as an animal model for observing hepatic steatosis. The aim of this study was to investigate whether metformin could improve the non-alcoholic fatty liver of laying hens and to examine the possible mechanisms of lipid-lowering effects. Forty-eight Leghorn laying hens of Hy-Line variety W-36 - 44 weeks with 64.8% hen-day egg production - were randomly assigned into 4 treatments, each receiving 0, 10, 30, or 100mg of metformin with saline per kg body weight by daily wing vein injection. Results showed that, compared with the control, significant decreases existed in the laying rates; plasma triglyceride, cholesterol, and insulin levels; body weights; abdominal fat weights; hepatic lipid contents; and hepatic fatty acid synthase expression of layers receiving 30 or 100mg per kg body weight, whereas significant increases in their hepatic 5'adenosine monophosphate-activated protein kinase, acyl-CoA carboxylase phosphorylation, adipose triglyceride lipase, and carnitine palmitoyl transferase-1 expression were observed. These data suggest that metformin could reduce lipid deposits in the liver and that the laying hen is a valuable animal model for studying hepatic steatosis.

  4. Hepatitis A

    MedlinePlus

    ... transaminase enzyme levels Treatment There is no specific treatment for hepatitis A. You should rest when the symptoms are ... and have not had hepatitis A or the hepatitis A vaccine. Common reasons for getting one or both of these treatments include: You live with someone who has hepatitis ...

  5. Fish oil at low dietary levels enhances physiological activity of sesamin to increase hepatic fatty acid oxidation in rats.

    PubMed

    Ide, Takashi

    2012-11-01

    We previously demonstrated that a diet containing fish oil at a level of 80 g/kg strongly stimulated the physiological activity of a sesame sesamin preparation containing sesamin and episesamin at equal amounts to increase hepatic fatty acid oxidation. This study was conducted to clarify whether fish oil at lower dietary levels enhances the physiological activity of sesamin to increase hepatic fatty acid oxidation. Rats were fed experimental diets supplemented with 0 or 2 g sesamin/kg, and containing 0, 15 or 30 g fish oil/kg for 15 days. Among rats fed sesamin-free diets, diets containing 15 and 30 g fish oil/kg slightly increased the activity of enzymes involved in hepatic fatty acid oxidation. Sesamin increased these values irrespective of the presence or absence of fish oil in diets; however, the extent of the increase of many parameters was much greater in rats given fish oil-containing diets than in those fed a fish oil-free diet. Diets simultaneously containing sesamin and fish oil increased the gene expression of various peroxisomal fatty acid oxidation enzymes in a synergistic manner; but they were ineffective in causing a synergistic increase in mRNA levels of mitochondrial fatty acid oxidation enzymes. The extent of the synergistic increase in the activity of hepatic fatty acid oxidation enzymes and mRNA levels of the peroxisomal enzymes was indistinguishable between diets containing 15 and 30 g fish oil/kg and appeared comparable to that observed previously with a diet containing 80 g fish oil/kg.

  6. 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...

  7. 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.

  8. 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.

  9. Enzyme activity control by responsive redoxpolymers.

    PubMed

    Nagel, Birgit; Warsinke, Axel; Katterle, Martin

    2007-06-05

    A new thermoresponsive poly-N-isopropylacrylamide (PNIPAM)-ferrocene polymer was synthesized and characterized. PNIPAMFoxy bears additional oxirane groups which were used for attachment by a self-assembly process on a cysteamine-modified gold electrode to create a thin hydrophilic film. The new redox polymer enabled electrical communication between the cofactor pyrrolinoquinoline quinone (PQQ) of soluble glucose dehydrogenase (sGDH) and the electrode for sensitive detection of this enzyme as a prospective protein label. The temperature influence on the redox polymer/enzyme complex was investigated. An inverse temperature response behavior of surface bound PNIPAMFoxy compared to the soluble polymer was found and is discussed in detail. The highest efficiency of mediated electron transfer for the immobilized PNIPAMFoxy with sGDH was observed at 24 degrees C, which was twice as high as that of its soluble counterpart. A steady-state electrooxidation current densitiy of 4.5 microA.cm-2 was observed in the presence of 10 nM sGDH and 5 mM glucose. A detection limit of 0.5 nM of soluble PQQ-sGDH was obtained.

  10. Feeding glycerol-enriched yeast culture improves lactation performance, energy status, and hepatic gluconeogenic enzyme expression of dairy cows during the transition period.

    PubMed

    Ye, G; Liu, J; Liu, Y; Chen, X; Liao, S F; Huang, D; Huang, K

    2016-06-01

    This study aimed to evaluate the effects of feeding glycerol-enriched yeast culture (GY) on feed intake, lactation performance, blood metabolites, and expression of some key hepatic gluconeogenic enzymes in dairy cows during the transition period. Forty-four multiparous transition Holstein cows were blocked by parity, previous 305-d mature equivalent milk yield, and expected calving date and randomly allocated to 4 dietary treatments: Control (no additive), 2 L/d of GY (75.8 g/L glycerol and 15.3 g/L yeast), 150 g/d of glycerol (G; 0.998 g/g glycerol), and 1 L/d of yeast culture (Y; 31.1 g/L yeast). All additives were top-dressed and hand mixed into the upper one-third of the total mixed ration in the morning from -14 to +28 d relative to calving. Results indicated that the DMI, NE intake, change of BCS, and milk yields were not affected by the treatments ( > 0.05). Supplementation of GY or Y increased milk fat percentages, milk protein percentages, and milk protein yields relative to the Control or G group ( < 0.05). Cows fed GY or G had higher glucose levels and lower β-hydroxybutyric acid (BHBA) and NEFA levels in plasma than cows fed the Control ( < 0.05) and had lower NEFA levels than cows fed Y ( < 0.05). On 14 d postpartum, cows fed GY or G had higher enzyme activities, mRNA, and protein expression of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; < 0.05); higher enzyme activities ( < 0.05) and a tendency toward higher mRNA expression ( < 0.10) of glycerol kinase (GK); and a tendency toward higher enzyme activities of pyruvate carboxylase (PC) in the liver ( < 0.10) when compared with cows fed Control or Y. The enzyme activities, mRNA, and protein expression of PEPCK-C, PC, and GK did not differ between cows fed GY and G ( > 0.10). In conclusion, dietary GY or Y supplementation increased the milk fat and protein content of the cows in early lactation and GY or G supplementation improved the energy status as indicated by greater plasma glucose and

  11. 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

  12. 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.

  13. Activation volumes of enzymes adsorbed on silica particles.

    PubMed

    Schuabb, Vitor; Czeslik, Claus

    2014-12-30

    The immobilization of enzymes on carrier particles is useful in many biotechnological processes. In this way, enzymes can be separated from the reaction solution by filtering and can be reused in several cycles. On the other hand, there is a series of examples of free enzymes in solution that can be activated by the application of pressure. Thus, a potential loss of enzymatic activity upon immobilization on carrier particles might be compensated by pressure. In this study, we have determined the activation volumes of two enzymes, α-chymotrypsin (α-CT) and horseradish peroxidase (HRP), when they are adsorbed on silica particles and free in solution. The experiments have been carried out using fluorescence assays under pressures up to 2000 bar. In all cases, activation volumes were found to depend on the applied pressure, suggesting different compressions of the enzyme-substrate complex and the transition state. The volume profiles of free and adsorbed HRP are similar. For α-CT, larger activation volumes are found in the adsorbed state. However, up to about 500 bar, the enzymatic reaction of α-CT, which is adsorbed on silica particles, is characterized by a negative activation volume. This observation suggests that application of pressure might indeed be useful to enhance the activity of enzymes on carrier particles.

  14. Effect of ketogenic diet on nucleotide hydrolysis and hepatic enzymes in blood serum of rats in a lithium-pilocarpine-induced status epilepticus.

    PubMed

    da Silveira, Vanessa Gass; de Paula Cognato, Giana; Müller, Alexandre Pastoris; Figueiró, Fabrício; Bonan, Carla Denise; Perry, Marcos L Santos; Battastini, Ana Maria Oliveira

    2010-06-01

    The ketogenic diet (KD) is a high-fat and low-carbohydrate diet, used for treating refractory epilepsy in children. We have previously shown alterations in nucleotidase activities from the central nervous system and blood serum of rats submitted to different models of epilepsy. In this study we investigated the effect of KD on nucleotidase activities in the blood serum, as well if KD has any influence in the activity of liver enzymes such as alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities in Wistar rats submitted to the lithium-pilocarpine model of epilepsy. At 21 days of age, rats received an injection of lithium chloride and, 18-19 h later, they received an injection of pilocarpine hydrochloride for status epilepticus induction. The results reported herein show that seizures induced by lithium-pilocarpine elicit a significant increase in ATP hydrolysis and alkaline phosphatase activity, as well as a decrease in ADP hydrolysis and aspartate aminotransferase activity. The KD is a rigorous regimen that can be associated with hepatic damage, as shown herein by the elevated activities of liver enzymes and 5'-nucleotidase in blood serum. Further studies are necessary to investigate the mechanism of inhibition of lithium on nucleotidases in blood serum.

  15. Effect of early feed restriction and enzyme supplementation on digestive enzyme activities in broilers.

    PubMed

    Pinheiro, D F; Cruz, V C; Sartori, J R; Vicentini Paulino, M L M

    2004-09-01

    The effect of feed restriction and enzymatic supplementation on intestinal and pancreatic enzyme activities and weight gain was studied in broiler chickens. Quantitative feed restriction was applied to chickens from 7 to 14 d of age. An enzyme complex mainly consisting of protease and amylase was added to the chicken ration from hatching to the end of the experiment. Birds subjected to feed restriction whose diet was not supplemented showed an increase in sucrase, amylase, and lipase activities immediately after the restriction period. Amylase, lipase, and chymotrypsin activities were higher in chickens subjected to feed restriction and fed a supplemented diet than in those only subjected to feed restriction. Trypsin activity increased after feed restriction and after supplementation, but there was no interaction between these effects. Early feed restriction had no effect on enzyme activity in 42-d-old chickens. Chickens subjected to early restriction and fed the supplemented diet presented higher sucrase, maltase, and lipase activities than nonsupplemented ones (P < 0.05). There was no effect of early feed restriction or diet supplementation on weight gain to 42 d. Percentage weight gain from 14 to 42 d of age was equivalent in feed-restricted and ad libitum fed birds. Feed-restricted broilers fed a supplemented diet showed a higher percentage weight gain than nonsupplemented birds. We conclude that enzymatic supplementation potentiates the effect of feed restriction on digestive enzyme activity and on weight gain.

  16. Compounds from Silicones Alter Enzyme Activity in Curing Barnacle Glue and Model Enzymes

    PubMed Central

    Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H.

    2011-01-01

    Background Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. Methodology/Principal Findings GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Conclusions/Significance Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management. PMID:21379573

  17. 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

  18. ENZYMIC ACTIVITY IN FREEZE DRIED FOODS

    DTIC Science & Technology

    and bananas. Factors studied include, polyphenol oxidase , peroxidase, sucrase, alpha and beta amylase, pectinesterase and ascorbase activity as well...storage of freeze-dried and frozen peas at different moisture was studied. Lipase activity and production of free fatty acid was following during long term

  19. Do Antioxidant Enzymes and Glutathione Play Roles in the Induction of Hepatic Oxidative Stress in Mice upon Subchronic Exposure to Mixtures of Dichloroacetate and Trichloroacetate?

    PubMed Central

    Hassoun, Ezdihar; Cearfoss, Jacquelyn

    2014-01-01

    Dichloroacetate (DCA) and trichloroacetate (TCA) are water chlorination byproducts, and their mixtures were previously found to induce additive to greater than additive effects on hepatic oxidative stress (OS) induction in mice after subchronic exposure. To investigate the roles of antioxidant enzymes and glutathione (GSH) in those effects, livers of B6C3F1 mice treated by gavage with 7.5, 15, or 30 mg DCA/kg/day, 12.5, 25, or 50 mg TCA/kg/day, and mixtures (Mix I, Mix II and Mix III) at DCA:TCA ratios corresponding to 7.5:12.5, 15:25 and 25:50 mg/kg/day, respectively, for 13 weeks. Livers were assayed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), as well as for GSH levels. In general, DCA suppressed SOD and GSH-Px activities and GSH levels but caused no changes in CAT activity; TCA increased SOD and CAT activities, suppressed GSH-Px activity, but did not change GSH levels; mixtures of DCA and TCA increased SOD and CAT activities and suppressed GSH-Px activity and GSH levels. In conclusion, antioxidant enzymes contribute to DCA-, TCA- and mixtures-induced OS, but not to changes from additive to greater than additive effects produced by different mixture compositions of the compounds. GSH on the hand may contribute to these changes. PMID:25530655

  20. 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.

  1. Investigation of enzyme activity by SERRS using poly-functionalised benzotriazole derivatives as enzyme substrates.

    PubMed

    Ingram, Andrew M; Stirling, Kirsten; Faulds, Karen; Moore, Barry D; Graham, Duncan

    2006-08-07

    New methods of measuring biologically relevant concentrations of enzymes are necessary to allow greater understanding of biological systems. We have previously shown that aryl azo benzotriazolyl alkyl esters can act as enzyme substrates, with the progress of the reaction being monitored using SERRS (see Nat. Biotechnol., 2004, 22, 1133, ref. ). This is a wholly novel analytical application of SERRS, and the low detection levels of the technique allow for an ultra-sensitive enzyme assay. Masked enzyme substrates are used that are invisible to SERRS until enzymatic hydrolysis. Turnover of the substrate by the enzyme leads to the release of the surface-seeking dye necessary for SERRS, and intense signals are produced. Here we report an improved synthesis of 2H-benzotriazolyl alkyl esters via nucleophilic substitution of a chloromethyl ester by benzotriazolyl azo dyes, giving up to a ten-fold increase on previously reported yields. Introduction of electron-withdrawing groups to the benzotriazole ring allows control over the SERRS properties of the compounds. This is of great significance in expanding the synthetic flexibility and subsequently the fundamental use of these compounds as ultra-sensitive and selective reporters of enzyme activity.

  2. Enzyme:nanoparticle bioconjugates with two sequential enzymes: stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles.

    PubMed

    Keighron, Jacqueline D; Keating, Christine D

    2010-12-21

    We report the synthesis and characterization of bioconjugates in which the enzymes malate dehydrogenase (MDH) and/or citrate synthase (CS) were adsorbed to 30 nm diameter Au nanoparticles. Enzyme:Au stoichiometry and kinetic parameters (specific activity, k(cat), K(M), and activity per particle) were determined for MDH:Au, CS:Au, and three types of dual-activity MDH/CS:Au bioconjugates. For single-activity bioconjugates (MDH:Au and CS:Au), the number of enzyme molecules adsorbed per particle was dependent upon the enzyme concentration in solution, with multilayers forming at high enzyme:Au solution ratios. The specific activity of adsorbed enzyme increased with increasing number adsorbed per particle for CS:Au, but was less sensitive to stoichiometry for MDH:Au. Dual activity bioconjugates were prepared in three ways: (1) by adsorption of MDH followed by CS, (2) by adsorption of CS followed by MDH, and (3) by coadsorption of both enzymes from the same solution. The resulting bioconjugates differed substantially in the number of enzyme molecules adsorbed per particle, the specific activity of the adsorbed enzymes, and also the enzymatic activity per particle. Bioconjugates formed by adding CS to the Au nanoparticles before MDH was added exhibited higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also had 3-fold higher per-particle sequential activity for conversion of malate to citrate, despite substantially fewer copies of both enzymes present.

  3. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  4. 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.

  5. 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.

  6. Enzyme activities in mitochondria isolated from ripening tomato fruit.

    PubMed

    Jeffery, D; Goodenough, P W; Weitzman, P D

    1986-09-01

    Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.

  7. 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

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

    PubMed

    Mingarro, I; Abad, C; Braco, L

    1995-04-11

    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.

  9. Chimeric enzymes with improved cellulase activities

    DOEpatents

    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.

  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. Catalytically active nanomaterials: a promising candidate for artificial enzymes.

    PubMed

    Lin, Youhui; Ren, Jinsong; Qu, Xiaogang

    2014-04-15

    Natural enzymes, exquisite biocatalysts mediating every biological process in living organisms, are able to accelerate the rate of chemical reactions up to 10(19) times for specific substrates and reactions. However, the practical application of enzymes is often hampered by their intrinsic drawbacks, such as low operational stability, sensitivity of catalytic activity to environmental conditions, and high costs in preparation and purification. Therefore, the discovery and development of artificial enzymes is highly desired. Recently, the merging of nanotechnology with biology has ignited extensive research efforts for designing functional nanomaterials that exhibit various properties intrinsic to enzymes. As a promising candidate for artificial enzymes, catalytically active nanomaterials (nanozymes) show several advantages over natural enzymes, such as controlled synthesis in low cost, tunability in catalytic activities, as well as high stability against stringent conditions. In this Account, we focus on our recent progress in exploring and constructing such nanoparticulate artificial enzymes, including graphene oxide, graphene-hemin nanocomposites, carbon nanotubes, carbon nanodots, mesoporous silica-encapsulated gold nanoparticles, gold nanoclusters, and nanoceria. According to their structural characteristics, these enzyme mimics are categorized into three classes: carbon-, metal-, and metal-oxide-based nanomaterials. We aim to highlight the important role of catalytic nanomaterials in the fields of biomimetics. First, we provide a practical introduction to the identification of these nanozymes, the source of the enzyme-like activities, and the enhancement of activities via rational design and engineering. Then we briefly describe new or enhanced applications of certain nanozymes in biomedical diagnosis, environmental monitoring, and therapeutics. For instance, we have successfully used these biomimetic catalysts as colorimetric probes for the detection of

  12. [Dosing time based on molecular mechanism of biological clock of hepatic drug metabolic enzyme].

    PubMed

    Matsunaga, Naoya

    2009-11-01

    The mammalian circadian pacemaker stays in the paired suprachiasmatic nuclei (SCN). Recent several studies reveal that the circadian rhythms of physiology and behavior are controlled by clock genes. In addition, the effectiveness and toxicity of many drugs vary depending on dosing time associated with 24-h rhythms of biochemical, physiological, and behavioral processes under the control of the circadian clock. Acetaminophen (APAP) is a widely used analgesic drug, and is mainly biotransformed and eliminated as nontoxic conjugates with glucuronic acid and sulfuric acid. Only a small portion of the dose is mainly bioactivated by CYP2E1 to N-acetyl-p-benzoquinone imine (NAPQI), a reactive toxic intermediate. For APAP overdose, glucuronidation and sulfation are saturated and the formation of NAPQI increases. However, the exact mechanisms underlying the chronotoxicity of APAP have not been clarified yet. In the present study, we have clarified that there was a significant dosing time-dependent difference in hepatotoxicity induced by APAP in mice. The mechanism may be related to the rhythmicity of CYP2E1 activity and GSH conjugation. In additon, we investigated whether the liver transcription factor hepatic nuclear factor-1alpha (HNF-1alpha) and clock genes undergoing astriking 24-h rhythm in mouse liver contribute to the 24-h regulation of CYP2E1 activity. A significant 24-h rhythmicity was demonstrated for CYP2E1 activity, protein levels and mRNA levels. HNF-1alpha and clock genes may contribute to produce the 24-h rhythm of CYP2E1 mRNA levels. Metabolism by CYP and GSH conjugation are common metabolic pathways for many drugs such as APAP. These findings support the concept that choosing the most appropriate time of day to administer the drugs associated with metabolic rhythmicity such as CYP and GSH conjugation may reduce hepatotoxicity in experimental and clinical situations. 24-h rhythm of CYP2E1 activity was controlled by HNF-1alpha and clock gene, in a

  13. Enzyme activation through the utilization of intrinsic dianion binding energy.

    PubMed

    Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

    2016-11-29

    We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol., 43: , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed.

  14. 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

  15. 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.

  16. [Activity of antioxidant enzymes in patients with liver cirrhosis].

    PubMed

    Czeczot, Hanna; Scibior, Dorota; Skrzycki, Michał; Podsiad, Małgorzata

    2006-01-01

    The aim of our studies was the estimation of activities of antioxidant enzymes in patients with liver cirrhosis. We investigated activities of superoxide dismutases (CuZnSOD, MnSOD), catalase (CAT), selenium dependent GSH peroxidase (Se-GSH-Px), selenium independent GSH peroxidase (non-Se-GSH-Px), GSH-S-transferase (GST), GSH reductase (GSHR) and the level ofreduced gutathione (GSH) in cirrhotic and healthy liver tissues. The activities of CuZnSOD, MnSOD, CAT and GSH-dependent enzymes (except GSHR) were found to be lower in cirrhotic tissue compared to healthy liver. Those changes were associated with decrease of GSH level in cirrhotic tissue compared with control liver tissue. Our results show that antioxidant barrier in liver cirrhosis is impaired. It is associated with decrease of glutathione level and changes of activities of antioxidant enzymes (SOD, CAT, GSHPx, GST, GSHR) in liver cirrhosis compared with healthy liver.

  17. Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes.

    PubMed

    Hutzler, J Matthew; Yang, Young-Sun; Albaugh, Daniel; Fullenwider, Cody L; Schmenk, Jennifer; Fisher, Michael B

    2012-02-01

    Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

  18. Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities

    PubMed Central

    Al-Abdullah, Ismail H.; Bagramyan, Karine; Bilbao, Shiela; Qi, Meirigeng; Kalkum, Markus

    2017-01-01

    A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells. PMID:28287171

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. Water modulation of stratum corneum chymotryptic enzyme activity and desquamation.

    PubMed

    Watkinson, A; Harding, C; Moore, A; Coan, P

    2001-09-01

    Exposure to a dry environment leads to depletion of water from the peripheral stratum corneum layers in a process dependent on the relative humidity (RH) and the intrinsic properties of the tissue. We hypothesized that by modulating the water content of the stratum corneum in the surface layers, RH effects the rate of desquamation by modulating the activity of the desquamatory enzymes, and specifically stratum corneum chymotryptic enzyme (SCCE). Using a novel air interface in vitro desquamatory model, we demonstrated RH-dependent corneocyte release with desquamatory rates decreasing below 80% RH. Application of 10% glycerol or a glycerol-containing moisturizing lotion further increased desquamation, even in humid conditions, demonstrating that water was the rate-limiting factor in the final stages of desquamation. Furthermore, even in humid conditions desquamation was sub-maximal. In situ stratum corneum SCCE activity showed a dependence on RH: activity was significantly higher at 100% than at 44% RH. Further increases in SCCE activity were induced by applying a 10% glycerol solution. Since SCCE, a water-requiring enzyme, must function in the water-depleted outer stratum corneum, we sought to determine whether this enzyme has a tolerance to lowered water activity. Using concentrated sucrose solutions to lower water activity, we analysed the activity of recombinant SCCE and compared it to that of trypsin and chymotrypsin. SCCE activity demonstrated a tolerance to water restriction, and this may be an adaptation to maintain enzyme activity even within the water-depleted stratum corneum intercellular space. Overall these findings support the concept that in the upper stratum corneum, RH modulates desquamation by its effect upon SCCE activity, and possibly other desquamatory hydrolases. In addition, SCCE may be adapted to function in the water-restricted stratum corneum intercellular space.

  5. Does diet influence salivary enzyme activities in elephant species?

    PubMed

    Boehlke, Carolin; Pötschke, Sandra; Behringer, Verena; Hannig, Christian; Zierau, Oliver

    2017-01-01

    Asian elephants (Elephas maximus) and African elephants (Loxodonta africana) are herbivore generalists; however, Asian elephants might ingest a higher proportion of grasses than Africans. Although some studies have investigated nutrition-specific morphological adaptations of the two species, broader studies on salivary enzymes in both elephant species are lacking. This study focuses on the comparison of salivary enzymes activity profiles in the two elephant species; these enzymes are relevant for protective and digestive functions in humans. We aimed to determine whether salivary amylase (sAA), lysozyme (sLYS), and peroxidase (sPOD) activities have changed in a species-specific pattern during evolutionary separation of the elephant genera. Saliva samples of 14 Asian and eight African elephants were collected in three German zoos. Results show that sAA and sLYS are salivary components of both elephant species in an active conformation. In contrast, little to no sPOD activity was determined in any elephant sample. Furthermore, sAA activity was significantly higher in Asian compared with African elephants. sLYS and sPOD showed no species-specific differences. The time of food provision until sample collection affected only sAA activity. In summary, the results suggest several possible factors modulating the activity of the mammal-typical enzymes, such as sAA, sLYS, and sPOD, e.g., nutrition and sampling procedure, which have to be considered when analyzing differences in saliva composition of animal species.

  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. Evolutionary transitions in enzyme activity of ant fungus gardens.

    PubMed

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

    2010-07-01

    Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

  8. 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

  9. Inducible trehalase enzyme activity of Morchella conica Persoon mycelium.

    PubMed

    Czövek, Pálma; Király, I

    2011-03-01

    Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.

  10. 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.

  11. Cell signalling and the hormonal stimulation of the hepatic glycine cleavage enzyme system by glucagon.

    PubMed Central

    Mabrouk, G M; Jois, M; Brosnan, J T

    1998-01-01

    The glycine cleavage enzyme system (GCS) is found in mitochondria. In liver it is activated by glucagon and other hormones but it is not known how the hormonal signal is transmitted to the mitochondria. We found that the cell-permeant protein phosphatase inhibitor okadaic acid stimulated flux through GCS and could induce a significant increase in the sensitivity of GCS and of glycogenolysis to glucagon. Half-maximal stimulation of GCS by glucagon occurred at 3.2+/-0.6 nM, whereas it was fully activated at 0.3 nM in the presence of 1 microM okadaic acid. The protein kinase A agonist adenosine-3',5'-cyclic monophosphorothioate, Sp isomer (10 microM) stimulated the GCS flux by approx. 100%. This stimulation was inhibited by the protein kinase A antagonist 8-bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS). Although Rp-8-Br-cAMPS significantly inhibited glucagon-stimulated glycogenolysis it had no effect on the glucagon-stimulated GCS flux. These results indicate that a cytoplasmic phosphorylated protein is involved in transmitting glucagon's effect to the mitochondria. However, protein kinase A does not have a necessary role in transmitting glucagon's signal. We also examined the role of protein kinase C because angiotensin II also stimulated flux through GCS. However, the phorbol ester PMA had no effect on either GCS or on glycogenolysis. PMID:9480887

  12. Miltefosine Suppresses Hepatic Steatosis by Activating AMPK Signal Pathway

    PubMed Central

    Zhu, Yaqin; Tong, Xing; Li, Kexue; Bai, Hui; Li, Xiaoyu; Ben, Jingjing; Zhang, Hanwen; Yang, Qing; Chen, Qi

    2016-01-01

    Background and Purpose It has been accepted that AMPK (Adenosine monophosphate–activated protein kinase) activation exhibits many beneficial effects on glucolipid metabolism. Lysophosphatidylcholine (LPC) is an important lysophospholipid which can improve blood glucose levels in diabetic mice and attenuate inflammation by activating AMPK signal pathway in macrophages. Synthetic alkylphospholipids (ALPs), such as miltefosine, is used as an alternate of LPC for the clinical application. Here, we investigated whether miltefosine could have an impact on hepatic steatosis and related metabolic disorders. Experimental Approach Mice were fed with high fat diet (HFD) for 16 weeks to generate an obese model. Next, the obese mice were randomly divided into three groups: saline-treated and miltefosine-treated (2.5 or 5 mg/kg/d) groups. Miltefosine was intraperitoneally administrated into mice for additional 4 weeks plus HFD treatment. Key Results It was shown that miltefosine treatment could substantially improve glucose metabolism, prevented hepatic lipid accumulation, and inhibited liver inflammation in HFD-fed mice by activating AMPK signal pathway. In vitro, miltefosine stimulated AMPKα phosphorylation both in time and dose dependent manner and decreased lipid accumulation in liver cells. When a specific AMPK inhibitor compound C was used to treat mice, the antagonistic effects of miltefosine on HFD-induced mouse hyperlipidaemia and liver steatosis were abolished. Treatment with miltefosine also dramatically inhibited the HFD-induced liver inflammation in mice. Conclusions and Implications Here we demonstrated that miltefosine might be a new activator of AMPK signal pathway in vivo and in vitro and be useful for treatment of hepatic steatosis and related metabolic disorders. PMID:27681040

  13. 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...

  14. Hepatitis B antigen in hepatocytes of chronic active liver disease.

    PubMed

    Kawanishi, H

    1979-04-01

    To study the morphologic interrelation of hepatocytes with the replication of hepatitis B vius (HBV) and immunocompetent cells in chronic active liver disease(CALD), organ cultures were prepared from liver biopsy specimens. Replication of hepatitis B core antigen (HBcAg) appears to occur in the nucleus of the hepatocyte in close association with intranuclear electron-dense strands and sometimes intranucleolar matrixes (likely HBcAg genomes), and cytoplasmic maturation of the HBcAg takes place in the preautolytic condition of host hepatocytes. Immunocompetent cells became progressively autolyzed in the early period of cultures. No difference in progression of hepatocyte injury in tissues from normal subjects and from hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative patients with CALD may suggest that intracellular synthesis of HBV alone is not cytopathic to host hepatocytes. This model is promising for the study of HBV replication and development, and also for testing the efficacy of new antiviral agents against the virus.

  15. Regulation of eNOS enzyme activity by posttranslational modification.

    PubMed

    Heiss, Elke H; Dirsch, Verena M

    2014-01-01

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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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...

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

    PubMed

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

    2015-09-01

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

  6. Viral hepatitis A, active component, U.S. Armed Forces, 2000-2010.

    PubMed

    2011-08-01

    From 2000 to 2010, there were 214 incident diagnoses of acute hepatitis A among active component members of the U.S. Armed Forces; the crude overall incidence rate during the period was 1.37 per 100,000 person-years. Rates of incident diagnoses of acute hepatitis A were relatively low throughout the period and much lower than during the pre-vaccine era (1990-1996). There were disproportionate numbers of diagnoses of acute hepatitis A among service members born in countries endemic for the infection. The low rates of acute hepatitis A among U.S. military members overall reflect the widespread use of hepatitis A virus vaccine.

  7. Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme.

    PubMed

    Love, Robert A; Parge, Hans E; Yu, Xiu; Hickey, Michael J; Diehl, Wade; Gao, Jingjin; Wriggers, Hilary; Ekker, Anne; Wang, Liann; Thomson, James A; Dragovich, Peter S; Fuhrman, Shella A

    2003-07-01

    The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the "thumb" domain, about 30 A from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization.

  8. 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

  9. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    PubMed

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry.

  10. RSPOs facilitated HSC activation and promoted hepatic fibrogenesis

    PubMed Central

    Yin, Xinguang; Yi, Huixing; Wang, Linlin; Wu, Wanxin; Wu, Xiaojun; Yu, Linghua

    2016-01-01

    Roof plate-specific spondin (RSPO) proteins are potent Wnt pathway agonists and involve in a broad range of developmental and physiological processes. This study investigated the activities and mechanisms of RSPOs in liver fibrogenesis, especially in hepatic stellate cell (HSC) activation. HSC activation was assessed by fibrosis biomarker (α-smooth muscle actin and Collagen-I), phenotypic change (accumulation of lipid droplets), and increased proliferation. Similarly, Wnt pathway activity was evaluated by the expression of nuclear β-catenin and T cell-specific transcription factors (TCF) activity. We found RSPOs were overexpressed in human fibrotic liver tissue and the expressions were correlated with liver fibrosis stages. In vitro studies showed RSPOs level increased during HSC activation, and stimuli with RSPOs enhanced Wnt pathway activity and promoted HSC activation subsequently. Furthermore, in vivo experiments demonstrated that the knockdown of RSPOs suppressed both Wnt pathway activity and HSC activation. Interestingly, the inhibitor of the Wnt signaling pathway Dickkopf1 impairs RSPOs effects on HSCs. Taken together, our results revealed that RSPOs facilitated HSC activation and promote liver fibrogenesis by enhancing the Wnt pathway. PMID:27572318

  11. Hepatitis B virus X protein activates human hepatic stellate cells through upregulating TGFβ1.

    PubMed

    Chen, H-Y; Chen, Z-X; Huang, R-F; Lin, N; Wang, X-Z

    2014-10-27

    We investigated the effects of the hepatitis B virus X gene (HBV X) on the activation of human hepatic stellate cells (HSCs) and the possible mechanisms underlying the pathway. Recombinant plasmid pHBV-X-IRES2-EGFP was constructed and transfected into HL-7702 cells using a lipid-mediated method. Transfected cells were screened by G418, which detected stable expression of the X gene by reverse transcription (RT)-PCR and Western blot analysis, and named L02/x. Cells not subjected to G418-selection were analyzed to confirm the transient expression of the X gene and named L02/48x. Subsequently, L02/x and L02/48x, together with non-HBx-expressing cells, were co-cultured with HSCs in a non-contact transwell system. After 36 h of co-culture, the proliferation and migration of HSCs was detected using different cell counting methods. Finally, the mRNA and protein levels of α-SMA, Col I, and TGFβ1 in HSCs were detected by real-time PCR and western blot analysis. RT-PCR and Western blot analysis showed that L02/x and L02/48x cells can express HBV X gene mRNA and protein. Additionally, HSCs co-cultured with L02/x or L02/48x cells showed significantly higher proliferation and migration levels than control groups. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of α-SMA, Col I, and TGFβ1 in HSCs co-cultured with HBx-expressing liver cells were higher than those in control groups. HBx protein activated HSCs in vitro, leading to increased proliferation and migration of HSCs and upregulation of α-SMA and Col I. The TGFβ1 gene may be involved in this pathway.

  12. Effects of dietary retinyl acetate on the promotion of hepatic enzyme-altered foci by polybrominated biphenyls in initiated rats.

    PubMed

    Rezabek, M S; Sleight, S D; Jensen, R K; Aust, S D

    1989-08-01

    Vitamin A inhibits the development of some chemically-induced tumours. Since polybrominated biphenyls (PBBs) are hepatic tumour promoters and they affect vitamin A homeostasis in rats, we put forward the hypothesis that dietary levels of vitamin A would influence tumour promotion by PBBs. In the study described here, female Sprague-Dawley rats were initiated on day 1 by ip administration of diethylnitrosamine. On day 7 after initiation, the rats were fed a vitamin A-deficient basal diet that was supplemented with either 2000 IU (low-vitamin A) or 200,000 IU (high-vitamin A) retinyl acetate/kg feed. From day 30 after initiation until the end of the study the following PBBs were added to the diets: Firemaster BP-6 (10 ppm), 2,4,5,2',4',5'-hexabromobiphenyl (10 ppm) or 3,4,5,3',4',5'-hexabromobiphenyl (1 ppm). The control animals received low- or high-vitamin A diets containing no PBBs. On day 180, the rats were necropsied, sections of various tissues were stained for histopathological examination and an evaluation of hepatic enzyme-altered foci was performed. Numbers of gamma-glutamyl transpeptidase-positive foci/cm3 liver and the mean volumes of these foci were lower in the high-vitamin A groups than those in the corresponding low-vitamin A groups, but these differences were not significant. The percentage of the liver volume occupied by foci was significantly greater in the low-vitamin A with 345-HBB group than in the corresponding high-vitamin A group. Thus, high dietary levels of vitamin A had some inhibitory effect on the promotion of hepatic-altered foci by 345-HBB in initiated rats.

  13. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

  14. Garlic oil attenuated nitrosodiethylamine-induced hepatocarcinogenesis by modulating the metabolic activation and detoxification enzymes.

    PubMed

    Zhang, Cui-Li; Zeng, Tao; Zhao, Xiu-Lan; Xie, Ke-Qin

    2013-01-01

    Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes.

  15. Garlic Oil Attenuated Nitrosodiethylamine-Induced Hepatocarcinogenesis by Modulating the Metabolic Activation and Detoxification Enzymes

    PubMed Central

    Zhang, Cui-Li; Zeng, Tao; Zhao, Xiu-Lan; Xie, Ke-Qin

    2013-01-01

    Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes. PMID:23494807

  16. 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

  17. Effects of neonatally administered chlorpromazine and reserpine on the responsiveness of rat hepatic drug-metabolising enzymes to testosterone in adult life.

    PubMed

    Finnen, M J; Hassall, K A

    1986-01-01

    The effects of neonatally administered chlorpromazine and reserpine on the response of rat hepatic drug-metabolising enzymes to testosterone in adult life have been investigated using the chlorinated cyclodiene substrate DME. Neonatal treatment with chlorpromazine and reserpine had effects on the metabolism of DME similar to, but not as pronounced as, those of castration when adult. The effects of adult castration of male rats on hepatic microsomal metabolism of DME were fully reversed by treatment with testosterone propionate, with metabolism being restored to that of a control intact male. However, testosterone propionate treatment of either intact or castrated adult males that had received neonatal reserpine or chlorpromazine did not restore levels of metabolism to those characteristic of control adult male rats. These results suggest that neonatally administered chlorpromazine and reserpine alter the sensitivity of hepatic drug-metabolising enzymes to the actions of testosterone in adult life.

  18. 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.

  19. Developmental changes in hepatic fructose 2,6-bisphosphate content and phosphofructokinase-1 activity in the transition of chicks from embryonic to neonatal nutritional environment.

    PubMed Central

    Hamer, M J; Dickson, A J

    1987-01-01

    Within 2 days of hatching in chicks, there are parallel increases in hepatic fructose 2,6-bisphosphate content and phosphofructokinase-1 activity. The changes observed are a consequence of feeding on the carbohydrate-rich diet of neonatal life: lack of access to food after hatching prevents changes for either parameter. The results are discussed in relation to changes in the activities of hepatic lipogenic enzymes during the embryonic/neonatal transition of chicks and the role of insulin in co-ordination of developmental processes. PMID:2959273

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

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2014-02-15

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

  1. A generic rate law for surface-active enzymes.

    PubMed

    Kartal, Onder; Ebenhöh, Oliver

    2013-09-02

    Many biochemical reactions are confined to interfaces, such as membranes or cell walls. Despite their importance, no canonical rate laws describing the kinetics of surface-active enzymes exist. Combining the approach chosen by Michaelis and Menten 100 years ago with concepts from surface chemical physics, we here present an approach to derive generic rate laws of enzymatic processes at surfaces. We illustrate this by a simple reversible conversion on a surface to stress key differences to the classical case in solution. The available area function, a concept from surface physics which enters the rate law, covers different models of adsorption and presents a unifying perspective on saturation effects and competition between enzymes. A remarkable implication is the direct dependence of the rate of a given enzyme on all other enzymatic species able to bind at the surface. The generic approach highlights general principles of the kinetics of surface-active enzymes and allows to build consistent mathematical models of more complex pathways involving reactions at interfaces.

  2. Extracellular enzyme activity and biogeochemical cycling in restored prairies

    NASA Astrophysics Data System (ADS)

    Lynch, L.; Hernandez, D.; Schade, J. D.

    2011-12-01

    Winter microbial activity in mid-latitude prairie ecosystems is thermally sensitive and significantly influenced by snow depth. Snow insulates the soil column facilitating microbial processing of complex organic substrates. Previous studies in forests and tundra ecosystems suggest patterns of substrate utilization and limitation are seasonal; above freezing, soil microbes access fresh litter inputs and sugar exudates from plant roots, while under frozen condition they recycle nutrients incorporated in microbial biomass. In order to liberate nutrients required for carbon degradation, soil microbes invest energy in the production of extracellular enzymes that cleave monomers from polymer bonds. The inverse relationship between relative enzyme abundance and substrate availability makes enzyme assays a useful proxy to assess changes in resources over time. Our objective in this study was to assess patterns in microbial biomass, nutrient availability, and extracellular enzyme activity in four snow exclosure sites over a seven-month period. Over the past three years, we have maintained a snow removal experiment on two restored prairies in central Minnesota. In each prairie, snow was continuously removed annually from two 4 x 4 m plots by shoveling after each snow event. Extractable C, N and P, and microbial C, N and P in soil samples were measured in samples collected from these snow removal plots, as well as in adjacent unmanipulated prairie control plots. Pools of C, N, and P were estimated using standard extraction protocols, and microbial pools were estimated using chloroform fumigation direct extraction (CFDE). We conducted fluorometric extracellular enzyme assays (EEA) to assess how the degradation potential of cellulose (cellobiohydrolase, CBH), protein (leucine aminopeptidase, LAP), and phosphate esters (phosphatase, PHOS) changed seasonally. Microbial C and N declined between October and June, while microbial P declined during the fall and winter, but increased

  3. 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

    2016-07-25

    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.

  4. 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

    2014-09-18

    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.

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

    PubMed

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

    2013-05-15

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

  6. 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

  7. Antioxidant enzyme activities in maize plants colonized with Piriformospora indica.

    PubMed

    Kumar, Manoj; Yadav, Vikas; Tuteja, Narendra; Johri, Atul Kumar

    2009-03-01

    The bioprotection performance of Piriformospora indica against the root parasite Fusarium verticillioides was studied. We found that maize plants first grown with F. verticillioides and at day 10 inoculated with P. indica showed improvements in biomass, and root length and number as compared with plants grown with F. verticillioides alone. To validate our finding that inoculation with P. indica suppresses colonization by F. verticillioides, we performed PCR analyses using P. indica- and F. verticillioides-specific primers. Our results showed that inoculation with P. indica suppresses further colonization by F. verticillioides. We hypothesized that as the colonization by P. indica increases, the presence of/colonization by F. verticillioides decreases. In roots, catalase (CAT), glutathione reductase (GR), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were found to be higher in F. verticillioides-colonized plants than in non-colonized plants. Increased activity of antioxidant enzymes minimizes the chances of oxidative burst (excessive production of reactive oxygen species), and therefore F. verticillioides might be protected from the oxidative defence system during colonization. We also observed decreased antioxidant enzyme activities in plants first inoculated with F. verticillioides and at day 10 inoculated with P. indica as compared with plants inoculated with F. verticillioides alone. These decreased antioxidant enzyme activities due to the presence of P. indica help the plant to overcome the disease load of F. verticillioides. We propose that P. indica can be used as a bioprotection agent against the root parasite F. verticillioides.

  8. Extracellular enzyme activity in a willow sewage treatment system.

    PubMed

    Brzezinska, Maria Swiontek; Lalke-Porczyk, Elżbieta; Kalwasińska, Agnieszka

    2012-12-01

    This paper presents the results of studies on the activity of extra-cellular enzymes in soil-willow vegetation filter soil which is used in the post-treatment of household sewage in an onsite wastewater treatment system located in central Poland. Wastewater is discharged from the detached house by gravity into the onsite wastewater treatment system. It flows through a connecting pipe into a single-chamber septic tank and is directed by the connecting pipe to a control well to be further channelled in the soil-willow filter by means of a subsurface leaching system. Soil samples for the studies were collected from two depths of 5 cm and 1 m from three plots: close to the wastewater inflow, at mid-length of the plot and close to its terminal part. Soil samples were collected from May to October 2009. The activity of the extra-cellular enzymes was assayed by the fluorometric method using 4-methylumbelliferyl and 7-amido-4-methylcoumarin substrate. The ranking of potential activity of the assayed enzymes was the same at 5 cm and 1 m soil depths, i.e. esterase > phosphmomoesterase > leucine-aminopeptidase > β-glucosidase > α-glucosidase. The highest values of enzymatic activity were recorded in the surface layer of the soil at the wastewater inflow and decreased with increasing distance from that point.

  9. Enzyme-like activities of algal polysaccharide - cerium complexes

    NASA Astrophysics Data System (ADS)

    Wang, Dongfeng; Sun, Jipeng; Du, Dehong; Ye, Shen; Wang, Changhong; Zhou, Xiaoling; Xue, Changhu

    2005-01-01

    Water-soluble algal polysaccharides (APS) (alginic acid, fucoidan and laminaran) possess many pharmacological activities. The results of this study showed that the APS-Ce4+ complexes have some enzyme-like activities. Fucoidan and its complex with Ce4+ have activities similar to those of SOD. The activities of laminaran, alginic acid and their complexes are not measurable. The APS do not show measurable activities in the digestion of plasmid DNA. In contrast, the APS - Ce4+ complexes show these measurable activities under the comparable condition when APS bind Ce4+ and form homogenous solutions. The laminaran - Ce4+ complex shows the most obvious activity in the digestion of plasmid DNA, pNPP and chloropy-rifos under neutral conditions.

  10. Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes.

    PubMed

    Adam, Nicole; Perner, Mirjam

    2017-01-01

    Here we outline how to identify hydrogenase enzymes from metagenomic libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis (ΔhyaB) via triparental mating. If a fosmid exhibits hydrogen uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. This new method enables screening of 48 metagenomic fosmid clones in parallel.

  11. Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae).

    PubMed

    Stygar, Dominika; Dolezych, Bogdan; Nakonieczny, Mirosław; Migula, Pawel; Michalczyk, Katarzyna; Zaak, Maria

    2010-10-01

    This article presents the activity of carbohydratases and proteases in the midgut of Cameraria ohridella larvae--an oligophagous pest whose preferred feeding is horse chestnuts leaves. Optimal media pH of the assayed enzymes were similar to those of other Lepidopterans. Relatively high amylase activity, as well as maltase and sucrase activities, indicates that starch and sucrose are the main digested saccharides. Trehalase activity was similar to that described in other Lepidopterans. Activities of glycosidases were significantly lower than those of disaccharidases what suggests that neither cellulose nor glycosides are important for C. ohridella. Trypsin is the main endoprotease of this pest. Like in other leaf-eaters carboxypeptidase activity was higher than that of aminopeptidase. The activity of the majority of examined enzymes increased in the following successive pest generations, which could be explained by the decreased nutritional value of older leaves. Probably this phenomenon in hydrolases activity in Cameraria is a nonspecific mechanism present at this stage of co-evolution of the horse chestnut and its pest.

  12. 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.

  13. 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...

  14. [Lysosomal enzyme activity in white blood cells in leukemias].

    PubMed

    Rybakova, L P; Kharchenko, M F

    1996-01-01

    Total enzyme activity of acidic hydrolases and total neutral proteinase were compared in the post-nuclear fraction of leukocytes from healthy subjects and leukemia patients. The levels of acidic phosphotase and neutral proteinase in lymphoid cells of healthy donors were 11 and 7 times lower than those in myeloid cells, respectively. Patients suffering chronic myeloid leukemia revealed enhanced levels of beta-glucuronidase and neutral proteinases whereas B-chronic lymphoid leukemia involved acidic hydrolase concentrations lower than normal. As chronic myeloid leukemia advanced, neutral proteinase activity dropped dramatically (2.5 times); an aggressive course of B-chronic lymphoid leukemia was accompanied by a 3-fold decrease in acidic hydrolase level. The results may be used as indirect evidence of differences in the role of lysosomal enzymes in the mechanism of protein processing involved in myeloid and lymphoid proliferative pathologies.

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

    PubMed

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

    2014-11-01

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

  16. 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.

  17. Sample storage for soil enzyme activity and bacterial community profiles.

    PubMed

    Wallenius, K; Rita, H; Simpanen, S; Mikkonen, A; Niemi, R M

    2010-04-01

    Storage of samples is often an unavoidable step in environmental data collection, since available analytical capacity seldom permits immediate processing of large sample sets needed for representative data. In microbiological soil studies, sample pretreatments may have a strong influence on measurement results, and thus careful consideration is required in the selection of storage conditions. The aim of this study was to investigate the suitability of prolonged (up to 16 weeks) frozen or air-dried storage for divergent soil materials. The samples selected to this study were mineral soil (clay loam) from an agricultural field, humus from a pine forest and compost from a municipal sewage sludge composting field. The measured microbiological parameters included functional profiling with ten different hydrolysing enzyme activities determined by artificial fluorogenic substrates, and structural profiling with bacterial 16S rDNA community fingerprints by amplicon length heterogeneity analysis (LH-PCR). Storage of samples affected the observed fluorescence intensity of the enzyme assay's fluorophor standards dissolved in soil suspension. The impact was highly dependent on the soil matrix and storage method, making it important to use separate standardisation for each combination of matrix type, storage method and time. Freezing proved to be a better storage method than air-drying for all the matrices and enzyme activities studied. The effect of freezing on the enzyme activities was small (<20%) in clay loam and forest humus and moderate (generally 20-30%) in compost. The most dramatic decreases (>50%) in activity were observed in compost after air-drying. The bacterial LH-PCR community fingerprints were unaffected by frozen storage in all matrices. The effect of storage treatments was tested using a new statistical method based on showing similarity rather than difference of results.

  18. 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.

  19. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

    SciTech Connect

    Parekkadan, Biju; Poll, Daan van; Megeed, Zaki; Kobayashi, Naoya; Tilles, Arno W.; Berthiaume, Francois; Yarmush, Martin L.

    2007-11-16

    Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-{alpha} abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.

  20. St. John's Wort increases brain serotonin synthesis by inhibiting hepatic tryptophan 2, 3 dioxygenase activity and its gene expression in stressed rats.

    PubMed

    Bano, Samina; Ara, Iffat; Saboohi, Kausar; Moattar, Tariq; Chaoudhry, Bushra

    2014-09-01

    We aimed to investigate the effects of herbal St. John's Wort (SJW) on transcriptional regulation of hepatic tryptophan 2, 3 - dioxygenase (TDO) enzyme activity and brain regional serotonin (5-HT) levels in rats exposed to forced swim test (FST). TDO mRNA expression was quantified using real-time reverse transcription polymerase chain (RT-PCR) reaction and brain regional indoleamines were determined by high performance liquid chromatography coupled to fluorescence detector. Behavioral analysis shows significant reduction in immobility time in SJW (500mg/kg/ml) administered rats. It was found that pretreatment of SJW to rats did not prevent stress-induced elevation in plasma corticosterone levels however it increases serotonin synthesis by virtue of inhibiting hepatic TDO enzyme activity and its gene expression, ascertaining the notion that there exists an inverse relationship between hepatic TDO enzyme activity and brain 5-HT. The drug also decreases serotonin turnover in all the brain areas (hypothalamus, hippocampus amygdala) in stressed rats endorsing its monoamine oxidase inhibition property. Inhibition of TDO enzyme activity and its gene expression by the drug provides new insights for the development of therapeutic interventions for stress related mental illnesses.

  1. Association of interleukin-15-induced peripheral immune activation with hepatic stellate cell activation in persons coinfected with hepatitis C virus and HIV.

    PubMed

    Allison, Robert D; Katsounas, Antonios; Koziol, Deloris E; Kleiner, David E; Alter, Harvey J; Lempicki, Richard A; Wood, Brad; Yang, Jun; Fullmer, Brandie; Cortez, Karoll J; Polis, Michael A; Kottilil, Shyam

    2009-08-15

    Hepatic stellate cells (HSCs) mediate hepatitis C virus (HCV)-related liver fibrosis, and increased HSC activation in human immunodeficiency virus (HIV)/HCV coinfection may be associated with accelerated fibrosis. We examined the level of HSC activation in HIV/HCV-coinfected and HCV-monoinfected subjects and its relationship to the level of activation and gene expression of peripheral immune cells in coinfected subjects. HSC activation levels positively correlated with peripheral CD4+ and CD8+ T cell immune activation and were associated with enhanced interleukin-15 (IL-15) gene expression, suggesting a pathogenic role for IL-15-driven immunomediated hepatic fibrosis. Future strategies that reduce immune activation and HSC activation may delay progression of liver fibrosis.

  2. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    PubMed

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

    2015-09-08

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical 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 four-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

  3. 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.

  4. 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.

  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. The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

    PubMed Central

    Tai, C L; Chi, W K; Chen, D S; Hwang, L H

    1996-01-01

    To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far. PMID:8970970

  7. Hepatic overexpression of a constitutively active form of liver glycogen synthase improves glucose homeostasis.

    PubMed

    Ros, Susana; Zafra, Delia; Valles-Ortega, Jordi; García-Rocha, Mar; Forrow, Stephen; Domínguez, Jorge; Calbó, Joaquim; Guinovart, Joan J

    2010-11-26

    In this study, we tested the efficacy of increasing liver glycogen synthase to improve blood glucose homeostasis. The overexpression of wild-type liver glycogen synthase in rats had no effect on blood glucose homeostasis in either the fed or the fasted state. In contrast, the expression of a constitutively active mutant form of the enzyme caused a significant lowering of blood glucose in the former but not the latter state. Moreover, it markedly enhanced the clearance of blood glucose when fasted rats were challenged with a glucose load. Hepatic glycogen stores in rats overexpressing the activated mutant form of liver glycogen synthase were enhanced in the fed state and in response to an oral glucose load but showed a net decline during fasting. In order to test whether these effects were maintained during long term activation of liver glycogen synthase, we generated liver-specific transgenic mice expressing the constitutively active LGS form. These mice also showed an enhanced capacity to store glycogen in the fed state and an improved glucose tolerance when challenged with a glucose load. Thus, we conclude that the activation of liver glycogen synthase improves glucose tolerance in the fed state without compromising glycogenolysis in the postabsorptive state. On the basis of these findings, we propose that the activation of liver glycogen synthase may provide a potential strategy for improvement of glucose tolerance in the postprandial state.

  8. The slowed brain: cortical oscillatory activity in hepatic encephalopathy.

    PubMed

    Butz, Markus; May, Elisabeth S; Häussinger, Dieter; Schnitzler, Alfons

    2013-08-15

    Oscillatory activity of the human brain has received growing interest as a key mechanism of large-scale integration across different brain regions. Besides a crucial role of oscillatory activity in the emergence of other neurological and psychiatric diseases, recent evidence indicates a key role in the pathophysiology of hepatic encephalopathy (HE). This review summarizes the current knowledge on pathological alterations of oscillatory brain activity in association with liver dysfunction and HE in the context of spontaneous brain activity, motor symptoms, sensory processing, and attention. The existing literature demonstrates a prominent slowing of the frequency of oscillatory activity as shown for spontaneous brain activity at rest, with respect to deficits of motor behavior and motor symptoms, and in the context of visual attention processes. The observed slowing extends across different subsystems of the brain and has been confirmed across different frequency bands, providing evidence for ubiquitous changes of oscillatory activity in HE. For example, the frequency of cortico-muscular coherence in HE patients appears at the frequency of the mini-asterixis (⩽12Hz), while cirrhotics without overt signs of HE show coherence similar to healthy subjects, i.e. at 13-30Hz. Interestingly, the so-called critical flicker frequency (CFF) as a measure of the processing of an oscillating visual stimulus has emerged as a useful tool to quantify HE disease severity, correlating with behavioral and neurophysiological alterations. Moreover, the CFF reliably distinguishes patients with manifest HE from cirrhotics without any signs of HE and healthy controls using a cut-off frequency of 39Hz. In conclusion, oscillatory activity is globally slowed in HE in close association with HE symptoms and disease severity. Although the underlying causal mechanisms are not yet understood, these results indicate that pathological changes of oscillatory activity play an important role in the

  9. Kinetics of conjugation and oxidation of nitrobenzyl alcohols by rat hepatic enzymes.

    PubMed

    Rickert, D E; deBethizy, J D; Glover, M R; Kedderis, G L

    1985-12-01

    Previous work has suggested that quantitative differences in the in vitro and in vivo metabolism of mononitrotoluene isomers are a result of differences in the hepatic conjugation and oxidation of the first metabolic intermediates, the mononitrobenzyl alcohols. We have determined the steady-state kinetic parameters, Vmax, Km and V/K, for the metabolism of the nitrobenzyl alcohols by rat hepatic alcohol dehydrogenase, glucuronyltransferase, and sulfotransferase. 3-Nitrobenzyl alcohol was the best substrate for cytosolic alcohol dehydrogenase (Vmax = 1.48 nmoles/min/mg protein, V/K = 3.15 X 10(-3) nmoles/min/mg protein/microM, Km = 503 microM). Vmax and Km values for 4-nitrobenzyl alcohol were similar, but V/K was about 60% of that for 3-nitrobenzyl alcohol. 2-Nitrobenzyl alcohol was not metabolized by the alcohol dehydrogenase preparation used here, but it was metabolized to 2-nitrobenzoic acid by a rat liver mitochondrial preparation. 2-Nitrobenzyl alcohol was the best substrate for microsomal glucuronyltransferase (Vmax = 3.59 nmoles/min/mg protein, V/K = 11.28 X 10(-3) nmoles/min/mg protein/microM, Km = 373 microM). The Vmax for 3-nitrobenzyl alcohol was similar, but the V/K was about half and the Km was about twice that for 2-nitrobenzyl alcohol. The Vmax for 4-nitrobenzyl alcohol was about 40% and the V/K was about half that for 2-nitrobenzyl alcohol. The best substrate for cytosolic sulfotransferase was 4-nitrobenzyl alcohol (Vmax = 1.69 nmoles/min/mg protein, V/K = 37.21 X 10(-3) nmoles/min/mg protein/microM, Km = 48 microM). The Vmax values for the other two benzyl alcohols were similar, but the V/K and Km values were about 11 and 400%, respectively, of those for 4-nitrobenzyl alcohol. These data are in qualitative agreement with results obtained when the nitrobenzyl alcohols were incubated with isolated hepatocytes, but they do not allow quantitative modeling of the data from hepatocytes.

  10. Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

    PubMed Central

    Takehara, Shizuka; Takahashi, Masaharu

    2013-01-01

    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

  11. Age-Dependent Hepatic UDP-Glucuronosyltransferase Gene Expression and Activity in Children

    PubMed Central

    Neumann, Elizabeth; Mehboob, Huma; Ramírez, Jacqueline; Mirkov, Snezana; Zhang, Min; Liu, Wanqing

    2016-01-01

    UDP-glucuronosyltransferases (UGTs) are important phase II drug metabolism enzymes. The aim of this study was to explore the relationship between age and changes in mRNA expression and activity of major human hepatic UGTs, as well as to understand the potential regulatory mechanism underlying this relationship. Using previously generated data, we investigated age-dependent mRNA expression levels of 11 hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17) and 16 transcription factors (AHR, AR, CAR, ESR2, FXR, GCCR, HNF1a, HNF3a, HNF3b, HNF4a, PPARA, PPARG, PPARGC, PXR, SP1, and STAT3) in liver tissue of donors (n = 38) ranging from 0 to 25 years of age. We also examined the correlation between age and microsomal activities using 14 known UGT drug substrates in the liver samples (n = 19) of children donors. We found a statistically significant increase (nominal p < 0.05) in the expression of UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT2B7, and UGT2B17, as well as glucuronidation activities of serotonin, testosterone, and vorinostat during the first 25 years of life. Expression of estrogen receptor 1 and pregnane X receptor, two strong UGT transcriptional regulators, were significantly correlated with both age and UGT mRNA expression (p ≤ 0.05). These results suggest that both UGT expression and activity increase during childhood and adolescence, possibly driven in part by hormonal signaling. Our findings may help explain inter-patient variability in response to medications among children. PMID:27899892

  12. Human T cell microparticles circulate in blood of hepatitis patients and induce fibrolytic activation of hepatic stellate cells

    PubMed Central

    Kornek, Miroslaw; Popov, Yury; Libermann, Towia A.; Afdhal, Nezam H.; Schuppan, Detlef

    2010-01-01

    Microparticles (MP) are small cell membrane vesicles which are released from cells during apoptosis or activation. While circulating platelet MP have been studied in some detail, the existence and functional role of T cell MP remain elusive. We show that blood from patients with active hepatitis C (ALT>100 IU/ml) contains elevated numbers of T cell MP compared to patients with mild hepatitis C (ALT<40 IU/ml) and healthy controls. T cell MP fuse with cell membranes of hepatic stellate cells (HSC), the major effector cells for excess matrix deposition in liver fibrosis and cirrhosis. MP uptake is partly ICAM-1 dependent and leads to activation of NFkB and ERK1/2 and subsequent upregulation of fibrolytic genes in HSC, to downregulation of procollagen α1(I) mRNA, and blunting of profibrogenic activities of TGFβ1. Ex vivo the induced fibrolytic activity is evident in MP derived from activated CD4+ T cells, and highest with MP from activated and apoptotic CD8+ T cells. Mass spectrometry, FACS analysis and function blocking antibodies revealed CD147/Emmprin as candidate transmembrane molecule in HSC fibrolytic activation by CD8+ T cell MP. We conclude that 1) circulating T cell MP are a novel diagnostic marker for inflammatory liver diseases, and 2) in vivo induction of T cell MP may be a novel strategy to induce regression of liver fibrosis. PMID:20979056

  13. Protoplast fusion enhances lignocellulolytic enzyme activities in Trichoderma reesei.

    PubMed

    Cui, Yu-xiao; Liu, Jia-jing; Liu, Yan; Cheng, Qi-yue; Yu, Qun; Chen, Xin; Ren, Xiao-dong

    2014-12-01

    Protoplast fusion was used to obtain a higher production of lignocellulolytic enzymes with protoplast fusion in Trichoderma reesei. The fusant strain T. reesei JL6 was obtained from protoplast fusion from T. reesei strains QM9414, MCG77, and Rut C-30. Filter paper activity of T. reesei JL6 increased by 18% compared with that of Rut C-30. β-Glucosidase, hemicellulase and pectinase activities of T. reesei JL6 were also higher. The former activity was 0.39 Uml(-1), while those of QM9414, MCG77, and Rut C-30 were 0.13, 0.11, and 0.16 Uml(-1), respectively. Pectinase and hemicellulase activities of JL6 were 5.4 and 15.6 Uml(-1), respectively, which were slightly higher than those of the parents. The effects of corn stover and wheat bran carbon sources on the cellulase production and growth curve of T. reesei JL6 were also investigated.

  14. Saturated fatty acids activate ERK signaling to downregulate hepatic sortilin 1 in obese and diabetic mice.

    PubMed

    Bi, Lipeng; Chiang, John Y L; Ding, Wen-Xing; Dunn, Winston; Roberts, Benjamin; Li, Tiangang

    2013-10-01

    Hepatic VLDL overproduction is a characteristic feature of diabetes and an important contributor to diabetic dyslipidemia. Hepatic sortilin 1 (Sort1), a cellular trafficking receptor, is a novel regulator of plasma lipid metabolism and reduces plasma cholesterol and triglycerides by inhibiting hepatic apolipoprotein B production. Elevated circulating free fatty acids play key roles in hepatic VLDL overproduction and the development of dyslipidemia. This study investigated the regulation of hepatic Sort1 in obesity and diabetes and the potential implications in diabetic dyslipidemia. Results showed that hepatic Sort1 protein was markedly decreased in mouse models of type I and type II diabetes and in human individuals with obesity and liver steatosis, whereas increasing hepatic Sort1 expression reduced plasma cholesterol and triglycerides in mice. Mechanistic studies showed that the saturated fatty acid palmitate activated extracellular signal-regulated kinase (ERK) and inhibited Sort1 protein by mechanisms involving Sort1 protein ubiquitination and degradation. Consistently, hepatic ERK signaling was activated in diabetic mice, whereas blocking ERK signaling by an ERK inhibitor increased hepatic Sort1 protein in mice. These results suggest that increased saturated fatty acids downregulate liver Sort1 protein, which may contribute to the development of dyslipidemia in obesity and diabetes.

  15. Effects of Sublethal Exposure to a Glyphosate-Based Herbicide Formulation on Metabolic Activities of Different Xenobiotic-Metabolizing Enzymes in Rats.

    PubMed

    Larsen, Karen; Najle, Roberto; Lifschitz, Adrián; Maté, María L; Lanusse, Carlos; Virkel, Guillermo L

    2014-07-01

    The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these

  16. An Extended Polyanion Activation Surface in Insulin Degrading Enzyme

    PubMed Central

    Song, Eun Suk; Ozbil, Mehmet; Zhang, Tingting; Sheetz, Michael; Lee, David; Tran, Danny; Li, Sheng; Prabhakar, Rajeev; Hersh, Louis B.; Rodgers, David W.

    2015-01-01

    Insulin degrading enzyme (IDE) is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ), glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use a computational approach to define three potential ATP binding sites and mutagenesis and kinetic studies to confirm the relevance of these sites. Mutations were made at four positively charged residues (Arg 429, Arg 431, Arg 847, Lys 898) within the polyanion-binding region, converting them to polar or hydrophobic residues. We find that mutations in all three ATP binding sites strongly decrease the degree of activation by ATP and can lower basal activity and cooperativity. Computational analysis suggests conformational changes that result from polyanion binding as well as from mutating residues involved in polyanion binding. These findings indicate the presence of multiple polyanion binding modes and suggest the anion-binding surface plays an important conformational role in controlling IDE activity. PMID:26186535

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. Application of capillary enzyme micro-reactor in enzyme activity and inhibitors studies of glucose-6-phosphate dehydrogenase.

    PubMed

    Camara, Mohamed Amara; Tian, Miaomiao; Guo, Liping; Yang, Li

    2015-05-15

    In this study, we present an on-line measurement of enzyme activity and inhibition of Glucose-6-phosphate dehydrogenase (G6PDH) enzyme using capillary electrophoresis based immobilized enzyme micro-reactor (CE-based IMER). The IMER was prepared using a two-step protocol based on electrostatic assembly. The micro-reactor exhibited good stability and reproducibility for on-line assay of G6PDH enzyme. Both the activity as well as the inhibition of the G6PDH enzyme by six inhibitors, including three metals (Cu(2+), Pb(2+), Cd(2+)), vancomycin, urea and KMnO4, were investigated using on-line assay of the CE-based IMERs. The enzyme activity and inhibition kinetic constants were measured using the IMERs which were found to be consistent with those using traditional off-line enzyme assays. The kinetic mechanism of each inhibitor was also determined. The present study demonstrates the feasibility of using CE-based IMERs for rapid and efficient on-line assay of G6PDH, an important enzyme in the pentosephosphate pathway of human metabolism.

  2. Protein stability and enzyme activity at extreme biological temperatures.

    PubMed

    Feller, Georges

    2010-08-18

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

  3. 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.

  4. Regulation of hepatic drug transporter activity and expression by organochlorine pesticides.

    PubMed

    Bucher, Simon; Le Vee, Marc; Jouan, Elodie; Fardel, Olivier

    2014-03-01

    Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides.

  5. Antioxidant Activity of Oat Proteins Derived Peptides in Stressed Hepatic HepG2 Cells

    PubMed Central

    Du, Yichen; Esfandi, Ramak; Willmore, William G.; Tsopmo, Apollinaire

    2016-01-01

    The purpose of this study was to determine, for the first time, antioxidant activities of seven peptides (P1–P7) derived from hydrolysis of oat proteins in a cellular model. In the oxygen radical absorbance capacity (ORAC) assay, it was found that P2 had the highest radical scavenging activity (0.67 ± 0.02 µM Trolox equivalent (TE)/µM peptide) followed by P5, P3, P6, P4, P1, and P7 whose activities were between 0.14–0.61 µM TE/µM). In the hepatic HepG2 cells, none of the peptides was cytotoxic at 20–300 µM. In addition to having the highest ORAC value, P2 was also the most protective (29% increase in cell viability) against 2,2′-azobis(2-methylpropionamidine) dihydrochloride -induced oxidative stress. P1, P6, and P7 protected at a lesser extent, with an 8%–21% increase viability of cells. The protection of cells was attributed to several factors including reduced production of intracellular reactive oxygen species, increased cellular glutathione, and increased activities of three main endogenous antioxidant enzymes. PMID:27775607

  6. Alteration of the mutagenicity 3,3'-dichlorobenzidine by modifiers of rat hepatic epoxide hydrolase activity

    SciTech Connect

    Iba, M.M.

    1986-03-05

    The involvement of arene oxides in the activation of benzidines was assessed by examining the effect of (I) the epoxide hydrolase inhibitor trichloropropylene oxide (TCPO), (II) purified rat liver microsomal (P) epoxide hydrolase (EH), and (III) pretreatment of rats with phenobarbital (PB) on hepatic Sg- or P-catalyzed mutagenicity of benzidine (BZ) and 3,3'-dichlorobenzidine (DCB) to Salmonella TA 98. When catalyzed by Sg from untreated rats, the mutagenicity of DCB and BZ was 601 +/- 101 and 79 +/- 25 (His/sup +/ revertants/plate) respectively, but was 345 +/- 55 and 226 +/- 30 respectively, when catalyzed by microsomes (P) from untreated rats. PB-pretreatment enhanced the Sg-catalyzed mutagenicity of DCB and BZ (2.3-fold and 1.7-fold, respectively) and the P-catalyzed mutagenicity of DCB (1.7-fold), but totally inhibited the P-catalyzed mutagenicity of BZ. In TCPO-supplemented activating systems from PB-pretreated rats, the mutagenicity of DCB was enhanced in both Sg and P (1.9-fold and 1.6-fold, respectively), whereas that of BZ was unchanged. Added EH enhanced the P-catalyzed mutagenicity of DCB (1.4-fold) but had no effect on that of BZ, suggesting that the activity of the enzyme on DCB metabolites may not be entirely detoxifying. The data suggest that epoxidation may contribute to the activation of DCB but not BZ.

  7. Hepatitis B virus stimulates G6PD expression through HBx-mediated Nrf2 activation

    PubMed Central

    Liu, B; Fang, M; He, Z; Cui, D; Jia, S; Lin, X; Xu, X; Zhou, T; Liu, W

    2015-01-01

    Metabolic reprogramming is a hallmark of physiological changes in cancer. Cancer cells primarily apply glycolysis for cell metabolism, which enables the cells to use glycolytic intermediates for macromolecular biosynthesis in order to meet the needs of cell proliferation. Here, we show that glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the pentose phosphate pathway, is highly expressed in chronic hepatitis B virus (HBV)-infected human liver and HBV-associated liver cancer, together with an elevated activity of the transcription factor Nrf2. In hepatocytes, HBV stimulates by its X protein (HBx) the expression of G6PD in an Nrf2 activation-dependent pathway. HBx associates with the UBA and PB1 domains of the adaptor protein p62 and augments the interaction between p62 and the Nrf2 repressor Keap1 to form HBx–p62–Keap1 complex in the cytoplasm. The aggregation of HBx–p62–Keap1 complexes hijacks Keap1 from Nrf2 leading to the activation of Nrf2 and consequently G6PD transcription. Our data suggest that HBV upregulates G6PD expression by HBx-mediated activation of Nrf2. This implies a potential effect of HBV on the reprogramming of the glucose metabolism in hepatocytes, which may be of importance in the development of HBV-associated hepatocarcinoma. PMID:26583321

  8. Serum angiotensin converting enzyme activity in chronic beryllium disease.

    PubMed

    Newman, L S; Orton, R; Kreiss, K

    1992-07-01

    Serum angiotensin converting enzyme (SACE) activity is used as a marker of sarcoidosis activity and severity, but in chronic beryllium disease (CBD) the studies of SACE give conflicting results. We examined SACE activity in 23 CBD patients, five patients with beryllium sensitization, and 25 beryllium-exposed control subjects. CBD patients underwent complete clinical evaluation, including physical examination, pulmonary function testing, exercise physiology testing, chest radiography, and bronchoscopy with bronchoalveolar lavage and biopsy. CBD SACE activity was systematically compared with these clinical markers of disease severity. Of CBD patients, 22% had elevated SACE activity. The test did not discriminate CBD patients from those in the beryllium-sensitized or beryllium-exposed groups. However, SACE activity in CBD correlated with the extent of pulmonary granulomatous inflammation as reflected by the symptom of breathlessness, the number of white cells in bronchoalveolar lavage (r = 0.44), the number of lavage lymphocytes (r = 0.58), the lavage lymphocyte percentage (r = 0.55), and the profusion of small opacities on chest radiograph (r = 0.41). The test-retest reliability of the assay was high (r = 0.84), as was the agreement between fresh and -70 degrees C frozen sera (r = 0.93). We conclude that SACE activity levels may reflect the extent of pulmonary granulomatous inflammation in CBD but that the test does not help discriminate disease from nondisease.

  9. Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes.

    PubMed

    Shi, Rong; Ma, Bingliang; Wu, Jiasheng; Wang, Tianming; Ma, Yueming

    2015-10-01

    The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high-throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin-O deethylation, coumarin-7 hydroxylation, bupropion hydroxylation, taxol-6 hydroxylation, omeprazole-5 hydroxylation, dextromethorphan-O demethylation, tolbutamide-4 hydroxylation, chlorzoxazone-6 hydroxylation, testosterone-6β hydroxylation, and midazolam-1 hydroxylation in rat liver microsomes.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Comparison of constitutive gene expression levels of hepatic cholesterol biosynthetic enzymes between Wistar-Kyoto and stroke-prone spontaneously hypertensive rats.

    PubMed

    Nemoto, Kiyomitsu; Ikeda, Ayaka; Ito, Sei; Miyata, Misaki; Yoshida, Chiaki; Degawa, Masakuni

    2013-01-01

    Serum total cholesterol amounts in the stroke-prone hypertensive rat (SHRSP) strain are lower than in the normotensive control strain, Wistar-Kyoto (WKY) rat. To understand the strain difference, constitutive gene expression levels of hepatic cholesterol biosynthetic enzymes in male 8-week-old SHRSP and WKY rats were comparatively examined by DNA microarray and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses. Of 22 cholesterol biosynthetic enzyme genes, expression levels of 8 genes, Pmvk, Idi1, Fdps, Fdft1, Sqle, Lss, Sc4mol, and Hsd17b7, in SHRSP were less than 50% those of the WKY rats; especially, the expression level of Sqle gene, encoding squalene epoxidase, a rate-limiting enzyme in cholesterol biosynthesis pathway, was about 20%. The gene expression level of sterol regulatory element-binding protein-2 (SREBP-2), which functions as a transcription factor upregulating gene expression of cholesterol biosynthetic enzymes, in SHRSP was about 70% of that in WKY rats. These results demonstrate the possibility that the lower serum total cholesterol level in SHRSP is defined by lower gene expression of most hepatic cholesterol biosynthetic enzymes. In particular, decreased gene expression level of Sqle gene might be the most essential factor. Moreover, the broad range of lowered rates of these genes in SHRSP suggests that the abnormal function and/or expression not only of SREBP-2 but also of one or more other transcription factors for those gene expressions exist in SHRSP.

  15. 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

  16. 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

  17. Characterization and chillproofing activity of two enzymes from Streptomyces species.

    PubMed

    Etok, C A; Eka, O U

    1996-01-01

    Two enzymes, amylase and protease of Streptomyces species were purified by a combination of ion exchange chromatography and gel filtration and characterized. The amylase had an exoaction on starch yielding maltose as a major end product and was identified as beta-amylase. The purified amylase had a molecular weight of 48,000 and was maximally active at 35 degrees C and at pH 6.0. On the other hand, protease had a molecular weight of 21,000 and was most active at pH 10.0 and at a temperature of 30 degrees C. The Km or MICHAELIS constant of amylase for maize starch was 0.333 mg/ml while that of protease for casein was 2.5 mg/ml. The feasibility of using the purified protease for various industrial application especially in the chillproofing of beer is discussed.

  18. 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.

  19. 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

  20. The use of selected plasma enzyme activities for the diagnosis of fatty liver-hemorrhagic syndrome in laying hens.

    PubMed

    Diaz, G J; Squires, E J; Julian, R J

    1999-01-01

    Profiles of plasma enzymes were compared in two strains of single comb white leghorn laying hens, a normal commercial strain and strain UCD-003, which is highly susceptible to fatty liver-hemorrhagic syndrome. Plasma activity of lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) averaged 194 +/- 27, 4.0 +/- 2.8, 146 +/- 20, 1.0 +/- 1.0, and 1041 +/- 268 U/liter, respectively in normal birds. Activities of LDH, GDH, AST, and ALT, but not CK, were significantly higher in UCD-003 than in normal hens. A bimodal distribution of activities of all enzymes was found in the UCD-003 hens, with some birds showing activities comparable with those of the normal hens and others with values that were 2-10 times greater than those found in normal hens. These results are consistent with the extensive hepatic lesions observed in the UCD-003 strain of birds. Average gross hemorrhagic scores from visual inspection (scale of 0-3) were 0.28 +/- 0.45 in normal birds and 1.63 +/- 0.94 in the UCD-003 birds. Even though no clear relationship was found between plasma enzyme activities and the extent of liver hemorrhage in individual birds, the UCD-003 hens consistently had average values significantly higher for plasma enzymes that indicate liver damage. The results suggest that measurement of enzyme activities indicative of liver damage in birds, particularly AST, LDH, and GDH, is a valuable tool in the diagnosis of fatty liver-hemorrhagic syndrome in a flock of layers.

  1. Induction of apoptosis by tanshinone I via cytochrome c release in activated hepatic stellate cells.

    PubMed

    Kim, Ji Young; Kim, Kyoung Mi; Nan, Ji-Xing; Zhao, Yu Zhe; Park, Pil-Hoon; Lee, Sang Jun; Sohn, Dong Hwan

    2003-04-01

    Hepatic stellate cells play central roles in hepatic fibrosis. The therapeutic goal in hepatic fibrosis is to halt or reverse fibrosis. Apoptosis is suggested to eliminate activated hepatic stellate cells in fibrosis. Salvia miltiorrhiza is a traditional medicine used to improve blood circulation and treat chronic hepatitis and hepatic fibrosis. We investigated the effect of tanshinone I, an ingredient of Salvia miltiorrhiza, on the apoptotic death of rat hepatic stellate cells transformed by simian virus 40 (T-HSC/Cl-6), which retains the features of activated stellate cells. Treatment of T-HSC/Cl-6 cells with tanshinone I resulted in the induction of typical DNA fragmentation and DNA ladder formation in a concentration- and time-dependent manner. The induction of apoptosis was confirmed by flow cytometric analysis. Treatment of T-HSC/Cl-6 cells with tanshinone I caused activation of caspase-3 and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. Tanshinone I induced mitochondrial membrane dipolarization and the release of cytochrome c from mitochondria into the cytosol. In conclusion, our results demonstrate that tanshinone I induces apoptosis of T-HSC/Cl-6 cells and that tanshinone I-induced apoptosis involves caspase activation through cytochrome c release and loss of mitochondrial membrane potential.

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

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-06-14

    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.

  3. 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.

  4. 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

  5. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    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.

  6. Comparative insecticide susceptibility and detoxification enzyme activities among pestiferous blattodea.

    PubMed

    Valles, S M; Koehler, P G; Brenner, R J

    1999-11-01

    Topical bioassays using propoxur, chlorpyrifos, and lambda-cyhalothrin were conducted on eight cockroach species. Based on lethal dose values, the relative toxicities of the insecticide classes were generally pyrethroid > carbamate > organophosphorous. Lambda-Cyhalothrin and propoxur were more toxic toward the Blattidae as compared with the Blattellidae. The order of lambda-cyhalothrin toxicity was Periplaneta americana > Periplaneta brunnea = Periplaneta australasiae = Periplaneta fuliginosa = Blatta orientalis > Blattella asahinai = Blattella germanica > Blattella vaga. The order of propoxur toxicity was B. orientalis > P. americana > P. brunnea = P. australasiae > B. asahinai > P. fuliginosa = B. germanica > B. vaga. The order of chlorpyrifos toxicity was P. americana > B. asahinai = B. vaga > B. orientalis = P. australasiae = P. brunnea > B. germanica = P. fuliginosa. Detoxification enzyme activities for each species also were measured and compared with insecticide toxicity. Propoxur LD50 was significantly (P = 0.01; r = 0.81) correlated with glutathione S-transferase activity. Lambda-Cyhalothrin LD50 correlated with methoxyresorufin O-demethylase activity (P = 0.01; r = 0.81), carboxylesterase activity (P = 0.03; r = - 0.75), general esterase activity (P = 0.02; r = - 0.79), and cockroach weight (P = 0.01; r = -0.95).

  7. Hepatitis B

    MedlinePlus

    ... times more infectious than HIV. Which adults need hepatitis B vaccine? Any sexually active adult who is not in ... share needles, syringes, or other drug-injection equipment. Hepatitis B vaccine is available alone or in a combination with ...

  8. Peroxisome proliferator-activated receptor alpha controls hepatic heme biosynthesis through ALAS1.

    PubMed

    Degenhardt, Tatjana; Väisänen, Sami; Rakhshandehroo, Maryam; Kersten, Sander; Carlberg, Carsten

    2009-05-01

    Heme is an essential prosthetic group of proteins involved in oxygen transport, energy metabolism and nitric oxide production. ALAS1 (5-aminolevulinate synthase) is the rate-limiting enzyme in heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target for the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). In primary human hepatocytes and in HepG2 cells, PPARalpha agonists induced an increase in ALAS1 mRNA levels, which was abolished by PPARalpha silencing. These effects are mediated by two functional PPAR binding sites at positions -9 and -2.3 kb relative to the ALAS1 transcription start site. PPARalpha ligand treatment also up-regulated the mRNA levels of the genes ALAD (5-aminolevulinate dehydratase), UROS (uroporphyrinogen III synthase), UROD (uroporphyrinogen decarboxylase), CPOX (coproporphyrinogen oxidase) and PPOX (protoporphyrinogen oxidase) encoding for enzymes controlling further steps in heme biosynthesis. In HepG2 cells treated with PPARalpha agonists and in mouse liver upon fasting, the association of PPARalpha, its partner retinoid X receptor, PPARgamma co-activator 1alpha and activated RNA polymerase II with the transcription start site region of all six genes was increased, leading to higher levels of the metabolite heme. In conclusion, these data strongly support a role of PPARalpha in the regulation of human ALAS1 and of five additional genes of the pathway, consequently leading to increased heme synthesis.

  9. 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

  10. Study on optimization of process parameters for enhancing the multi-hydrolytic enzyme activity in garbage enzyme produced from preconsumer organic waste.

    PubMed

    Arun, C; Sivashanmugam, P

    2017-02-01

    The garbage enzymes produced from preconsumer organic waste containing multi hydrolytic enzyme activity which helps to solubilize the waste activated sludge. The continuous production of garbage enzyme and its scaling up process need a globe optimized condition. In present study the effect of fruit peel composition and sonication time on enzyme activity were investigated. Garbage enzyme produced from 6g pineapple peels: 4g citrus peels pre-treated with ultrasound for 20min shows higher hydrolytic enzymes activity. Simultaneously statistical optimization tools were used to model garbage enzyme production with higher activity of amylase, lipase and protease. The maximum activity of amylase, lipase and protease were predicted to be 56.409, 44.039, 74.990U/ml respectively at optimal conditions (pH (6), temperature (37°C), agitation (218 RPM) and fermentation duration (3days)). These optimized conditions can be successfully used for large scale production of garbage enzyme with higher hydrolytic enzyme activity.

  11. Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese.

    PubMed

    Torres-Llanez, M J; González-Córdova, A F; Hernandez-Mendoza, A; Garcia, H S; Vallejo-Cordoba, B

    2011-08-01

    The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC(50); <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α(S1)-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.

  12. 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

  13. 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

  14. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    NASA Astrophysics Data System (ADS)

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-05-01

    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 (A549) 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.

  15. Pentoxifylline aggravates fatty liver in obese and diabetic ob/ob mice by increasing intestinal glucose absorption and activating hepatic lipogenesis

    PubMed Central

    Massart, J; Robin, MA; Noury, F; Fautrel, A; Lettéron, P; Bado, A; Eliat, PA; Fromenty, B

    2012-01-01

    BACKGROUND AND PURPOSE Pentoxifylline is in clinical trials for non-alcoholic fatty liver disease and diabetic nephropathy. Metabolic and hepatic effects of pentoxifylline were assessed in a murine model of obesity and type 2 diabetes. EXPERIMENTAL APPROACH Pentoxifylline (100 mg·kg−1·day−1) was administered for 4 days or 3 weeks in lean and obese/diabetic ob/ob mice. Plasma lipids, glucose, other metabolites and relevant enzymes were measured by standard assays. Hepatic lipids in vivo were assessed with magnetic resonance spectroscopy and by histology. Hepatic extracts were also analysed with RT-PCR and Western blotting. KEY RESULTS Four days of pentoxifylline treatment slightly increased liver lipids in ob/ob mice. After 3 weeks, pentoxifylline exacerbated fatty liver and plasma transaminases in ob/ob mice but did not induce liver steatosis in lean mice. Plasma glucose was highest in fed, but not fasted, ob/ob mice treated with pentoxifylline. During the first 10 min of an oral glucose tolerance test, blood glucose increased more rapidly in pentoxifylline-treated mice. Jejunal expression of glucose transporter 2 isoform was increased in pentoxifylline-treated obese mice. Hepatic activity of carbohydrate response element binding protein (ChREBP) increased after pentoxifylline in ob/ob, but not lean, mice. Hepatic expression of lipogenic enzymes was highest in pentoxifylline-treated ob/ob mice. However, pentoxifylline reduced markers of oxidative stress and inflammation in ob/ob liver. CONCLUSION AND IMPLICATIONS Pentoxifylline exacerbated fatty liver in ob/ob mice through enhanced intestinal glucose absorption, increased postprandial glycaemia and activation of hepatic lipogenesis. Long-term treatment with pentoxifylline could worsen fatty liver in some patients with pre-existing hyperglycaemia. PMID:21740407

  16. Endothelin-converting enzyme 2 differentially regulates opioid receptor activity

    PubMed Central

    Gupta, A; Fujita, W; Gomes, I; Bobeck, E; Devi, L A

    2015-01-01

    BACKGROUND AND PURPOSE Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co-endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin-converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization. EXPERIMENTAL APPROACH The effect of ECE2 inhibition on hydrolysis of the endocytosed peptide was examined using thin-layer chromatography and on μ opioid receptor trafficking using either elisa or microscopy. The effect of ECE2 inhibition on receptor signalling was measured using a cAMP assay and, in vivo, on antinociception induced by intrathecally administered opioids by the tail-flick assay. KEY RESULTS The highly selective ECE2 inhibitor, S136492, significantly impaired μ receptor recycling and signalling by only those ligands that are ECE2 substrates and this was seen both in heterologous cells and in cells endogenously co-expressing μ receptors with ECE2. We also found that ECE2 inhibition attenuated antinociception mediated only by opioid peptides that are ECE2 substrates. CONCLUSIONS AND IMPLICATIONS These results suggest that ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating opioid receptor activity. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24990314

  17. 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

  18. Cerebrospinal fluid enzymes in acute brain injury. 1. Dynamics of changes in CSF enzyme activity after acute experimental brain injury.

    PubMed Central

    Maas, A I

    1977-01-01

    Changes in CSF enzyme activity were studied after brain trauma for their prognostic value. Raised values of CPK and HBDH were demonstrated in the CSF of patients with severe brain injuries. Standardised cold lesions of the brain were induced in cats. The activities of the enzymes CPK, HBDH, LDH, GOT, GPT, and pseudocholinesterase were studied at half hour intervals in the cerebrospinal fluid and at hourly intervals in the serum. A statistically highly significant increase of all enzymes studied developed in the CSF. The greatest changes occurred within four hours of freezing. Large increases could occur in half an hour. Isoenzyme studies demonstrated that CPK and LDH were of cerebral origin. No consistently significant changes could be shown in the serum enzyme activity. It is concluded that after brain injuries, enzymes are released into the extracellular fluid of the brain and transported to the CSF. The limited value of a single enzyme estimation is emphasised. The results described seem to provide indirect evidence for transependymal flow of extracellular fluid in brain oedema. Images PMID:915509

  19. 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

    2014-10-05

    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.

  20. Amphiregulin activates human hepatic stellate cells and is upregulated in non alcoholic steatohepatitis

    PubMed Central

    McKee, Chad; Sigala, Barbara; Soeda, Junpei; Mouralidarane, Angelina; Morgan, Maelle; Mazzoccoli, Gianluigi; Rappa, Francesca; Cappello, Francesco; Cabibi, Daniela; Pazienza, Valerio; Selden, Claire; Roskams, Tania; Vinciguerra, Manlio; Oben, Jude A.

    2015-01-01

    Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways, and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD. PMID:25744849

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

    PubMed

    Pavelka, S

    2014-01-01

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

  2. 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...

  3. Influence of dietary nutritional composition on caterpillar salivary enzyme activity.

    PubMed

    Babic, Branislav; Poisson, Alexandre; Darwish, Shireef; Lacasse, Jean; Merkx-Jacques, Magali; Despland, Emma; Bede, Jacqueline C

    2008-01-01

    Caterpillars are faced with nutritional challenges when feeding on plants. In addition to harmful secondary metabolites and protein- and water-limitations, tissues may be carbohydrate-rich which may attenuate optimal caterpillar performance. Therefore, caterpillars have multiple strategies to cope with surplus carbohydrates. In this study, we raise the possibility of a pre-ingestive mechanism to metabolically deal with excess dietary sugars. Many Noctuid caterpillars secrete the labial salivary enzyme glucose oxidase (GOX), which oxidizes glucose to hydrogen peroxide and gluconate, a nutritionally unavailable carbohydrate to the insect. Beet armyworm, Spodoptera exigua, larvae were restricted to diets varying in protein to digestible carbohydrate (P:C) ratio (42p:21c; 33p:30c; 21p:42c) and total nutrient concentration (42% and 63%). High mortality and longer developmental time were observed when caterpillars were reared on the C-biased, P-poor diet (21p:42c). As the carbohydrate content of the diet increased, caterpillars egested excess glucose and a diet-dependent difference in assimilated carbohydrates and pupal biomass was not observed, even though caterpillars restricted to the C-biased diet (21p:42c) accumulated greater pupal lipid reserves. Larval labial salivary GOX activity was also diet-dependent and gluconate, the product of GOX activity, was detected in the frass. Unexpectedly, GOX activity was strongly and positively correlated with dietary protein content.

  4. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes.

    PubMed

    Campbell, D R; Kurzer, M S

    1993-09-01

    Eleven flavonoid compounds were compared with aminoglutethimide (AG), a pharmaceutical aromatase inhibitor, for their abilities to inhibit aromatase enzyme activity in a human preadipocyte cell culture system. Flavonoids exerting no effect on aromatase activity were catechin, daidzein, equol, genistein, beta-naphthoflavone (BNF), quercetin and rutin. The synthetic flavonoid, alpha-naphthoflavone (ANF), was the most potent aromatase inhibitor, with an I50 value of 0.5 microM. Three naturally-occurring flavonoids, chrysin, flavone, and genistein 4'-methyl ether (Biochanin A) showed I50 values of 4.6, 68, and 113 microM, respectively, while AG showed an I50 value of 7.4 microM. Kinetic analyses showed that both AG and the flavonoids acted as competitive inhibitors of aromatase. The Ki values, indicating the effectiveness of inhibition, were 0.2, 2.4, 2.4, 22, and 49 microM, for ANF, AG, chrysin, flavone, and Biochanin A, respectively. Chrysin, the most potent of the naturally-occurring flavonoids, was similar in potency and effectiveness to AG, a pharmaceutical aromatase inhibitor used clinically in cases of estrogen-dependent carcinoma. These data suggest that flavonoid inhibition of peripheral aromatase activity may contribute to the observed cancer-preventive hormonal effects of plant-based diets.

  5. 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

  6. Inhibition of cytochrome P450 enzymes by saturated and unsaturated fatty acids in human liver microsomes, characterization of enzyme kinetics in the presence of bovine serum albumin (0.1 and 1.0% w/v) and in vitro - in vivo extrapolation of hepatic clearance.

    PubMed

    Palacharla, Raghava Choudary; Uthukam, Venkatesham; Manoharan, Arunkumar; Ponnamaneni, Ranjith Kumar; Padala, Nagasurya Prakash; Boggavarapu, Rajesh Kumar; Bhyrapuneni, Gopinadh; Ajjala, Devender Reddy; Nirogi, Ramakrishna

    2017-04-01

    The objective of the study was to determine the effect of fatty acids on CYP enzymes and the effect of BSA on intrinsic clearance of probe substrates. The inhibitory effect of thirteen fatty acids including saturated, mono-unsaturated and polyunsaturated fatty acids on CYP enzymes, kinetic parameters and intrinsic clearance values of nine CYP marker probe substrate reactions in the absence and presence of BSA (0.1 and 1.0% w/v) were characterized in human liver microsomes. The results demonstrate that most of the unsaturated fatty acids showed marked inhibition towards CYP2C8 mediated amodiaquine N-deethylation followed by inhibition of CYP2C9 and CYP2B6 mediated activities. The addition of 0.1% BSA in the incubation markedly improved the unbound intrinsic clearance values of probe substrates by reducing the Km values with little or no effect on maximal velocity. The addition of BSA (0.1 and 1.0% w/v) did not influence the unbound intrinsic clearance of marker reactions for CYP2A6, and CYP3A4 enzymes. The addition of 0.1% w/v BSA is sufficient to determine the intrinsic clearance of marker probe reactions by metabolite formation approach. The predicted hepatic clearance values for the substrates using the well-stirred model, in the presence of BSA (0.1% BSA), are comparable to the in vivo hepatic clearance values.

  7. Metabolic Activation of the Anti-Hepatitis C Virus Nucleotide Prodrug PSI-352938

    PubMed Central

    Niu, Congrong; Tolstykh, Tatiana; Bao, Haiying; Park, Yeojin; Babusis, Darius; Lam, Angela M.; Bansal, Shalini; Du, Jinfa; Chang, Wonsuk; Reddy, P. Ganapati; Zhang, Hai-Ren; Woolley, Joseph; Wang, Li-Quan; Chao, Piyun B.; Ray, Adrian S.; Otto, Michael J.; Sofia, Michael J.

    2012-01-01

    PSI-352938 is a novel cyclic phosphate prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methylguanosine-5′-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3′,5′-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O6-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5′-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically. PMID:22526308

  8. 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.

  9. Diurnal locomotor activity and oxidative metabolism of the suprachiasmatic nucleus in two models of hepatic insufficiency.

    PubMed

    Lopez, Laudino; Cimadevilla, Jose M; Aller, Maria A; Arias, Jaime; Nava, M Paz; Arias, Jorge L

    2003-08-15

    Subjects with hepatic cirrhosis develop alterations of several rhythmic behavioural and biochemical patterns. Since most cirrhotic patients combine portal hypertension and hepatic impairment, our work aims to assess the extent to which rhythmical changes can be due to hepatic insufficiency or portal hypertension. This was done using two experimental models in rats, portacaval shunt model (PC) and portal hypertension by a triple stenosing ligature of the portal vein (PH). We assess diurnal locomotor activity and determine the oxidative metabolism of the suprachiasmatic nucleus (SCN) by histochemical determination of cytochrome oxidase (COX). The results show that animals with PC have altered diurnal locomotor rhythm compared to control and PH rats (p<0.001). They also present lower COX activity in the SCN (p<0.05). We conclude that rhythmic alterations are due to hepatic insufficiency and not to portal hypertension.

  10. 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.

  11. Phosphate-Modified Nucleotides for Monitoring Enzyme Activity.

    PubMed

    Ermert, Susanne; Marx, Andreas; Hacker, Stephan M

    2017-04-01

    Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

  12. Activated Notch signaling is required for hepatitis B virus X protein to promote proliferation and survival of human hepatic cells.

    PubMed

    Wang, Fan; Zhou, Haiyan; Xia, Xiumei; Sun, Qian; Wang, Ying; Cheng, Bin

    2010-12-01

    Hepatitis B virus X protein (HBx) is a multifunctional oncoprotein which plays a crucial role in the pathogenesis of hepatocellular carcinoma (HCC). However, the exact mechanisms remain controversial. Here we show that HBx strongly stimulated cell growth, promoted cell cycle progression and inhibited apoptosis of human non-tumor hepatic cell line L02 cells. It also accelerated tumor formation of L02 cells in BALB/c nude mice. Furthermore, Notch signaling components were upregulated in HBx-expressing L02 cells compared to normal L02 cells. However, blocking Notch signaling with a γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) attenuated cell growth, shortened the S phase of cell cycle and promoted apoptosis of HBx-expressing L02 cell in a dose- and time-dependent manner, but normal L02 cells were not significantly affected by Notch signaling blocking. Therefore, our findings demonstrate that HBx could promote the growth of human non-tumor hepatic cell line L02 cells both in vitro and in vivo, which may require the activation of Notch signaling pathway.

  13. 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.

  14. Ultrasonic Monitoring of Enzyme Catalysis; Enzyme Activity in Formulations for Lactose-Intolerant Infants.

    PubMed

    Altas, Margarida C; Kudryashov, Evgeny; Buckin, Vitaly

    2016-05-03

    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.

  15. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities.

    PubMed

    Meneely, Kathleen M; Luo, Qianyi; Lamb, Audrey L

    2013-11-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.

  16. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

    PubMed Central

    Meneely, Kathleen M.; Luo, Qianyi; Lamb, Audrey L.

    2013-01-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities. PMID:24055536

  17. Hepatic (Liver) Function Panel

    MedlinePlus

    ... related side effects. The hepatic function panel evaluates: Alanine aminotransferase (ALT). This enzyme, found in the liver, ... MORE ON THIS TOPIC Mononucleosis Hepatitis Blood Test: Alanine Aminotransferase (ALT, or SGPT) Blood Test: Aspartate Aminotransferase ( ...

  18. Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis.

    PubMed

    Song, Zhenyuan; Deaciuc, Ion; Zhou, Zhanxiang; Song, Ming; Chen, Theresa; Hill, Daniell; McClain, Craig J

    2007-10-01

    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.

  19. Activity-based profiling of the proteasome pathway during hepatitis C virus infection.

    PubMed

    Nasheri, Neda; Ning, Zhibin; Figeys, Daniel; Yao, Shao; Goto, Natalie K; Pezacki, John Paul

    2015-11-01

    Hepatitis C virus (HCV) infection often leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The stability of the HCV proteins is controlled by ubiquitin-dependent and ubiquitin-independent proteasome pathways. Many viruses modulate proteasome function for their propagation. To examine the interrelationship between HCV and the proteasome pathways we employed a quantitative activity-based protein profiling method. Using this approach we were able to quantify the changes in the activity of several proteasome subunits and found that proteasome activity is drastically reduced by HCV replication. The results imply a link between the direct downregulation of the activity of this pathway and chronic HCV infection.

  20. PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro

    PubMed Central

    Kan, Chin Fung Kelvin; Singh, Amar Bahadur; Dong, Bin; Shende, Vikram Ravindra; Liu, Jingwen

    2017-01-01

    The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells. PMID:25645621

  1. PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro.

    PubMed

    Kan, Chin Fung Kelvin; Singh, Amar Bahadur; Dong, Bin; Shende, Vikram Ravindra; Liu, Jingwen

    2015-05-01

    The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

  2. 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.

  3. 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.

  4. 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.

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

    PubMed

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

    2016-02-10

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

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

    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

  7. 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.

  8. Substrate supply, fine roots, and temperature control proteolytic enzyme activity in temperate forest soils.

    PubMed

    Brzostek, Edward R; Finzi, Adrien C

    2011-04-01

    Temperature and substrate availability constrain the activity of the extracellular enzymes that decompose and release nutrients from soil organic matter (SOM). Proteolytic enzymes are the primary class of enzymes involved in the depolymerization of nitrogen (N) from proteinaceous components of SOM, and their activity affects the rate of N cycling in forest soils. The objectives of this study were to determine whether and how temperature and substrate availability affect the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ectomycorrhizal or arbuscular mycorrhizal fungi. Temperature limitation of proteolytic enzyme activity was observed only early in the growing season when soil temperatures in the field were near 4 degrees C. Substrate limitation to proteolytic activity persisted well into the growing season. Ligninolytic enzyme activity was higher in soils dominated by ectomycorrhizal associated tree species. In contrast, the activity of proteolytic and chitinolytic enzymes did not differ, but there were differences between mycorrhizal association in the control of roots on enzyme activity. Roots of ectomycorrhizal species but not those of arbuscular mycorrhizal species exerted significant control over proteolytic, chitinolytic, and ligninolytic enzyme activity; the absence of ectomycorrhizal fine roots reduced the activity of all three enzymes. These results suggest that climate warming in the absence of increases in substrate availability may have a modest effect on soil-N cycling, and that global changes that alter belowground carbon allocation by trees are likely to have a larger effect on nitrogen cycling in stands dominated by ectomycorrhizal fungi.

  9. Controlled exogenous enzyme imbibition and activation in whole chickpea seed enzyme reactor (SER).

    PubMed

    Kliger, Eynav; Fischer, Lutz; Lutz-Wahl, Sabine; Saguy, I Sam

    2011-05-01

    Chickpeas are of excellent quality (protein, vitamins, minerals, unsaturated fatty acids) and very low in phytoestrogen, making them a potentially promising source for vegetarian-based infant formula (VBIF). However, their high starch and fiber concentration could hinder their utilization for infants. To overcome this natural shortcoming, a solid-state "enzymation" (SSE) process was developed in which imbibition of exogenous enzyme facilitates hydrolysis within the intact chickpea seed. The process was termed seed enzyme reactor (SER). Liquid imbibition data of dry chickpeas during soaking were fitted with the Weibull distribution model. The derived Weibull shape parameter, β, value (0.77 ± 0.11) indicated that the imbibition mechanism followed Fickian diffusion. Imbibition occurred through the coat and external layers. The process was tested using green fluorescent protein (GFP) as an exogenous marker, and involved soaking, thermal treatment, peeling, microwave partial drying, rehydration in enzyme solution, and SSE at an adjusted pH, time, and temperature. Amylases, or a combination of amylases and cellulases, resulted in significant carbohydrate hydrolysis (23% and 47% of the available starch, respectively). In addition, chickpea initial raffinose and stachyose concentration was significantly reduced (91% and 92%, respectively). The process could serve as a proof of concept, requiring additional development and optimization to become a full industrial application.

  10. Significant interspecies differences in induction profiles of hepatic CYP enzymes by TCDD in bank and field voles.

    PubMed

    Murtomaa-Hautala, Mari; Korkalainen, Merja; Pelkonen, Olavi; Hegde, Nagabhooshan; Pohjanvirta, Raimo; Huitu, Otso; Henttonen, Heikki; Rautio, Arja; Viitala, Pirkko; Viluksela, Matti

    2012-03-01

    The gene expression and induction of cytochrome P450 (CYP)-enzymes following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) peroral administration was studied in the livers of two wild vole species--the bank vole (Myodes glareolus) and the field vole (Microtus agrestis). The dioxin-sensitive C57BL/6 mouse was used as a reference. Doses of 0.05, 0.5, 5.0, and 50 µg/kg were applied to ascertain a dose-response relationship, and the dose of 50 µg/kg was applied to the study time course for up to 96 h. The cytochrome P450 1A1 (CYP1A1) mRNA expression showed an expected dose-dependent increase equally in both vole species. Bank voles expressed notably higher CYP2A mRNA levels as compared with field voles. Both species exhibited dose-dependent increases in putative CYP1A-, CYP2B-, and CYP2A-associated activities as measured by fluorometric assays for ethoxyresorufin-O-deethylase (EROD), penthoxyresorufin-O-depenthylase (PROD), and 7-ethoxycoumarin-O-deethylase (ECOD), respectively. Putative CYP2A-associated coumarin-7-hydroxylase (COH) activity showed a slight increase at the two highest doses of TCDD in field voles but not in bank voles, and their basal COH activity was only one-fourth or less of that in field voles. Overall, however, bank voles tended to exhibit higher CYP-associated enzyme activities measured at the two largest doses of TCDD than field voles. A western blot analysis of aryl hydrocarbon receptor (AhR) revealed that the two vole species had differential band patterns, suggesting dissimilar structures for their AhRs.

  11. Chaperone-like activities of {alpha}-synuclein: {alpha}-Synuclein assists enzyme activities of esterases

    SciTech Connect

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo . E-mail: ywryu@ajou.ac.kr; Doohun Kim, T. . E-mail: doohunkim@ajou.ac.kr

    2006-08-11

    {alpha}-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of {alpha}-synuclein has not yet been known. Here we have shown that {alpha}-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of {alpha}-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with {alpha}-synuclein. Our results indicate that {alpha}-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.

  12. The association of phosphoinositide 3-kinase enhancer A with hepatic insulin receptor enhances its kinase activity.

    PubMed

    Chan, Chi Bun; Liu, Xia; He, Kunyan; Qi, Qi; Jung, Dae Y; Kim, Jason K; Ye, Keqiang

    2011-07-01

    Dysfunction of hepatic insulin receptor tyrosine kinase (IRTK) causes the development of type 2 diabetes. However, the molecular mechanism regulating IRTK activity in the liver remains poorly understood. Here, we show that phosphoinositide 3-kinase enhancer A (PIKE-A) is a new insulin-dependent enhancer of hepatic IRTK. Liver-specific Pike-knockout (LPKO) mice display glucose intolerance with impaired hepatic insulin sensitivity. Specifically, insulin-provoked phosphoinositide 3-kinase/Akt signalling is diminished in the liver of LPKO mice, leading to the failure of insulin-suppressed gluconeogenesis and hyperglycaemia. Thus, hepatic PIKE-A has a key role in mediating insulin signal transduction and regulating glucose homeostasis in the liver.

  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. 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.

  15. 2.3.7.8-Tetrachlorodibenzo-p-Dioxin Induced Immunosuppression: Its Possible Alteration by In Vivo Administration of Specific Hepatic Enzyme Inducers.

    DTIC Science & Technology

    1987-06-27

    England .2 LIST OF ACOREVIAT:CNS AND SYMBOLS TCDD =2.3.7.8-tetrachlorodibenzo-p-dioxtn TCDF = 2.3.7 8-IOetrachlo’dibenzofuran 3MC =3-methyicholanthrene...this research program was to clarify whether drugs that in vitro inhibit TCDD binding to the hepatic cytosolic Ah "rec3ptor" such as 3MC , PNF and TCDF...these enzymes has also been performed. The study of this year has been concentrated on the effects of only two inducers, that is TCDF and 3MC , since

  16. Lipopolysaccharide, immune activation, and liver abnormalities in HIV/hepatitis B virus (HBV)-coinfected individuals receiving HBV-active combination antiretroviral therapy.

    PubMed

    Crane, Megan; Avihingsanon, Anchalee; Rajasuriar, Reena; Velayudham, Pushparaj; Iser, David; Solomon, Ajantha; Sebolao, Baotuti; Tran, Andrew; Spelman, Tim; Matthews, Gail; Cameron, Paul; Tangkijvanich, Pisit; Dore, Gregory J; Ruxrungtham, Kiat; Lewin, Sharon R

    2014-09-01

    We investigated the relationship between microbial translocation, immune activation, and liver disease in human immunodeficiency virus (HIV)/hepatitis B virus (HBV) coinfection. Lipopolysaccharide (LPS), soluble CD14, CXCL10, and CCL-2 levels were elevated in patients with HIV/HBV coinfection. Levels of LPS, soluble CD14, and CCL-2 declined following receipt of HBV-active combination antiretroviral therapy (cART), but the CXCL10 level remained elevated. No markers were associated with liver disease severity on liver biopsy (n = 96), but CXCL10, interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α, and interferon γ (IFN-γ) were all associated with elevated liver enzyme levels during receipt of HBV-active cART. Stimulation of hepatocyte cell lines in vitro with IFN-γ and LPS induced a profound synergistic increase in the production of CXCL10. LPS may contribute to liver disease via stimulating persistent production of CXCL10.

  17. Activating nuclear xenobiotic receptors and triggering ER stress and hepatic cytochromes P450 systems in quails (Coturnix C. coturnix) during atrazine exposure.

    PubMed

    Du, Zheng-Hai; Qin, Lei; Lin, Jia; Sun, Yan-Chun; Xia, Jun; Zhang, Cong; Li, Xue-Nan; Li, Jin-Long

    2017-02-10

    Atrazine (ATR) is one of the most widely detected contaminant in the ecosystem. Nuclear xenobiotic receptors are activated by herbicides and induce the transcription of CYP450 isoforms involved in xenobiotic metabolism and transport. However, little is known about hepatic nuclear xenobiotic receptors in birds are responsible for ATR-induced hepatotoxicity via regulating the cytochrome P450 enzyme systems (CYP450s). The objective of this study was to investigate the mechanism of ATR hepatotoxicity in quails. For this purpose, male quails were dosed by oral gavage from sexual immaturity to maturity with 0, 50, 250, and 500 mg/kg/day ATR for 45 days. The results showed that ATR exposure caused the hepatotoxicity damage and endoplasmic reticulum (ER) degeneration. It suggested that ER is a target organelle of ATR toxicity in hepatocytes. ATR exposure disrupted the hepatic CYP450s homeostasis. This study also demonstrated that ATR triggered the CYP450 isoforms transcription via activating the hepatic CAR/PXR pathway. The present study provides new insights regarding the mechanism of the ATR-induced hepatotoxicity through activating nuclear xenobiotic receptors and triggering ER stress and hepatic CYP450s in quails.

  18. Mouse Hepatitis Virus Infection Induces a Toll-Like Receptor 2-Dependent Activation of Inflammatory Functions in Liver Sinusoidal Endothelial Cells during Acute Hepatitis

    PubMed Central

    Bleau, Christian; Filliol, Aveline; Samson, Michel

    2016-01-01

    ABSTRACT Under physiological conditions, the liver sinusoidal endothelial cells (LSECs) mediate hepatic immune tolerance toward self or foreign antigens through constitutive expression of anti-inflammatory mediators. However, upon viral infection or Toll-like receptor 2 (TLR2) activation, LSECs can achieve proinflammatory functions, but their role in hepatic inflammation during acute viral hepatitis is unknown. Using the highly virulent mouse hepatitis virus type 3 (MHV3) and the attenuated variants 51.6-MHV3 and YAC-MHV3, exhibiting lower tropism for LSECs, we investigated in vivo and in vitro the consequence of LSEC infection on their proinflammatory profiles and the aggravation of acute hepatitis process. In vivo infection with virulent MHV3, in comparison to attenuated strains, resulted in fulminant hepatitis associated with higher hepatic viral load, tissue necrosis, and levels of inflammatory mediators and earlier recruitment of inflammatory cells. Such hepatic inflammatory disorders correlated with disturbed production of interleukin-10 (IL-10) and vascular factors by LSECs. We next showed in vitro that infection of LSECs by the virulent MHV3 strain altered their production of anti-inflammatory cytokines and promoted higher release of proinflammatory and procoagulant factors and earlier cell damage than infection by attenuated strains. This higher replication and proinflammatory activation in LSECs by the virulent MHV3 strain was associated with a specific activation of TLR2 signaling by the virus. We provide evidence that TLR2 activation of LSCEs by MHV3 is an aggravating factor of hepatic inflammation and correlates with the severity of hepatitis. Taken together, these results indicate that preservation of the immunotolerant properties of LSECs during acute viral hepatitis is imperative in order to limit hepatic inflammation and damage. IMPORTANCE Viral hepatitis B and C infections are serious health problems affecting over 350 million and 170 million

  19. 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.

  20. Serum hepatic biochemical activity in two populations of workers exposed to styrene

    PubMed Central

    Brodkin, C; Moon, J; Camp, J; Echeverria, D; Redlich, C; Willson, R; Checkoway, H

    2001-01-01

    OBJECTIVE—To determine whether hepatic biochemical changes, as measured by routinely available tests indicative of hepatocellular necrosis, cholestasis, or altered hepatic clearance of bilirubin, occur in association with low to moderate exposure to styrene commonly experienced in industrial production.
METHODS—Two independent cross sectional studies were performed comparing serum hepatic transaminases (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)), cholestatic enzymes (alkaline phosphatase (AP) and γ glutamyl transpeptidase (GGT)), and bilirubin in (a) 47 workers of fibreglass reinforced plastics who were exposed to styrene and (b) 21 boat and tank fabricators, with separate referent groups of unexposed workers. Exposure to styrene was assessed in air by dosimetry, and in venous blood by headspace analysis. Hepatic biochemical variables were assessed across strata of exposure to styrene defined as 25 ppm in air, or 0.275 mg/l in blood, adjusting for age, sex, body mass index, and ethanol consumption.
RESULTS—A consistent and significant linear trend for increasing direct bilirubin and direct/total bilirubin ratio was found in association with increasing exposure to styrene, by both air and blood monitoring, in both studies. Mean direct bilirubin concentrations increased from 0.05-0.08 mg% in referents to 0.12-0.19 in workers exposed above 25 ppm, with a significant exposure-response trend (p<0.005). Significantly increased direct/total bilirubin ratios, ranging from 0.22 to 0.35 were associated with exposure to styrene (p<0.001), indicating diminished hepatic clearance of conjugated bilirubin. Also, a significant linear association between the hepatic transaminases ALT and AST and exposure to styrene was found in pooled regression analyses, with an increase in AP of about 10 IU/ml in workers exposed above 25 ppm air or 0.275 mg/l blood styrene in pooled analyses from both studies.
CONCLUSIONS—The consistent finding

  1. Activity of purified hepatitis C virus protease NS3 on peptide substrates.

    PubMed Central

    Steinkühler, C; Urbani, A; Tomei, L; Biasiol, G; Sardana, M; Bianchi, E; Pessi, A; De Francesco, R

    1996-01-01

    The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation. PMID:8794305

  2. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

    PubMed

    Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si

    2017-04-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

  3. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

    NASA Astrophysics Data System (ADS)

    Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

    2017-01-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

  4. Activation of the aryl hydrocarbon receptor induces hepatic steatosis via the upregulation of fatty acid transport.

    PubMed

    Kawano, Yuki; Nishiumi, Shin; Tanaka, Shinwa; Nobutani, Kentaro; Miki, Akira; Yano, Yoshihiko; Seo, Yasushi; Kutsumi, Hiromu; Ashida, Hitoshi; Azuma, Takeshi; Yoshida, Masaru

    2010-12-15

    The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix/Per-ARNT-Sim domain transcription factor, which is activated by various xenobiotic ligands. AHR is known to be abundant in liver tissue and to be associated with hepatic steatosis. However, it has not yet been elucidated how the activation of AHR promotes hepatic steatosis. The aim of this study is to clarify the role of AHR in hepatic steatosis. The intraperitoneal injection of 3-methylcholanthrene (3MC), a potent AHR ligand, into C57BL/6J mice significantly increased the levels of triglycerides and six long-chain monounsaturated fatty acids in the livers of mice, resulting in hepatic microvesicular steatosis. 3MC significantly enhanced the expression level of fatty acid translocase (FAT), a factor regulating the uptake of long-chain fatty acids into hepatocytes, in the liver. In an in vitro experiment using human hepatoma HepG2 cells, 3MC increased the expression level of FAT, and the downregulation of AHR by AHR siRNA led to the suppression of 3MC-induced FAT expression. In addition, the mRNA level of peroxisome proliferator-activated receptor (PPAR) α, an upstream factor of FAT, was increased in the livers of 3MC-treated mice. Taking together, AHR activation induces hepatic microvesicular steatosis by increasing the expression level of FAT.

  5. 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.

  6. Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

    PubMed Central

    Valero, E; Varón, R; García-Carmona, F

    1995-01-01

    A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

  7. Annexation of a high-activity enzyme in a synthetic three-enzyme complex greatly decreases the degree of substrate channeling.

    PubMed

    You, Chun; Zhang, Y-H Percival

    2014-06-20

    The self-assembled three-enzyme complex containing triosephosphate isomerase (TIM), aldolase (ALD), and fructose 1,6-biphosphatase (FBP) was constructed via a mini-scaffoldin containing three different cohesins and the three dockerin-containing enzymes. This enzyme complex exhibited 1 order of magnitude higher initial reaction rates than the mixture of noncomplexed three enzymes. In this enzyme cascade reactions, the reaction mediated by ALD was the rate-limiting step. To understand the in-depth role of the rate-limiting enzyme ALD in influencing the substrate channeling effect of synthetic enzyme complexes, low-activity ALD from Thermotoga maritima was replaced with a similar-size ALD isolated from Thermus thermophilus, where the latter had more than 5 times specific activity of the former. The synthetic three-enzyme complexes annexed with either low-activity or high-activity ALDs exhibited higher initial reaction rates than the mixtures of the two-enzyme complex (TIM-FBP) and the nonbound low-activity or high activity ALD at the same enzyme concentration. It was also found that the annexation of more high-activity ALD in the synthetic enzyme complexes drastically decreased the degree of substrate channeling from 7.5 to 1.5. These results suggested that the degree of substrate channeling in synthetic enzyme complexes depended on the enzyme choice. This study implied that the construction of synthetic enzyme enzymes in synthetic cascade pathways could be a very important tool to accrelerate rate-limiting steps controlled by low-activity enzymes.

  8. The effect of the P1 side chain on the binding of optimized carboxylate and activated carbonyl inhibitors of the hepatitis C virus NS3 protease.

    PubMed

    Kawai, Stephen H; LaPlante, Steven R; Llinàs-Brunet, Montse; Hucke, Oliver

    2010-07-01

    Peptidyl inhibitors of the hepatitis C virus NS3 protease hold much promise as direct-acting antiviral agents against hepatitis C infection. The optimization of N-terminal cleavage products, found to exhibit activity (product inhibition) against the enzyme, has led to potent tripeptide inhibitors that bear free C-terminal carboxylate groups. An analogous activated carbonyl compound (pentafluoroethyl ketone) bearing a P1 norvaline (Nva) was found to possess comparable activity against hepatitis C virus protease. However, an analogue bearing an aminocyclopropylcarboxylic acid (Acca) P1 residue exhibited very poor activity. (19)F-NMR studies indicate that the propensity of the Acca-derived activated carbonyl to form hemiketals is only slightly reduced compared with that of a P1 Nva equivalent. These results, as well as molecular modeling studies, argue against steric hindrance of the nucleophilic attack of Ser-139 accounting for the poor mechanism-based inhibition by the former. We hypothesize that the conformational properties of the respective C-termini in the context of an adaptable active site account for the divergent P1 structure-activity relationships.

  9. Acetylcholinesterase activity in an experimental rat model of Type C hepatic encephalopathy.

    PubMed

    Méndez, Marta; Méndez-López, Magdalena; López, Laudino; Aller, María A; Arias, Jaime; Arias, Jorge L

    2011-05-01

    Patients with liver malfunction often suffer from hepatic encephalopathy, a neurological complication which can affect attention and cognition. Diverse experimental models have been used to study brain alterations that may be responsible for hepatic encephalopathy symptoms. The aim of the study was to determine whether cognitive impairment found in cirrhosis could be due to disturbance of acetylcholinesterase activity. Acetylcholinesterase activity was assessed in the brains of Wistar rats with thioacetamide-induced cirrhosis. The cirrhotic group displayed up-regulation of acetylcholinesterase levels in the entorrhinal cortex, anterodorsal and anteroventral thalamus and accumbens, whereas down-regulation was found in the CA1, CA3 and dentate gyrus of the hippocampus. Our results indicate that the experimental model of hepatic encephalopathy by chronic administration of thioacetamide presents alterations of acetylcholinesterase activity in brain limbic system regions, which play a role in attention and memory.

  10. Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

    PubMed Central

    Funk, Michael A.; Judd, Evan T.; Marsh, E. Neil G.; Elliott, Sean J.; Drennan, Catherine L.

    2014-01-01

    Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic α-subunit of BSS with its accessory subunits β and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded β/α-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits β and γ display folds related to high potential iron–sulfur proteins but differ substantially from each other in how they interact with the α-subunit. BSSγ binds distally to the active site, burying a hydrophobic region of BSSα, whereas BSSβ binds to a hydrophilic surface of BSSα that is proximal to the active site. To further investigate the function of BSSβ, we determined the structure of a BSSαγ complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSSα that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSSαγ complex center around the crucial glycyl radical domain, thus suggesting a role for BSSβ in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations. PMID:24982148

  11. 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.

  12. Guanidinylated Neomycin Mediates Heparan Sulfate–dependent Transport of Active Enzymes to Lysosomes

    PubMed Central

    Sarrazin, Stéphane; Wilson, Beth; Sly, William S; Tor, Yitzhak; Esko, Jeffrey D

    2010-01-01

    Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, β--glucuronidase (GUS) and α--iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells. PMID:20442709

  13. Characterization of E-NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes.

    PubMed

    Doleski, Pedro H; Adefegha, Stephen A; Cabral, Fernanda L; Leal, Daniela B R

    2017-03-01

    The activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) was characterized in hepatic lymphocytes (HL) of rats. For this purpose, a specific method for the isolation of lymphocytes from hepatic tissue was developed. Subsequently, E-NTPDase activity of rat HL was compared with that of rat peripheral lymphocytes. The HL showed high cell count and viability. Also, the characterization test revealed that the optimal E-NTPDase activities were attained at 37°C and pH 8.0 in the presence of Ca(2+) . In addition, in the presence of specific E-NTPDase inhibitors (20mM sodium azide and 0.3mM suramin), there were significant inhibitions in nucleotide hydrolysis. However, there was no significant change in adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolysis in the presence of inhibitors of other E-ATPase (0.1mM Ouabain, 0.5mM orthovanadate, and 1mM, 5mM, and 10mM sodium azide). Furthermore, the kinetic behavior of the enzyme in HL showed apparent Km of 134.90 ± 0.03μM and 214.40 ± 0.06μM as well as Vmax of 345.0 ± 28.32 and 242.0 ± 27.55 ƞmol Pi/min/mg of protein for ATP and ADP, respectively. The Chevillard plot revealed that ATP and ADP were hydrolyzed at the same active site of the enzyme. Our results suggest that the degradation of extracellular nucleotides in HL may have been primarily accomplished by E-NTPDase. The higher E-NTPDase activity observed in HL may be attributed to the important physiological functions of ATP and ADP in HL.

  14. Erythropoietin alleviates hepatic insulin resistance via PPARγ-dependent AKT activation

    PubMed Central

    Ge, Zhijuan; Zhang, Pengzi; Hong, Ting; Tang, Sunyinyan; Meng, Ran; Bi, Yan; Zhu, Dalong

    2015-01-01

    Erythropoietin (EPO) has beneficial effects on glucose metabolism and insulin resistance. However, the mechanism underlying these effects has not yet been elucidated. This study aimed to investigate how EPO affects hepatic glucose metabolism. Here, we report that EPO administration promoted phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation in palmitic acid (PA)-treated HepG2 cells and in the liver of high-fat diet (HFD)-fed mice, whereas adenovirus-mediated silencing of the erythropoietin receptor (EPOR) blocked EPO-induced AKT signalling in HepG2 cells. Importantly, a peroxisome proliferator-activated receptor γ (PPARγ) antagonist and PPARγ small interfering RNA (siRNA) abrogated the EPO-induced increase in p-AKT in HepG2 cells. Lentiviral vector-mediated hepatic PPARγ silencing in HFD-fed C57BL/6 mice impaired EPO-mediated increases in glucose tolerance, insulin sensitivity and hepatic AKT activation. Furthermore, EPO activated the AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signalling pathway, and AMPKα and SIRT1 knockdown each attenuated the EPO-induced PPARγ expression and deacetylation and PPARγ-dependent AKT activation in HepG2 cells. In summary, these findings suggest that PPARγ is involved in EPO/EPOR-induced AKT activation, and targeting the PPARγ/AKT pathway via EPO may have therapeutic implications for hepatic insulin resistance and type 2 diabetes. PMID:26643367

  15. Copper-induced changes in tissue enzyme activity in a freshwater mussel.

    PubMed

    Rajalakshmi, S; Mohandas, A

    2005-09-01

    Changes in enzyme activity levels are of great diagnostic value. Lysosomal membrane is often the target of injury by xenobiotics, resulting in destabilization. Variations in the activity of acid phosphatase (ACP) a marker enzyme, in gills and hepatopancreas of the freshwater mussel Lamellidens corrianus (Lea) exposed to different concentrations of copper for 24, 120, and 168 h are discussed. The aim was to determine if the metal caused any variation in enzyme activity in the two tissues studied and, if so, whether the length of exposure had any influence on enzyme activity. ACP activity was determined as described in Sigma Technical Bulletin No. 104 and expressed as micromoles of p-nitrophenol liberated per milligram of protein per hour. Both concentration of the metal and length of exposure were found to influence enzyme activity. Higher concentrations of metals are assumed to induce stress proteins like metallothioneins.

  16. 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.

  17. Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME.

    PubMed

    Golbeck, J H; Cammarata, K V

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14.18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.Sonication releases polyphenol oxidase from the membrane largely in the latent state. C(18) fatty acids, especially linolenic acid, are potent activators of the enzymic activity. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time.Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. The K(m) values for 3,4-dihydroxyphenylalanine and O(2) are 6.5 and 0.065 millimolar, respectively. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K(m) A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  18. Molecular architectures and functions of radical enzymes and their (re)activating proteins.

    PubMed

    Shibata, Naoki; Toraya, Tetsuo

    2015-10-01

    Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes—i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here.

  19. Reversal of arsenic-induced hepatic apoptosis with combined administration of DMSA and its analogues in guinea pigs: role of glutathione and linked enzymes.

    PubMed

    Mishra, Deepshikha; Mehta, Ashish; Flora, Swaran J S

    2008-02-01

    Arsenicosis, due to contaminated drinking water in the Indo-Bangladesh region, is a serious health hazard in terms of morbidity and mortality. Reactive oxygen species (ROS) generated due to arsenic toxicity have been attributed as one of the initial signals that impart cellular toxicity, which is controlled by the internal antioxidant glutathione (GSH). In the present study, we investigated (i) the role of GSH and its linked enzymes, glutathione peroxidase and glutathione reductase, in reversing chronic arsenic toxicity using a thiol chelating agent, meso-2,3-dimercaptosuccinic acid (DMSA), or one of its analogues individually or in combination; (ii) if alterations in the carbon side chain of DMSA increased efficacy; and (iii) whether the combination therapy enhance arsenic removal from hepatic tissue and prevent hepatic apoptosis. Results indicated that chronic arsenic exposure led to a ROS-mediated, mitochondrial-driven, caspase-dependent apoptosis in hepatic cells with a significant increase in glutathione disulfide (GSSG) levels and decreased glutathione reductase levels. Monotherapy with DMSA and its analogues did show minimal recovery postchelation. However, the combination of DMSA with long carbon chain analogues like monoisoamyl DMSA (MiADMSA) or monocyclohexyl DMSA (MchDMSA) showed a better efficacy in terms of reducing the arsenic burden as well as reversing altered biochemical variables indicative of oxidative stress and apoptosis. We also observed that GSH and its linked enzymes, especially glutathione reductase, play a vital role in scavenging ROS, maintaining GSH pools, and providing clinical recoveries. On the basis of the above observations, we recommend that combinational therapy of DMSA and its long carbon chain analogues MiADMSA or MchDMSA would be more effective in arsenic toxicity.

  20. 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

  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. IRF5 governs liver macrophage activation that promotes hepatic fibrosis in mice and humans

    PubMed Central

    Alzaid, Fawaz; Lagadec, Floriane; Albuquerque, Miguel; Ballaire, Raphaëlle; Orliaguet, Lucie; Hainault, Isabelle; Blugeon, Corinne; Lemoine, Sophie; Lehuen, Agnès; Saliba, David G.; Udalova, Irina A.; Paradis, Valérie; Foufelle, Fabienne

    2016-01-01

    Hepatic fibrosis arises from inflammation in the liver initiated by resident macrophage activation and massive leukocyte accumulation. Hepatic macrophages hold a central position in maintaining homeostasis in the liver and in the pathogenesis of acute and chronic liver injury linked to fibrogenesis. Interferon regulatory factor 5 (IRF5) has recently emerged as an important proinflammatory transcription factor involved in macrophage activation under acute and chronic inflammation. Here, we revealed that IRF5 is significantly induced in liver macrophages from human subjects developing liver fibrosis from nonalcoholic fatty liver disease or hepatitis C virus infection. Furthermore, IRF5 expression positively correlated with clinical markers of liver damage, such as plasma transaminase and bilirubin levels. Interestingly, mice lacking IRF5 in myeloid cells (MKO) were protected from hepatic fibrosis induced by metabolic or toxic stresses. Transcriptional reprogramming of macrophages lacking IRF5 was characterized by immunosuppressive and antiapoptotic properties. Consequently, IRF5 MKO mice respond to hepatocellular stress by promoting hepatocyte survival, leading to complete protection from hepatic fibrogenesis. Our findings reveal a regulatory network, governed by IRF5, that mediates hepatocyte death and liver fibrosis in mice and humans. Therefore, modulating IRF5 function may be an attractive approach to experimental therapeutics in fibroinflammatory liver disease. PMID:27942586

  3. Effects of thyroid hormone (thyroxine) and testosterone on hepatic 11beta-hydroxysteroid dehydrogenase mRNA and activity in pubertal hypothyroid male rats.

    PubMed

    Liu, Y J; Nakagawa, Y; Toya, K; Saegusa, H; Nasuda, K; Endoh, A; Ohzeki, T

    1998-04-01

    To investigate the effects of thyroid hormone and testosterone on 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), we measured changes in hepatic 11beta-dehydrogenase activity and its mRNA levels in pubertal methimazole (MMI)-induced hypothyroid male rats following treatment with thyroxine ([T4] 50 microg/kg/d) or testosterone (250 microg/d) for 14 days. Hypothyroidism in male rats markedly reduced hepatic 11beta-HSD1 mRNA levels and serum testosterone concentrations (P < .01). Subcutaneous injection of T4 in the hypothyroid rats significantly (P < .01) increased hepatic 11beta-HSD1 mRNA to approximately normal levels and simultaneously increased serum testosterone levels. However, the same daily dose of T4 administered to castrated male hypothyroid rats for 14 days did not elevate hepatic 11beta-HSD1 activity. Treatment with testosterone for 14 days in castrated hypothyroid male rats and rats without gonadectomy significantly (P < .01) increased the enzyme activity without administration of T4. Variations in hepatic 11beta-HSD1 activity were demonstrated to be accompanied by changes in serum testosterone levels in the rats following alteration of the thyroid hormone state. These results suggest that the effect of T4 in increasing the subnormal 11beta-HSD1 gene expression in hypothyroid male rats is mediated by its ability to increase testosterone production in these rats, because in castrated hypothyroid rats, T4 does not elevate 11beta-HSD1 gene expression.

  4. Epigallocatechin-3-gallate enhances key enzymatic activities of hepatic thioredoxin and glutathione systems in selenium-optimal mice but activates hepatic Nrf2 responses in selenium-deficient mice.

    PubMed

    Dong, Ruixia; Wang, Dongxu; Wang, Xiaoxiao; Zhang, Ke; Chen, Pingping; Yang, Chung S; Zhang, Jinsong

    2016-12-01

    Selenium participates in the antioxidant defense mainly through a class of selenoproteins, including thioredoxin reductase. Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechin in green tea. Depending upon the dose and biological systems, EGCG may function either as an antioxidant or as an inducer of antioxidant defense via its pro-oxidant action or other unidentified mechanisms. By manipulating the selenium status, the present study investigated the interactions of EGCG with antioxidant defense systems including the thioredoxin system comprising of thioredoxin and thioredoxin reductase, the glutathione system comprising of glutathione and glutathione reductase coupled with glutaredoxin, and the Nrf2 system. In selenium-optimal mice, EGCG increased hepatic activities of thioredoxin reductase, glutathione reductase and glutaredoxin. These effects of EGCG appeared to be not due to overt pro-oxidant action because melatonin, a powerful antioxidant, did not influence the increase. However, in selenium-deficient mice, with low basal levels of thioredoxin reductase 1, the same dose of EGCG did not elevate the above-mentioned enzymes; intriguingly EGCG in turn activated hepatic Nrf2 response, leading to increased heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 protein levels and thioredoxin activity. Overall, the present work reveals that EGCG is a robust inducer of the Nrf2 system only in selenium-deficient conditions. Under normal physiological conditions, in selenium-optimal mice, thioredoxin and glutathione systems serve as the first line defense systems against the stress induced by high doses of EGCG, sparing the activation of the Nrf2 system.

  5. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation

    PubMed Central

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis. PMID:27679638

  6. Queuine promotes antioxidant defence system by activating cellular antioxidant enzyme activities in cancer.

    PubMed

    Pathak, Chandramani; Jaiswal, Yogesh K; Vinayak, Manjula

    2008-04-01

    Constant generation of Reactive oxygen species (ROS) during normal cellular metabolism of an organism is generally balanced by similar rate of consumption by antioxidants. Imbalance between ROS production and antioxidant defense results in increased level of ROS causing oxidative stress which leads to promotion of malignancy. Queuine is a hyper modified base analogue of guanine, found at first anti-codon position of Q- family of tRNAs. These tRNAs are completely modified with respect to queuosine in terminally differentiated somatic cells, however hypomodification of Q-tRNAs is close association with cell proliferation. Q-tRNA modification is essential for normal development, differentiation and cellular functions. Queuine is a nutrient factor to eukaryotes. It is found to promote cellular antioxidant defense system and inhibit tumorigenesis. The activities of antioxidant enzymes like catalase, SOD, glutathione peroxidase and glutathione reductase are found to be low in Dalton's lymphoma ascites transplanted (DLAT) mouse liver compared to normal. However, exogenous administration of queuine to DLAT mouse improves the activities of antioxidant enzymes. The results suggest that queuine promotes antioxidant defense system by increasing antioxidant enzyme activities and in turn inhibits oxidative stress and tumorigenesis.

  7. An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases.

    PubMed

    Chen, Jieliang; Zhang, Wen; Lin, Junyu; Wang, Fan; Wu, Min; Chen, Cuncun; Zheng, Ye; Peng, Xiuhua; Li, Jianhua; Yuan, Zhenghong

    2014-02-01

    The hepatitis B virus (HBV) is a DNA virus that can cause chronic hepatitis B (CHB) in humans. Current therapies for CHB infection are limited in efficacy and do not target the pre-existing viral genomic DNA, which are present in the nucleus as a covalently closed circular DNA (cccDNA) form. The transcription activator-like (TAL) effector nucleases (TALENs) are newly developed enzymes that can cleave sequence-specific DNA targets. Here, TALENs targeting the conserved regions of the viral genomic DNA among different HBV genotypes were constructed. The expression of TALENs in Huh7 cells transfected with monomeric linear full-length HBV DNA significantly reduced the viral production of HBeAg, HBsAg, HBcAg, and pgRNA, resulted in a decreased cccDNA level and misrepaired cccDNAs without apparent cytotoxic effects. The anti-HBV effect of TALENs was further demonstrated in a hydrodynamic injection-based mouse model. In addition, an enhanced antiviral effect with combinations of TALENs and interferon-α (IFN-α) treatment was observed and expression of TALENs restored HBV suppressed IFN-stimulated response element-directed transcription. Taken together, these data indicate that TALENs can specifically target and successfully inactivate the HBV genome and are potently synergistic with IFN-α, thus providing a potential therapeutic strategy for treating CHB infection.

  8. 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.

  9. Altered hyperlipidemia, hepatic steatosis, and hepatic peroxisome proliferator-activated receptors in rats with intake of tart cherry.

    PubMed

    Seymour, E Mitchell; Singer, Andrew A M; Kirakosyan, Ara; Urcuyo-Llanes, Daniel E; Kaufman, Peter B; Bolling, Steven F

    2008-06-01

    Elevated plasma lipids, glucose, insulin, and fatty liver are among components of metabolic syndrome, a phenotypic pattern that typically precedes the development of Type 2 diabetes. Animal studies show that intake of anthocyanins reduces hyperlipidemia, obesity, and atherosclerosis and that anthocyanin-rich extracts may exert these effects in association with altered activity of tissue peroxisome proliferator-activated receptors (PPARs). However, studies are lacking to test this correlation using physiologically relevant, whole food sources of anthocyanins. Tart cherries are a rich source of anthocyanins, and whole cherry fruit intake may also affect hyperlipidemia and/or affect tissue PPARs. This hypothesis was tested in the Dahl Salt-Sensitive rat having insulin resistance and hyperlipidemia. For 90 days, Dahl rats were pair-fed AIN-76a-based diets supplemented with either 1% (wt:wt) freeze-dried whole tart cherry or with 0.85% additional carbohydrate to match macronutrient and calorie provision. After 90 days, the cherry-enriched diet was associated with reduced fasting blood glucose, hyperlipidemia, hyperinsulinemia, and reduced fatty liver. The cherry diet was also associated with significantly enhanced hepatic PPAR-alpha mRNA, enhanced hepatic PPAR-alpha target acyl-coenzyme A oxidase mRNA and activity, and increased plasma antioxidant capacity. In conclusion, physiologically relevant tart cherry consumption reduced several phenotypic risk factors that are associated with risk for metabolic syndrome and Type 2 diabetes. Tart cherries may represent a whole food research model of the health effects of anthocyanin-rich foods and may possess nutraceutical value against risk factors for metabolic syndrome and its clinical sequelae.

  10. 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

  11. Growing steers grazing high versus low endophyte (Neotyphodium coenophialum)-infected tall fescue have reduced serum enzymes, increased hepatic glucogenic enzymes, and reduced liver and carcass mass.

    PubMed

    Brown, K R; Anderson, G A; Son, K; Rentfrow, G; Bush, L P; Klotz, J L; Strickland, J R; Boling, J A; Matthews, J C

    2009-02-01

    It is well established that grazing Neotyphodium coenophialum-infected forages results in reduced BW gain and serum prolactin concentrations of cattle. The objective of this study was to determine the potential effects of toxic endophyte-infected tall fescue consumption on blood metabolites, carcass characteristics, and content of proteins critical for AA metabolism in the liver, kidney, and LM tissue of growing steers. Steers grazed a low toxic endophyte (LE; 0.023 microg/g ergot alkaloids) tall fescue-mixed grass pasture (n = 9; BW = 266 +/- 10.9 kg; 5.7 ha) or a high toxic endophyte (HE; 0.746 microg/g of ergot alkaloids) tall fescue pasture (n = 10; BW = 267 +/- 14.5 kg; 5.7 ha) from June 14 through at least September 11 (> or =89 d). No difference was observed for BW (P < 0.10) for the overall 85-d growth period. Also, no differences were observed for ribeye area/100 kg of HCW (P > 0.91), backfat (P > 0.95), or backfat/100 kg of HCW (P > 0.67). However, ADG (P < 0.01), final BW (P < 0.05), HCW (P < 0.01), dressing percentage (P < 0.01), ribeye area (P < 0.01), whole liver wet weight (P < 0.01), and whole liver wet weight/100 kg of end BW (P < 0.01) were greater for LE steers than HE steers. After 85 d of grazing, serum concentrations of alkaline phosphatase (P < 0.05), alanine aminotransferase (P < 0.01), aspartate aminotransferase (P < 0.03), cholesterol (P < 0.01), lactate dehydrogenase (P < 0.01), and prolactin (P < 0.01) were less for HE than LE steers. At slaughter, hepatic content of cytosolic phosphoenolpyruvate carboxykinase (P < 0.01) was greater in HE steers than LE steers. Hepatic content of aspartate aminotransferase (P < 0.01) also was greater, whereas renal and LM content were not (P > or = 0.42). No differences (P > or = 0.15) were observed for hepatic, renal, and LM content of alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase, and 3 glutamate transport proteins. These data indicate that the HE steers displayed classic

  12. 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

    2015-04-03

    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.

  13. 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

  14. New hydroxamate inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme active-site recognition.

    PubMed

    Bourdel, E; Doulut, S; Jarretou, G; Labbe-Jullie, C; Fehrentz, J A; Doumbia, O; Kitabgi, P; Martinez, J

    1996-08-01

    Selective and mixed inhibitors of the three zinc metallopeptidases that degrade neurotensin (NT), e.g. endopeptidase 24-16 (EC 3.4.24.16), endopeptidase 24-11 (EC 3.4.24.11 or neutral endopeptidase, NEP) and endopeptidase 24-15 (EC 3.4.24.15), and leucine-aminopeptidase (type IV-S), that degrades the NT-related peptides, Neuromedin N (NN), are of great interest. On the structural basis of compound JMV 390-1 (N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)-benzylpropyl]-L- isoleucyl-L-leucine), which was a full inhibitor of the major NT degrading enzymes, several hydroxamate inhibitors corresponding to the general formula HONHCO-CH2-CH(CH2-C6H5)CO-X-Y-OH (with X-Y = dipeptide) have been synthesized. Compound 7a (X-Y = Ile-Ala) was nearly 40-times more potent in inhibiting EC 24-16 than NEP and more than 800-times more potent than EC 24-15, with an IC50 (12 nM) almost equivalent to that of compound JMV 390-1. Therefore, this compound is an interesting selective inhibitor of EC 24-16, and should be an interesting probe to explore the physiological involvement of EC 24-16 in the metabolism of neurotensin.

  15. 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

  16. 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.

  17. 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

  18. Secretion of an articular cartilage proteoglycan-degrading enzyme activity by murine T lymphocytes in vitro.

    PubMed Central

    Kammer, G M; Sapolsky, A I; Malemud, C J

    1985-01-01

    Destruction of articular cartilage is the hallmark of inflammatory arthritides. Enzymes elaborated by mononuclear cells infiltrating the synovium mediate, in part, the degradation of the cartilage extracellular matrix. Since mononuclear cells are the dominant cell type found in chronic inflammatory synovitis, we investigated whether interaction of immune mononuclear cells with antigen initiated the synthesis and secretion of a proteoglycan-degrading enzyme activity. Proteoglycan-degrading enzyme activity was monitored by the capacity of murine spleen cell conditioned medium to release [3H]serine/35SO4 incorporated into rabbit cartilage proteoglycan monomer fraction (A1D1), and by the relative change in specific viscosity of bovine nasal cartilage proteoglycan monomer. The results demonstrated that both virgin and immune mononuclear cells spontaneously generated proteoglycan-degrading enzyme activity and that cellular activation and proliferation induced by the antigen keyhole limpet hemocyanin or the mitogen phytohemagglutinin was not required. Kinetic studies demonstrated stable release of the enzyme activity over 72 h. Cell separation studies showed that T lymphocytes, a thymoma line, and macrophages separately produced proteoglycan-degrading enzyme activity. The enzyme activity has been partially characterized and appears to belong to a class of neutral pH metal-dependent proteinases. These observations, the first to demonstrate that T lymphocytes secrete an enzyme capable of degrading cartilage proteoglycan, raise the possibility that this enzyme activity contributes to cartilage extracellular matrix destruction in vivo. Moreover, these data support the conclusion that production of this enzyme by T lymphocytes is independent of an antigen-specific stimulus. PMID:3897284

  19. Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity.

    PubMed Central

    Dobransky, T; Davis, W L; Xiao, G H; Rylett, R J

    2000-01-01

    Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity. PMID:10861222

  20. Anti-hepatitis B activity of isoquinoline alkaloids of plant origin.

    PubMed

    Aljofan, Mohamad; Netter, Hans J; Aljarbou, Ahmed N; Hadda, Taibi Ben; Orhan, Ilkay Erdogan; Sener, Bilge; Mungall, Bruce A

    2014-05-01

    Hepatitis B virus (HBV) is the causative agent of B-type hepatitis in humans, a vaccine-preventable disease. Despite the availability of effective vaccines, globally, 2 billion people show evidence of past or current HBV infection, of which 350 million people are persistently infected, with an estimated annual increase of 1 million. There is no cure for chronic HBV infections, which are associated with cirrhotic liver failure and with an increased risk of developing hepatocellular carcinoma. Hepatitis antiviral research has focused primarily on the development of inhibitors of viral polymerase through the use of nucleoside analogues. Therefore, there is an urgent need for the development of non-nucleoside compounds to be used as an alternative or to complement the current therapy. To address this need, 18 isoquinoline alkaloids were evaluated for their potential antiviral activity against HBV in vitro.

  1. Hepatic ethoxy-, methoxy- and pentoxyresorufin O-dealkylase activities in Landrace and Duroc pigs stimulated with HCG.

    PubMed

    Zamaratskaia, G; Zlabek, V; Ropstad, E; Tajet, H; Andresen, Ø

    2010-12-01

    The effect of human chorionic gonadotropin (hCG) stimulation on the activities of ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD) and pentoxyresorufin O-depentylase (PROD) was studied in intact male pigs of purebred Landrace and Duroc breeds. Pigs were divided into four groups: two control groups of each breed, without hCG stimulation (n = 20 for each breed), and two experimental groups (n = 18 for each breed), with hCG stimulation (Pregnyl(®); N.V. Organon, Oss, The Netherlands, 30 IU/kg live weight). Pigs were slaughtered 3 days after hCG stimulation and enzyme activities were measured in hepatic microsomes using two approaches. First, only one substrate concentration was used for the analysis of each enzyme activity. We found that EROD activity was suppressed by hCG-stimulation in Landrace (p = 0.004), but not Duroc pigs (p > 0.05). Generally, EROD activity was higher in Duroc pigs compared with Landrace (p = 0.017). Methoxyresorufin O-demethylase and PROD activities did not differ between groups (p > 0.05). To further characterize EROD, MROD and PROD, enzyme kinetic studies were performed. V(max) values for EROD and MROD in both breeds were lower after hCG stimulation (p < 0.001 for Landrace and p < 0.05 for Duroc). Additionally, V(max) values for EROD significantly differed between Landrace and Duroc pigs being higher in Duroc pigs (p < 0.05). We concluded that both hCG stimulation and breed differences may be important in the regulation of EROD and MROD activities. This study provides the first data on the effect of hCG stimulation and thus high testicular steroids, on EROD, MROD and PROD activities. Further studies are needed to investigate individual CYP450 enzymes and their regulation in porcine tissues.

  2. Angiotensin-converting enzyme inhibitors improve hepatic steatosis by modulating expression of tumour necrosis factor-alpha, interleukin-6 and adiponectin receptor-2 in rats with type 2 diabetes.

    PubMed

    Zhang, Xia; Li, Zhong-Zhuan; Liu, Dong-Fang; Xu, Xin; Mei, Zhe-Chuan; Shen, Wei

    2009-07-01

    1. Angiotensin-converting enzyme inhibitors (ACEI) are hypotensive drugs that have been shown to prevent Type 2 diabetes mellitus (T2DM) in high-risk individuals. However, in T2DM, the effects of ACEI on hepatic steatosis are not known. The aim of the present study was to examine the effects of ACEI on changes in liver histology and hepatic mRNA expression of adipokines in rats with T2DM. 2. Thirty-six rats were divided into a normal control group, a T2DM group and a fosinopril-treated group. After six weeks of treatment with 5 mg/kg per day fosinopril, an ACEI, changes in liver histology, serum fasting glucose (FG), insulin, triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, adiponectin were evaluated, as was hepatic TNF-alpha, IL-6 and adiponectin receptor-2 (adipoR2) mRNA expression. 3. The degree of hepatic steatosis and inflammation, serum FG, insulin, TG, TC, ALT, TNF-alpha and IL-6 concentrations and hepatic TNF-alpha and IL-6 mRNA expression were significantly higher in rats with T2DM than in normal controls. Serum adiponectin concentrations and hepatic adipoR2 mRNA expression in rats with T2DM were significantly lower than in normal controls. Fosinopril significantly reduced the degree of hepatic steatosis, serum FG, insulin, ALT, TNF-alpha and IL-6 concentrations and hepatic TNF-alpha and IL-6 mRNA expression. Fosinopril significantly increased serum adiponectin concentrations and hepatic adipoR2 mRNA expression. 4. In conclusion, the ACEI improved insulin sensitivity and hepatic steatosis in rats with T2DM by increasing circulating adiponectin and hepatic adipoR2 levels, in addition to reducing pro-inflammatory cytokine levels in the circulation and liver.

  3. FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis.

    PubMed

    Kir, Serkan; Beddow, Sara A; Samuel, Varman T; Miller, Paul; Previs, Stephen F; Suino-Powell, Kelly; Xu, H Eric; Shulman, Gerald I; Kliewer, Steven A; Mangelsdorf, David J

    2011-03-25

    Fibroblast growth factor (FGF) 19 is an enterokine synthesized and released when bile acids are taken up into the ileum. We show that FGF19 stimulates hepatic protein and glycogen synthesis but does not induce lipogenesis. The effects of FGF19 are independent of the activity of either insulin or the protein kinase Akt and, instead, are mediated through a mitogen-activated protein kinase signaling pathway that activates components of the protein translation machinery and stimulates glycogen synthase activity. Mice lacking FGF15 (the mouse FGF19 ortholog) fail to properly maintain blood concentrations of glucose and normal postprandial amounts of liver glycogen. FGF19 treatment restored the loss of glycogen in diabetic animals lacking insulin. Thus, FGF19 activates a physiologically important, insulin-independent endocrine pathway that regulates hepatic protein and glycogen metabolism.

  4. High-throughput fluorometric measurement of potential soil extracellular enzyme activities.

    PubMed

    Bell, Colin W; Fricks, Barbara E; Rocca, Jennifer D; Steinweg, Jessica M; McMahon, Shawna K; Wallenstein, Matthew D

    2013-11-15

    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

  5. Lupinus albus Conglutin Gamma Modifies the Gene Expressions of Enzymes Involved in Glucose Hepatic Production In Vivo.

    PubMed

    González-Santiago, Ana E; Vargas-Guerrero, Belinda; García-López, Pedro M; Martínez-Ayala, Alma L; Domínguez-Rosales, José A; Gurrola-Díaz, Carmen M

    2017-01-18

    Lupinus albus seeds contain conglutin gamma (Cγ) protein, which exerts a hypoglycemic effect and positively modifies proteins involved in glucose homeostasis. Cγ could potentially be used to manage patients with impaired glucose metabolism, but there remains a need to evaluate its effects on hepatic glucose production. The present study aimed to analyze G6pc, Fbp1, and Pck1 gene expressions in two experimental animal models of impaired glucose metabolism. We also evaluated hepatic and renal tissue integrity following Cγ treatment. To generate an insulin resistance model, male Wistar rats were provided 30% sucrose solution ad libitum for 20 weeks. To generate a type 2 diabetes model (STZ), five-day-old rats were intraperitoneally injected with streptozotocin (150 mg/kg). Each animal model was randomized into three subgroups that received the following oral treatments daily for one week: 0.9% w/v NaCl (vehicle; IR-Ctrl and STZ-Ctrl); metformin 300 mg/kg (IR-Met and STZ-Met); and Cγ 150 mg/kg (IR-Cγ and STZ-Cγ). Biochemical parameters were assessed pre- and post-treatment using colorimetric or enzymatic methods. We also performed histological analysis of hepatic and renal tissue. G6pc, Fbp1, and Pck1 gene expressions were quantified using real-time PCR. No histological changes were observed in any group. Post-treatment G6pc gene expression was decreased in the IR-Cγ and STZ-Cγ groups. Post-treatment Fbp1 and Pck1 gene expressions were reduced in the IR-Cγ group but increased in STZ-Cγ animals. Overall, these findings suggest that Cγ is involved in reducing hepatic glucose production, mainly through G6pc inhibition in impaired glucose metabolism disorders.

  6. Activation of the farnesoid X receptor induces hepatic expression and secretion of fibroblast growth factor 21.

    PubMed

    Cyphert, Holly A; Ge, Xuemei; Kohan, Alison B; Salati, Lisa M; Zhang, Yanqiao; Hillgartner, F Bradley

    2012-07-20

    Previous studies have shown that starvation or consumption of a high fat, low carbohydrate (HF-LC) ketogenic diet induces hepatic fibroblast growth factor 21 (FGF21) gene expression in part by activating the peroxisome proliferator-activated receptor-α (PPARα). Using primary hepatocyte cultures to screen for endogenous signals that mediate the nutritional regulation of FGF21 expression, we identified two sources of PPARα activators (i.e. nonesterified unsaturated fatty acids and chylomicron remnants) that induced FGF21 gene expression. In addition, we discovered that natural (i.e. bile acids) and synthetic (i.e. GW4064) activators of the farnesoid X receptor (FXR) increased FGF21 gene expression and secretion. The effects of bile acids were additive with the effects of nonesterified unsaturated fatty acids in regulating FGF21 expression. FXR activation of FGF21 gene transcription was mediated by an FXR/retinoid X receptor binding site in the 5'-flanking region of the FGF21 gene. FGF19, a gut hormone whose expression and secretion is induced by intestinal bile acids, also increased hepatic FGF21 secretion. Deletion of FXR in mice suppressed the ability of an HF-LC ketogenic diet to induce hepatic FGF21 gene expression. The results of this study identify FXR as a new signaling pathway activating FGF21 expression and provide evidence that FXR activators work in combination with PPARα activators to mediate the stimulatory effect of an HF-LC ketogenic diet on FGF21 expression. We propose that the enhanced enterohepatic flux of bile acids during HF-LC consumption leads to activation of hepatic FXR and FGF19 signaling activity and an increase in FGF21 gene expression and secretion.

  7. 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.

  8. 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

  9. The Enterococcus hirae Mur-2 enzyme displays N-acetylglucosaminidase activity

    PubMed Central

    Eckert, Catherine; Magnet, Sophie; Mesnage, Stéphane

    2007-01-01

    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

  10. 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...

  11. Temperature-responsive enzyme-polymer nanoconjugates with enhanced catalytic activities in organic media.

    PubMed

    Zhu, Jingying; Zhang, Yifei; Lu, Diannan; Zare, Richard N; Ge, Jun; Liu, Zheng

    2013-07-11

    A general approach for preparing enzyme-polymer nanoconjugates that respond to temperature in organic media is presented. These nanoconjugates readily dissolve in organic solvents for homogenous catalysis at 40 °C and showed greatly enhanced apparent catalytic activities. The recovery of the soluble enzyme-polymer nanoconjugates is accomplished by temperature-induced precipitation.

  12. 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...

  13. 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)

  14. Mechanism of allopurinol-mediated increase in enzyme activity in man

    PubMed Central

    Beardmore, Thomas D.; Cashman, Jay S.; Kelley, William N.

    1972-01-01

    Allopurinol therapy in man interferes with pyrimidine biosynthesis de novo by inhibition of one or both of the two enzymes, orotate phosphoribosyltransferase (OPRT) and orotidylic decarboxylase (ODC), responsible for the conversion of orotic acid to uridine-5′-monophosphate. Inhibition of this pathway in vivo is followed in 1-3 wk by an increase in the activity of both of these enzymes in erythrocytes and of ODC in circulating leukocytes. This drug-mediated increase in enzyme activity in erythrocytes could not be attributed to enzyme stabilization or induction in vivo but appeared to be due to enzymeactivation.” “Activation” of the OPRT enzyme was directly demonstrated in erythrocytes studied in vitro after incubation with oxipurinol, and to a lesser extent, with allopurinol. No evidence for “activation” of the ODC enzyme was demonstrated in vitro. This response to allopurinol therapy provides an excellent model for examining the mechanism of increased enzyme activity in response to drug administration. PMID:5032526

  15. 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...

  16. 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.

  17. Sediment Microbial Enzyme Activity as an Indicator of Nutrient Limitation in Great Lakes Coastal Wetlands

    EPA Science Inventory

    This study, the first to link microbial enzyme activities to regional-scale anthropogenic stressors, suggests that microbial enzyme regulation of carbon and nutrient dynamics may be sensitive indicators of nutrient dynamics in aquatic ecosystems, but further work is needed to elu...

  18. 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…

  19. Activity of an enzyme immobilized on superparamagnetic particles in a rotational magnetic field

    SciTech Connect

    Mizuki, Toru; Watanabe, Noriyuki; Nagaoka, Yutaka; Fukushima, Tadamasa; Morimoto, Hisao; Usami, Ron; Maekawa, Toru

    2010-03-19

    We immobilize {alpha}-amylase extracted from Bacillus Iicheniformis on the surfaces of superparamagnetic particles and investigate the effect of a rotational magnetic field on the enzyme's activity. We find that the activity of the enzyme molecules immobilized on superparamagnetic particles increases in the rotational magnetic field and reaches maximum at a certain frequency. We clarify the effect of the cluster structures formed by the superparamagnetic particles on the activity. Enzyme reactions are enhanced even in a tiny volume of solution using the present method, which is very important for the development of efficient micro reactors and micro total analysis systems ({mu}-TAS).

  20. Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells.

    PubMed

    Tylicki, Adam; Czerniecki, Jan; Dobrzyn, Pawel; Matanowska, Agnieszka; Olechno, Anna; Strumilo, Slawomir

    2005-10-01

    Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.

  1. 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.

  2. EZH2-mediated repression of Dkk1 promotes hepatic stellate cell activation and hepatic fibrosis.

    PubMed

    Yang, Yang; Chen, Xiao-Xia; Li, Wan-Xia; Wu, Xiao-Qin; Huang, Cheng; Xie, Juan; Zhao, Yu-Xin; Meng, Xiao-Ming; Li, Jun

    2017-03-23

    EZH2, a histone H3 lysine-27-specific methyltransferase, is involved in diverse physiological and pathological processes including cell proliferation and differentiation. However, the role of EZH2 in liver fibrosis is largely unknown. In this study, it was identified that EZH2 promoted Wnt pathway-stimulated fibroblasts in vitro and in vivo by repressing Dkk-1, which is a Wnt pathway antagonist. The expression of EZH2 was increased in CCl4 -induced rat liver and primary HSCs as well as TGF-β1-treated HSC-T6, whereas the expression of Dkk1 was reduced. Silencing of EZH2 prevented TGF-β1-induced proliferation of HSC-T6 cells and the expression of α-SMA. In addition, knockdown of Dkk1 promoted TGF-β1-induced activation of HSCs. Moreover, silencing of EZH2 could restore the repression of Dkk-1 through trimethylation of H3K27me3 in TGF-β1-treated HSC-T6 cells. Interestingly, inhibition of EZH2 had almost no effect on the activation of HSC when Dkk1 was silenced. Collectively, EZH2-mediated repression of Dkk1 promotes the activation of Wnt/β-catenin pathway, which is an essential event for HSC activation.

  3. 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

  4. Chronic hepatitis C infection–induced liver fibrogenesis is associated with M2 macrophage activation

    PubMed Central

    Bility, Moses T.; Nio, Kouki; Li, Feng; McGivern, David R.; Lemon, Stanley M.; Feeney, Eoin R.; Chung, Raymond T.; Su, Lishan

    2016-01-01

    The immuno-pathogenic mechanisms of chronic hepatitis C virus (HCV) infection remain to be elucidated and pose a major hurdle in treating or preventing chronic HCV-induced advanced liver diseases such as cirrhosis. Macrophages are a major component of the inflammatory milieu in chronic HCV–induced liver disease, and are generally derived from circulating inflammatory monocytes; however very little is known about their role in liver diseases. To investigate the activation and role of macrophages in chronic HCV–induced liver fibrosis, we utilized a recently developed humanized mouse model with autologous human immune and liver cells, human liver and blood samples and cell culture models of monocyte/macrophage and/or hepatic stellate cell activation. We showed that M2 macrophage activation was associated with liver fibrosis during chronic HCV infection in the livers of both humanized mice and patients, and direct-acting antiviral therapy attenuated M2 macrophage activation and associated liver fibrosis. We demonstrated that supernatant from HCV-infected liver cells activated human monocytes/macrophages with M2-like phenotypes. Importantly, HCV-activated monocytes/macrophages promoted hepatic stellate cell activation. These results suggest a critical role for M2 macrophage induction in chronic HCV-associated immune dysregulation and liver fibrosis. PMID:28000758

  5. Chronic hepatitis C infection-induced liver fibrogenesis is associated with M2 macrophage activation.

    PubMed

    Bility, Moses T; Nio, Kouki; Li, Feng; McGivern, David R; Lemon, Stanley M; Feeney, Eoin R; Chung, Raymond T; Su, Lishan

    2016-12-21

    The immuno-pathogenic mechanisms of chronic hepatitis C virus (HCV) infection remain to be elucidated and pose a major hurdle in treating or preventing chronic HCV-induced advanced liver diseases such as cirrhosis. Macrophages are a major component of the inflammatory milieu in chronic HCV-induced liver disease, and are generally derived from circulating inflammatory monocytes; however very little is known about their role in liver diseases. To investigate the activation and role of macrophages in chronic HCV-induced liver fibrosis, we utilized a recently developed humanized mouse model with autologous human immune and liver cells, human liver and blood samples and cell culture models of monocyte/macrophage and/or hepatic stellate cell activation. We showed that M2 macrophage activation was associated with liver fibrosis during chronic HCV infection in the livers of both humanized mice and patients, and direct-acting antiviral therapy attenuated M2 macrophage activation and associated liver fibrosis. We demonstrated that supernatant from HCV-infected liver cells activated human monocytes/macrophages with M2-like phenotypes. Importantly, HCV-activated monocytes/macrophages promoted hepatic stellate cell activation. These results suggest a critical role for M2 macrophage induction in chronic HCV-associated immune dysregulation and liver fibrosis.

  6. 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

  7. [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.

  8. 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.

  9. Array-on-Array Strategy For Activity-Based Enzyme Profiling.

    PubMed

    Sieow, Brendan Fu-Long; Uttamchandani, Mahesh

    2017-01-01

    We describe a novel array on array strategy intended to enhance the throughput of enzymatic activity screening using microarrays. This strategy consists of spotting a first array with large droplets of enzymes with varying concentrations and subsequently spotting a second array with small droplets of fluorogenic substrate on top of the enzyme array. By varying the array on array spotting patterns of different classes of enzyme (e.g., proteases, phosphatases, kinases) and their corresponding fluorogenic substrates, we have the unprecedented ability for testing enzymes and mixed samples in a multiplexed fashion within a single microarray slide. This new approach enables rapid enzyme characterization building upon a one enzyme on one slide droplet-based screening concept previously established.

  10. Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities

    PubMed Central

    Helmerhorst, Eva J.; Wei, Guoxian

    2015-01-01

    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. PMID:26113763

  11. Lectin-directed enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs.

    PubMed

    Garnier, Philippe; Wang, Xiang-Tao; Robinson, Mark A; van Kasteren, Sander; Perkins, Alan C; Frier, Malcolm; Fairbanks, Antony J; Davis, Benjamin G

    2010-12-01

    The lectin-directed enzyme activated prodrug therapy (LEAPT) bipartite drug delivery system utilizes glycosylated enzyme, localized according to its sugar pattern, and capped prodrugs released by that enzyme. In this way, the sugar coat of a synthetic enzyme determines the site of release of a given drug. Here, prodrugs of doxorubicin and 5-fluorouracil capped by the nonmammalian l-rhamnosyl sugar unit have been efficiently synthesized and evaluated for use in the LEAPT system. Both are stable in blood, released by synthetically d-galactosylated rhamnosidase enzyme, and do not inhibit the uptake of the synthetic enzyme to its liver target. These results are consistent with their proposed mode of action and efficacy in models of liver cancer, and confirm modular flexibility in the drugs that may be used in LEAPT.

  12. Aerobic and anaerobic bioprocessing of activated sludge: floc disintegration by enzymes.

    PubMed

    Ayol, Azize; Filibeli, Ayse; Sir, Diclehan; Kuzyaka, Ersan

    2008-11-01

    Hydrolytic enzymes such as glucosidases, lipases, and proteases have an imperative function at the hydrolysis stage of complex organic structures in the degradation of biodegradable particulate organic matter. As a key factor, extracellular polymeric substances (EPS) control the extracellular hydrolytic enzymes in this degradation mechanism. A flocculated matrix of EPS bridging with bacteria holds back the dewaterability properties of the bioprocessed sludges. Disruption of the flocculated matrix leads to improved solubilization of sludge solids by attacking the hydrolytic enzymes to polymeric substances forming enzyme-substrate complexes. To determine the floc disintegration mechanisms by enzymes during aerobic and anaerobic bioprocessing of sludges, experimental data obtained from three aerobic digesters and three anaerobic digesters were evaluated. As part of a broader project examining the overall fate and effects of hydrolytic enzymes in biological sludge stabilization, this paper compares the performances of aerobic and anaerobic reactors used in this study and reports significant improvements in enzymatic treatment of activated sludge.

  13. 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

  14. An evaluation of hepatic enzyme elevations among HIV-infected released prisoners enrolled in two randomized placebo-controlled trials of extended release naltrexone.

    PubMed

    Vagenas, Panagiotis; Di Paola, Angela; Herme, Maua; Lincoln, Thomas; Skiest, Daniel J; Altice, Frederick L; Springer, Sandra A

    2014-07-01

    Extended-release naltrexone (XR-NTX), an approved treatment for opioid or alcohol dependence, is a once-monthly injectable formulation of naltrexone. Hepatotoxicity concerns have limited its use, necessitating further investigation. This study aims to examine hepatic enzyme levels in participants of 2 randomized placebo-controlled trials (RCTs) of XR-NTX. Hepatic transaminases were measured in 85 patients enrolled in RCTs of XR-NTX among HIV-infected prisoners, transitioning to the community and receiving treatment for either dependence on alcohol (52.9%), opioids (44.7%) or both (16.5%). Baseline characteristics included HCV co-infection (55.7%), antiretroviral therapy (81%), mental illness (39%) and receiving psychiatric medications (34.1%). Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transferase (GGT) were not statistically different between persons randomized to placebo (N=24) and XR-NTX (N=61) arms. These results confirm that XR-NTX is safe to use among opioid and alcohol dependent HIV-infected released prisoners receiving ART with high rates of co-morbid HCV infection and mental illness.

  15. Inhibition of lipopolysaccharide-induced liver injury in rats treated with a hepatic drug-metabolizing enzyme inducer p,p'-DDT.

    PubMed

    Shimada, Yuko; Tomita, Mariko; Yoshida, Toshinori; Fukuyama, Tomoki; Katoh, Yoshitaka; Ohnuma-Koyama, Aya; Takahashi, Naofumi; Soma, Katsumi; Kojima, Sayuri; Ohtsuka, Ryoichi; Takeda, Makio; Kuwahara, Maki; Harada, Takanori

    2015-03-01

    Hepatocellular hypertrophy in association with drug-metabolizing enzyme induction is considered to be an adaptive change associated with drug metabolism. To improve our understanding of liver hypertrophy, we determined the effect of a single ip injection of either lipopolysaccharide (LPS) or vehicle in male F344 rats with hepatocellular hypertrophy induced by oral delivery of p,p'-DDT for 2 weeks. The rats were sacrificed 3h or 24h after LPS or vehicle injection. LPS induced a focal hepatocellular necrosis in rats fed the control diet. When rats pre-treated with p,p'-DDT were injected with LPS, necrotic foci surrounded by ballooned hepatocytes were observed in the liver. The change was consistent with reduced LPS-mediated increases in plasma hepatic biomarkers, neutrophil influx, and apoptosis, and also associated with hepatic mRNA levels of TNF-α, CYPs, and NOS2. By contrast, when combined with p,p'-DDT and LPS, faint hepatocellular fatty change was extended, together with a synergistic increase in total blood cholesterol. These results suggest that hepatocytes exposed to p,p'-DDT are protected from the cell-lethal toxic effects of an exogenous stimulus, resulting in cell ballooning rather than necrosis in association with reduced inflammation and apoptosis, but compromised by an adverse effect on lipid metabolism.

  16. Activation of a Covalent Enzyme-Substrate Bond by Noncovalent Interaction with an Effector

    PubMed Central

    Malhotra, O. P.; Bernhard, Sidney A.

    1973-01-01

    The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon β-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD+, even where the “redox” properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H2O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native “active-thiol” furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the “two equivalent

  17. Histidine augments the suppression of hepatic glucose production by central insulin action.

    PubMed

    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

    2013-07-01

    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.

  18. 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.

  19. Physics-based enzyme design: predicting binding affinity and catalytic activity.

    PubMed

    Sirin, Sarah; Pearlman, David A; Sherman, Woody

    2014-12-01

    Computational enzyme design is an emerging field that has yielded promising success stories, but where numerous challenges remain. Accurate methods to rapidly evaluate possible enzyme design variants could provide significant value when combined with experimental efforts by reducing the number of variants needed to be synthesized and speeding the time to reach the desired endpoint of the design. To that end, extending our computational methods to model the fundamental physical-chemical principles that regulate activity in a protocol that is automated and accessible to a broad population of enzyme design researchers is essential. Here, we apply a physics-based implicit solvent MM-GBSA scoring approach to enzyme design and benchmark the computational predictions against experimentally determined activities. Specifically, we evaluate the ability of MM-GBSA to predict changes in affinity for a steroid binder protein, catalytic turnover for a Kemp eliminase, and catalytic activity for α-Gliadin peptidase variants. Using the enzyme design framework developed here, we accurately rank the most experimentally active enzyme variants, suggesting that this approach could provide enrichment of active variants in real-world enzyme design applications.

  20. Role of enzyme-substrate flexibility in catalytic activity: an evolutionary perspective.

    PubMed

    Demetrius, L

    1998-09-21

    Site-directed mutagenesis has proved an effective experimental technique to investigate catalytic mechanisms and to determine relations between enzyme structure and function. This article invokes an analytical model based on evolution by mutation and natural selection-Nature's analogue of site-directed mutagenesis-to derive a set of general rules relating enzyme structure and activity. The catalysts are described in terms of the structural parameters, rigidity and flexibility, and the functional variables, reaction rate and substrate specificity. The evolutionary model predicts the following structure-activity relations: (a) rigid enzyme-flexible substrate: large variation in reaction rates, broad substrate specificity; (b) rigid enzyme-rigid substrate: diffusion controlled rates, absolute specificity; (c) flexible enzyme-rigid substrate: intermediate reaction rates, group specificity; (d) flexible enzyme-flexible substrate: slow rates, absolute specificity. Spectroscopic methods and X-ray crystallography now yield important characteristics of enzyme-substrate complexes such as molecular flexibility. The evolutionary analysis we have exploited provides general principles for inferring catalytic activity from structural studies of enzyme-substrate complexes.

  1. Dosage Compensation in Drosophila: Nadp-Enzyme Activities and Cross-Reacting Material

    PubMed Central

    Williamson, John H.; Bentley, Michael M.

    1983-01-01

    The relationships between gene dosage, enzyme activities and CRM levels have been determined for G6PD and 6PGD. Enzyme activities and CRM levels were directly proportional and increased in genotypes carrying duplications of the respective structural genes. When a duplication consisting of the distal 45% of the X chromosome was used to duplicate Pgd+, 6PGD activity and CRM increased and G6PD activity decreased. When the proximal 55% of the X chromosome was duplicated, G6PD activity and CRM increased whereas 6PGD activity and CRM levels decreased. These observations support the model of dosage compensation of X-linked genes that invokes an autosomal activator in limited concentrations for which X-linked loci compete. The distal 45% of the X chromosome, when duplicated, caused a significant increase in NADP-malic enzyme activity and CRM levels, as if a structural gene for NADP-ME is sex-linked. PMID:6406296

  2. Cholesterol overloading leads to hepatic L02 cell damage through activation of the unfolded protein response.

    PubMed

    Li, Qi; Liu, Zhiguo; Guo, Jianli; Chen, Jiangyuan; Yang, Pu; Tian, Jun; Sun, Jun; Zong, Yiqiang; Qu, Shen

    2009-10-01

    Reported data indicate that cholesterol loading in the liver can cause hepatic injury. To explore the possible mechanisms of cell damage resulting from cholesterol overloading in hepatocytes, cell apoptosis, the unfolded protein response (UPR) and the correlation between them were assessed in the cholesterol-overloaded normal human hepatic cell line L02. L02 cells were incubated with 200 microg/ ml of low density lipoprotein (LDL) for 24 h with or without 20 microg/ml 58035, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). In the LDL+58035 group, the intracellular cholesterol level was dramatically increased, which was measured by an enzymatic combined high performance liquid chromatography assay. Expression of immunoglobulin-binding protein, X-box binding protein 1, activating transcription factor 6, activating transcription factor 4, CCAAT/enhancer-binding protein homologous protein-10, markers of endoplasmic reticulum stress (ERS)/ UPR, were up-regulated as determined using reverse transcription-polymerase chain reaction (RT-PCR) or Western blot analysis. The rate of cell apoptic death increased 21.3+/-2.4%. Meanwhile, the active caspase-3 protein expression was increased 8.4-fold compared to the active caspase-3 protein expression in the controls. Furthermore, 4-phenylbutyric acid, an inhibitor of UPR, partly reduced cell apoptosis and activation of caspase-3. This study suggests that cholesterol overloading in hepatic L02 cells induces ERS and activates the UPR which, in part, leads to the apoptotic damage of cells.

  3. 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.

  4. Investigations on the activity of poly(2-oxazoline) enzyme conjugates dissolved in organic solvents.

    PubMed

    Konieczny, Stefan; Krumm, Christian; Doert, Dominik; Neufeld, Katharina; Tiller, Joerg C

    2014-07-10

    The use of enzymes in organic solvents offers a great opportunity for the highly selective synthesis of complex organic compounds. In this study we investigate the POXylation of several enzymes with different polyoxazolines ranging from the hydrophilic poly(2-methyl-oxazoline) (PMOx) to the hydrophobic poly(2-heptyl-oxazoline) (PHeptOx). As reported previously on the examples of model enzymes POXylation mediated by pyromellitic acid dianhydride results in highly modified, organosoluble protein conjugates. This procedure is here extended to a larger number of proteins and optimized for the different polyoxazolines. The resulting polymer-enzyme conjugates (PEC) became soluble in different organic solvents ranging from hydrophilic DMF to even toluene. These conjugates were characterized regarding their solubility and especially their activity in organic solvents and in some cases the PECs showed significantly (up to 153,000 fold) higher activities than the respective native enzymes.

  5. Mesoporous silica-encapsulated gold nanoparticles as artificial enzymes for self-activated cascade catalysis.

    PubMed

    Lin, Youhui; Li, Zhenhua; Chen, Zhaowei; Ren, Jinsong; Qu, Xiaogang

    2013-04-01

    A significant challenge in chemistry is to create synthetic structures that mimic the complexity and function of natural systems. Here, a self-activated, enzyme-mimetic catalytic cascade has been realized by utilizing expanded mesoporous silica-encapsulated gold nanoparticles (EMSN-AuNPs) as both glucose oxidase- and peroxidase-like artificial enzymes. Specifically, EMSN helps the formation of a high degree of very small and well-dispersed AuNPs, which exhibit an extraordinarily stability and dual enzyme-like activities. Inspired by these unique and attractive properties, we further piece them together into a self-organized artificial cascade reaction, which is usually completed by the oxidase-peroxidase coupled enzyme system. Our finding may pave the way to use matrix as the structural component for the design and development of biomimetic catalysts and to apply enzyme mimics for realizing higher functions.

  6. 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.

  7. 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 %.

  8. Detection of Extracellular Enzyme Activity in Penicillium using Chromogenic Media.

    PubMed

    Yoon, Ji Hwan; Hong, Seung Beom; Ko, Seung Ju; Kim, Seong Hwan

    2007-09-01

    A total of 106 Penicillium species were tested to examine their ability of degrading cellobiose, pectin and xylan. The activity of β-glucosidase was generally strong in all the Penicillium species tested. P. citrinum, P. charlesii, P. manginii and P. aurantiacum showed the higher ability of producing β-glucosidase than other tested species. Pectinase activity was detected in 24 Penicillium species. P. paracanescens, P. sizovae, P. sartoryi, P. chrysogenum, and P. claviforme showed strong pectinase activity. In xylanase assay, 84 Penicillium species showed activity. Strong xylanase activity was detected from P. megasporum, P. sartoryi, P. chrysogenum, P. glandicola, P. discolor, and P. coprophilum. Overall, most of the Penicillium species tested showed strong β-glucosidase activity. The degree of pectinase and xylanase activity varied depending on Penicillium species.

  9. 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

  10. 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.

  11. Defying the activity-stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability.

    PubMed

    Siddiqui, Khawar Sohail

    2017-05-01

    The biotechnological applications of enzymes are limited due to the activity-stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity-stability trade-off can be overcome. In this review, the origin of the activity-stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity-stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel-Danson equilibrium model, catalytic antibodies, and life on cold planets.

  12. Ligand activation of peroxisome proliferator-activated receptor-β/δ suppresses liver tumorigenesis in hepatitis B transgenic mice.

    PubMed

    Balandaram, Gayathri; Kramer, Lance R; Kang, Boo-Hyon; Murray, Iain A; Perdew, Gary H; Gonzalez, Frank J; Peters, Jeffrey M

    2016-07-01

    Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits steatosis and inflammation, known risk factors for liver cancer. In this study, the effect of ligand activation of PPARβ/δ in modulating liver tumorigenesis in transgenic hepatitis B virus (HBV) mice was examined. Activation of PPARβ/δ in HBV mice reduced steatosis, the average number of liver foci, and tumor multiplicity. Reduced expression of hepatic CYCLIN D1 and c-MYC, tumor necrosis factor alpha (Tnfa) mRNA, serum levels of alanine aminotransaminase, and an increase in apoptotic signaling was also observed following ligand activation of PPARβ/δ in HBV mice compared to controls. Inhibition of Tnfa mRNA expression was not observed in wild-type hepatocytes. Ligand activation of PPARβ/δ inhibited lipopolysaccharide (LPS)-induced mRNA expression of Tnfa in wild-type, but not in Pparβ/δ-null Kupffer cells. Interestingly, LPS-induced expression of Tnfa mRNA was also inhibited in Kupffer cells from a transgenic mouse line that expressed a DNA binding mutant form of PPARβ/δ compared to controls. Combined, these results suggest that ligand activation of PPARβ/δ attenuates hepatic tumorigenesis in HBV transgenic mice by inhibiting steatosis and cell proliferation, enhancing hepatocyte apoptosis, and modulating anti-inflammatory activity in Kupffer cells.

  13. Enzyme assays.

    PubMed

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

    2009-01-07

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

  14. High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

    PubMed Central

    Lakaye, Bernard; Servais, Anne-Catherine; Scholer, Georges; Fillet, Marianne; Elias, Benjamin; Derochette, Jean-Michel; Crommen, Jacques; Wins, Pierre; Bettendorff, Lucien

    2012-01-01

    Background We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. PMID:22984449

  15. 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.

  16. Development of Activity-based Cost Functions for Cellulase, Invertase, and Other Enzymes

    NASA Astrophysics Data System (ADS)

    Stowers, Chris C.; Ferguson, Elizabeth M.; Tanner, Robert D.

    As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

  17. Improved complementary polymer pair system: switching for enzyme activity by PEGylated polymers.

    PubMed

    Kurinomaru, Takaaki; Tomita, Shunsuke; Kudo, Shinpei; Ganguli, Sumon; Nagasaki, Yukio; Shiraki, Kentaro

    2012-03-06

    The development of technology for on/off switching of enzyme activity is expected to expand the applications of enzyme in a wide range of research fields. We have previously developed a complementary polymer pair system (CPPS) that enables the activity of several enzymes to be controlled by a pair of oppositely charged polymers. However, it failed to control the activity of large and unstable α-amylase because the aggregation of the complex between anionic α-amylase and cationic poly(allylamine) (PAA) induced irreversible denaturation of the enzyme. To address this issue, we herein designed and synthesized a cationic copolymer with a poly(ethylene glycol) backbone, poly(N,N-diethylaminoethyl methacrylate)-block-poly(ethylene glycol) (PEAMA-b-PEG). In contrast to PAA, α-amylase and β-galactosidase were inactivated by PEAMA-b-PEG with the formation of soluble complexes. The enzyme/PEAMA-b-PEG complexes were then successfully recovered from the complex by the addition of anionic poly(acrylic acid) (PAAc). Thus, dispersion of the complex by PEG segment in PEAMA-b-PEG clearly plays a crucial role for regulating the activities of these enzymes, suggesting that PEGylated charged polymer is a new candidate for CPPS for large and unstable enzymes.

  18. Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21.

    PubMed

    Cornu, Marion; Oppliger, Wolfgang; Albert, Verena; Robitaille, Aaron M; Trapani, Francesca; Quagliata, Luca; Fuhrer, Tobias; Sauer, Uwe; Terracciano, Luigi; Hall, Michael N

    2014-08-12

    The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level.

  19. Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21

    PubMed Central

    Cornu, Marion; Oppliger, Wolfgang; Albert, Verena; Robitaille, Aaron M.; Trapani, Francesca; Quagliata, Luca; Fuhrer, Tobias; Sauer, Uwe; Terracciano, Luigi; Hall, Michael N.

    2014-01-01

    The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)–dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level. PMID:25082895

  20. Associations between PON1 enzyme activities in human ovarian follicular fluid and serum specimens

    PubMed Central

    Kim, Keewan; Fujimoto, Victor Y.; Browne, Richard W.

    2017-01-01

    The importance of high-density lipoprotein (HDL) particle components to reproduction is increasingly recognized, including the constituent paraoxonase 1 (PON1). However, the reliability characteristics of PON1 enzymes in ovarian follicular fluid (FF) as biomarkers for clinical and epidemiologic studies have not been described. Therefore, we characterized PON1 enzymes in FF and serum and assessed the impact of the PON1 Q192R polymorphism on associations between enzyme activities in two compartments. We also evaluated associations between HDL particle size and enzyme activities. We collected FF and serum from 171 women undergoing in vitro fertilization. PON1 activities were measured as paraoxonase and arylesterase activities, and HDL particle size was determined by 1H NMR spectrometry. Reliability indices for PON1 activities were characterized and we evaluated HDL particle sizes as predictors of PON1 enzyme activities. We found that PON1 enzyme activities were correlated between compartments, but higher in serum than in FF. For FF, the index of individuality (II) was low and the coefficient of variation (CV%) was high for paraoxonase activity overall (0.12 and 11.51%, respectively). However, IIs increased (0.33–1.30) and CV%s decreased (5.58%-8.52%) when stratified by PON1 Q192R phenotype. The intraclass correlation coefficient (ICC) for FF paraoxonase activity was high overall (0.89) but decreased when stratified by PON1 Q192R phenotype (0.43–0.75). We found similar, although more modest, patterns for FF arylesterase activity. For enzyme activities in serum, ICCs were close to 1.00 across all phenotypes. Additionally, different HDL particle sizes predicted PON1 enzyme activities according to PON1 Q192R phenotype. Overall, stratification by PON1 Q192R phenotype improved the reliability characteristics of FF PON1 enzymes as biomarkers for use in clinical investigations but diminished usefulness for epidemiologic studies. Thus, we recommend stratification by PON1 Q

  1. [Soil enzyme activities under two forest types as affected by different levels of nitrogen deposition].

    PubMed

    Zhao, Yu-tao; Li, Xue-feng; Han, Shi-jie; Hu, Yan-ling

    2008-12-01

    A simulation test was conducted to study the change trends of soil cellulase, polyphenol oxidase, and sucrase activities under natural broadleaf-Korean pine (Pinus koraiensis) and secondary poplar (Populus davidiana) -birch (Betula platyphylla) mixed forests as affected by 0, 25, and 50 kg x hm(-2) x a(-1) of N deposition. The results showed that the effects of elevated N deposition on test enzyme activities varied with forest type, and short-term nitrogen addition could significantly affect the test enzyme activities. High N deposition decreased soil polyphyneol oxidase activity, and correspondingly, soil cellulase and sucrase activities also had a trend of decrease.

  2. Effects of deep tillage and straw returning on soil microorganism and enzyme activities.

    PubMed

    Ji, Baoyi; Hu, Hao; Zhao, Yali; Mu, Xinyuan; Liu, Kui; Li, Chaohai

    2014-01-01

    Two field experiments were conducted for two years with the aim of studying the effects of deep tillage and straw returning on soil microorganism and enzyme activity in clay and loam soil. Three treatments, (1) conventional tillage (CT), shallow tillage and straw returning; (2) deep tillage (DT), deep tillage and straw returning; and (3) deep tillage with no straw returning (DNT), were carried out in clay and loam soil. The results showed that deep tillage and straw returning increased the abundance of soil microorganism and most enzyme activities. Deep tillage was more effective for increasing enzyme activities in clay, while straw returning was more effective in loam. Soil microorganism abundance and most enzyme activities decreased with the increase of soil depth. Deep tillage mainly affected soil enzyme activities in loam at the soil depth of 20-30 cm and in clay at the depth of 0-40 cm. Straw returning mainly affected soil microorganism and enzyme activities at the depths of 0-30 cm and 0-40 cm, respectively.

  3. Induction of antioxidant enzyme activity and lipid peroxidation level in ion-beam-bombarded rice seeds

    NASA Astrophysics Data System (ADS)

    Semsang, Nuananong; Yu, LiangDeng

    2013-07-01

    Low-energy ion beam bombardment has been used to mutate a wide variety of plant species. To explore the indirect effects of low-energy ion beam on biological damage due to the free radical production in plant cells, the increase in antioxidant enzyme activities and lipid peroxidation level was investigated in ion-bombarded rice seeds. Local rice seeds were bombarded with nitrogen or argon ion beams at energies of 29-60 keV and ion fluences of 1 × 1016 ions cm-2. The activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST) and lipid peroxidation level were assayed in the germinated rice seeds after ion bombardment. The results showed most of the enzyme activities and lipid peroxidation levels in both the argon and nitrogen bombarded samples were higher than those in the natural control. N-ion bombardment could induce higher levels of antioxidant enzyme activities in the rice samples than the Ar-ion bombardment. Additional effects due to the vacuum condition were found to affect activities of some antioxidant enzymes and lipid peroxidation level. This study demonstrates that ion beam bombardment and vacuum condition could induce the antioxidant enzyme activity and lipid peroxidation level which might be due to free radical production in the bombarded rice seeds.

  4. Effects of age, species difference, antibiotics and toxicants on intestinal enzyme activity and genotoxicity

    SciTech Connect

    Chadwick, R.W.; George, S.E.; Kohan, M.J.; Allison, J.C. . Health Effects Research Lab.); Chang, J.; Long, J.E.; Duffy, M.C. ); Dekker, J.P.; Forehand, L.R. )

    1993-08-01

    Altered intestinal enzyme activity significantly affects the biotransformation and toxicity of many xenobiotics. This article summarizes research, supported by the US Air Force Bioenvironmental Hazards Research Program, that employs a novel gas-liquid chromatographic assay to investigate the effects of age, species difference, antibiotics, and environmental chemicals on enzyme activity in various regions of the intestinal tract. Significant research findings include the following: (a) age-dependent alterations in enzyme activity in the gastrointestinal (GI) tract of the developing animal that suggest a changing susceptibility to toxicants during this period; (b) discovery of previously unreported mucosal enzymes in the small intestine that are present in germ-free rats and are not susceptible to antibiotics; (c) markedly greater intestinal nitroreductase activity and significantly higher bioactivation of the procarcinogen 2,6-dinitrotoluene (DNT) in CD-1 mice than in Fischer 344 rats; (d) significantly altered intestinal enzyme activity in rats pretreated with lindane, pentachlorophenol, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or Aroclor 1254; (e) potentiated DNT genotoxicity by Aroclor 1254 and pentachlorophenol pretreatment; and (f) a transient antagonism of DNT genotoxicity by 2,4,5-T pretreatment. Enzyme activity in the small intestine may have greater toxicological importance than previously thought in the biotransformation of environmental chemicals and as an indicator of change in the microbial flora.

  5. Purine enzyme activities in recent onset rheumatoid arthritis: are there differences between patients and healthy controls?

    PubMed Central

    Stolk, J N; Boerbooms, A M; De Abreu, R A; Kerstens, P J; de Koning, D G; de Graaf, R; Mulder, J; van de Putte, L B

    1996-01-01

    OBJECTIVE: Purine enzyme activities may predict the effectiveness of azathioprine treatment and be associated with increased deaths from infectious diseases. In rheumatoid arthritis, patients show variable responses to azathioprine and a higher percentage of death is caused by infections. The aim of the study was to investigate possible rheumatoid arthritis associated abnormalities of purine enzyme activities by measuring several of these enzymes in patients with recent onset rheumatoid arthritis before treatment with disease modifying antirheumatic drugs or prednisone. METHODS: 23 patients with recent onset rheumatoid arthritis and 28 healthy controls were studied. Activities of the enzymes 5'-nucleotidase, purine nucleoside phosphorylase (PNP), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and thiopurine methyltransferase (TPMT) were measured. Assessment of disease activity and blood sampling for routine measurements and HLA typing were done simultaneously. RESULTS: Purine enzyme activities did not differ between patients and healthy controls. Enzyme activities had no significant relations with indices of disease activity or rheumatoid factor titre or with the rheumatoid arthritis associated HLA types. Activity of 5'nucleotidase decreased with age (P < or = 0.05) and was lower by about 27% (P = 0.007) in males than in females. CONCLUSIONS: In rheumatoid arthritis patients, neither the variability in azathioprine effectiveness nor the increased death rate from infections can be explained by pre-existing abnormalities in the activities of the purine enzymes 5'-nucleotidase, PNP, HGPRT, or TPMT at an early stage of the disease, before disease modifying antirheumatic drugs or prednisone treatment. Besides adjustment for age, results of studies involving purine 5' nucleotidase activity should also be adjusted for sex. PMID:8984938

  6. Tropisetron Protects Against Acetaminophen-Induced Liver Injury via Suppressing Hepatic Oxidative Stress and Modulating the Activation of JNK/ERK MAPK Pathways

    PubMed Central

    Lee, Hung-Chen; Liao, Chia-Chih; Li, Allen H.

    2016-01-01

    Objectives. To investigate the protective effects of tropisetron on acetaminophen- (APAP-) induced liver injury